UNIVERSIDAD COMPLUTENSE DE MADRID · una gran variedad de patógenos, y distinguirlos de las...
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UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas Dpto. de Bioquímica y Biología Molecular I Marcadores de activación alternativa de macrófagos: DC-SIGN y FRβ Tesis Doctoral Elena Sierra Filardi Madrid, 2010
Transcript of UNIVERSIDAD COMPLUTENSE DE MADRID · una gran variedad de patógenos, y distinguirlos de las...
UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas
Dpto. de Bioquímica y Biología Molecular I
Marcadores de activación alternativa de macrófagos:
DC-SIGN y FRβ
Tesis Doctoral Elena Sierra Filardi
Madrid, 2010
UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas
Dpto. de Bioquímica y Biología Molecular I
Marcadores de activación alternativa de macrófagos:
DC-SIGN y FRβ
Este trabajo ha sido realizado por Elena Sierra Filardi para optar al grado de Doctor,
en el Centro de Investigaciones Biológicas de Madrid (CSIC), bajo la dirección del
Dr. Ángel Luis Corbí López.
Fdo. Dr. Ángel Luis Corbí López
A mis padres,
Rafael y María
“La felicidad humana generalmente no se logra con grandes golpes de suerte, que pueden ocurrir pocas veces, sino con pequeñas cosas que ocurren todos los días”
Benjamin Franklin
Agradecimientos
Pues sí, aunque parece mentira, por fin ha llegado el momento... Y lo primero que quiero es dar las
gracias a todas las personas que de una forma u otra me han ayudado a llegar hasta aquí.
Ante todo quiero dar las gracias a Ángel, por darme la oportunidad de formar parte de su equipo, de
realizar este trabajo y por toda su ayuda. A Vicente y a Luis, que me ayudaron en mis primeros contactos con el
mundo de la biología. Y a María, porque gracias a ella pude empezar este trabajo.
Y siguiendo el orden cronológico, tengo que dar las gracias a las personas que me ayudaron en mis
comienzos en el laboratorio. Esther, gracias por recibirme cada mañana con una sonrisa, así todo era más fácil.
Diego, gran experto en DC-SIGN, gracias por tu ayuda y tu compañía en tantos y tantos experimentos. Amaya,
gracias por tu ayuda y colaboración durante todos estos años.
Ahora toca el turno de mis compañeras actuales, por las que creo que he sido capaz de llegar hasta el
final. Ángeles, con la que llevo desde el principio y a la que he visto doctorarse, casarse y ahora ser mamá,...
cómo pasa el tiempo! Gracias por tu ayuda. Laura, gracias por tu apoyo y por tus ánimos, y por esas risas, que
al final es con lo que hay que quedarse. Ya pronto te toca a tí! Noemí, gracias por todo, por aguantarme en los
buenos y malos momentos, has sido un gran apoyo para mí. Sonia, gracias por tu ayuda en esta última etapa,
por tu paciencia y tus valiosos consejos.
También quiero dar las gracias a las personas con las que he compartido menos tiempo, a los llegados
recientemente, Mateo, Concha y María, y a los que han ido pasando durante estos años por el laboratorio, Idoia
y Rocío. Carmen, gracias por tus consejos y por tu dulzura. Tilman, gracias por poner siempre una sonrisa a la
vida aún en los momentos más duros, por escucharme y confiar en mí, y por todos los buenos momentos de las
comidas. Aún se te echa de menos!
A los vecinos, las Cristinas, Ángela y Miguel, por sus consejos y críticas en los seminarios. José Luis,
muchas gracias por estar siempre dispuesto a escuchar, por tus ánimos y por hacerme ver la parte positiva de
las cosas. Pilar, gracias por todo tu apoyo, tus ánimos y tus gestos de cariño cuando más se necesitan.
Y ahora a los de siempre, a los viejos amigos del CIB. Luque, qué decir de tí. Muchas gracias por todo,
por escucharme, por intentar hacerme ver que las cosas no son tan malas y, sobre todo, gracias por hacerme
reír tanto. José, poco tiempo pero valioso. Gracias también por esos momentos tan divertidos. A mis grandes
compañeras de gymkana, Marta y Leo, gracias por estar ahí todos estos años. No sé si os prefiero de pitufos,
de pulpo, de pez amarillo, o mejor tal y como sois. Y gracias a todas las personas que en estos años han
compartido alguna hora de comida conmigo (sois tantos que se me olvidaría algún nombre). Gracias a todos.
Y ahora a los últimos y no menos importantes. A mis padres, Rafael y María, gracias a vosotros he llegado
hasta aquí, no sólo en lo profesional, sino también y más importante, en lo personal. Esto es una pequeña
forma de agradeceros todo lo que habéis y seguís haciendo por mí. A mi hermana Mª Luisa y a mis sobrinos
Alejandro y Aitana, por todas las sonrisas que me habéis regalado. Y a Luis, el gran sufridor de mis gruñidos.
Gracias por escucharme y por intentar aconsejarme siempre, y sobre todo, gracias por tu paciencia!
Y a todos lo que están o estarían orgullosos de que haya llegado hasta aquí.
Gracias
Índice
ABREVIATURAS ......................................................................................................................... 1 INTRODUCCIÓN ......................................................................................................................... 5
1. El sistema inmunitario y sus componentes celulares .............................................................. 7
2. Monocitos ................................................................................................................................. 8
3. Células dendríticas.................................................................................................................... 9
4. Macrófagos ............................................................................................................................. 11
4.1 Diferenciación de macrófagos ................................................................................... 12
4.1.1 Citoquinas implicadas ................................................................................... 12
4.1.1.1 Macrófagos generados en presencia de GM-CSF y M-CSF .......... 13
4.1.1.2 Fenotipo y función de macrófagos M1 y M2 ................................... 14
4.1.2 Tejido-especificidad ...................................................................................... 16
4.1.2.1 Macrófagos intestinales ................................................................... 16
4.1.2.2 Macrófagos peritoneales ................................................................. 16
4.2 Activación de macrófagos .......................................................................................... 17
4.2.1 Activación clásica vs. activación alternativa ................................................. 17
4.2.2 Características fenotípicas de macrófagos activados .................................. 19
4.2.3 Macrófagos asociados a tumores ................................................................. 21
4.3 Estudios de expresión génica en diferenciación y activación de macrófagos ........... 23
5. El receptor de patógenos DC-SIGN ....................................................................................... 24
5.1 Expresión y localización tisular .................................................................................. 25
5.2 Estructura y dominios funcionales ............................................................................. 25
5.3 Estructura génica, isoformas y polimorfismos ........................................................... 26
5.4 Función y señalización ............................................................................................... 28
OBJETIVOS ............................................................................................................................... 31 RESULTADOS ........................................................................................................................... 35
1. El receptor de folato β se expresa en macrófagos asociados a tumores y constituye un
marcador de macrófagos anti-inflamatorios/reguladores M2 ..................................................... 39
2. Activina A previene la adquisición de marcadores anti-inflamatorios/M2 y sesga la
secreción de citoquinas por los macrófagos .............................................................................. 53
3. Requerimientos estructurales para la multimerización del receptor de patógenos DC-
SIGN (CD209) en la superficie celular ....................................................................................... 79
4. Identificación de epítopos en la molécula de DC-SIGN ......................................................... 99
Índice
DISCUSIÓN .............................................................................................................................. 111
El receptor de folato β es un marcador de macrófagos anti-inflamatorios M2 y TAM, cuya
expresión es regulada por activina A ....................................................................................... 113
Requerimientos estructurales de DC-SIGN para su multimerización. Influencia de la
presencia de variantes con menor tamaño en la región del cuello .......................................... 125
Identificación epítopos en la molécula de DC-SIGN ................................................................. 130
CONCLUSIONES ..................................................................................................................... 133 BIBLIOGRAFÍA ........................................................................................................................ 137 ANEXO ..................................................................................................................................... 157
Abreviaturas
Abreviaturas
Gran parte de las abreviaturas y acrónimos empleados en esta Tesis Doctoral proceden del inglés y como tal se han mantenido: AAMØ Alternative activated macrophage
APC Antigen presenting cell
CAMØ Classical activated macrophage CD Cluster of differentiation
CEA Carcinoembryonic antigen
CEACAM Carcinoembryonic antigen-related cell adhesion molecule
CLEC C-type lectin-like receptor
CLP Common lymphoid progenitor
CLR C-type lectin receptor
CMP Common myeloid progenitor
CRD Carbohydrate recognition domain
DAMP Danger-associated molecular patterns
DC Dendritic cell
DC-SIGN Dendritic cell-specific ICAM-3 grabbing nonintegrin
DC-SIGNR DC-SIGN related
DNA Deoxyribonucleic acid
ECD Extracellular domain
FR Folate receptor
FSH Follicle-stimulating hormone
G-CSF Granulocyte colony-stimulating factor
GM-CSF Granulocyte-macrophage colony-stimulating factor
HIV Human immunodeficiency virus HSC Hematopoietic stem cells
HTLV-1 Human T-cell lymphotropic virus type 1
ICAM Intracellular adhesion molecule
IFN Interferon
IL Interleukin
iNOS Inducible nitric oxide synthase
ITIM Tyrosine-based inhibitory motif
Lea, Leb, Lex, Ley LewisA, LewisB, LewisX, LewisY
LPS Lipopolysaccharide
M-CSF Macrophage colony-stimulating factor MDDC Monocyte-derived dendritic cell
MDM Monocyte-derived macrophage
MHC Major histocompatibility complex
MPS Mononuclear phagocyte system
3
Abreviaturas
4
MyD88 Myeloid differentiation primary response gene (88)
NFκB Nuclear factor-kappaB
NK Natural killer
NO Nitric oxide
NOD Nucleotide-binding oligomerization domain
PAMP Pathogen-associated molecular patterns
PBMC Peripheal blood-mononuclear cells
PPARγ Peroxisome proliferator-activated receptor gamma
PRR Pattern recognition receptor
RA Rheumatoid arthritis RNA Ribonucleic acid
RNS Reactive nitrogen species
ROS Reactive oxygen species SARS Severe acute respiratory syndrome
SCF Stem cell factor
TAM Tumor-associated macrophages
TCR T cell receptor
TGFβ Transforming growth factor-beta
Th T helper
TLR Toll-like receptor
TNFα Tumor necrosis factor-alpha
Introducción
Introducción
1. El sistema inmunitario y sus componentes celulares
La función esencial del sistema inmunitario es proteger al organismo de agentes infecciosos y
microorganismos presentes en el ambiente. Para ser eficaz, el sistema inmunitario debe detectar
una gran variedad de patógenos, y distinguirlos de las células y tejidos del propio organismo. En
vertebrados, en este sistema de defensa colaboran el sistema inmunitario innato y el sistema
inmunitario adaptativo [1].
El sistema inmunitario innato constituye la primera línea de defensa que limita la infección tras
la exposición a microorganismos, y proporciona una respuesta inmediata e inespecífica, pues
reconoce y responde a los patógenos de forma genérica y sin conferir inmunidad duradera contra
ellos [2]. Este sistema de defensa incluye componentes celulares (células epiteliales, células
dendríticas, macrófagos, neutrófilos y células NK), moléculas del sistema del complemento y
citoquinas. Sus células están equipadas con receptores de reconocimiento de patrones (PRR), que
reconocen patrones moleculares asociados a patógenos (PAMP) y señales endógenas asociadas a
daño tisular (DAMP). El sistema inmunitario innato es capaz de “activarse” únicamente frente a estas
“señales de peligro” detectadas por los PRR de forma específica [3]. Por contra, el sistema
inmunitario adaptativo genera respuestas antígeno-específicas y confiere memoria inmunológica tras
el primer contacto con el antígeno. La respuesta inmunitaria adaptativa está mediada por
componentes celulares (linfocitos T y B) y humorales (anticuerpos). Las células presentadoras de
antígeno (APC), y en especial células dendríticas y macrófagos, juegan un papel fundamental en la
conexión entre la inmunidad innata y la inmunidad adaptativa, ya que son las responsables de
procesar y presentar antígenos a los linfocitos T en el contexto de las moléculas del complejo de
histocompatibilidad (MHC) presentes en su superficie [1]. En consecuencia, el sistema de defensa
innato tiene como segunda función estimular y polarizar la respuesta inmunitaria adaptativa con
objeto de optimizar la eliminación del patógeno y minimizar los daños tisulares colaterales [4].
El sistema inmunitario de los vertebrados superiores está compuesto por gran variedad de
células funcionalmente diferentes que derivan de células madre hematopoyéticas (HSC) [5]. Las
HSC se renuevan a sí mismas y dan lugar a células progenitoras mieloides (CMP) y linfoides (CLP),
con potencial más limitado y que dan origen a granulocitos, monocitos, macrófagos, células
dendríticas y mastocitos [6], o linfocitos B y T, y células NK [7], respectivamente.
7
Introducción
2. Monocitos
Los monocitos se originan en la médula ósea a partir de un precursor mieloide y se liberan
posteriormente al torrente sanguíneo, donde constituyen un 10% de los leucocitos circulantes en
humanos [8]. Los monocitos de sangre periférica tienen una vida media relativamente corta (24-72
horas) [9], y contribuyen a la renovación de los macrófagos y células dendríticas tisulares [10]. Los
monocitos son heterogéneos en términos de morfología, marcadores de superficie y capacidad
fagocítica [11], y exhiben una elevada plasticidad en su proceso de diferenciación, que es tejido y/o
estímulo dependiente [12]. Como consecuencia, el fenotipo y las funciones efectoras de los
macrófagos residentes en los diferentes tejidos (macrófagos alveolares, células de Kupffer,
microglía, osteoclastos) varían considerablemente. La plasticidad del sistema de diferenciación
mieloide se refleja en la capacidad de “transdiferenciación” que exhiben los distintos tipos celulares
derivados de monocitos. Así, por ejemplo, los macrófagos pueden ser inducidos a adquirir
propiedades fenotípicas y funcionales de células dendríticas, mientras que las células dendríticas
derivadas de monocitos (MDDC) in vitro pierden sus funciones efectoras al retirar las citoquinas que
promueven su generación [13] (Figura 1). Dicha plasticidad también se refleja en procesos
fisiológicos como la resolución de la inflamación, donde la presencia de células apoptóticas facilita la
transformación de macrófagos citotóxicos/pro-inflamatorios en macrófagos promotores de
crecimiento/anti-inflamatorios encargados de reparar y limitar el daño tisular asociado al proceso
inflamatorio [14].
GM-CSF M-CSF
GM-CSF + IL-4/IL-13/IFNα IL-3 + IL-4
Célula dendrítica Macrófago
Monocito
IL-6 / IL-10 / IFNγ
IL-4
GM-CSF + IL-4
“cytokine remove” + M-CSF
Figura 1.- Diferenciación in vitro de monocitos. Esquema ilustrativo de la plasticidad y la estímulo-dependencia de la diferenciación de monocitos de sangre periférica.
Las citoquinas son el estímulo crítico para que los monocitos progresen hacia cada una de sus
alternativas de diferenciación. De hecho, la primera citoquina con la que los monocitos entran en
8
Introducción
contacto determina su programa de diferenciación y su perfil de respuesta a otras citoquinas [15]. La
diferenciación in vitro de monocitos a macrófagos o células dendríticas es un ejemplo de dicha
dependencia (Figura 1). Las citoquinas comúnmente empleadas para generar MDDC in vitro son
GM-CSF e IL-4 [16-18], mientras que los macrófagos se diferencian en presencia de GM-CSF o M-
CSF [19]. En humanos, IL-4 favorece la diferenciación a células dendríticas e impide la generación
de macrófagos [16, 20], mientras que la presencia de IL-6 limita la generación de estas células y
promueve la diferenciación a macrófagos de manera dependiente de M-CSF [21]. Por otra parte, el
entorno celular y la presencia de estímulos externos también condiciona la diferenciación del
monocito inducida por citoquinas [10].
Por lo que se refiere a factores de transcripción, el factor PU.1 junto con C/EBPα, RUNX1 y AP-
1, es crítico en la diferenciación monocítica, ya que ratones deficientes en PU.1 carecen de linaje
mielomonocítico, lo que es debido fundamentalmente a su papel esencial en la regulación de los
genes que codifican los receptores de GM-CSF, M-CSF y G-CSF [22].
3. Células dendríticas
En 1973 Ralph M. Steinman y Zanvil A. Cohn describieron un tipo celular presente en los
órganos linfoides periféricos de ratón y al que denominaron “célula dendrítica” (DC) [23].
Posteriormente las DC fueron identificadas como un componente minoritario de las células
mononucleares de sangre periférica (PBMC) en humanos [24], y se caracterizaron por ser las
células estimuladoras más potentes en cultivos leucocitarios mixtos y en la activación de linfocitos
citotóxicos [25, 26]. En la actualidad, las DC se consideran centinelas del sistema inmunitario y APC
“profesionales”, ya que son las únicas APC eficaces en la activación de linfocitos T naive, debido a
su elevada expresión de moléculas de MHC, coestimuladoras y de adhesión en su superficie. Las
DC son capaces de presentar antígenos exógenos en el contexto de MHC-II y MHC-I (“cross-
priming”), lo que justifica su capacidad de inducción de respuestas inmunitarias primarias [27].
En función de su linaje o de su estado de activación, las DC tienen la capacidad de iniciar una
respuesta inmunitaria o promover tolerancia [28]. Aún más, las DC determinan el tipo de respuesta
inmunitaria que se genera frente a un antígeno, pues son ellas quienes determinan la polarización
de los linfocitos Th naive hacia Th1 (productores de IFNγ y eficaces en la eliminación de patógenos
intracelulales), Th2 (productores de IL-4 y efectivos en la eliminación de patógenos extracelulares),
Th17 (productores de IL-17 e implicados en respuestas autoinmunes) o Treg (células T reguladoras
implicadas en procesos inmunosupresores) [29].
9
Introducción
Las DC humanas son una población heterogénea en cuanto a fenotipo, localización anatómica
y función, y se clasifican en dos grupos según su grado de parentesco con linajes celulares bien
establecidos: DC mieloides y DC plasmacitoides [30]. Las DC mieloides (CD11c+ CD123-) se
distribuyen prácticamente en todos los tejidos y se denominan de formas diversas dependiendo de
su localización tisular: células de Langerhans (en epidermis y mucosas), DC dérmicas, DC tímicas,
DC intersticiales (en casi la totalidad de órganos), etc. [29]. Las DC mieloides circulantes
representan sólamente un 0.5% de las PBMC totales [31]. Por el contrario, las DC plasmacitoides o
linfoides (CD11c- CD123+) proceden de progenitores distribuidos en el timo y en áreas T de los
órganos linfoides secundarios [32], y residen en nódulos linfáticos, bazo, timo, médula ósea y sangre
periférica [33]. Las DC plasmacitoides son importantes mediadores de la inmunidad anti-viral,
produciendo grandes cantidades de IFNα al ser estimuladas [34].
SSiisstteemmaa cciirrccuullaattoorriioo
Figura 2.- “Ciclo vital” de las células dendríticas. Las células dendríticas se diferencian a partir de progenitores de médula ósea que llegan a los tejidos a través del sistema circulatorio, y donde residen como DC inmaduras hasta que reciben señales que promueven su migración y maduración. Las DC maduras migran a los ganglios linfáticos, donde activan y polarizan a los linfocitos T naive hacia los diferentes tipos de células Th.
DIFERENCIACIÓN
Progenitores de médula ósea
DC de sangre periférica DC inmaduras
TTe
MMéédduullaa óósseeaa
ejjiiddoo
VVííaa lliinnffááttiiccaa
NNóódduulloo lliinnffááttiiccoo
MADURACIÓNMIGRACIÓN
DC maduras
Señales de peligro
Antígeno soluble
Célula T efectora
Célula T naive
10
Introducción
Las DC mieloides se originan a partir de progenitores de médula ósea, que generan
precursores circulantes cuya extravasación a los tejidos da lugar a las DC inmaduras residentes
(Figura 2). La elevada capacidad fagocítica de estas células les permite captar y procesar
contínuamente antígenos que son cargados en moléculas de MHC [30]. La detección de “señales de
peligro” a través de los receptores tipo Toll (TLR) y proteínas NOD hace que las DC “maduren” y
migren hacia los órganos linfoides secundarios. Durante ese trayecto, estas células disminuyen su
capacidad de captura y procesamiento de antígenos, y aumentan los niveles de expresión de
moléculas coestimulatorias y MHC en membrana. En las áreas T de los nódulos linfáticos, las DC
acaban interaccionando con linfocitos T que portan TCR específicos para los antígenos que las DC
capturaron en los tejidos de origen, iniciando así la respuesta inmunitaria adaptativa [35]. Las DC
maduras presentan antígenos a los linfocitos T CD8+ y CD4+, y estos últimos a su vez regulan a
otras células del sistema inmunitario, como células T citotóxicas CD8 y células B específicas de
antígeno, o células no específicas de antígeno como macrófagos, eosinófilos y células NK [36].
Como se ha comentado anteriormente, las DC están especializadas en la presentación de
antígeno a células T naive, y se diferencian de los macrófagos por su eficiente capacidad de
presentación de antígeno. Recientemente se ha planteado que las DC no constituyen una población
celular diferente de los macrófagos, ya que prodecen de un mismo precursor común, son sensibles
a los mismos factores de crecimiento, y no existen marcadores específicos ni funciones efectoras
únicas de las DC que justifiquen su distinción de los macrófagos [37].
4. Macrófagos
Metchnikoff, Premio Nobel de Fisiología y Medicina en el año 1908 por sus trabajos sobre el
sistema inmunitario, identificó células capaces de digerir partículas exógenas en el tubo digestivo de
las larvas de peces. A estas células las llamó “fagocitos”, y más tarde las definió como glóbulos
blancos integrantes de la primera línea de defensa contra las infecciones en los seres vivos [38]. El
término “macrófago” (MØ; del griego makros "grande" y phago "comer") fue asignado en 1924 por
Aschoff a un conjunto de células del sistema retículo-endotelial, que incluía monocitos, macrófagos,
histiocitos, fibroblastos, células endoteliales y células reticulares [39]. Posteriormente se reemplazó
este término por el de sistema fagocítico mononuclear (MPS), que comprende monoblastos y
promonocitos de médula ósea, monocitos de sangre periférica y macrófagos tisulares.
Los macrófagos juegan un papel crítico en el desarrollo de la respuesta inmunitaria, debido a
que actúan como primera barrera de defensa, al detectar y eliminar partículas “extrañas”
(microorganismos, macromoléculas tóxicas, células propias dañadas o muertas) mediante
11
Introducción
fagocitosis o secreción de enzimas, citoquinas o producción de especies reactivas de oxígeno
(ROS) y nitrógeno (RNS) [40]. Durante la respuesta inmunitaria adaptativa los macrófagos presentan
antígenos a los linfocitos T en el contexto de MHC-II y/o MHC-I, y colaboran con la respuesta
humoral en la eliminación de agentes extraños [41]. Además, los macrófagos tienen un papel
importante en procesos de reparación de heridas y resolución de la inflamación, promoviendo el
reclutamiento de otras células inflamatorias hacia los focos de inflamación, así como a remodelación
de matriz extracelular y angiogénesis. En consecuencia, el término “macrófago” agrupa una
multiplicidad de células cuya finalidad es el mantenimiento de la homeostasis y la integridad tisular
[12].
4.1 Diferenciación de macrófagos
Los macrófagos se originan a partir de HSC, y derivan en su mayoría de monocitos circulantes
que se extravasan a los tejidos por el influjo de citoquinas y quimioquinas [19]. A pesar de ello, un
pequeño porcentaje de macrófagos (aprox. 5%) derivan de la división local de fagocitos
mononucleares en los tejidos [42]. Como se comentó anteriormente, el fenotipo de los macrófagos
residentes en tejidos está determinado por el microambiente tisular, la matriz extracelular y los
productos de secreción y moléculas de superficie de las células próximas [8].
4.1.1 Citoquinas implicadas
Las principales citoquinas que determinan la supervivencia, diferenciación y quimiotaxis de los
macrófagos son GM-CSF, M-CSF e IL-3 [12] [43]. El M-CSF es sintetizado constitutivamente por
numerosos tipos celulares (macrófagos, células endoteliales, fibroblastos, osteoblastos, células del
estroma, etc.), y su concentración en suero oscila entre de 3-8 ng/ml [44]. Además, su producción es
inducida por la activación de células hematopoyéticas y fibroblastos con GM-CSF, TNFα [45], IL-1 e
IFNγ [46]. La síntesis de M-CSF es regulada de manera tejido-específica [43] y sus niveles son
elevados en estados de inmunosupresión (embarazo, tumores), siendo su papel importante en el
establecimiento de la tolerancia materna hacia el embrión [47]. A diferencia del GM-CSF, esta
citoquina juega un papel fundamental en el desarrollo mieloide, ya que ratones M-CSF-/- exhiben una
generación deficiente de macrófagos [48], mientras que los ratones GM-CSF-/- sólo muestran
alterada la maduración de macrófagos alveolares [49]. El receptor de M-CSF de alta afinidad (CSF-
1R, M-CSFR, c-fms, CD115) se expresa principalmente en células del linaje monocítico, como
monocitos, DC, macrófagos y sus precursores [43, 50].
12
Introducción
Por otro lado, el GM-CSF es producido por diferentes tipos celulares, incluyendo linfocitos T y
B, macrófagos, mastocitos, eosinófilos, neutrófilos y células endoteliales [43]. En condiciones
fisiológicas el GM-CSF se encuentra en suero a una concentración de 20-100 pg/ml y, aunque
puede ser producida constitutivamente por células tumorales, en la mayoría de los casos se requiere
activación de las células productoras [18]. El GM-CSF promueve viabilidad, proliferación y
maduración de precursores de neutrófilos, eosinófilos y macrófagos, y sus funciones dependen de
su concentración, ya que efectos en viabilidad celular requieren menores concentraciones que las
precisas para afectar a la proliferación celular [18]. Los efectos biológicos del GM-CSF están
mediados por el receptor de GM-CSF que, a diferencia del receptor homodimérico del M-CSF (M-
CSFR), está compuesto por una cadena α de unión a GM-CSF, y una cadena β necesaria para la
transducción de señales [51].
4.1.1.1 Macrófagos generados en presencia de GM-CSF y M-CSF
GM-CSF y M-CSF presentan una modulación cruzada de sus respectivas actividades
funcionales: mientras que el M-CSF aumenta la generación de macrófagos en presencia de bajos
niveles de GM-CSF [52], altas concentraciones de esta última impiden el desarrollo de macrófagos
mediado por M-CSF, debido a la acción inhibitoria de GM-CSF sobre la expresión de M-CSFR [53,
54]. Aunque los macrófagos humanos derivados de monocitos (MDM) diferenciados en presencia de
GM-CSF o M-CSF in vitro se consideran equivalentes a los macrófagos residentes en los tejidos en
condiciones homeostáticas [19], ambas citoquinas se usan indistíntamente en la generación in vitro
de MDM, dando lugar a poblaciones fenotípica y funcionalmente diferentes [19] (Figura 3). Así, en
presencia de GM-CSF se generan macrófagos, denominados M1, que producen citoquinas pro-
inflamatorias (IL-23, IL-12, IL-1β, IL-6, TNFα) en respuesta a Mycobacterium y promueven
inmunidad de tipo Th1 (pro-Th1) [55, 56]. Por contra, los macrófagos inducidos por M-CSF o M2
secretan IL-10 en respuesta a estímulos externos, inhiben respuestas Th1, y se han implicado en la
inducción de tolerancia [55-57]. Los macrófagos M2 actúan como moduladores de autoinmunidad,
ya que inducen células Treg e inhiben la diferenciación de linfocitos Th1 y Th17 [58]. Por todo ello,
los macrófagos M1 y M2 juegan papeles opuestos durante la respuesta inmunitaria, y son
considerados como macrófagos pro- y anti-inflamatorios, respectivamente (Figura 3). Del mismo
modo, GM-CSF y M-CSF se emplean para la generación in vitro de macrófagos a partir de
precursores de médula ósea de ratón, y sus propiedades pro- y anti-inflamatorias se ajustan a las de
los macrófagos M1 y M2 derivados de monocitos humanos [59, 60].
13
Introducción
Macrófago pro-inflamatorio (M1)
Macrófago anti-inflamatorio (M2)
IL-23, IL-12, IL-1β, IL-6, TNFα
GM-CSF
M-CSF
IL-10 Th1
Figura 3.- Macrófagos diferenciados en presencia GM-CSF y M-CSF. Esquema ilustrativo de los macrófagos generados en presencia de GM-CSF (M1 o pro-inflamatorios) o M-CSF (M2 o anti-inflamatorios) y sus diferencias en la respuesta inmunitaria.
4.1.1.2 Fenotipo y función de macrófagos M1 y M2
Además de diferencias en la producción de citoquinas en respuesta a LPS o Mycobacterium,
los macrófagos generados en presencia de GM-CSF (M1) y M-CSF (M2) tienen características
fenotípicas diferentes (Tabla 1). Los macrófagos M2 presentan una morfología elongada en forma
de huso, mientras los macrófagos M1 son más redondeados [19]. Por otro lado, los macrófagos M2
presentan mayor expresión de CD14, M-CSFR y del receptor “scavenger” CD163, mientras los
macrófagos M1 expresan mayores niveles de HLA-DQ y HLA-DR [19, 56]. Respecto a la expresión
de PRR, ambos tipos de macrófagos expresan niveles similares de TLR2 y TLR4, y la expresión de
DC-SIGN es baja pero significativa en macrófagos M1 y mayor en macrófagos M2 [56].
Desde el punto de vista funcional, ambas poblaciones de macrófagos también se comportan de
forma diferente (Tabla 1). Los macrófagos M2 presentan mayor capacidad de fagocitosis mediada
por receptores de Fcγ [61], mayor actividad fungicida debida a la producción de ROS [62], y mayor
producción de H2O2 en respuesta a estímulos fagocíticos [63]. Por su parte, los macrófagos
generados en presencia de GM-CSF tienen mayor capacidad de presentación de antígeno que los
macrófagos M2 [56]. Aunque ambos tipos de macrófagos son diana para la infección inicial por HIV-
1, convirtiéndose en reservorios virales, los macrófagos M2 tienen mayor capacidad de producción
de partículas virales, mientras que los macrófagos M1 inhiben la replicación viral a nivel post-
transcripcional [64].
14
Introducción
Características M1 M2
Antígenos de superficie CD11b ++ ++ CD11c ++ ++ CD14 - ++ CD71 + - CD163 - + CD209 - + HLA-DR ++ + HLA-DQ + - 710F + -
Receptores FcγR I (CD64) + + FcγR II (CD32) + + FcγR III (CD16) - + Receptor “scavenger” tipo A + + M-CSFR (c-fms) + +++ Integrinas αvβ3 αvβ5 Funciones Fagocitosis mediada por FcγR Débil Fuerte Producción de H2O2 Débil Fuerte Sensibilidad a H2O2 Resistente Sensible Actividad catalasa Alta Baja Susceptibilidad a HIV-1 Resistente Susceptible Susceptibilidad a M. tuberculosis Susceptible Resistente Producción de IL-10 Débil Fuerte
Tabla 1.- Características fenotípicas y funcionales de los macrófagos generados in vitro en presencia de GM-CSF (M1) o M-CSF (M2). [19, 56].
Otra de las diferencias existentes entre los macrófagos generados en presencia de GM-CSF y
M-CSF es la secreción de quimioquinas. Los macrófagos M2 sólo son capaces de producir CCL18
(PARC) tras estimulación, mientras que los macrófagos M1 secretan niveles constitutivos de CCL22
(MDC), CCL17 (TARC) y CCL18, que mantienen al ser estimulados [56]. A pesar de que los
macrófagos M2 producen niveles bajos de citoquinas pro-inflamatorias y altos niveles de IL-10 tras
estimulación, son capaces de secretar quimioquinas atrayentes de otros tipos celulares (neutrófilos,
monocitos y linfocitos T), lo contribuye a su fenotipo anti-inflamatorio/regulador. En ese sentido,
CXCL8 (IL-8) es producida tanto por macrófagos M1 como M2, mientras que sólo los macrófagos
M2 secretan constitutivamente CCL2 (MCP-1). A su vez, ambos tipos de macrófagos son capaces
de secretar CXCL10, CCL3 (MIP-1α), CCL4 (MIP-1β) y CCL5 (RANTES) tras estimulación con LPS
[56].
15
Introducción
4.1.2 Tejido-especificidad
La heterogeneidad y plasticidad funcional de los macrófagos se refleja en su especialización en
las diferentes localizaciones anatómicas [65]. Los macrófagos localizados en tejidos en contacto con
el entorno exterior (pulmón, placenta, mucosas intestinales) se encuentran continuamente expuestos
a patógenos y desafíos ambientales. Por ello existen mecanismos de inhibición “temporal” de las
funciones de estos macrófagos, lo que evita daños colaterales en el tejido y permite que sólo se
generen reacciones pro-inflamatorias cuando son absolutamente requeridas. Los macrófagos
peritoneales y los situados en el intestino son ejemplos de macrófagos que han desarrollado
estrategias para regular a la baja sus funciones efectoras [66].
4.1.2.1 Macrófagos intestinales
Los macrófagos del tracto digestivo se encuentran estratégicamente localizados en la lámina
propia [67], y en tejidos linfoides secundarios asociados al sistema digestivo, como amígdalas y
placas de Peyer [68]. Funcionalmente, los macrófagos intestinales carecen de actividad
presentadora de antígeno y actividad “respiratory burst”, pero poseen gran capacidad fagocítica y
bactericida [69]. Estas células tienen reducida la producción de citoquinas pro-inflamatorias debido a
la inhibición de NFκB por el TGFβ liberado por las células del estroma [70]. Este estado de falta
parcial de respuesta a estímulos externos ha sido definido como “anergia inflamatoria”, y explica la
incapacidad de los macrófagos intestinales de mediar en la inflamación de la mucosa [71]. De
hecho, en pacientes con enfermedad inflamatoria intestinal se han descrito alteraciones en la vía de
señalización de TGFβ, lo que hace que un gran porcentaje de macrófagos sean capaces de liberar
citoquinas pro-inflamatorias [72, 73]. En consecuencia, los macrófagos intestinales son un claro
ejemplo de macrófagos anti-inflamatorios in vivo [70].
4.1.2.2 Macrófagos peritoneales
En humanos, la concentración de M-CSF en el fluido peritoneal es muy elevada y se
correlaciona con el número de macrófagos peritoneales [74]. Estudios realizados con macrófagos
aislados de muestras de diálisis peritoneal han mostrado que dichas células son fenotípica y
funcionalmente similares a los macrófagos anti-inflamatorios generados in vitro, por cuanto exhiben
alta capacidad de fagocitosis, endocitosis y macropinocitosis, producción de elevadas cantidades de
IL-10 tras estimulación, y una disminuida capacidad de estimulación de células T [75].
16
Introducción
4.2 Activación de macrófagos
4.2.1 Activación clásica vs. activación alternativa
La variedad de estímulos de activación/desactivación de macrófagos [43], combinado con la
heterogeneidad y plasticidad de los macrófagos residentes en tejidos en condiciones homeostáticas,
permite la existencia de numerosos estados de activación de macrófagos [8]. Así, el IFNγ producido
por células Th1, T citotóxicas CD8+ y células NK, convierte a los macrófagos en células con elevada
capacidad citotóxica, microbicida (especialmente de patógenos intracelulares) y anti-proliferativa. La
adquisición de estas propiedades es debida a la producción de mediadores tóxicos (ROS, RNS) y
citoquinas pro-inflamatorias [75]. Este tipo de activación, denominada clásica (CAMØ, M1) [76], da
lugar a macrófagos que secretan altos niveles de IL-12 e IL-23 y muy bajos niveles de IL-10 en
respuesta a Mycobacterium [77], y promueven fuertes respuestas inmunitarias Th1 (Figura 4).
Activación clásica (CAMØ)
IL-12
IFNγ
NK
IL-4
Th2 Th1
IL-13
IL-10
Activación alternativa (AAMØ) Basófilo
Eosinófilo NK
Figura 4.- Tipos de activación de macrófagos. Representación esquemática de la activación de macrófagos mediante estimulación con IFNγ (activación clásica) o citoquinas Th2 como IL-4 e IL-13 (activación alternativa).
Las funciones inflamatorias y citotóxicas de los macrófagos activados contribuyeron a la
percepción de que sólo citoquinas Th1 promovían activación de macrófagos, mientras que
citoquinas de tipo Th2 las bloqueaban o desactivaban [78]. Sin embargo, además de inhibir
respuestas Th1, las citoquinas Th2 provocan un aumento de las funciones de los macrófagos como
presentación de antígeno, reparación tisular y capacidad endocítica [77]. Por ello, los factores que
inhiben la generación y actividad de los CAMØ (citoquinas Th2 como IL-4 e IL-13, citoquinas
desactivadoras como IL-10 y TGFβ, hormonas como glucocorticoides y la vitamina D3), e incluso las
células apoptóticas, han sido agrupados como inductores de una forma “alternativa” de activación de
macrófagos (AAMØ, M2) [77] (Figura 4). Los AAMØ producen grandes cantidades de IL-10 y TGFβ
y niveles muy bajos de IL-12 bajo estimulación [79], y pueden presentar funciones
inmunosupresoras e inhibir la proliferación de células T [80].
17
Introducción
Las diferencias en las funciones de CAMØ y AAMØ han sido demostradas en numerosos
ensayos in vitro, donde los AAMØ inducen mayor proliferación celular y deposición de colágeno de
células fibroblásticas [81], e inhiben la proliferación de linfocitos inducida por mitógenos [82]. Al
mismo tiempo, los AAMØ contribuyen a la vascularización in vivo y exhiben actividad angiogénica in
vitro [83], similar a la de MDDC maduras en presencia de citoquinas como IL-10, TGFβ, o
glucocorticoides [84]. Por otro lado, existen numerosos estudios que ponen de manifiesto que los
AAMØ activados con IL-4 son esenciales en la eliminación y control de la infección por patógenos
extracelulares [77].
Aunque el término AAMØ fue inicialmente propuesto para identificar exclusivamente a
macrófagos activados por IL-4/IL-13 [85], la variedad de estímulos anti-inflamatorios que provocan
una activación “no clásica” de macrófagos ha hecho necesario establecer una nomenclatura más
precisa. Mantovani y colaboradores han clasificado estas formas de activación alternativa de
acuerdo con el estímulo inductor: los macrófagos estimulados por las citoquinas Th2 IL-4/IL-13 son
denominados M2a, los activados por complejos inmunes y ligandos de TLR son denominados M2b,
y los macrófagos activados en presencia de IL-10 son denominados M2c [86] (Figura 5, izquierda).
Recientemente se ha propuesto otra clasificación de macrófagos activados de acuerdo con sus
funciones en el mantenimiento de la homeostasis: macrófagos involucrados en la defensa del
organismo, en reparación de heridas y en regulación inmunitaria. Sin embargo, es preciso enfatizar
que además de estos tres grupos es posible definir numerosos estados funcionales intermedios, lo
que avala la existencia de un amplio rango de estados de activación de macrófagos [87] (Figura 5, derecha).
M1
M2b
M2a
M2c
INFγ IL-4
IL-13
LPS
Inmuno-complejos
IL-10
TGFβ
Defensa
Reparación de heridas Reguladores
Figura 5.- Propuestas de clasificación de macrófagos activados. Los macrófagos polarizados se pueden clasificar en función del estímulo de activación (izquierda) [86] o de su función efectora primordial (derecha) [87]. Los tres colores primarios (rojo, amarillo, azul) representan las tres poblaciones de macrófagos definidas, mientras que los colores secundarios representan macrófagos con funciones intermedias.
18
Introducción
4.2.2 Características fenotípicas de macrófagos activados
Los macrófagos activados presentan diferentes propiedades fenotípicas y funcionales en
función del estímulo de activación (Figura 6). Aunque los macrófagos M2a y M2b exhiben niveles de
expresión de moléculas de adhesión (CD11a, CD54, CD58) y coestimuladoras (CD40, CD80, CD86)
similares a los CAMØ, la activación alternativa de macrófagos en respuesta a IL-4 e IL-13 va unida a
la adquisición de un repertorio de receptores fagocitarios característicos. Estos receptores dotan a
los macrófagos M2a de potentes actividades endocitotóxicas y fagocíticas, y entre ellos son
destacables: 1) el receptor de manosa (MR1, CD206), cuya señalización intracelular está asociada a
la producción de IL-10, la expresión de IL-1Rα, y a la inhibición de la producción IL-12 en respuesta
a endotoxina [79, 88]; 2) el receptor “scavenger” 1 de macrófagos (MSR1, CD204), con un claro
papel en el reconocimiento y eliminación de lipoproteínas [89]; 3) el receptor de β-glucanos Dectin-1,
con especificidad por glucanos β-1,3 y β-1,6, típicos de hongos y algunas bacterias, y que colabora
funcionalmente con TLR2 en la respuesta inflamatoria anti-fúngica [88, 90]; y 4) DC-SIGN, con un
amplio espectro de reconocimiento de patógenos [88, 91, 92] (Figura 6). Otros marcadores de
activación alternativa de macrófagos humanos son DCIR y DCL-1 [93-95], CD23 [77] y el receptor
“scavenger” CD163 [85].
M1
M2b
M2a
M2c
MHCII
CD16CD32CD64
TLR2TLR4
CD163
CD163
Dectin-1DC-SIGN
Crecimiento tumoralInmunoregulaciónDeposición de matrizRemodelación de tejido
Respuesta Th2Inmunoregulación
Crecimiento tumoralRespuesta Th2AlergiaEliminación de parásitosEncapsulación de parásitos
Resistencia al tumorRespuesta Th1Eliminación de patógenos intracelulares
CD80CD86 CD23CCL2-5
CCL11CCL17CCL22CXCL1-5CXCL8-11CXCL16
IL-1IL-6IL-12IL-23TNFα
IL-1IL-6IL-10TNFα
CCL1CCL11CCL17CCL22CCL24
CCL11CCL17CCL18CCL22CCL24
CCL16CCL18CXCL13
IL-10
IL-10TGFβ
CD206MSR1
Figura 6.- Características fenotípicas y funcionales de macrófagos polarizados en función del estímulo de activación. En el dibujo se representan las principales características fenotípicas (secreción de quimioquinas (cuadro gris) y citoquinas (cuadro amarillo), y expresión de receptores de membrana), así como las características funcionales que diferencian los diversos estados de polarización de macrófagos.
19
Introducción
La expresión de genes que controlan el metabolismo celular también se utiliza para discernir
entre los diferentes tipos de macrófagos activados. Así, la expresión de genes que participan en el
metabolismo de la arginina, permite diferenciar CAMØ y AAMØ en ratón, pero no en macrófagos
humanos [96, 97]. La arginasa 1 (Arg1) es un marcador prototípico de activación alternativa, ya que
su expresión es dependiente de IL-4/IL-13, mientras que la óxido nítrico sintasa (iNOS) es inducida
por IFNγ. Los CAMØ metabolizan arginina vía iNOS, generando óxido nítrico, que posee elevada
actividad microbicida. Por el contrario, la expresión de Arg1 permite a los AAMØ producir poliaminas
y prolina, que son esenciales para la proliferación celular y la producción de colágeno,
respectivamente [98]. Otros marcadores de AAMØ en ratón, y que carecen de homólogos en
humanos, son los miembros de la familia quitinasa Ym1 y Ym2 (Chi3l3 y Chi3l4), y Fizz1,
involucrado en el metabolismo de lípidos [99].
Por otro lado, la polarización del macrófago hacia un fenotipo alternativo lleva asociada un
aumento en la expresión de genes relacionados con el metabolismo de lípidos, especialmente de
aquellos implicados en la captación y oxidación de ácidos grasos [100]. Así, además de Fizz1, Stab-
1 y la lipoxigenasa ALOX15 presentan mayor expresión en AAMØ [77, 93]. A diferencia de AAMØ,
los CAMØ sobre-expresan genes involucrados en el metabolismo del colesterol como ABCA1 y
apolipoproteínas L (APOL1-3,6), involucrados en su transporte y en el desarrollo de aterosclerosis
[93, 101]. A su vez, genes que codifican para las enzimas implicadas en el metabolismo de
mediadores lipídicos (eicosanoides, leucotrienos, esfingosina y ceramida) también se expresan
diferencialmente entre CAMØ y AAMØ. Más concretamente, la expresión de COX-2 está asociada
con el metabolismo de ácido araquidónico en CAMØ, mientras que las enzimas esfingosina y
ceramida quinasas, que catalizan el equilibrio ceramida-esfingosina, están más expresadas en
CAMØ y AAMØ, respectivamente [93].
El receptor PPARγ, y alguno de sus genes diana (FABP4), también se incluyen dentro de los
genes con mayor expresión en AAMØ, ya que IL-4 es un inductor de este receptor y de sus
activadores metabólicos [102]. Los ratones deficientes en PPARγ tienen disminuidos los niveles de
mRNA y la actividad de Arg1, no presentan macrófagos con fenotipo alternativo y, dado su papel en
el metabolismo de ácidos grasos, tienen mayor tendencia a la obesidad [103]. Además, se ha
descrito a PPARγ como regulador negativo de la activación clásica del macrófago [104]. Por tanto,
PPARγ regula las respuestas dependientes de IL-4, y es requerido para la adquisición y
mantenimiento del fenotipo alternativo en macrófagos activados [103].
Mientras que PPARγ es un factor crítico para la activación alternativa inducida por IL-4, la
activación de los factores de transcripción NFκB, STAT-1 y AP-1 son esenciales para la polarización
clásica del macrófago [105]. Estímulos inflamatorios como LPS, activan rutas de señalización
dependientes de MyD88, que llevan a la activación de NFκB y AP-1, y rutas independientes de este
adaptador intracelular, con la activación de IRF3 y STAT-1 [106]. Por el contrario, la IL-10 liberada
20
Introducción
por algunos AAMØ inhibe la activación de NFκB y mantiene su fenotipo inmunosupresor [107-109].
De hecho, la pérdida de expresión de IRF3, STAT-1 y NFκB en macrófagos derivados de médula
ósea de ratón está asociada a la supresión de la polarización pro-inflamatoria [110].
4.2.3 Macrófagos asociados a tumores
Los macrófagos asociados a tumores (TAM) constituyen un ejemplo paradigmático de la
plasticidad del proceso de activación de macrófagos y de su repercusión fisiológica y patológica. En
los tumores existe una gran infiltración de leucocitos inflamatorios [111], cuyo estado de maduración
y localización espacial determina su influencia sobre el tumor. Los macrófagos son el componente
mayoritario de dicho infiltrado tumoral [112], y constituyen un claro ejemplo de activación alternativa
patológica de macrófagos.
Los TAM se originan a partir de monocitos de sangre periférica reclutados hacia el tumor, en su
fase inicial de formación, por factores como M-CSF, MCP-1, VEGF y Angiopoietina-2 [113-117]
(Figura 7). La diferenciación intratumoral da lugar a macrófagos con niveles reducidos de receptores
de quimioquinas, lo que evita su migración desde los tejidos tumorales. Los TAM regulan varios
pasos clave en el desarrollo del tumor, y su abundancia se correlaciona con la progresión tumoral,
remodelación de matriz extracelular, estimulación de la proliferación, migración e invasión de las
células cancerosas, e inhibición de la inmunidad adaptativa (inmunosupresión) [117]. La elevada
densidad de macrófagos en zonas metastáticas, como los nódulos linfoides regionales, favorece el
crecimiento del tumor [113].
Anergia, supresión, respuesta Th2
TAM Célula tumoral
IL-10, TGFβ
Factores de crecimiento
Reclutamiento/ supervivencia
M-CSF, VEGF, MCP-1
VEGF, FGF, TGFβ Quimioquinas
IL-10, TGFβ
Angiogénesis y remodelación de matriz
MMP-9, uPA
Figura 7.- Interacción entre macrófagos y células tumorales. Las células tumorales secretan factores que atraen y determinan la polarización de los macrófagos en los tumores. A su vez, los TAM producen factores de crecimiento que promueven angiogénesis y remodelación del tejido, y contribuyen a la progresión y diseminación del tumor [118].
21
Introducción
El fenotipo y función de los TAM está determinado por los factores microambientales presentes
en el tumor [118, 119] (Figura 7). Citoquinas y factores de crecimiento derivadas del tumor (IL-10,
TGFβ, M-CSF, VEGF, MCP-1) aumentan la generación de macrófagos y reducen la diferenciación
de DC y, en consecuencia, determinan los niveles relativos de APC en el tumor y en los tejidos
cercanos [21]. Junto con TGFβ, M-CSF es el mayor responsable del ambiente inmunosupresor
intratumoral [111]. De hecho, en un modelo de carcinoma mamario espontáneo, los ratones M-CSF-/-
presentan una progresión tumoral más lenta que los ratones normales [120]. La IL-10 presente en el
tumor induce en los TAM la adquisición de funciones asociadas a macrófagos M2 [121]. Por ello, los
TAM tienen reducida la capacidad de producir moléculas anti-tumorales (TNFα, IL-1, ROS, NO) y
citoquinas inflamatorias (IL-12, IL-1β, TNFα, IL-6) [122], y no presentan activación de NFκB [111].
La producción de mediadores inmunosupresores (prostaglandinas, IL-10 y TGFβ) permite a los
TAM inducir la diferenciación de células Treg, que suprimen la actividad de los linfocitos T efectores
y de otras células inflamatorias [111], favoreciendo por tanto el crecimiento tumoral [123, 124]. La
actividad angiogénica del tumor está asimismo favorecida por la acumulación de TAM en regiones
de hipoxia poco vascularizadas, a las que se adaptan por la activación de factores como HIF-1 y
HIF-2 [125]. Los TAM también promueven angiogénesis a través de la liberación de factores de
crecimiento (VEGF, FGF y HGF), metalo-proteasas (MMP-9) y el activador de plasminógeno (uPA),
todos los cuales contribuyen a degradar la matriz extracelular, facilitando por tanto la migración e
invasión de células tumorales [126] (Figura 7).
La expresión de marcadores típicos de macrófagos M2 de ratón como Arg1, Ym1, Fizz1 y Mgl2
se observa en TAM procedentes de fibrosarcoma y de linfoma T BW-Sp3, lo que corrobora el
fenotipo alternativo de estos macrófagos [127, 128]. Sin embargo, en ese mismo modelo se
observan también altos niveles de quimioquinas Th1 como CCL5, CXCL9 y CXCL10, lo que sugiere
la desviación de las características típicas de macrófagos M2 [127]. Aunque los TAM son
considerados macrófagos con fenotipo anti-inflamatorio por su secreción de citoquinas y la deficiente
activación de NFκB, también contribuyen a la angiogénesis y crecimiento tumoral mediante la
secreción de mediadores típicos de macrófagos M1 y reguladores de NFκB, como TNFα, IL-1β y
MMP-9. Por otro lado, en un estado tumoral avanzado, los TAM de ratón expresan constitutivamente
NOS2 y Arg1 que, implicados en el metabolismo de la arginina, producen liberación de NO y
aumento en la producción de ROS (O2- y H2O2) y RNS (ONOO-), deteniendo la proliferación y,
eventualmente, provocando la muerte de células T [116]. En consecuencia, los TAM son capaces de
expresar características pro-inflamatorias y supresoras, existiendo un equilibrio en su polarización
entre un fenotipo M1 y M2. Esta versatilidad en el fenotipo de los TAM es posiblemente debida al
cambio dinámico existente en el microambiente tumoral desde eventos tempranos hasta los estados
avanzados del tumor, y está regulada por mecanismos moleculares, como la modulación de la
actividad de NFκB o las vías de señalización activadas por hipoxia [129]. En los casos que la
22
Introducción
presencia de TAM se correlaciona con un buen pronóstico del tumor, el GM-CSF podría ser
responsable de la adquisición de un fenotipo citotóxico por los macrófagos intratumorales [130].
4.3 Estudios de expresión génica en diferenciación y activación de macrófagos
La identificación de genes diferencialmente expresados en distintas poblaciones de macrófagos
activados permite determinar su papel en la adquisición de un fenotipo de polarización concreto, y
su posible participación en determinados procesos celulares o fisiológicos [131-133]. En este
sentido, estudios realizados en macrófagos peritoneales tratados con IL-4 han permitido identificar
marcadores de activación alternativa de macrófagos en ratón, como Ym1 y Arg1 [134]. La expresión
diferencial de estos genes dependientes de IL-4 se ha corroborado en un modelo de infección con el
nematodo Brugia malayi [135]. La identificación de genes asociados a los diferentes estados de
polarización de macrófagos puede proporcionar nuevas dianas terapéuticas en patologías
inflamatorias y/o autoinmunes.
Respecto a los estudios realizados en macrófagos humanos polarizados en presencia de
citoquinas, Mantovani y colaboradores han determinado los cambios génicos inducidos en la
diferenciación de monocitos CD14+ en presencia de M-CSF, y las diferencias existentes entre
macrófagos polarizados por LPS e IFNγ o IL-4 [93]. Posteriormente, se han identificado genes cuya
expresión se modifica en monocitos expuestos a GM-CSF o GM-CSF e IL-4 [136], o a estímulos
“alternativos” como IL-13 [101] o IL-10 [137]. Por otro lado, Hamilton y colaboradores han analizado
macrófagos de ratón generados en presencia de GM-CSF (M1) o M-CSF (M2), y han evidenciado la
contribución de IFN de tipo I en las diferencias fenotípicas de ambas poblaciones [138]. Según estos
autores, la expresión diferencial de citoquinas y quimioquinas en respuesta a LPS se justifica porque
la señalización desde TLR4 se lleva a cabo de forma distinta en ambos tipos de macrófagos, por la
ruta MyD88-independiente (caso de los M2) o MyD88-dependiente (en los M1) [138].
Estos estudios de expresión génica han permitido identificar marcadores moleculares asociados
a respuestas inmunitarias frente a infecciones bacterianas [139, 140], patologías como la
enfermedad pulmonar obstructiva crónica (EPOC), y el desarrollo de tumores [141, 142]. Por otro
lado, estudios realizados sobre la interacción macrófago-patógeno han identificado estrategias de
defensa del hospedador y de evasión por parte del patógeno [143]. En consecuencia, todas estas
aproximaciones han hecho posible diseccionar la polarización de macrófagos frente a estímulos
patogénicos concretos, lo que ha permitido establecer que los procesos de activación/polarización
de macrófagos y de maduración de MDDC son específicos del estímulo que los provoca [140, 141,
144].
23
Introducción
5. El receptor de patógenos DC-SIGN
Las lectinas son proteínas que reconocen de manera específica carbohidratos presentes en
antígenos propios y patógenos [145]. En vertebrados, las lectinas se clasifican en diferentes
subgrupos, siendo los receptores lectina de tipo C (CLR) uno de los mejor estudiados. Los CLR se
caracterizan por tener al menos un dominio de reconocimiento de carbohidratos (CRD) a través del
cual unen carbohidratos de forma dependiente de Ca2+ [146]. Los CLR pueden ser proteínas
solubles o proteínas transmembrana, y se han definido siete sub-grupos en función de su homología
de secuencia, estructura y disposición del CRD respecto al resto de la molécula [147] (Tabla 2). Los
grupos I, III y VII engloban lectinas solubles, mientras que el resto de grupos corresponden a
lectinas de membrana, que a su vez pueden ser proteínas de tipo I, como el receptor de manosa
(MR), o de tipo II, como DC-SIGN [148].
Grupo Moléculas representativas Características
I Agrecano, versicano, neurocano Proteoglicanos, glicoproteínas de matriz extracelular
II Receptor de asialoglicoproteína, CD23, DC-SIGN, LSECtin Receptores de membrana tipo II
III Proteína de unión a manosa, SP-A, SP-D Colectinas. Oligómeros asociados por un dominio tipo colágeno. Extracelulares y solubles
IV Selectinas L, P y E Glicoproteínas de membrana de tipo I, implicadas en adhesión leucocitaria
V NKG2, LY49, CD69 Antígenos linfocitarios de tipo II
VI Receptor de manosa, DEC-205 Receptores de membrana de tipo I con varios CRD extracelulares
VII Proteína asociada a pancreatitis/hepatoma Extracelulares y solubles
Tabla 2.- Clasificación de las lectinas de tipo C.
DC-SIGN (“Dendritic cell-specific ICAM-3 grabbing nonintegrin”, CD209, CLEC4L) fue descrito
en 1992 por Curtis y colaboradores como una lectina de tipo C que reconoce la proteína gp120 de la
envuelta del HIV-1 [149]. Posteriormente se caracterizó como un receptor presente en MDDC que
participa en la interacción DC-célula T mediante el reconocimiento de la molécula de adhesión
intracelular ICAM-3 [150]. En la actualidad y, como se ha mencionado anteriormente, DC-SIGN
constituye un marcador de macrófagos anti-inflamatorios M2 y AAMØ [56, 151].
24
Introducción
5.1 Expresión y localización tisular
Aunque descrita como específica de células dendríticas, DC-SIGN no sólo se expresa in vivo en
DC de tejidos periféricos y linfoides [152], sino que también se expresa en poblaciones CD14+ de
sangre periférica [153] y en determinadas subpoblaciones de macrófagos presentes en sinusoides
medulares de nódulos linfáticos [151], intestino [154], pulmón [152], placenta [152, 155, 156] y
macrófagos sinoviales [157]. La expresión de DC-SIGN se induce in vitro por IL-4 en monocitos [92,
150], en macrófagos [91, 158] y en la línea celular mieloide THP-1 [91], y sus niveles de expresión
son controlados por el factor de transcripción PU.1 [159]. Estudios de localización subcelular han
situado a DC-SIGN en “lipid rafts”, microdominios de membrana ricos en colesterol y esfingolípidos,
lo que puede favorecer a su capacidad de unión e internalización de partículas víricas, así como a
su capacidad señalizadora tras el reconocimiento de ligandos [160, 161].
5.2 Estructura y dominios funcionales
Estructuralmente, DC-SIGN es una proteína transmembrana tipo II de 404 aminoácidos, cuya
región extracelular incluye un CRD, un cuello o “stalk” que le separa de la zona transmembrana, y
con una corta región citoplásmica de 42 aminoácidos [162] (Figura 8).
DOMINIO FUNCIÓN ESTRUCTURAL
Figura 8.- Estructura y función de los dominios de DC-SIGN. Ct, extremo carboxilo-terminal; Nt, extremo amino-terminal; NLT, motivo de glicosilación; EEE, dominio triacídico; LL, motivo dileucina; Y, tirosina del motivo YKSL.
YLL
EEE
Y
LL EEE
Y
LL EEE
Nt
NLTNLT
NLT
Ct
Dominio Interacción lectina con ligandos
Cuello Multimerización(dominios
repetidos)
Internalización,Dominio tráfico y citoplásmico señalización
25
Introducción
El CRD de DC-SIGN es una estructura globular que consta de 12 cadenas β, 2 hélices α y 3
puentes disulfuro, además de 2 sitios de unión a Ca2+ [162] . Uno de esos sitios es esencial para la
conformación del CRD, mientras que el otro participa en la interacción con los ligandos
carbohidratados y determina su especificidad. La secuencia de aminoácidos de este segundo sitio
contiene un motivo EPN que confiere a DC-SIGN especificidad por manosa. El cuello de DC-SIGN
está compuesto por 8 dominios repetidos de 23 aminoácidos ricos en leucinas, el primero de los
cuales contiene un motivo de glicosilación (NLT) (Figura 8) [163]. Esta región es fundamental para
la formación de estructuras multiméricas, más concretamente tetrámeros, lo que incrementa
considerablemente la avidez de interacción de DC-SIGN por sus ligandos [164-166]. La región
transmembrana comprende desde Leu43 a Ser61 [163]. La zona amino-terminal constituye la cola
citoplásmica, que posee un motivo dileucina (LL) que promueve la rápida internalización de DC-
SIGN tras interaccionar con ligandos solubles, un motivo triacídico (EEE), que determina que los
complejos DC-SIGN-ligando sean dirigidos a compartimentos lisosomales [163, 167], y un motivo
basado en tirosina (ITIM-like), que capacita a esta lectina para transmitir señales intracelulares [161]
(Figura 8).
5.3 Estructura génica, isoformas y polimorfismos
El gen de DC-SIGN mapea en la región p13 del cromosoma 19, y consta de 7 exones [168]
(Figura 9). Los exones 1a y 1c codifican la cola citoplásmica, el exón 3 codifica la región del cuello,
y los exones 4, 5 y 6 codifican el CRD [163]. En ratón no existe un gen ortólogo de DC-SIGN
humano, aunque existen moléculas homólogas dentro de la familia SIGN: mDC-SIGN (murine DC-
SIGN) o SIGNR5, SIGNR1 (SIGN related), SIGNR2, SIGNR3, SIGNR4, el pseudogen SIGNR6,
SIGNR7 y SIGNR8 [169, 170].
El gen de DC-SIGN está sometido a un complejo sistema de “splicing” alternativo, que origina
un gran número de transcritos con estructuras diferentes de la prototípica [168]. Entre estas
variantes se incluyen isoformas con una cola citoplásmica alternativa, isoformas sin región
transmembrana e isoformas con CRD incompletos, así como una gran variedad de transcritos con
un número variable de repeticiones en la región del cuello. El patrón de isoformas y los niveles de
expresión de cada una de ellas es variable, tanto en individuos de una población como en los
distintos estadios de diferenciación de un mismo tipo celular [168].
26
Introducción
Figura 9.- Estructura génica de DC-SIGN. En el esquema se representan los exones que codifican para cada una de las regiones que forman DC-SIGN (números romanos) y su tamaño (números arábigos), así como la localización y tamaño de los intrones (números romanos y arábigos en gris).
La variabilidad estructural del gen de DC-SIGN a nivel poblacional puede tener importantes
repercusiones patológicas, ya que se han descrito polimorfismos en la región codificante y
reguladora que se asocian con susceptibilidad alterada a infecciones como tuberculosis o HIV-1
[171, 172]. Existen discrepancias entre el posible papel protector de las variantes génicas de DC-
SIGN, que pueden ser debidas a las diferentes poblaciones estudiadas, e incluso a la existencia de
otros polimorfismos. La mayoría de estos estudios se han centrado en un cambio en el nucleótido
-336 (variante G o A) en la región promotora de esta lectina, que afecta al sitio de unión del factor de
transcripción Sp1 [173]. Martin y colaboradores asocian la presencia de la variante DC-SIGN-336G
con una mayor susceptibilidad a la infección por HIV-1 por vía parenteral pero no por vía mucosa
[174], mientras que otros autores encuentra asociación únicamente entre la variante DC-SIGN-139C
y una progresión acelerada del SIDA en individuos hemofílicos japoneses infectados por HIV-1
[175].
Respecto a la infección por M. tuberculosis, las variantes DC-SIGN-336A y -871G se asocian a
una protección frente a la infección en una población en el sur de África [176], mientras que en la
población sub-Sahariana el alelo -336G está asociado a una mayor protección [177]. Sin embargo,
otros trabajos posteriores en pacientes colombianos [178], tunecinos [179] y africanos [180], no han
observado asociación entre los polimorfismos en la posición DC-SIGN-336 y la susceptibilidad a
tuberculosis. Recientemente se ha analizado la frecuencia de la variante DC-SIGN-336G en
individuos de India infectados con HIV-1 y/o tuberculosis. Al ser menos frecuente en individuos
infectados por HIV-1, se especula que la presencia de esta variante protege frente a la infección por
HIV-1 y, sin embargo, aumenta la susceptibilidad a tuberculosis [181].
La presencia de polimorfismos en la región promotora de DC-SIGN también se ha asociado con
susceptibilidad alterada frente a otras infecciones y patologías. De hecho, la variante DC-SIGN-
336G está asociada con mayor protección frente a la fiebre del Dengue, pero no frente a la fiebre
hemorrágica del Dengue en individuos de Tailandia [182]. Por otro lado, no se ha encontrado
+ 46 206 981 1052 1994 2420 2571 3293 3405 4272 1425 4470 (425) (774) (100) (372) (721) (866)
Ia Ic II III IV V VIIb I II III IV V
1 2 3 4 5 6 7 8
27
Introducción
asociación entre la variante DC-SIGN-336A/G y la susceptibilidad a la enfermedad celiaca, aunque
la variante DC-SIGN-336G sí está asociada a dicha enfermedad dentro del grupo de pacientes HLA-
DQ2(-) [183]. La enorme variabilidad en el gen de DC-SIGN se puso de manifiesto en un estudio que
analizó la presencia de variantes en las posiciones -336, -332, -201 y -139 en cuatro grupos étnicos
de Brasil, y su posible correlación con la infección por HTLV-1 [184]. Según este estudio, las
variantes -336A y -139A son más comunes en individuos asiáticos, y la variante -201T no se
observa en caucásicos, asiáticos ni amerindios. Por otro lado, la variante -336A es más frecuente en
pacientes infectados por HTLV-1 y el alelo -139A está asociado con la protección frente a la
infección por este virus.
De todos estos estudios se concluye que DC-SIGN puede contribuir a la
susceptibilidad/transmisibilidad de las infecciones provocadas por numerosos patógenos. Además
de estas variantes en la región reguladora, existen polimorfismos en la región codificante que se
localizan principalmente en el exón 3 que codifica el cuello de DC-SIGN. De ellos y de las
discrepancias sobre su posible asociación con susceptibilidad a infecciones en diferentes grupos
étnicos, se profundizará en el apartado de “Discusión”.
5.4 Función y señalización
DC-SIGN es, probablemente, la lectina con el mayor rango de ligandos descrito, siendo capaz
de actuar como receptor de adhesión celular y de reconocer estructuras de carbohidratos presentes
en antígenos propios y en patógenos (Tabla 3). DC-SIGN presenta una alta afinidad por
carbohidratos con dimanosas terminales y estructuras internas de manosas ramificadas
(manotriosas α1→3, α1→6) [185, 186], y por carbohidratos que contienen fucosa, en concreto por
los trisacáridos que constituyen los antígenos de los grupos sanguíneos de Lewis (Lex, Ley, Lea, Leb)
[187-189].
Como receptor de patógenos, DC-SIGN interacciona con sus PAMP y el complejo DC-SIGN-
patógeno se internaliza, promoviendo el procesamiento y la posterior presentación de antígenos a
los linfocitos T, para acabar induciendo respuestas inmunitarias frente a dichos microorganismos
[167, 190]. Dentro del amplio rango de patógenos reconocidos por DC-SIGN [191], se encuentran
bacterias [192-194], hongos [195, 196], parásitos [197] y virus [149, 198, 199]. Recientemente
incluso se ha descrito la interacción de DC-SIGN con alérgenos comunes [200] (Tabla 3).
28
Introducción
Patógeno Ligando de DC-SIGN
Virus HIV-1 gp120 CMV gB
Ébola GP de la envuelta
Margburg GP de la envuelta
Dengue gE
HCV gE1/gE2
SARS proteína S
Herpesvirus humano ?
H5N1 (cepa del virus de la gripe aviar) ?
Bacterias cepas patogénicas de Mycobacterium ManLAM Helicobacter pilori LPS
Klebsiella pneumonia LPS
Neisseria meningitidis LPS
Neisseria gonorrhoeae LPS
Lactobacillus acidophilus NCFM SlpA
Parásitos Leishmania LPG? Schistosoma mansoni SEA
Hongos Candida albicans ? Aspergillus fumigatus Galactomanano
Tabla 3.- Patógenos y ligandos que se unen a DC-SIGN. HIV: virus de la inmunodeficiencia humana; CMV: citomegalovirus; HCV: virus de la hepatitis C; SARS: síndrome respiratorio agudo severo; gB, gE, gE1, gE2: glicoproteínas B, E, E1, E2; GP: glicoproteína; LPG: lipofosfoglicano; LPS: lipopolisacárido; ManLAM: lipoarabinomanano recubierto de manosas; SEA: antígeno soluble de los huevos; SlpA: proteína A de la capa superficial; Lex: Lewisx; Ley: Lewisy.
Por su capacidad de reconocer ligandos endógenos, DC-SIGN también puede mediar procesos
de adhesión intercelular (Figura 10). Así, DC-SIGN podría intervenir en la migración transendotelial
de DC gracias a la interacción con ICAM-2 presente en células endoteliales [153]. La unión de DC a
neutrófilos tiene lugar a través del reconocimiento por DC-SIGN de los carbohidratos ricos en Lex de
la integrina Mac-1 (CD11b/CD18) [201, 202] y de CEACAM-1 [202-204]. DC-SIGN también reconoce
el antígeno carcinoembrionario (CEA) de células de cáncer colorrectal, caracterizado por una mayor
presencia de Lex y Ley [191]. Otro de los ligandos endógenos propuestos para DC-SIGN es ICAM-3.
Aunque en un principio se propuso que la adhesión inicial entre DC y linfocitos T vírgenes estaba
mediada por la interacción DC-SIGN/ICAM-3 [150], esta hipótesis no ha podido ser corroborada por
otros autores [151, 205, 206].
29
Introducción
30
Célula endotelial
ICAM-2
ICAM-3
CE
Figura 10.- Ligandos endógenos de DC-SIGN. Representación esquemática de las interacciones de DC-SIGN con sus ligandos endógenos: ICAM-2 de células endoteliales, ICAM-3 de células T, CEA de células tumorales, y las moléculas CEACAM-1 y Mac-1 en neutrófilos.
Como se ha comentado anteriormente, DC-SIGN es capaz de transmitir señales intracelulares
específicas tras su interacción con carbohidratos presentes en patógenos, señales que a su vez se
interrelacionan con las señales procedentes de TLR [160]. En función de la naturaleza del
carbohidrato reconocido por DC-SIGN, las MDDC secretan un patrón diferente de citoquinas [207].
Así, la unión de patógenos que expresan manosas en su superficie, como M. tuberculosis o HIV-1,
conduce a un aumento en la producción de IL-10, IL12 e IL-6 de forma dependiente de Raf-1 [207].
Sin embargo, la unión de ligandos que contienen fucosa, como Ley de H. pilori, disminuye la
secreción de IL-12 e IL-6 de manera dependiente de Raf-1 mientras que se incrementa la
producción de IL-10 de forma independiente de Raf-1. El mecanismo molecular responsable del
aumento en la producción de IL-10 de forma Raf-1-dependiente implica la posterior acetilación de
p65 de NFκB, que conlleva a un incremento en la actividad transcripcional de IL-10 [208]. Por otro
lado, la activación de ERK en la ruta de señalización de DC-SIGN parece ser dependiente del
ligando involucrado. Así, la activación de DC-SIGN con anticuerpos específicos frente al CRD, la
unión de gp120 de HIV-1, o la unión del alergeno Ara h1, induce fosforilación de ERK1/2 [160, 209,
210]. Sin embargo, otros estudios han demostrado que la unión de ligandos patogénicos a DC-
SIGN, como ManLAM de M. tuberculosis o la proteína Salp15 de Ixodes scapularis, no provoca
activación de ERK [208, 211]. En consecuencia, DC-SIGN es considerado un modulador de la
respuesta inmune al ser capaz de alterar el balance Th1/Th2 y de modificar las señales procedentes
de otros PRR como TLR4 [212].
A
Célula tumoral
Célula T
Mac-1
CEACAM-1
Neutrófilo
Objetivos
Objetivos
El objetivo general de esta Tesis Doctoral consistió en la identificación y caracterización de marcadores de macrófagos activados con un fenotipo anti-inflamatorio/alternativo, y en
concreto el estudio de dos esos marcadores, el receptor de folato β (FRβ) y DC-SIGN:
1. Análisis de la expresión del FRβ en macrófagos anti-inflamatorios M2 y macrófagos
asociados a tumores.
2. Búsqueda de factores que regulan la expresión y función del FRβ en macrófagos M2.
3. Caracterización estructural y funcional de isoformas y polimorfismos de DC-SIGN en células
dendríticas derivadas de monocitos.
4. Identificación de epítopos estructurales y funcionales en la molécula de DC-SIGN mediante el
empleo de anticuerpos monoclonales.
33
Resultados
Resultados
Esta Tesis Doctoral se presenta en formato de artículos publicados. La sección de resultados incluye los artículos que dan respuesta a los objetivos planteados:
1. Los resultados del análisis de la expresión del FRβ en macrófagos anti-inflamatorios y
macrófagos asociados a tumores se presentan en el siguiente artículo:
Sierra-Filardi E, et al. Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res,
2009 Dec 15;69(24):9395-403.
2. Los resultados obtenidos de la búsqueda de factores que regulan la expresión y función del
FRβ en macrófagos M2 se recogen en el siguiente artículo:
Sierra-Filardi E, et al. Activin prevents the acquisition of M2/anti-inflammatory markers and skews the macrophage cytokine profile. Manuscrito en preparación.
3. Los resultados generados tras la caracterización de isoformas y polimorfismos de DC-SIGN
se publicaron en el artículo:
Sierra-Filardi E, et al. Structural requirements for multimerization of the pathogen receptor DC-SIGN (CD209) on the cell surface. J Biol Chem, 2008 Feb 15;283(7):3889-903.
4. Los resultados obtenidos tras el análisis estructural de la molécula de DC-SIGN se recogen
en el siguiente artículo:
Sierra-Filardi E, et al. Epitope mapping on the dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor. Mol Immunol, 2010 Jan;47(4):840-848.
37
Resultados
1. El receptor de folato β se expresa en macrófagos asociados a tumores y
constituye un marcador de macrófagos anti-inflamatorios/reguladores M2
La activación de macrófagos comprende un amplio espectro de estados funcionales
dependientes del microambiente de citoquinas. Los macrófagos activados se han agrupado
funcionalmente según su respuesta a estímulos pro-Th1/pro-inflamatorios (LPS, IFNγ, GM-CSF) (M1)
o pro-Th2/anti-inflamatorios (IL-4, IL-10, M-CSF) (M2). En el presente manuscrito demostramos que
el receptor de folato β (FRβ), codificado por el gene FOLR2, es un marcador de macrófagos
generados en presencia de M-CSF (M2), pero no de GM-CSF (M1), y que su expresión se
correlaciona con un aumento de la captación de folato. La capacidad de captar folato por los
macrófagos es promovida por M-CSF, mantenida por IL-4, prevenida por GM-CSF y reducida por
IFNγ, lo que indica una relación entre la expresión del FRβ y la polarización M2. De acuerdo con los
datos in vitro, la expresión del FRβ se detecta en macrófagos asociados a tumores (TAM), que
exhiben un perfil funcional de tipo M2 y ejercen potentes funciones inmunosupresoras dentro del
ambiente tumoral. El FRβ se expresa y media la captación de folato por TAM CD163+ CD14+ IL-10+,
y su expresión es inducida de una manera dependiente de M-CSF por líquido ascítico tumoral y por
el medio condicionado de fibroblastos y líneas tumorales. Estos resultados definen al FRβ como un
marcador de la polarización M2 de macrófagos, e indican que los conjugados de folato con drogas
terapéuticas son una potente herramienta en inmunoterapia frente a los TAM.
39
Immunology
Folate Receptor β Is Expressed by Tumor-Associated Macrophagesand Constitutes a Marker for M2 Anti-inflammatory/Regulatory Macrophages
Amaya Puig-Kröger,1,2 Elena Sierra-Filardi,1 Angeles Domínguez-Soto,1 Rafael Samaniego,3
María Teresa Corcuera,4 Fernando Gómez-Aguado,4 Manohar Ratnam,5
Paloma Sánchez-Mateos,2 and Angel L. Corbí1
d,
1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas; 2Unidad de Inmuno-Oncología and 3Unidad deMicroscopía Confocal, Hospital General Universitario Gregorio Marañón; 4Servicio de Anatomía Patológica, Hospital Carlos III, MadriSpain; and 5University of Toledo College of Medicine, Toledo, Ohio
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AbstractMacrophage activation comprises a continuum of functionstates critically determined by cytokine microenvironmenActivated macrophages have been functionally groupeaccording to their response to pro-Th1/proinflammatorstimuli [lipopolysaccharide, IFNγ, granulocyte macrophagcolony-stimulating factor (GM-CSF); M1] or pro-Th2/antinflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2We report that folate receptor β (FRβ), encoded by the FOLRgene, is a marker for macrophages generated in the presencof M-CSF (M2), but not GM-CSF (M1), and whose expressiocorrelates with increased folate uptake ability. The acquisitioof folate uptake ability by macrophages is promoted by M-CSmaintained by IL-4, prevented by GM-CSF, and reduced bIFNγ, indicating a link between FRβ expression and M2 polaization. In agreement with in vitro data, FRβ expressiondetected in tumor-associated macrophages (TAM), whicexhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRβexpressed, and mediates folate uptake, by CD163+ CD68+ CD14IL-10–producing TAM, and its expression is induced by tumoderived ascitic fluid and conditioned medium from fibroblasand tumor cell lines in an M-CSF–dependent manner. Thesresults establish FRβ as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates otherapeutic drugs are a potential immunotherapy tool ttarget TAM. [Cancer Res 2009;69(24):9395–403]
IntroductionMacrophages exhibit a continuum of functional activatio
states under homeostatic and pathologic conditions (1, 2). Dpending on the stimulus, activated macrophages acquire microbicidal, pro-inflammatory, and antitumor activities, but mighalso contribute to tissue repair, resolution of inflammatioand tumor cell growth and metastasis (1). These two extreme
-s
s.
Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).
A. Puig-Kröger and E. Sierra-Filardi are co-first authors. P. Sánchez-Mateos andA.L. Corbí contributed equally to this work. The order of authors should be consideredarbitrary.
Requests for reprints: Amaya Puig-Kröger, Laboratorio de Inmuno-Oncología,Hospital General Universitario Gregorio Marañón, Doctor Esquerdo 46, 28007Madrid, Spain. Phone: 34-91-5868750; Fax: 34-91-5868052; E-mail: [email protected].
©2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-09-2050
93www.aacrjournals.org
of the spectrum of macrophage activation have been coined a“classic”/M1 and “alternative”/M2 (3) and play opposing roleduring immune and inflammatory responses. Although granulocyte macrophage colony-stimulating factor (GM-CSF) and M-CSFcontribute to macrophage differentiation, each cytokine promotethe acquisition of distinct pathogen susceptibility (4) and inflammatory functions (5–8). GM-CSF–derived macrophages (M1) are proinflammatory and potentiate Th1 responses, whereas M-CSF–drivenmacrophages (M2) secrete IL-10 in response to pathogens and donot activate Th1 responses (8).Tumor-associated macrophages (TAM) are abundant immuno
suppressive cells recruited into the tumor microenvironment bcytokines such as M-CSF and CCL2 (9). The relevance of M-CSFand TAM in tumor progression and metastasis is now well established (10, 11). TAM represent a unique type of M2-polarizedmacrophages, as they promote angiogenesis, tissue remodelingand repair (2, 12). In fact, clinical studies have revealed a correlation between high tumor macrophage content and poor patienprognosis. Because TAM are potential targets for anticancer therapy (13, 14), identification of TAM-specific markers constitutesvery active area of research.The folate receptor gene family includes four members (FRα o
FOLR1, FRβ or FOLR2, FRγ or FOLR3, and FRδ or FOLR4), whose encoded products bind folic acid with high affinity (15). FOLR1 andFOLR2 encode glycosyl phosphatidylinositol–anchored endocytic receptors expressed in certain epithelial tissues and various tumor(FOLR1; refs. 16, 17) or in normal myeloid cells and acute myelogenous leukemias (FOLR2; refs. 18–20). Within the myeloid lineage, folate receptor β (FRβ) is expressed in a nonfunctional state in CD34bone marrow cells (21, 22) and neutrophils (18), whereas it mediatefolate binding in activated synovial macrophages from rheumatoidarthritis (23) and in ovarian cancer–associated murine macrophage(24). The high affinity of FRα and FRβ for folate binding, their endocytic capacity, and their restricted expression have prompted thevaluation of the potential therapeutic value of folate-drug conjugates in cancer and inflammatory pathologies (25, 26).In the present article, we describe that functional FRβ is specif
ically expressed by M-CSF–polarized (M2) macrophages as well aby ex vivo isolated TAM, and that tumors induce its expression in anM-CSF–dependent manner, thus supporting folate-drug conjugateas valuable tools to target TAM in tumor immunotherapy protocols
-).
Materials and MethodsCell culture and treatments. Human monocytes were purified by mag
netic cell sorting using CD14microbeads (Miltenyi Biotech) as described (27
95 Cancer Res 2009; 69: (24). December 15, 2009
41
MGMreor(AteliptuDMes
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Cancer Research
Ca
1 or M2 monocyte-derived macrophages were generated in the presence of-CSF (1,000 units/mL, ImmunoTools GmbH) or M-CSF (10 ng/mL),
spectively. When indicated, macrophages were treated for 72 h with IL-6IL-10 (50 ng/mL), and anti-M-CSF blocking monoclonal antibodybingdon) was used at 0.5 μg/mL. For activation, macrophages were trea-d with IL-4 (1,000 units/mL), IL-10 (50 ng/mL), IFNγ (500 units/mL), oropolysaccharide (LPS; 50 ng/mL; E. coli 055:B5, Sigma) for 48 h. Humanmor cell lines (JAR, JEG-3, NIH-OVCAR-3, and Colo320) were cultured inEM containing 10% FCS. Cultures of tumor-associated fibroblasts were
tablished from primary melanoma according to standard procedures.Human TAM were obtained from melanoma and breast adenocarcino-a patients after obtaining written informed consent and following Med-l Ethics committee procedures (Hospital General Universitario Gregorioarañón). Histopathologic diagnosis was confirmed for each specimen.M were isolated by Ficoll gradient cell separation and subsequent mag-tic cell sorting using CD14 microbeads. Phenotypic analysis was carriedt by indirect immunofluorescence (28) using rabbit polyclonal antiserati-human FRβ (18). Folate-FITC binding and endocytosis assays werene as reported (26). Flow cytometry on permeabilized ex vivo isolatedM was done using phycoerythrin (PE)-labeled anti-CD68 monoclonal an-ody (clone Y1/82A, Biolegend), Alexa Fluor 647–labeled anti-CD163onoclonal antibody (clone RM3/1, Biolegend), and a polyclonal antiserumainst human FRβ followed by incubation with FITC-labeled goat anti-bbit affinity-purified antibody. The presence of Tie2-positive FRβ-positiveacrophages was evaluated using a PE-labeled anti-Tie2 monoclonal anti-dy (clone 33.1, Biolegend). Isotype-matched monoclonal antibodies (PE-
Cous
cralcous
(RceanG(2GAGESCAG5′CAfobptaFORoin
9396ncer Res 2009; 69: (24). December 15, 2009
42
ol, Alexa 647-Control) and a preimmune rabbit antiserum (29) weres negative controls.stern blot. Western blot was carried out with 10 μg of lysates fromplasma membranes (30). Protein detection was done with a polyclon-isera against FRβ (18) or a monoclonal antibody against CD29. Forl purposes, a previously described rabbit pre-immune antiserum was29).R. Total RNA from solid tumor tissue and TAM was extractedasy kit, Qiagen), retrotranscribed, and amplified using standard pro-es. Oligonucleotides specific for FOLR2, MAFB, IL10, ESR1, MAGEA3,APDH were as follows: FRBs, 5′-AGAAAGACATGGTCTGGAAATG--3′, and FRBas, 5′-GACTGAACTCAGCCAAGGAGCCAGAGTT-3′af-Bs, 5′-CCCGGCTGGCCCGCGAGAGAC-3′, and Maf-Bas, 5′-CTAG-CGGCGCTGGCGT-3′ (31); IL10s, 5′-ATGCCCCAAGCTGAGAACCAA-CA-3, and IL10as, 5-TCTCAAGGGGCTGGGTCAGCTATCCCA-3;, 5′-TCAGATAATCGACGCCAGG-3′, and ESR1as, 5′-GGCTCAGCATC-AAGG-3′; MAGEA3s, 5′-GAAGCCGGCCCAGGCTCG-3′, and MA-as, 5 ′-GGAGTCCTCATAGGATTGGCTCC-3 ′ ; and GAPDHs,CTGAGAACGGGAAGCTTGTCA-3′, and GAPDHas, 5′-CGGCCAT-CCACAGTTTC-3′. Amplified fragments (783 bp for FOLR2, 347 bpAFB, 352 bp for IL10, 511 bp for ESR1, 457 bp for GAPDH, and 423MAGEA3) were resolved by agarose gel electrophoresis. For quanti-reverse transcription-PCR (RT-PCR), oligonucleotides for FOLR1,
2, FOLR3, JDP2, NRAMP1, and IL10 were designed according to thesoftware for quantitative real-time PCR, and RNA was amplified us-e Universal Human Probe Roche library (Roche Diagnostics). Assays
gure 1. FOLR2 mRNA and FRβ protein expression and function in M1 and M2 macrophages. A, FOLR2, JDP2, SLC11A1, and IL10 are differentially expressedM1 and M2 macrophages, as determined by microarray DNA analysis and quantitative RT-PCR. B, right, FRβ expression in cell membrane extracts, as determinedWestern blot using an antihuman FRβ polyclonal antiserum (18). As a control, CD29 expression levels were determined in parallel. Left, cell surface expressionFRβ on M1 and M2 macrophages, determined by flow cytometry using a polyclonal antiserum against human FRβ (ref. 18; empty histogram). As a controlled histogram), a previously described rabbit preimmune antiserum (29) was used. C, FRβ function in M1 and M2 macrophages, as shown by binding (4°C) andtake (37°C) of folate-FITC (empty histogram, black line). Transferrin-FITC internalization (empty histogram, gray line) was determined in parallel on both macrophagees. Each experiment was done three times, and a representative experiment is shown. D, binding (4°C) and internalization (37°C) of folate-FITC by M2crophages, in the absence (empty histograms, black line) or the presence (empty histograms, gray line) of a 100 mol/L excess of folic acid. The experiment wasne four times, and one of the experiments is shown. Representative confocal sections of M2 macrophages incubated with folate FITC for 1 h at 37°C, and theirrresponding differential interference contrast images, are shown. The percentage of marker-positive cells and the mean fluorescence intensity (in parentheses)e indicated in flow cytometry experiments (B–D), and filled histograms indicate cell autofluorescence (C and D).
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s-T
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were made in triplicates and results normalized according to the expresion levels of 18S RNA and GAPDH. Results were obtained using the ΔΔCmethod for quantitation and expressed as normalized fold expression.
Confocal microscopy and immunohistochemistry. Human melanma tissues (subcutaneous tissue, lymph node, and lung metastasis) weobtained from patients with primary and metastatic lesions undergoinsurgical treatment. Thick sections (4 μm in depth) of cryopreserved tissuwere first blocked for 10 min with 1% human immunoglobulins and theincubated for 1 h with a rabbit polyclonal antiserum against human FR(18), anti-CD163 or HMB-45 monoclonal antibodies, or isotype-matchecontrol antibodies. All primary antibodies were used at 1 to 5 μg/mL, folowed by incubation with FITC-labeled antimouse and Texas red–labeleantirabbit secondary antibodies. Samples were imaged using a confocscanning inverted AOBS/SP2 microscope (Leica Microsystems) with63× PL-APO NA 1.3 immersion objective. Image processing and colocaliztion analyses (scatter plots) were assessed with the Leica Confocal SoftwaLCS-15.37. Tissue microarrays (TMAH-MTC-01, RayBiotech) were prcessed according to the manufacturer's recommendations.
ResultsFRβ is expressed in macrophages generated in the presenc
of M-CSF. Gene expression profiling on macrophages generatedthe presence of GM-CSF (M1) or M-CSF (M2) resulted in the idetification of more than 250 differentially expressed genes (>2-fodifferences, P < 0.05; data not shown). Among them, FOLR2, whiccodes for FRβ, was preferentially expressed in M2 macrophag(P = 1.3 × 10−7; Fig. 1A). The JDP2 gene, which encodes an activatoprotein-1 repressor, also showed higher expression in M2 macrphages (P = 0.02), whereas SLC11A1, which encodes the NRAMPprotein associated with classic macrophage activation, was epressed at higher levels in M1 macrophages (P = 0.029; Fig. 1AInterestingly, and in agreement with their anti-inflammatory actiity, the expression of IL10 was considerably higher in M-CSFprimed macrophages (P = 1.2 × 10−4). The differential expressioof FOLR2, JDP2, SLC11A1, and IL10 in both types of macrophagwas confirmed by real-time RT-PCR on mRNA from independendonors (Fig. 1A). Besides, FRβ expression was exclusively detecte
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in membrane lysates and on the cell surface of M2 macrophages(Fig. 1B), thus validating the transcriptome data.Because FRβ binds folic acid and folate conjugates (32), the abil-
ity of FRβ to mediate folate-FITC uptake by M2 macrophages wasassessed. Whereas both macrophage types endocytosed transferrin-FITC, M-CSF–polarized macrophages displayed folate binding andinternalization ability, and GM-CSF–induced macrophages showedno folate uptake capacity, in agreement with their lack of FRβ ex-pression (Fig. 1C). Folate binding and uptake by M-CSF macro-phages were specific, as both were inhibited by a 100 mol/Lexcess of folic acid (Fig. 1D). Moreover, folate conjugates enteredcells by endocytosis because most of the folate-FITC fluorescencecould not be stripped from the cell surface by an acid wash step(Supplementary Fig. S1). Considering that neither FOLR1 norFOLR3 was expressed by M-CSF macrophages (SupplementaryFig. S2), FOLR2-encoded FRβ protein must be responsible forthe folate binding ability of M2 macrophages. Kinetic studies re-vealed that FOLR2 mRNA and FRβ protein are initially detected 48to 72 hours after M-CSF addition, and that their levels dramatical-ly increase at later incubation times (Fig. 2A and B). Acquisition offolate uptake ability correlated with protein expression at all timepoints and showed its highest level at the end of the culture period(Fig. 2C). Therefore, M-CSF promotes the expression of a func-tional FRβ protein, which constitutes a marker of M-CSF–polarizedM2 macrophages.Expression of FRβ in TAM. TAM are an M2-skewed macro-
phage population that exhibits immunosuppressive activity withinthe tumor microenvironment, and whose recruitment and differ-entiation is influenced by M-CSF (9). Given the preferential expres-sion of FRβ in M-CSF–polarized M2 macrophages, its presencewas evaluated in TAM. Immunohistochemistry revealed that FRβis frequently coexpressed with CD163 in TAM from primary andmetastatic melanoma (Figs. 3A and 4A) but is absent from mela-noma HMB-45+ cells (Figs. 3A and 4A). In fact, FOLR2 mRNA couldbe detected in three melanoma samples (Fig. 3B). Ex vivo isolatedCD14+ TAM from the pleural fluid of a metastatic melanoma
Folate Receptor β Is an M2 Macrophage Marker
Figure 2. Acquisition of FRβ expressioon monocyte treatment with M-CSF.A, FOLR2 mRNA expression levelsalong M-CSF–induced polarization ofmacrophages, as determined byquantitative RT-PCR. Columns, meannormalized fold expression (relative to 1rRNA levels) from triplicate determinatiobars, SD. B, FRβ expression along M1and M2 macrophage polarization, asdetermined by Western blot at the indicatime points. As a control, CD29expression levels were also determinedC, internalization of folate-FITC duringM-CSF–induced macrophage polarizatio(empty histograms), as determined by fcytometry at the indicated time points.Filled histograms, cell autofluorescenceThe percentage of marker-positive cellsand the mean fluorescence intensity(in parentheses) are indicated in eachcase.
97 Cancer Res 2009; 69: (24). December 15, 2009
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pressed mRNA for FOLR2, IL10, and the macrophage-specificAFB (31), whereas they lacked expression of the melanoma-ecific marker MAGEA3 mRNA (Fig. 3B, lanes 4) and were de-oid of FOLR1 and FOLR3 mRNA (Supplementary Fig. S3).hree-color analysis on isolated melanoma TAM indicated thatll FRβ+ macrophages are CD68+, and that the percentage ofRβ+ CD163+ macrophages (87%) is similar to that of CD163+
D68+ cells (88%; Fig. 3C). Thus, most melanoma TAM frome analyzed sample coexpress CD163, CD68, and FRβ and ex-ibit folate-FITC internalization ability (Fig. 3D). It is also worthoting that a percentage of FRβ+ macrophages coexpress Tie26%; Fig. 3C). Altogether, these results indicate that FRβ isnctionally expressed on IL10 mRNA–expressing CD14+ CD68+
D163+ melanoma TAM.Evaluation of FRβ expression on other tumor tissues indicatedat FRβ is detected in the stroma of lung, ovary, colon, gastric,nd breast cancers, where numerous CD68+ TAM were also pres-t (Fig. 4B). Analysis of ex vivo isolated CD14+ TAM from a meta-atic breast adenocarcinoma also revealed the coexpression ofD68 and FRβ, and that 80% of the cells exhibited a CD163+
Rβ+ phenotype (Fig. 5A). Importantly, primary and metastaticreast adenocarcinoma tissues were found to contain both FOLR2
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9398ancer Res 2009; 69: (24). December 15, 2009
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MAFB mRNA (Fig. 5B), and ex vivo isolated CD14+ metastaticst adenocarcinoma TAM expressed FOLR2, IL10, and MAFBA (Fig. 5B, lane 5). CD14+ CD163+ TAM also exhibited specifice-FITC binding and uptake (Fig. 5C) and produced IL-10 in re-se to LPS stimulation (Fig. 5D). Because FOLR2 and IL10A are coexpressed in M2 macrophages in vitro (Fig. 1), andfrom metastatic breast adenocarcinoma express functionaland produce IL-10, these results indicate that FRβ activitys anti-inflammatory M2-like TAM.rameters affecting FRβ expression on human macro-es. GM-CSF and M-CSF are tumor-derived factors that modu-myeloid cell differentiation (33). Unlike M-CSF, GM-CSFated the aquisition of FOLR2 mRNA during in vitromonocyte-to-ophage differentiation, even in the presence of M-CSF (Fig. 6A).result explains the differential expression of FRβ on both typesacrophages, and suggests that the relative levels of tissueCSF and M-CSF determine macrophage FRβ expression. Otherines commonly released by tumors (33) also affected FOLR2A; IL-6 alone and IL-10 in combination with M-CSF upregu-FOLR2 mRNA expression (Fig. 6B). Therefore, tumor-derivedkines (M-CSF, GM-CSF, IL-6, and IL-10) modulate FRβession in human macrophages.
igure 3. Expression and function of FRβ in TAM isolated from melanoma. A, confocal sections of infiltrating macrophages on a subcutaneous primary melanomasue sample, as determined by double immunofluorescence analysis of FRβ (green) and the macrophage marker CD163 (red; top), or FRβ and the melanomaarker HMB-45 (red; bottom). The corresponding scatter plots are shown, and colocalizing pixels (blue rectangles) are displayed on the merge images as whiteasks. Magnification of a FRβ/CD163 colocalizing area appears enlarged in the top image, and the enlarged area is depicted in white. In the top image, note theexpression of FRβ by tumor-infiltrating macrophages (CD163+), whereas nonstained areas correspond to tumor cells (CD163−). Nuclei were counterstained with,6-diamidino-2-phenylindole (DAPI). B, detection of FOLR2, MAFB, IL10, MAGEA3, and GAPDH mRNA by RT-PCR on RNA from two different primary melanomasues (lanes 2 and 3) and from CD14+ cells isolated from the pleural fluid of a metastatic melanoma (lane 4). Control RT-PCR reactions were loaded in lane 1,xt to the lane containing the molecular size markers. C, expression of CD68, CD163, Tie2, and FRβ in CD14+ TAM isolated from a melanoma pleural fluid, astermined by three-color flow cytometry analysis on permeabilized cells. Isotype-matched monoclonal antibodies and a preimmune rabbit antiserum were used asgative controls. The percentages of single- and double-positive cells are indicated. D, binding (4°C) and internalization (37°C) of folate-FITC by CD14+ TAMolated from a metastatic melanoma (empty histograms). Filled histograms, cell autofluorescence. The percentage of marker-positive cells and the mean fluorescencetensity (in parentheses) are indicated.
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en-tsdgd).d),ni–no-daess,
Expression of FRβ on TAM led us to analyze the nature of thstimuli that might control its presence in the tumor microenviroment. FOLR2 mRNA was variably upregulated by supernatanfrom tumor cell lines, with placenta choriocarcinoma JAR anJEG-3 cells and ovary carcinoma NIH-OVCAR-3 cells promotinthe highest level of upregulation (Fig. 6C). In contrast, conditionemedia from colon carcinoma Colo320 cells had no effect (Fig. 6CMore importantly, ascitic fluid from the breast carcinoma analyzein Fig. 5 promoted a strong upregulation of FOLR2 mRNA (Fig. 6Cconfirming that tumor cells release factors that upregulate humamacrophage FRβ expression. The addition of a blocking antM-CSF monoclonal antibody greatly reduced the upregulatioof FOLR2 mRNA promoted by ascitic fluid from breast carcinma (Fig. 6D) or by conditioned medium from tumor-associatefibroblasts or JEG-3 tumor cells (Fig. 6D). Therefore, M-CSF ismajor determinant for FRβ expression on human macrophagand contributes, alone or in combination with other cytokine
to FRβ cell surface expression on TAM.93www.aacrjournals.org
DiscussionGM-CSF and M-CSF contribute to the generation of different
macrophage subsets and enhance myeloid cell survival and prolif-eration (9). However, GM-CSF promotes the generation of myeloidcells with potent antigen presentation activity, whereas M-CSFleads to the generation of macrophage cells with regulatory prop-erties (9). Gene expression profiling allowed us to identify FRβ aspreferentially expressed by macrophages generated under the in-fluence of M-CSF, which display FRβ-dependent folate bindingability. FRβ expression on in vitro differentiating macrophageswas enhanced by M-CSF and by tumor cell-conditioned mediumin an M-CSF–dependent manner. Conversely, GM-CSF preventedthe acquisition of FRβ expression. Importantly, FRβ was detectedin TAM, where FRβ-mediated folate binding activity correlates withthe presence of IL10mRNA. Therefore, FRβ constitutes a marker forM-CSF–primed IL-10–expressing M2-polarized macrophages, pro-viding a molecular basis for the value of folate-conjugated drugs
Folate Receptor β Is an M2 Macrophage Marker
in cancer therapy approaches.
Figure 4. Expression of FRβ in TAM from primary and metastatic melanoma. A, expression of FRβ in melanoma-infiltrating macrophages on a primary melanoma(#130, bottom) or two metastatic melanomas (#98 and #146, top and middle), as determined by double immunofluorescence analysis of FRβ and the macrophagemarker CD163 (top rows) or FRβ and the melanoma tumor marker HMB-45 (bottom rows). B, expression of FRβ in tumors of distinct tissue origins. Light microscopyimages of the macrophage marker CD68 (middle) and FRβ (right) staining of tumor tissue from lung squamous cancer (1; magnification, ×20), ovariancystadenoma-mucous (2; magnification, ×20), rectal colon adenocarcinoma (3; magnification, ×20), gastric adenocarcinoma (4; magnification, ×40), and breastinvasive ductal cancer (5; magnification, ×40). Left, staining yielded by normal rabbit serum, used as a control.
99 Cancer Res 2009; 69: (24). December 15, 2009
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Because macrophage polarization is stimulus dependent (1), al-rnatively activated M2 macrophages have been further classifieds M2a, M2b, or M2c in an effort to link genetic markers to specificacrophage-activating stimuli (34). The expression of FRβ in-CSF–generated macrophages indicates that it is preferentiallypressed by IL-10–producing M2 macrophages and, therefore,entifies a population of macrophages with anti-inflammatory/gulatory properties. The presence of FRβ in M-CSF–primedvitro macrophages is in agreement with its upregulation in hu-an decidual macrophages, which exhibit an immunosuppressivehenotype and whose gene expression profile closely correspondsthat of M2-polarized macrophages (35). Further supporting its
resence on M2 macrophages, FRβ has been detected on F4/80+
D68+ murine peritoneal macrophages (36), where its mRNA levelsn be further upregulated by IL-4 (37). Therefore, FRβ expressionems not to be restricted to anti-inflammatory/regulatory IL-10–roducing M2 macrophages and marks a wider range of alterna-vely activated macrophages in the human and murine systems.owever, the functional state of FRβ on murine peritoneal macro-hages is still not clear because folate binding ability is only de-cted after stimulation with inflammatory stimuli (26).The expression of FRβ on TAM from primary and metastaticelanoma and breast carcinoma (Figs. 3–5) is also in agreementith a previous report describing the presence of FRβ in CD68+
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3+ cells within human and rat glioblastoma (36). In an appar-ontradiction, gene expression profiling has revealed downre-ed FRβ mRNA levels in murine fibrosarcoma TAM relative toevels detected in thioglycollate-elicited peritoneal macro-es (12). However, because the latter exhibit functional charac-tics of M-CSF–driven M2 macrophages (38), these results doule out the presence of detectable levels of FRβ in murine. Besides, it is also possible that FRβ is expressed by TAMtumor-dependent manner, a phenomenon which would bereement with its differential upregulation by distinct tumor-itioned media (Fig. 6) and the variable levels of FRβ in TAMa variety of human tumors (Fig. 4). Finally, it is also possibledifferences might exist between murine and human TAM,is already evident that paradigmatic M2 murine macrophageers (Arginase and Ym1) are not useful to identify human al-tively activated macrophages (39). Whether the acquisitionβ expression by tumor-infiltrating macrophages is detri-
tal for the tumor (e.g., by removing folate) or favors tumorgrowth is a matter that deserves further investigation. Re-less of the precise role of FRβ on TAM, the presence ofon their cell surface provides an opportunity for depletionM through the use of folate-conjugated drugs. As an exam-and while this article was being completed, Nagai and co-ers have shown the feasibility of reducing tumor growth by
igure 5. Expression and function of FRβ in TAM isolated from breast adenocarcinoma. A, expression of FRβ, CD68, and CD163 in permeabilized CD14+ TAMm a metastatic breast adenocarcinoma, as determined by flow cytometry using PE-labeled anti-CD68, Alexa Fluor 647–labeled anti-CD163, and a polyclonaltiserum against human FRβ (18), followed by FITC-labeled goat anti-rabbit antibodies. Isotype-matched monoclonal antibodies and a preimmune rabbit antiserum9) were used as negative controls (top). The percentages of single-positive and double-positive cells are indicated. B, detection of the indicated mRNA byT-PCR on RNA from three different primary breast adenocarcinoma tissues (lanes 2–4) and from CD14+ cells isolated from ascitic fluid from a metastatic breastenocarcinoma (lane 5). Control RT-PCR reactions were loaded in lane 1, next to the lane containing the molecular size markers. C, binding (4°C; top) andternalization (37°C; bottom) of folate-FITC by CD14+ TAM isolated from a metastatic breast adenocarcinoma, in the absence (empty histograms, black line) or theesence (empty histograms, gray line) of a 100 mol/L excess of folic acid. Filled histograms, cell autofluorescence. The percentage of marker-positive cells ande mean fluorescence intensity (in parentheses) are indicated. The experiment was done two times, and one of the experiments is shown. Representative confocalctions and differential interference contrast microscopy images of macrophages incubated with folate-FITC. D, CD14+ TAM isolated from a metastatic breastenocarcinoma were either untreated or stimulated with LPS (50 ng/mL) for 24 h, and IL-10 release was determined by ELISA. Columns, mean of triplicateterminations; bars, SD.
www.aacrjournals.org
β
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Folate Receptor β Is an M2 Macrophage Marker
targeting an immunotoxin to TAM using an antimouse FRmonoclonal antibody (36).Given the tumor influence on macrophage functions (40), FR
might specifically mark tumor-infiltrating human macrophagwhose effector functions have been already skewed by tumor-derivefactors. In this regard, our data also suggest that tumor-deriveM-CSF, which recruits and shapes macrophage functions (33would be the primary determinant for FRβ expression. HoweveFRβ expression is also detected in resident macrophages withnontumor tissue (data not shown). This fact, together with the icrease in FRβ expression during the in vitro macrophage differetiation that takes place in the absence of exogenous cytokinemight indicate that FRβ could be a macrophage differentiatiomarker under homeostatic conditions, and whose levels could bmaintained or upregulated by anti-inflammatory cytokines andownregulated by pro-inflammatory stimuli. In this regard, cytkines such as IL-4, IL-13, and IL-10, which promote macrophagalternative activation, trigger a transient increase of FOLR2 mRN
94www.aacrjournals.org
levels in M2 macrophages (Supplementary Fig. S4A and B). By con-trast, LPS greatly downregulates FOLR2 mRNA levels (Supplemen-tary Fig. S4A and B) although it does not lead to a great decrease incell surface FRβ (Supplementary Fig. S4C). This divergence mightbe explained by the fact that FOLR2 is an endocytic receptor whoseprotein levels are higher than those present on the cell surface. Infact, flow cytometry on permeabilized cells showed that a largeproportion of FRβ is located intracellularly in both in vitro M2macrophages and TAM (Supplementary Fig. S5).The presence of functional FRβ on M-CSF–primed macrophages
and the detection of FRβ mRNA in other types of M2-polarizedmacrophages (12, 35, 37) are difficult to reconcile with its expres-sion (25) and function (26) in synovial macrophages from rheuma-toid arthritis patients, which are embedded in a inflammatorypro-M1 environment. It could be speculated that synovial macro-phages might exhibit a mixed M1/M2 phenotype, similar to whatoccurs with myeloid populations within tumors (41), an expla-nation that would be compatible with the high levels of M-CSF
Figure 6. Parameters affecting FRβ expression on human macrophages. A and B, FOLR2 mRNA expression in macrophages exposed for 72 h to the indicatedcytokines, as determined by quantitative RT-PCR. Results are expressed as normalized fold expression relative to 18S rRNA levels and the FOLR2 RNA levels inperipheral blood monocytes (Mon.). Columns,mean of triplicate determinations; bars, SD. C, FOLR2mRNA expression in macrophages exposed for 72 h to conditionedmedia from the ascitic fluid of a breast carcinoma (ABC) or tumor cell lines, as determined by quantitative RT-PCR. Results are expressed as normalized foldexpression (relative to 18S rRNA levels). Columns, mean of triplicate determinations from three independent macrophage preparations; bars, SD. D, inhibitory effect ofanti–M-CSF on FOLR2 mRNA levels induced by ascitic fluid from metastatic breast carcinoma (ABC) or conditioned-medium from JEG-3 placenta choriocarcinoma(JEG-3) or tumor-associated fibroblasts (Fibroblast). The results are depicted as the FOLR2 mRNA levels detected in the presence of the anti–M-CSF antibody relativeto the levels seen in untreated cells (set to 100 in the three cases).
01 Cancer Res 2009; 69: (24). December 15, 2009
47
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resent in rheumatoid arthritis synovia (42). M-CSF is produced bynovial fibroblasts, and administration of M-CSF is known to ex-erbate arthritis in some settings (9, 43). Therefore, the levels of-CSF within the synovia of rheumatoid arthritis might suffice toromote FRβ expression on surrounding macrophages, althoughe concomitant presence of extremely high levels of tumor necro-s factor αmight override its immunosuppressive actions. Alterna-vely, because M-CSF contributes to macrophage recruitment, FRβxpression might mark macrophages newly recruited into therthritic synovia, whose later levels of FRβ expression would beetermined by the pro-inflammatory environment. In this regard,is worth noting that (a) FRβ+ macrophages are more prominentlyetected at early stages during development of animal models ofherosclerosis and muscle injury and in rheumatoid arthritis inumans;6 (b) in vitro acute (48 hours) exposure of FRβ-expressing2 macrophages to M1-polarizing stimuli (e.g., LPS, GM-CSF,d IFNγ) does not result in loss of FRβ expression, which is onlyoderately downregulated by IFNγ (Supplementary Fig. S4C).herefore, folate-targeted killing of FRβ+ macrophages in inflamma-ry disease murine models might contribute to inflammation reso-tion by preferentially eliminating newly recruited macrophages.
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9402ancer Res 2009; 69: (24). December 15, 2009
48
though further studies are needed to correlate FRβ expressionunction in macrophages within inflamed tissues, our results in-e that cytokines favoring the generation of anti-inflammatory/latory macrophages, and known to shape TAM effector func-(M-CSF and IL-10), promote and are permissive for FRβ
ession, whereas factors skewing macrophage polarizationrd the proinflammatory branch either inhibit (IFNγ) or abro-FRβ expression.
losure of Potential Conflicts of Interestpotential conflicts of interest were disclosed.
nowledgmentsed 6/4/09; revised 10/9/09; accepted 10/15/09; published OnlineFirst 12/8/09.ant support: Ministerio de Educación y Ciencia (grant BFU2008-01493-BMC),erio de Sanidad y Consumo, Instituto de Salud Carlos III (Spanish Network forsearch in Infectious Diseases, REIPI RD06/0008), and Fundación para la Inves-ón y Prevención del SIDA en España (FIPSE 36663/07; A.L. Corbí); grant06-08615 from Ministerio de Educación y Ciencia and Fundación Ramón Arecesoncurso Nacional, 2007; P. Sánchez-Mateos); and grant PI08/1208 from Institu-Salud Carlos III (A. Puig-Kröger). A. Puig-Kröger is supported by Ministerio ded y Consumo, Instituto de Salud Carlos III (CP06/00199).e costs of publication of this article were defrayed in part by the payment of pages. This article must therefore be hereby marked advertisement in accordance8 U.S.C. Section 1734 solely to indicate this fact.thank Dr. Philip S. Low for reagent supply and valuable advice and discussions,abel Treviño and Julia Villarejo for help with immunohistochemistry samplesing.
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late Receptor β Is an M2 Macrophage Marker
2008;3:e2078. Arginase-1 and Ym1
ancer Res 2009; 69: (24). December 15, 2009
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Supplementary Figures
Folate-FITCFolate-FITC + Acidic wash
1.0 (4.7)94 (35)
74 (13.5)
Folate-FITCFolate-FITC + Acidic washFolate-FITCFolate-FITC + Acidic wash
1.0 (4.7)94 (35)
74 (13.5)
Supplementary Figure 1.- Folate-FITC internalization ability of M-CSF-primed M2 macrophages. Internalization was done for 1 hour at 37ºC and cells were subsequently either untreated (empty histogram, black line) or subjected to an acidic cold wash with PBS 50 mM Glycine pH 3.2 (empty histograms, grey line) to eliminate cell surface-bound fluorecence. Autofluorescence of cells is indicated (grey histogram). In all cases, the percentage of fluorescence-positive cells and the mean fluorescence intensity (between parenthesis) are indicated.
FOLR1
M2 (MCSF)HeLa
Monocytes
20
15
10
5
Nor
mal
ized
Fold
Exp
ress
ion
FOLR2 FOLR3 Donor# 1
Donor# 2
Donor# 3
2.0
1.5
1.0
0.5Nor
mal
ized
Fold
Exp
ress
ion
FOLR2FOLR1
FOLR3
BA
FOLR1
M2 (MCSF)HeLa
Monocytes
20
15
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5
Nor
mal
ized
Fold
Exp
ress
ion
FOLR2 FOLR3 Donor# 1
Donor# 2
Donor# 3
2.0
1.5
1.0
0.5Nor
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Fold
Exp
ress
ion
FOLR2FOLR1
FOLR3
BA
Supplementary Figure 2.- A. FOLR1, FOLR2 and FOLR3 mRNA expression levels determined by qRT-PCR in HeLa cells, M2 (MCSF) macrophages and peripheral blood monocytes, and expressed as Normalized Fold Expression (relative to 18S rRNA levels). Shown is the mean and standard deviation of triplicate determinations for each gene. B. FOLR1, FOLR2 and FOLR3 mRNA expression levels determined by qRT-PCR in three different M2 macrophage preparations, and expressed as Normalized Fold Expression (relative to 18S rRNA levels). Shown is the mean and standard deviation of triplicate determinations for each gene.
51
2
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MCSF LPS IL-4 IL-13 IL-10
0.2
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Nor
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Fold
Exp
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ionFOLR2 FOLR2
M2 + IFN-γ M2 + IL-4M2 M2 + GM-CSF M2 + LPS
1.0 (3.4)82 (13.5)FRβ
expression
1.0 (3.7)34 (8.7)
1.0 (3.3)44 (9.2)
1.0 (4.3)71 (15)
1.0 (3.6)62 (12)
Folatecapture
Folate-FITCFolate-FITC + 100X Folic Acid
C2.0 (3)96 (33)
6.0 (5.6)
5.0 (4)87 (34)23 (7)
1.0 (3)74 (14)
3.0 (4.3)
3.0 (4.3)95 (33)24 (7)
1.0 (3.5)78 (15)5 (5.5)
2
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MCSF LPS IL-4 IL-13 IL-10
0.2
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Nor
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Exp
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ionFOLR2 FOLR2
M2 + IFN-γ M2 + IL-4M2 M2 + GM-CSF M2 + LPS
1.0 (3.4)82 (13.5)FRβ
expression
1.0 (3.7)34 (8.7)
1.0 (3.3)44 (9.2)
1.0 (4.3)71 (15)
1.0 (3.6)62 (12)
Folatecapture
Folate-FITCFolate-FITC + 100X Folic Acid
C2.0 (3)96 (33)
6.0 (5.6)
5.0 (4)87 (34)23 (7)
1.0 (3)74 (14)
3.0 (4.3)
3.0 (4.3)95 (33)24 (7)
1.0 (3.5)78 (15)5 (5.5)
Supplementary Figure 3.- A-B. FOLR2 mRNA expression in day-7 M2 macrophages exposed for 24 (A) or 48 (B) hours to LPS, IL-4, IL-13 or IL-10, as determined by qRT-PCR. Results are expressed as Normalized Fold Expression (relative to GAPDH mRNA levels and the FOLR2 mRNA levels in macrophages exposed to M-CSF). Shown is the mean and standard deviation of triplicate determinations. C. Folate-FITC capture ability (upper panels) and FRβ cell surface expression (lower panels) in M-CSF-primed M2 macrophages exposed to the indicated cytokines for the last 48 hours of the 7-day differentiation process. Internalization was done either in the absence (empty histograms, black line) or the presence (empty histograms, grey line) of a 100-molar excess of folic acid. Filled histograms (thin line) indicate cell autofluorescence in each case. Cell surface expression was determined by flow cytometry using a polyclonal antiserum against human FRβ (empty histograms with thick lines) and a previously reported pre-immune rabbit antiserum as negative control (filled histograms with thin lines). The percentage of marker-positive cells and the mean fluorescence intensity (between parenthesis) are indicated. Each experiment was performed twice, and a representative one is shown.
2 (3,5)93 (14)
2 (4)100 (84)
11 (4)99 (76)
2 (4,4)67 (24)
CD14+ TAMs(Breast Adenocarcinoma)
Non-permeabilized
M2
ControlFRβ
Permeabilized
2 (3,5)93 (14)
2 (4)100 (84)
11 (4)99 (76)
2 (4,4)67 (24)
CD14+ TAMs(Breast Adenocarcinoma)
Non-permeabilized
M2
ControlFRβ
Permeabilized
Supplementary Figure 4.- FRβ expression in M-CSF-primed M2 macrophages (left panels) and isolated CD14+ TAM from a breast adenocarcinoma (right panels) as determined by flow cytometry using a polyclonal antiserum against human FRβ (empty histograms with thick lines) and a previously reported pre-immune rabbit antiserum as negative control (filled histograms with thin lines). In both cases cells were analyzed before (upper panels) or after permeabilization (lower panels), to detect only cell surface or total content of FRβ. The percentage of marker-positive cells and the mean fluorescence intensity (between parenthesis) are indicated in each case.
52
Resultados
2. Activina A previene la adquisición de marcadores anti-inflamatorios/M2 y sesga la secreción de citoquinas por los macrófagos.
La progresión tumoral está favorecida por el cambio en la polarización de los macrófagos
asociados a tumores hacia la adquisición de funciones efectoras inmunoreguladoras y anti-
inflamatorias. A diferencia del GM-CSF que polariza los macrófagos hacia un fenotipo inflamatorio
M1, el M-CSF genera macrófagos inmunosupresores M2 que expresan el receptor de folato β (FRβ)
y producen IL-10. Debido a que la depleción de macrófagos FRβ+ ha sido utilizado en terapia frente
a tumores, hemos buscado factores que controlan la expresión del FRβ en macrófagos. En este
sentido, hemos identificado a la activina A como una citoquina producida por los macrófagos M1
(GM-CSF), y cuya presencia limita la adquisición de la expresión del FRβ y otros marcadores M2 (M-
CSF). De hecho, el GM-CSF promueve la expresión de activina A, mientras que es inhibida por M-
CSF incluso en macrófagos M1 (GM-CSF). La activina A secretada por los macrófagos M1 (GM-
CSF) realza la actividad de los promotores génicos dependientes de Smad, explicando así la
activación diferencial de Smad2 en los macrófagos M1 (GM-CSF) y M2 (M-CSF), lo que contribuye a
la inhibición del crecimiento de células tumorales por el medio condicionado de macrófagos M1 (GM-
CSF). Además, la activina A modula la producción de citoquinas por los macrófagos M2, ya que
reduce la producción de IL-10, aunque no modifica la secreción de TNFα, en respuesta a LPS. Por
lo tanto, la activina A sesga la polarización del macrófago contribuyendo a la generación de
macrófagos inflamatorios en respuesta a GM-CSF y limitando la generación de macrófagos anti-
inflamatorios.
53
Activin prevents the acquisition of M2/anti-inflammatory markers and skews the
macrophage cytokine profile
Running head: Activin shapes macrophage polarization
Elena Sierra-Filardi ∗,‡, Amaya Puig-Kröger ∗,‡, Francisco J. Blanco*, Carmelo Bernabéu*,
Miguel A. Vega ∗ and Angel L. Corbí ∗
∗ Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
‡ Both authors contributed equally and the order of authors should be considered arbitrary.
Corresponding author: Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, CSIC. Ramiro de
Maeztu, 9. Madrid 28040; Phone: 34-91-8373112, ext. 4376; FAX: 34-91-5627518.
E-mail: [email protected]
1 This work was supported by the Ministerio de Ciencia e Innovación (Grant BFU2008-01493-BMC), Instituto de Salud Carlos III (Spanish Network for the Research in Infectious Diseases REIPI RD06/0008, and Red de Investigación en SIDA RIS RD06/0006/1016), Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36663/07), and Fundación Mutua Madrileña, to ALC, and grant PI08/1208 from Instituto de Salud Carlos III to APK. APK is supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (CP06/00199). 2 Address correspondence and reprint requests to: Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, CSIC. Ramiro de Maeztu, 9, 28040 Madrid. Tel. +34-91-8373112 + 4376; FAX: 34-91-5627518; E-mail addresses: [email protected]
55
ABSTRACT
Tumor progression is favored by the shift in the polarization state of tumor-associated macrophages
towards the acquisition of immunoregulatory and anti-inflanmatory effector functions. Unlike GM-
CSF, which polarizes macrophages towards a pro-inflammatory M1 phenotype, M-CSF generates IL-
10-producing Folate Receptor β (FRβ)-expressing immunosuppressive M2 macrophages. Since
depletion of FRβ-expressing macrophages has proven successful in cancer therapy strategies, we
sought to identify the factors controlling macrophage FRβ expression. The search identified Activin
A as a cytokine produced by M1 (GM-CSF) macrophages, and whose presence limits the acquisition
of FRβ and other M2 (M-CSF)-specifc markers. In fact, GM-CSF promotes Activin A expression,
whereas M-CSF downregulates its expression even in fully polarized M1 (GM-CSF) macrophages.
M1 (GM-CSF) macrophage-derived Activin A enhances the activity of Smad-dependent gene
promoters, thus explaining the differential Smad2 activation of M1 (GM-CSF) and M2 (M-CSF)
macrophages, and contributes to the tumor cell growth inhibitory activity of M1 (GM-CSF)
Macrophage-conditioned medium. Besides, Activin A modulates cytokine production by M2
macrophages, as it reduces their LPS-induced IL-10 production and it had no effect on the TNFα
release in response to LPS. Therefore, Activin A skews macrophage polarization by contributing to
the generation of pro-inflammatory macrophages in response to GM-CSF and limiting the generation
of anti-inflammatory macrophages.
56
INTRODUCTION
Tissue resident macrophages are phenotypically and functionally heterogeneous under homeostatic
conditions because of their extreme sensitivity to the extracellular cytokine millieu 1-3. Although GM-
CSF and M-CSF contribute to cell survival, proliferation and macrophage development, they exert
distinct actions during macrophage differentiation in vivo and in vitro. Deficiency of M-CSF alters
the development of various macrophage populations 4, whereas GM-CSF-deficient mice only exhibit
altered maturation of alveolar macrophages 5. Along the same line, both cytokines promote the in
vitro differentiation of macrophages with distinct morphology, pathogen susceptibility 6 and
inflammatory function 7-10. GM-CSF gives rise to monocyte-derived macrophages with high antigen-
presenting properties and which produce pro-inflammatory cytokines in response to LPS, whereas
M-CSF leads to the generation of macrophages with high phagocytic activity and IL-10-producing
ability in response to pathogens 10,11. Based on their respective cytokine profiles, human macrophages
generated in the presence of GM-CSF or M-CSF are considered as representative of the classically
(M1) or alternatively activated (M2) macrophage polarization states, respectively 10,12,13. Moreover,
since they might play opposite roles during immune and inflammatory responses, M1 (GM-CSF) and
M2 (M-CSF) macrophages are now considered as pro- and anti-inflammatory macrophages 10,12,
respectively.
Activins are pluripotent and ubiquitous growth and differentiation factors, which are structurally
composed of two β subunits (activin-A, βAβA; activin-AB, βAβB; activin-B, βBβB) linked by a
single covalent disulfide bond 14,15. Activin biological activities are mediated by signal transduction
molecules shared by TGFβ (Smad2,3) 16. Like TGFβ, activins exert both immunostimulatory and
immunosuppressive functions at the T cell level 16, and their effects on myeloid cells include
promotion of macrophage alternative activation 17 and inhibition of CD40L-induced cytokine
production by monocyte-derived dendritic cells 18. Activin A expression has been detected in many
immune cell types 16, and is upregulated upon activation and in response to inflammatory mediators
both in vitro 18 and in vivo 19, what has led to the suggestion that it functions as a modulator of
inflammatory responses by limiting cytokine and chemokine release.
We have recently dissected the differences in gene expression between M1 (GM-CSF) and M2 (M-
CSF) macrophages, and described the preferential expression of Folate Receptor β (FRβ) on in vitro
derived M2 (M-CSF) macrophages and ex vivo Tumor-Associated Macrophages (TAM) 20. To
identify factors mediating the acquisition of their respective profiles, we analyzed whether M1-
derived factors influenced the acquisition of M2-specific markers. M1 macrophages were found to
secrete large amounts of functional Activin A, whose presence conditions the activation state of the
57
TGFβ signaling system, impairs the acquisition of M2 (M-CSF) markers, and modulates the
production of IL-10. These results place Activin A as a factor that contributes to macrophage
polarization and shapes the inflammatory behaviour of macrophages. Moreover, given the
macrophage ability for re-polarization under appropriate cytokine conditions 21, Activin A might
function, in an autocrine or paracrine manner, by halting macrophage switch between polarization
states.
58
MATERIALS AND METHODS
Cell culture and flow cytometry.- Human peripheral blood mononuclear cells (PBMC) were isolated
from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway)
gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell
sorting using CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes (>95%
CD14+ cells) were cultured at 0.5 x 106 cells/ml for 7 days in RPMI supplemented with 10% fetal
calf serum (FCS) (completed medium), at 37ºC in a humidified atmosphere with 5% CO2, and
containing 1000U/ml GM-CSF or M-CSF (10 ng/ml, ImmunoTools GmbH, Friesoythe, Germany) to
generate M1 and M2 monocyte-derived macrophages, respectively. Cytokines were added every two
days. When indicated, recombinant human Activin A (2.5-25 ng/ml, Miltenyi Biotech, Bergisch
Gladbach, Germany) was added together with the indicated cytokine. To generate monocyte-derived
dendritic cells (MDDC), monocytes were cultured at 0.7 x 106 cells/ml in complete medium
containing GM-CSF (1000 U/ml) and IL-4 (1000U/ml, ImmunoTools GmbH, Friesoythe, Germany)
for 5-7 days, with cytokine addition every second days. When indicated, M1 macrophages were
treated with IL-4 (1000U/ml) or IFNγ (500U/ml) for 48 hours. The mink lung epithelial cell line
Mv1Lu 22 was maintained in DMEM supplemented with 10% fetal calf serum. Phenotypic analysis
was carried out by flow cytometry as previously reported 20, and using rabbit polyclonal antisera anti-
human FRβ 23 or a previously described preimmune serum 24, and FITC-labelled Fab goat anti-rabbit
IgG. All incubations were done in the presence of 50 µg/ml of human IgG to prevent binding through
the Fc portion of the antibodies.
Western blot.- Cell lysates were obtained in 10mM Tris-HCl pH 8, 150mM NaCl, 1%NP-40 (NP-40
lysis buffer) containing 2 mM Pefabloc and 2 µg/ml of aprotinin, antipain, leupeptin and pepstatin.
Ten µg of cell or membrane lysates was subjected to SDS-PAGE and transferred onto an Immobilon
polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking of the unoccupied
sites with 5% non-fat dry milk in 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20, protein
detection was performed using the Supersignal West Pico Chemiluminescent system (Pierce,
Rockford, IL). Protein detection was carried out using polyclonal antisera against phosphorylated
Smad2 (pSmad2 (Ser465/467), clone A5S, Millipore), Smad2 (anti-Smad2/3, Millipore), GAPDH
(sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin (Sigma-Aldrich, UK ).
ELISA.- Supernatants from M1 and M2 macrophages were tested for the presence of cytokines and
growth factors using commercially available ELISA for TNF-α (ImmunoTools), IL-6
(Immunotools), IL-12p40 (OptEIATM IL-12p40 set, BD Pharmingen, San Diego, CA), IL-10
59
(ImmunoTools), and Activin A (R&D Systems, Inc, Minneapolis, USA) following the protocols
supplied by the manufacturers.
Reporter Gene Assays.- The effect of macrophage culture supernatants on the Activin signaling
pathway was analyzed by transfecting 0.5 μg of the p3TP-Lux reporter construct 25 in Mv1Lu cells, a
well stablished cellular model to study the signaling of the TGF-β superfamily 22, using Superfect
(Qiagen). After transfections, cells were washed, cultured in DMEM plus 0.2% FCS, and treated with
undiluted condition media from M1 (GM-CSF) or M2 (M-CSF) macrophages, 25 ng/ml rhActivin A
(Miltenyi Biotec) or 10 ng/ml TGF-β1 (R&D Systems) for 24 hours. When indicated, cells were
preincubated for 30 minutes with 10 μM SB431542 (Sigma), an ALK4, 5 and 7 inhibitor, before
treatment. Activin A activity in M1 supernatants was neutralized using 0.1 μg/ml of a blocking
antibody (R&D Systems). In some experiments, cells were cotransfected with 0.4 μg of expression
vectors for a dominant negative mutant of either Smad2 26 or Smad3 27. To normalize transfection
efficiency, cells were co-transfected with an SV40 promoter-based β-galactosidase expression
plasmid (RSV-βgal). Measurement of relative luciferase units and β-galactosidase activity were
performed using the Dual-Glo Luciferase Assay System (Promega) and the Galacto-Ligth kit
(Tropix), respectively, in a Varioskan Flash spectral scanning multimode reader (Thermo Scientific).
Quantitative real-time RT-PCR.- Oligonucleotides for selected genes were designed according to
the Roche software for quantitative real time PCR. Total RNA from M1 and M2 macrophages was
extracted using the RNAeasy kit (Qiagen), retrotranscribed and amplified using the Universal Human
Probe Roche library (Roche Diagnostics). Assays were made in triplicates and results normalized
according to the expression levels of 18S RNA and GAPDH. Results were expressed using
the ΔΔCT method for quantitation.
60
RESULTS
M1 (GM-CSF)-conditioned medium prevents acquisition of Folate Receptor β (FRβ) expression.-
Expression of cell surface FRβ, encoded by the FOLR2 gene, identifies IL-10-producing ex vivo
isolated Tumor-Associated Macrophages (TAM) 20, as well as M2 (M-CSF), but not M1 (GM-CSF),
in vitro polarized macrophages (Figure 1A). FRβ mediates the capture of Folate-FITC by M2 (M-
CSF) polarized macrophages, but not GM-CSF-polarized (M1) macrophages (Figure 1B). That the
differential expression FRβ in M1 and M2 macrophages is due to the opposite effect of GM-CSF and
M-CSF on FOLR2 gene expression was indicated by the dramatic downregulation of FOLR2 RNA
levels in FRβ-positive M2 (M-CSF) macrophages after exposure to GM-CSF (Figure 1C), and by the
inhibitory effect of M1 (GM-CSF) macrophage-conditiones medium on the FOLR2 RNA induction
that takes place in cytokine-free medium 20 (Figure 1D). In fact, this inhibitory activity was evident
even after a 1/10 dilution of the M1 (GM-CSF)-conditioned medium, which reduced FOLR2 RNA
induction by more than 90% (Figure 1D). Therefore, M1 (GM-CSF) macrophages secrete factor(s)
that prevent the acquisition of the M2 (M-CSF)-specific marker FRβ.
R
elat
ive
mR
NA
leve
ls
4
810
6
2
0 10Mo. 50 100% SN M1
FOLR2D
1% (3)82% (13)
1% (3)1% (4)
M1 M2
A
M1 M2
B
0.2
0.40.6
0.8
1.01.2
FOLR2
Rel
ativ
e m
RN
A le
vels
CGM-CSFM-CSF
FRβControl
Transferrin-Texas Red
Folate-FITC
Rel
ativ
e m
RN
A le
vels
4
810
6
2
0 10Mo. 50 100% SN M1
FOLR2D
1% (3)82% (13)
1% (3)1% (4)
M1 M2
A
M1 M2
B
0.2
0.40.6
0.8
1.01.2
FOLR2
Rel
ativ
e m
RN
A le
vels
CGM-CSFM-CSF
FRβControl
Transferrin-Texas Red
Folate-FITC
Figure 1.- M1 (GM-CSF)-conditioned medium inhibits the M-CSF-induced Folate Receptor β (FRβ) expression.- A. Cell surface expression of FRβ on M1 and M2 macrophages, as determined by flow cytometry using a polyclonal antiserum against human FRβ 23 (empty histogram). As a control, a previously described rabbit pre-immune antiserum 24 was used (filled histogram). The percentage of marker-positive cells and the mean fluorescence intensity (in parenthesis) are indicated. B. FRβ function in M1 and M2 macrophages, as demonstrated by confocal microscopy on cells incubated at 37ºC with Folate-FITC (green fluoresence) and Transferrin-Texas red (red fluorescence). C. FOLR2 mRNA expression levels, as determined by qRT-PCR on M2 (M-CSF) macrophages after replacement of the culture supernatant by either M-CSF- (grey histograms) or GM-CSF-containing complete medium (empty histograms) for 24 hours. Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the RNA levels in cells maintained in M-CSF-containing medium). Mean and standard deviation of triplicate determinations are shown. D. FOLR2 mRNA expression levels determined by qRT-PCR on monocytes (Mo.) and macrophages exposed to M-CSF for 7 days and either in the absence (0)
61
or in the presence of different concentrations of M1 (GM-CSF) macrophage-conditioned media (% SN M1). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to levels detected in peripheral blood monocytes). Mean and standard deviation of triplicate determinations are shown.
M1 (GM-CSF) Macrophages secrete Activin A, which downregulates FOLR2 gene expression.- To
identify M1 (GM-CSF)-derived factors that prevent FRβ induction, we searched for soluble factors
preferentially produced by M1 macrophages. Gene expression profiling 20 revealed that expression of
the INHBA gene, which codes for the Inhibin βA subunit 14,15, is >30-times higher in M1 than in M2
macrophages (log2 M1/M2 = 6.1; p = 5.3 x 10-8, Figure 2A), a difference further verified by qRT-
PCR on independent samples (Figure 2A). In fact, and unlike FOLR2, INHBA RNA expression was
induced in M2 macrophages after exposure to GM-CSF (Figure 2B), and abrogated in fully polarized
M1 (GM-CSF) macrophages upon replacement of their conditioned medium by M-CSF (Figure 2C).
In agreement with RNA data and its inducibility by GM-CSF 28, Activin A protein levels were
considerably higher in M1 (GM-CSF)-conditioned media (Figure 2D), where Activin A levels
continuously increased from the initial stages of the M1 differentiation/polarization process (Figure
2E). Moreover, and although LPS increases circulating Activin A levels in vivo 19, the differential
production of Activin A by both types of macrophages was maintained after LPS stimulation (Figure
2F). Altogether, these results indicate that Activin A expression is differentially regulated by GM-
CSF and M-CSF, and that Activin A expression inversely correlates with FOLR2 gene expression.
1.0
2.0
3.0
4.0
Act
ivin
A (n
g/m
l) M1
M2
#1 #2 #3 #4 #5 #6
Donor
D
1
2
6
Act
ivin
A (n
g/m
l)
Donor # 1 Donor # 2
0 1 3 7 0 1 3 7 days
EM1
M2
log 2
M1
/ M2
INHBA
10
2468
A
qPCRMicroarray
4
812
16
M1 M1+LPS
M2 M2+LPS
Act
ivin
A (n
g/m
l)
FM1
M2
10203040506070 INHBA
Rel
ativ
e m
RN
A le
velsB
0.2
0.40.6
0.8
1.0
INHBA
Rel
ativ
e m
RN
A le
velsC
GM-CSFM-CSF
Addition ReplaceGM-CSF
M-CSF
1.0
2.0
3.0
4.0
Act
ivin
A (n
g/m
l) M1
M2
#1 #2 #3 #4 #5 #6
Donor
D
1
2
6
Act
ivin
A (n
g/m
l)
Donor # 1 Donor # 2
0 1 3 7 0 1 3 7 days
EM1
M2
log 2
M1
/ M2
INHBA
10
2468
A
qPCRMicroarrayqPCRMicroarray
4
812
16
M1 M1+LPS
M2 M2+LPS
Act
ivin
A (n
g/m
l)
FM1
M2
10203040506070 INHBA
Rel
ativ
e m
RN
A le
velsB
0.2
0.40.6
0.8
1.0
INHBA
Rel
ativ
e m
RN
A le
velsC
GM-CSFM-CSF GM-CSFM-CSF
Addition ReplaceGM-CSF
M-CSF
Figure 2.- Activin A is differentially produced by M1 (GM-CSF) and M2 (M-CSF) polarized macrophages.- A. Relative INHBA gene expression in M1 and M2 macrophages, as determined by microarray DNA analysis (empty histograms) and quantitative RT-PCR (grey histograms). B. INHBA mRNA expression levels as determined by qRT-PCR on M2 (M-CSF) macrophages after replacement of the culture supernatant by either M-CSF- (grey histograms) or GM-CSF-containing complete medium (empty histograms) for 24 hours. Results are expressed as Relative mRNA levels (relative to 18S rRNA
62
levels and referred to the RNA levels in cells maintained in M-CSF-containing medium). Mean and standard deviation of triplicate determinations are shown. C. INHBA mRNA expression levels as determined by qRT-PCR on M1 (GM-CSF) macrophages treated with M-CSF (grey histograms) or GM-CSF (empty histograms) for 24 hours and either in their own conditioned medium (Addition) or after replacement of the culture supernatant (Replace). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the RNA levels determined in cells cultured with GM-CSF). D. Determination of Activin A levels released by M1 (GM-CSF) and M2 (M-CSF) macrophages generated from peripheral blood monocytes of six independent donors, as determined by ELISA. Each determination was performed in triplicate, and mean and standard deviations are shown. E. Determination of Activin A release during the differentiation of M1 (GM-CSF) and M2 (M-CSF) macrophages from two independent donors, as determined by ELISA on culture supernatants removed at the indicated time points. Each determination was performed in triplicate, and mean and standard deviations are shown. F. Determination of Activin A levels released by M1 (GM-CSF) and M2 (M-CSF) macrophages either untreated or stimulated with 10 ng/ml LPS for 24 hours. Each determination was performed in triplicate, and mean and standard deviations are shown.
To determine whether INHBA-encoded Activin A affects FOLR2 gene expression, M2 polarization
was accomplished in the presence of recombinant human Activin A. The M-CSF-dependent
acquisition of FOLR2 RNA expression was dose-dependently reduced in the presence of Activin A,
an effect that could be observed both during (3 days) and at the end (7 days) of the
macrophage/polarization process (Figure 3A,B). Moreover, Activin A also reduced the FOLR2 RNA
upregulation that takes place in the absence of exogenous M-CSF (Figure 3B). Therefore, Activin A
inhibits the acquisition of FOLR2 RNA expression in differentiating macrophages. Consequently, it
can be concluded that the differential expression of FRβ on M1 and M2 macrophages is a
consequence of the opposite actions of GM-CSF and M-CSF on INHBA gene expression.
M-CSFGM-CSFAct. A 2.5 ng/mlAct. A 25 ng/ml
100
200
150
50
FOLR2
Mo.Rel
ativ
e m
RN
A le
velsBA
Rel
ativ
e m
RN
A le
vels FOLR2
0.4
0.8
1.21.0
0.6
0.2
0.4
0.8
1.0
0.6
0.2
FOLR2
M-CSF GM-CSF M-CSF+Act. A
3 days 7 days M-CSFGM-CSFAct. A 2.5 ng/mlAct. A 25 ng/ml
100
200
150
50
FOLR2
Mo.Rel
ativ
e m
RN
A le
velsBA
Rel
ativ
e m
RN
A le
vels FOLR2
0.4
0.8
1.21.0
0.6
0.2
0.4
0.8
1.0
0.6
0.2
FOLR2
M-CSF GM-CSF M-CSF+Act. A
3 days 7 days
Figure 3.- Activin A inhibits Folate Receptor β (FRβ) expression.- A. FOLR2 mRNA expression levels as determined by qRT-PCR on macrophages differentiated for 3 days (left panel) or 7 days (right panel) in the presence of M-CSF, GM-CSF or M-CSF plus Activin A (Act. A). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the presence of M-CSF), and shown is the mean and standard deviation of triplicate determinations. B. FOLR2 mRNA expression levels determined by qRT-PCR on monocytes (Mo.) or macrophages differentiated in the presence of the indicated cytokine combinations. Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level in monocytes), and shown is the mean and standard deviation of triplicate determinations.
63
Activin A contributes to M1 (GM-CSF) Macrophage polarization.- Considering the above results,
we assessed the influence of Activin A on genes whose expression, like FOLR2, is significantly
higher in M2 (MCSF) than in M1 (GM-CSF) macrophages 20 (Figure 4A). After a 7-day M-CSF-
driven polarization, Activin A was capable of inhibiting the M-CSF-dependent induction of MAF,
HTR2B, SEPP1, IGF1 and F13A1, and abrogating that of SERPINB2, whereas it had no inhibitory
effect on HS3ST1 gene expression (Figure 4B). Therefore, Activin A inhibits the expression of genes
preferentially upregulated during M-CSF-dependent polarization. On the other hand, and since the
upregulation of other M2 (M-CSF)-specific genes like MAFB or HMOX1 is not affected by Activin
A but prevented by GM-CSF (Supplementary Figure 1), it is tempting to conclude that the combined
action of both GM-CSF and Activin A limits the acquisition of M2 (M-CSF) macrophage markers
during the M-CSF-dependent polarization.
B
Rel
ativ
e m
RN
A le
vels
0.4
0.8
1.0
0.6
0.2
0.4
0.81.0
0.6
0.2
HTR2B
0.4
0.8
1.0
0.6
0.2
MAF SEPP1
0.4
0.8
1.0
0.6
0.2
IGF1
0.4
0.81.0
0.6
0.2
SERPINB2
1.0
2.0
1.5
0.5
HS3ST1
0.4
0.8
1.0
0.6
0.2
F13A1
Rel
ativ
e m
RN
A le
vels
M-CSF GM-CSF M-CSF+Act. A
F13A1 SERPINE1SERPINB2
log 2
M1
/ M2
10
-4-2
2468
-8-6
MAFHTR2BSEPP1IGF1
A
qPCRMicroarray
4.4 x 10-7 3.9 x 10-12 2.0 x 10-2 4.2 x 10-4 3.6 x 10-7 5.4 x 10-3
1.2 x 10-2
B
Rel
ativ
e m
RN
A le
vels
0.4
0.8
1.0
0.6
0.2
0.4
0.81.0
0.6
0.2
HTR2B
0.4
0.8
1.0
0.6
0.2
MAF SEPP1
0.4
0.8
1.0
0.6
0.2
IGF1
0.4
0.81.0
0.6
0.2
SERPINB2
1.0
2.0
1.5
0.5
HS3ST1
0.4
0.8
1.0
0.6
0.2
F13A1
Rel
ativ
e m
RN
A le
vels
M-CSF GM-CSF M-CSF+Act. AM-CSF GM-CSF M-CSF+Act. A
F13A1 SERPINE1SERPINB2
log 2
M1
/ M2
10
-4-2
2468
-8-6
MAFHTR2BSEPP1IGF1
A
qPCRMicroarray
4.4 x 10-7 3.9 x 10-12 2.0 x 10-2 4.2 x 10-4 3.6 x 10-7 5.4 x 10-3
1.2 x 10-2
Figure 4.- Effect of Activin A on the acquisition of M2 (M-CSF)-specific markers.- A. Relative expression of the indicated genes in M1 (GM-CSF)- and M2 (M-CSF)-polarized macrophages, as determined by microarray DNA analysis (empty histograms) and quantitative RT-PCR (grey histograms). B. MAF, HTR2B, SEPP1, IGF1, SERPINB2, F13A1 and HS3ST1 mRNA expression levels as determined by qRT-PCR on macrophages differentiated for 7 days in the presence of M-CSF, GM-CSF or M-CSF plus Activin A (Act. A). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the presence of M-CSF), and shown is the mean and standard deviation of triplicate determinations.
64
Because of its continuous presence during GM-CSF-mediated polarization, we hypothesized that
Activin A could shape the phenotypic and functional polarization state of macrophages in an
autocrine/paracrine manner. It has been proposed that Activin A is a Th2-polarizing cytokine 17 and,
therefore, its effects on the expression of genes preferentially found in both M1 (GM-CSF) and IL-4-
activated macrophages 29 was evaluated. Activin A did not modify the expression of TM4SF1,
MMP12 and CCL17, which are preferentially expressed by M1 (GM-CSF) macrophages
(Supplementary Figure 2A), or ILR1N, a known Actinin A target gene 30 (Supplementary Figure 2B).
By contrast, the presence of Activin A enhanced SERPINE1 RNA levels, which are significantly
higher in M1 (GM-CSF) macrophages (Figure 5A). Therefore Activin A contributes to shaping the
phenotypic polarization of M1 (GM-CSF) macrophages.
Next, conditioned medium from M1 (GM-CSF) macrophages was analyzed for Activin A-dependent
functions. Activin A has tumour suppressive properties and contributes to cancer cell growth arrest 31,32, and results indicated that M1 (GM-CSF) macrophage-conditioned medium inhibits the growth
of K562 leukemic cells (Figure 5B) and promotes their differentiation into Hemoglobin-expressing
cells (Figure 5C). Moreover, both activities were reduced in the presence of a blocking anti-Activin
A monoclonal antibody (Figure 5B,C). Therefore, M1 (GM-CSF) macrophages release functional
Activin A, what might endow them with the tumor-resistance capability that characterizes M1-
polarized macrophages 33.
20406080
100
M1 M1+
antiActAActA- M2
Rel
ativ
e ce
ll Pr
olife
ratio
n (9
6h)
51
7381 80
106
M1 ActA- M2 M1 M2
Donor # 1 Donor # 2
10
20
30
Ben
zidi
ne+
cells
ActA+
antiActAM1+
antiActAActA
+antiActA
B C
Donor # 1 Donor # 2
0.20.40.60.81.0
SERPINE1
M-CSF
GM-CSF
Activin A
Rel
ativ
e m
RN
A le
vels
A
20406080
100
M1 M1+
antiActAActA- M2
Rel
ativ
e ce
ll Pr
olife
ratio
n (9
6h)
51
7381 80
106
M1 ActA- M2 M1 M2
Donor # 1 Donor # 2
10
20
30
Ben
zidi
ne+
cells
ActA+
antiActAM1+
antiActAActA
+antiActA
B C
Donor # 1 Donor # 2
0.20.40.60.81.0
SERPINE1
M-CSF
GM-CSF
Activin A
Rel
ativ
e m
RN
A le
vels
A
Figure 5.- Effect of Activin A on the acquisition of M1 (M-CSF)-specific markers and effector functions.- A. SERPINE1 mRNA expression levels as determined by qRT-PCR on macrophages from two independent donors, and treated for 7 days with M-CSF, GM-CSF or Activin A. Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the presence of GM-CSF), and shown is the mean and standard deviation of triplicate determinations. B. Proliferation of K562 cells exposed for 96 hours to the indicated conditioned media (M1 or M2) or Activin A, and either in the absence or presence or a blocking anti-Activin A monoclonal antibody. Results are expressed relative to the proliferation observed in untreated cells (Relative cell proliferation). C. Differentiation of K562 cells into Hemoglobin-containing cells (Benzidine+ cells) after exposure for 96 hours to Activin A or the indicated conditioned media (M1 or M2) from two independent donors, and either in the absence or presence or a blocking anti-Activin A monoclonal antibody.
65
Activin A modulates the cytokine-producing profile of M2 (M-CSF) macrophages.- The above
results imply that Activin A actively participates in shaping macrophage polarization. Therefore, the
influence of Activin A on the paradigmatic effector functions of M1 (GM-CSF) and M2 (M-CSF)
macrophages was studied. As reported 10,12, M1 and M2 macrophages differ in terms of T cell
stimulayory activity and cytokine/chemokine release in response to pathogenic stimulation 9,10.
Although M1 (GM-CSF) induced considerable higher T cell proliferation than M2 (M-CSF) in
allogeneic MLR, exposure of the latter to Activin A did not modify their T cell stimulatory ability
(Supplementary Figure 3), indicating that M1 (GM-CSF)-derived Activin A is not responsible for the
high T cell stimulary activity of M1 (GM-CSF) macrophages. Regarding cytokine release, and in
agreement with previous reports 10, LPS stimulation led to production of significant levels of pro-
inflammatory cytokines (TNFα, IL-12p40, IL-6) and acquisition of dendritic cell maturation ability
(Supplementary Figure 4) by M1 (GM-CSF) macrophages, whereas M2 (M-CSF) produced high
levels of IL-10 and low (TNFα) or undetectable (IL-6, IL12p40) levels of pro-inflammatory
cytokines (Figure 6A). However, the presence of Activin A significantly reduced the release of IL-10
from LPS-stimulated M2 (M-CSF) macrophages, although it had no effect on the production of
TNFα (Figure 6B). This result indicates that Activin A negatively regulates IL-10 production from
M2 (M-CSF) macrophages, and suggests that Activin A contributes to the potent pro-inflammatory
nature of M1 (GM-CSF) macrophages by inhibiting IL-10 production. Moreover, macrophages
exposed to Activin A during the M-CSF-driven polarization process exhibited a highly diminished
production of IL-10 in response to LPS (Figure 6B). Therefore, Activin A also interferes with the
acquisition of the IL-10-producing ability by M2 (M-CSF) macrophages. This result further supports
the involvement of Activin A in shaping macrophage polarization by impairing the acquisition of an
anti-inflammatory phenotype and cytokine profile.
66
A
50100150200250300
IL-1
0 (p
g/m
l)
50010001500200025003000
IL-1
2p40
(pg/
ml)
100200300400500600700
TN
Fα(p
g/m
l)
M1
IL-6
(pg/
ml)
1000200030004000500060007000
M1/LPS M2 M2/LPS
B
LPS LPSActA
M2
LPS LPSActA
M2 ActA
20406080
100
Rel
ativ
e le
vels
of
LPS
-indu
ced
IL-1
0
4080
120160200
Rel
ativ
e le
vels
of
LPS
-indu
ced
TN
Fα
p=0.003
p=0.01
A
50100150200250300
IL-1
0 (p
g/m
l)
50010001500200025003000
IL-1
2p40
(pg/
ml)
100200300400500600700
TN
Fα(p
g/m
l)
M1
IL-6
(pg/
ml)
1000200030004000500060007000
M1/LPS M2 M2/LPS
B
LPS LPSActA
M2
LPS LPSActA
M2 ActA
20406080
100
Rel
ativ
e le
vels
of
LPS
-indu
ced
IL-1
0
4080
120160200
Rel
ativ
e le
vels
of
LPS
-indu
ced
TN
Fα
p=0.003
p=0.01
Figure 6.- Effect of Activin A on the LPS-induced cytokine profile of polarized macrophages.- A. Determination of IL-12p40, IL-6, IL-10 and TNFα release by ELISA in culture supernatants of M1 and M2 macrophages either untreated or stimulated with LPS (10 ng/ml) for 24 hours. Each determination was performed in triplicate, and mean and standard deviations are shown. B. Determination of IL-10 and TNFα release by ELISA in culture supernatant of M2 macrophages differentiated in the absence (M2) or presence of Activin A (M2 ActA), and either unstimulated or stimulated with LPS (10 ng/ml) for 24 hours in the presence or absence of Activin A. Each determination was performed in triplicate, and mean and standard deviations are shown.
Activin A-initiated signaling during macrophage polarization.- Since Activin A activates Smad2 16,
Smad2 phosphorylation was determined in unstimulated and LPS-stimulated macrophages. Smad2
was constitutively phosphorylated in M1 (GM-CSF) macrophages, and LPS treatment did not modify
its phosphorylation state (Figure 7A). By contrast, no Smad2 phosphorylation was detected in either
untreated or after LPS-stimulated M2 (M-CSF) macrophages (Figure 7A). The absence of Smad2
activation in M2 macrophages was not due to a defective Activin/Smad signaling pathway, as
Activin A treatment of M2 macrophages readily led to overt Smad2 phosphorylation (Figure 7B). A
definitive support for an Activin A role in shaping M1 (GM-CSF) macrophage polarization was
obtained through evaluation of the transcriptional effects of M1 (GM-CSF)-derived Activin A. Like
TGFβ and recombinant Activin A, M1 (GM-CSF)-conditioned medium transactivated the Smad2-
dependent p3TP-Lux reporter construct (p = 0.0008), whereas supernatants from M2 (M-CSF)
macrophages had no effect (Figure 7C). Importantly, the transactivation ability of the M1 (GM-
CSF)-conditioned medium was abolished (p = 0.015) by SB431542, an inhibitor of ALK4, ALK5
67
and ALK7 receptors which prevents Smad2 phosphorylation 34 (Figure 7C), by cotransfection of a
dominant negative form of Smad2 (Figure 7C) and by a blocking antibody against human Activin A
(p = 0.0001) (Figure 7C). Altogether, this set of results demonstrate that M1 (GM-CSF) macrophage-
derived Activin A activates Smad2, and modulates gene expression in macrophages and other cell
types, thus providing a molecular basis for its macrophage polarization shaping ability.
β-actin
M2/LPSM1/LPShours0 0.5 2 0 0.5 2pSmad2
Smad2
A
n.s.
C
RL
U (L
uc/β
-Gal
)x10
0
Act. AM1 SNM2 SN
---
SB431542 anti.-Act.A
Smad2(d.n.)
Smad3(d.n.)
TGF-β1
2468
10121416
p=0.0008
p=0.0001
p=0.015
pSmad2
GAPDH
M2- +Act.A
B
β-actin
M2/LPSM1/LPShours0 0.5 2 0 0.5 2pSmad2
Smad2
A
n.s.
C
RL
U (L
uc/β
-Gal
)x10
0
Act. AM1 SNM2 SN
---
SB431542 anti.-Act.A
Smad2(d.n.)
Smad3(d.n.)
TGF-β1
2468
10121416
p=0.0008
p=0.0001
p=0.015
pSmad2
GAPDH
M2- +Act.A
B
Figure 7.- Smad2 is constitutively phosphorylated in M1 (GM-CSF) macrophages, whose Activin A release activates Smad-dependent reporter genes.- A. Detection of activated and total Smad2 on lysates of untreated or LPS-treated M1 and M2 macrophages, as determined by Western blot. β-actin expression levels were determined in parallel as a loading control. B. Detection of activated Smad2 on lysates of untreated or Activin A-treated M2 macrophages, as determined by Western blot. GAPDH expression levels were determined in parallel as a loading control. C. Transcriptional activity of the p3TP-Lux reporter construct in Mv1Lu cells either unstimulated or exposed to 10 ng/ml TGFβ1, 25 μg/ml Activin A, or conditioned medium from M1 (M1 SN) or M2 (M2 SN) macrophages. When indicated, cells were either preincubated for 30 minutes with 10 μM SB431542 before treatment, maintained in culture medium with 0.1 μg/ml of an anti-Activin A (anti-Act.A) or cotransfected with expression vectors coding for dominant negative mutants of either Smad2 (Smad2 d.n.) or Smad3 (Smad3 d.n.). For normalization purposes, cells were co-transfected with the RSV-β-gal expression plasmid, and results are presented as RLU (Relative Light Units), which indicate the units of luciferase activity per unit of β-galactosidase activity for each assay condition. Experiments were performed in triplicate, and shown are the mean and standard deviation.
Tumor-conditioned media modulates Activin A expression.- Since INHBA mRNA levels pro-
inflammatory macrophages and tumors-derived factors polarize macrophages towards an
alternative/anti-inflammatory state 35, we hypothesized that the expression of INHBA and FOLR2
mRNA could be also oppositely modulated in the presence of tumor-conditioned media. To test this
hypothesis, Activin A-expressing M1 (GM-CSF) macrophages were exposed to ascitic fluid from
three independent metastatic gastric carcinomas or a metastatic colon carcinoma. As shown in Figure
8, the presence of the tumor-derived media caused a great reduction in the levels of INHBA mRNA
expression, that is compatible with tumor-derived factors promoting a shift in macrophage
polarization. In fact, and in agreement with its preferential expression in anti-inflammatory
macrophages 20, FOLR2 mRNA levels exhibited a concomitant increase in those M1 (GM-CSF)
macrophages that had been exposed to the ascitic fluid with a more potent inhibitory action on
68
INHBA mRNA expression (Figure 8). Altogether these results confirm the modulatory action that
tumor-derived factors have on macrophage polarization, and indicate that both INHBA and FOLR2
mRNA levels are adequate markers for pro-inflammatory M1 and anti-inflammatory M2
macrophages.
0,16
0,47
0,07 0,1
0,56
INHBA1.0
1
Nor
mal
ized
rel
ativ
e m
RN
A le
vels
11,02
7,46
1.01
5
10
0,03
Colon# 2# 1
Gastric carcinoma
# 3
FOLR2
0,160,16
0,470,47
0,07 0,1
0,560,56
INHBA1.0
1
Nor
mal
ized
rel
ativ
e m
RN
A le
vels
11,02
7,46
1.01
5
10
0,030,03
Colon# 2# 1
Gastric carcinoma
# 3
FOLR2
Figure 8.- Tumor-conditioned media oppositely modulates INHBA and FOLR2 mRNA in pro-inflammatory M1 (GM-CSF) macrophages.- INHBA and FOLR2 mRNA levels were determined in M1 (GM-CSF) macrophages exposed for 24 hours to a 1:1 dilution of ascitic fluid from the indicated metastatic tumors. Results are expressed as Normalized Relative mRNA levels according to GAPDH mRNA levels and relative to the respective mRNA levels present in M1 (GM-CSF) macrophages maintained under normal culture conditions. Shown is the mean and standard deviation of triplicate determinations.
69
DISCUSSION
Macrophage differentiation and polarization are critically determined by the cellular environment,
which also dictates cytokine responsiveness 36. The search for the mechanisms underlying GM-CSF-
and M-CSF-driven macrophage differentiation and the acquisition of anti-inflammatory M2 (M-CSF)
macrophage markers has led to the identification of Activin A as a factor that shapes macrophage
polarization in response to GM-CSF, and whose presence limits the production of IL-10 and prevents
the expression of genes associated to M2 macrophage polarization. The relevance of the Activin A
expression by macrophages is underscored by its downregulation in the presence of tumor-
conditioned media. Since tumors skew macrophage polarization towards the acquisition of
alternative/M2 phenotypic and functional characteristics 37, the downmodulation of Activin A by
tumor-derived ascitic fluids strongly suggests that Activin A constitutes a useful marker for the
identification of TAM whose polarization state has not yet been fully modulated by the tumor
microenvironment. Whereas the identity of the tumor-derived factors that downmodulate Activin A
is currently unknown, it is tempting to speculate that M-CSF might be a contributing factor, specially
considering its negative regulatory effect on INHBA mRNA expression in vitro (Figure 2) and its the
positive regulatory action on FOLR2 mRNA levels.
The ability of Activin A to trigger Arginase-1 expression and inhibit IFNγ-induced NO synthase
expression has led to the suggestion that it functions as a Th2 cytokine that promotes alternative
murine macrophage activation 17. However, although M1 macrophages release high levels of Activin
A, they do not display any of the phenotypic makers that characterize alternatively activated human
macrophages 29 (Puig-Kröger, Sierra-Filardi, Vega and Corbí, unpublished). Activin exhibits both
pro- or anti-inflammatory activities 19, and is synthesized by monocytes/macrophages in response to
inflammatory stimuli. Although it does not elicit TNF-α release by itself, Activin A stimulates the
production of IL-1, IL-6 and TNF-α by human monocytes and macrophages, inhibits IL-10 effects
on prostatic epithelial cells 38, and its inhibition by follistatin leads to reduced levels of LPS-induced
IL-1 and TNF-α 19. Activin A production by activated monocytes/macrophages is PKC-dependent 39,
and is promoted by LPS 19 and pro-inflammatory agents like TNFα 40 or IL1β 41. The link between
pro-inflammatory macrophage polarization and Activin A is further illustrated by the fact that
Activin A expression is inhibited by anti-inflammatory agents like glucorticoids and retinoic acid 28.
Since factors promoting M1/classical macrophage polarization enhance Activin A production, its
expression might be a common parameter of M1-polarized macrophages, as well as a critical
contributor to their phenotype and effector functions. In this regard, since M1 (GM-CSF)
macrophages express type I (ALK4, ACR1B) and type II (ACVR2A, ACVR2B) Activin A receptors
mRNA (data not shown), it can be predicted that Activin A influences the gene expression profile of
70
M1 polarized macrophages in a paracrine/autocrine manner. This appears to be true, at least
considering the state of phosphorylation od Smad2 in M1 (GM-CSF) macrophages. Smad2
phosphorylation is induced upon Activin A binding to its cell surface receptors 14, and Smad2 was
found to be phosphorylated in unstimulated M1 macrophages, whereas no Smad2 activation was
observed in M2 macrophages (Figure 6C). The constitutive activation of Smad2 in M1 (GM-CSF)
macrophages undoubtfully suggests that a prominent role for Activin A (and/or TGFβ family factors)
in shaping the inflammatory response of M1 macrophages to exogenous stimuli.
The identity of the genes specifically upregulated by Activin A in M1 (GM-CSF) macrophages
remains to be determined, although we have alredy shown that SERPINE1 expression is clearly
enhanced when macrophage polarization takes place in the presence of Activin A. Conversely,
various M2 (M-CSF)-specific markers have already been identified whose expression is either
prevented or downmodulated by Activin A (Figure 4), thus leading to the conclusion that Activin A
negatively affects the acquisition of genes preferentially expressed by anti-inflammatory
macrophages. This effect is particularly relevtant in the case of IL-10, whose release constitutes a
hallmark of stimulated M2 (M-CSF) macrophages 10. In the specific case of IL-10, it is worth noting
that its transcription in macrophages is dependent on the cMaf transcription factor42, that also
suppresses IL-12p70 production 43. The expression of the cMaf transcription factor is significantly
higher in M2 (M-CSF) than in M1 (GM-CSF) macrophages, both the RNA (Figure 4) and protein
level (data not shown), and, like in the case of Th2 lymphocyte polarization 44, its expression seems
to mark the acquisition of the anti-inflammatory phenotype by M2 macrophages. Importantly,
Activin A inhibits the M-CSF-dependent acquisition of MAF RNA expression, what might constitute
the molecular basis for its negative effect on the production of IL-10 by LPS-stimulated M2 (M-CSF)
macrophages.
In summary, the present manuscript identifies Activin A as a relevant contributor to the differential
gene expression profile exhibited by pro-inflammatory and anti-inflammatory macrophages. The
importance of Activin A in macrophage polarization is supported by its ability to reduce IL-10
production by anti-inflammatory macrophages, and to inhibit the acquisition of the IL-10-producing
ability during M-CSF-driven polarization. The identification of a set of M2 (M-CSF) macrophage-
specific genes whose expression is found in Tumor-Associated Macrophages 20 (and data not shown)
and negatively affected by Activin provides potential therapeutic targets for the modulation of the
macrophage inflammatory response under pathological conditions.
71
ACKNOWLEDGMENTS
The autors gratefully acknowledge Dr. Carmen Sánchez-Torres for valuable discussions and
suggestions.
72
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Supplementary Figures
M-CSFM-CSF+GM-CSFM-CSF+Act. A
Rel
ativ
e m
RN
A le
vels MAFB
0.4
0.8
1.21.0
0.6
0.2
MAF
0.4
0.8
1.21.0
0.6
0.2
HMOX1
0.4
0.8
1.21.0
0.6
0.2
M-CSFM-CSF+GM-CSFM-CSF+Act. A
M-CSFM-CSF+GM-CSFM-CSF+Act. A
Rel
ativ
e m
RN
A le
vels MAFB
0.4
0.8
1.21.0
0.6
0.2
MAFB
0.4
0.8
1.21.0
0.6
0.2
MAF
0.4
0.8
1.21.0
0.6
0.2
HMOX1
0.4
0.8
1.21.0
0.6
0.2
Supplementary Figure 1.- MAF, MAFB and HMOX1 mRNA expression levels as determined by qRT-PCR on macrophages exposed for 3 days to M-CSF, M-CSF plus GM-CSF or M-CSF plus Activin A (Act. A). Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the presence of M-CSF). Shown is the mean and standard deviation of triplicate determinations.
2000
4000
6000
8000
CCL17 IL1RN TM4SF1MMP12
10
20
30
100
200
300
20
40
60
B
RPMIGM-CSFActivin A
Rel
ativ
e m
RN
A le
vels
log 2
M1
/ M2
TM4SF1 MMP12
10
2
468
12CCL17A
qPCRMicroarray
2000
4000
6000
8000
CCL17 IL1RN TM4SF1MMP12
10
20
30
100
200
300
20
40
60
B
RPMIGM-CSFActivin A
Rel
ativ
e m
RN
A le
vels
log 2
M1
/ M2
TM4SF1 MMP12
10
2
468
12CCL17A
qPCRMicroarray
Supplementary Figure 2.- A. Relative expression of the indicated genes in M1 and M2 macrophages, as determined by microarray analysis (empty histograms) and quantitative RT-PCR (grey histograms). B. CCL17, IL1RN, MMP12 and TM4SF1 mRNA expression levels determined by qRT-PCR on macrophages differentiated for 3 days in the absence or in the presence of GM-CSF or Activin A. Results are expressed as Relative mRNA levels (relative to 18S rRNA levels and referred to the expression level observed in the absence of cytokines), and shown is the mean and standard deviation of triplicate determinations.
77
2468
101214161820
M1 M2 M2+Act.A
Prol
ifera
tion
(cpm
x 1
0-3)B
10
20
30
40
M1 M2
Prol
ifera
tion
(cpm
x 1
0-3)
1:2001:401:8
A1:401:8
2468
101214161820
M1 M2 M2+Act.A
Prol
ifera
tion
(cpm
x 1
0-3)B
10
20
30
40
M1 M2
Prol
ifera
tion
(cpm
x 1
0-3)
1:2001:401:8
A1:401:8
Supplementary Figure 3.- M1 or M2 macrophages (A,B), or M2 macrophages differentiated with M-CSF in the presence of Activin A (Act. A) (B) were irradiated and used to stimulate 2 x 105 allogeneic peripheral blood T lymphocytes at the indicated Macrophage/T cell ratios. After a 5 day co-culture, 3H-thymidine was added to the culture for 16 hours and T cell proliferation determined by measuring the incorporated thymidine. Each experiment was performed in triplicate, and mean and standard deviations are shown.
3.6 (0.4)
8.9 (0.4)
5.3 (0.4)
11.7 (0.5)
11.2 (0.5)
53.3 (1.2)
33.4 (0.8)
28.5 (0.7)
64.7 (1.9)
8.0 (0.4)
5.1 (0.4)
13.4 (0.5)
16.4 (0.5)
26.0 (0.6)
36.3 (0.9)
-
M1
M1/LPS
M2
M2/LPS
P3X63 CD83 CD863.6
(0.4)
8.9 (0.4)
5.3 (0.4)
11.7 (0.5)
11.2 (0.5)
53.3 (1.2)
33.4 (0.8)
28.5 (0.7)
64.7 (1.9)
8.0 (0.4)
5.1 (0.4)
13.4 (0.5)
16.4 (0.5)
26.0 (0.6)
36.3 (0.9)
3.6 (0.4)
8.9 (0.4)
5.3 (0.4)
11.7 (0.5)
11.2 (0.5)
53.3 (1.2)
33.4 (0.8)
28.5 (0.7)
64.7 (1.9)
8.0 (0.4)
5.1 (0.4)
13.4 (0.5)
16.4 (0.5)
26.0 (0.6)
36.3 (0.9)
-
M1
M1/LPS
M2
M2/LPS
P3X63 CD83 CD86
Supplementary Figure 4.- Immature monocyte-derived dendritic cells (MDDC) were exposed to conditioned media from either untreated or LPS (10 ng/ml)-treated M1 and M2 macrophages. After 48 hours, MDDC cell surface expression of CD83 and CD86 was determined by flow cytometry (P3X63, isotype-matched control). The percentage of marker-positive cells (upper number) and the mean fluorescence intensity (lower number) are indicated in each case.
78
Resultados
3. Requerimientos estructurales para la multimerización del receptor de patógenos DC-SIGN (CD209) en la superficie celular
DC-SIGN (Dendritic cell-specific ICAM3- grabbing non-integrin, CD209) es una lectina tipo C
que reconoce oligosacáridos presentes en patógenos con gran relevancia clínica (HIV,
Mycobacterium, Aspergillus). Mediante “splicing” alternativo y polimorfismos genéticos se generan
variantes de DC-SIGN que se detectan a nivel de mRNA en los sitios de entrada y transmisión de
patógenos. En este estudio se demuestra que las células mieloides expresan variantes de DC-SIGN
con diferentes tamaños de cuello, y que la multimerización de DC-SIGN en el contexto celular
depende del dominio lectina y del número y disposición de las repeticiones de la región del cuello,
cuya glicosilación afecta negativamente a la formación de oligómeros. Variantes de la región del
cuello de DC-SIGN que ocurren de forma natural difieren en su capacidad de mediar multimerización
en la membrana celular, exhiben una habilidad alterada de unión de azúcares, y conservan su
capacidad de interacción con patógenos. En consecuencia, se puede concluir que la formación de
agregados de moléculas de DC-SIGN inducida por patógenos predomina sobre la capacidad de
multimerización basal. El análisis de polimorfismos en el cuello de DC-SIGN indica que el número de
variantes alélicas en la población es mayor de lo esperado, y que la multimerización de la molécula
prototípica es modulada por la presencia de variantes alélicas con una estructura de cuello diferente.
Estos resultados demuestran que la presencia de variantes alélicas o la expresión de isoformas de
“splicing” del dominio del cuello pueden influir en la presencia y estabilidad de multímeros de DC-
SIGN en la superficie celular, lo que proporciona una explicación molecular para la asociación entre
polimorfismos de DC-SIGN y la susceptibilidad alterada a HIV y otros patógenos.
79
Structural Requirements for Multimerization of the PathogenReceptor Dendritic Cell-specific ICAM3-grabbingNon-integrin (CD209) on the Cell Surface*□S
Received for publication, July 23, 2007, and in revised form, November 26, 2007 Published, JBC Papers in Press, December 11, 2007, DOI 10.1074/jbc.M706004200
Diego Serrano-Gomez‡1,2, Elena Sierra-Filardi‡1, Rocıo T. Martınez-Nunez‡, Esther Caparros‡, Rafael Delgado§,Mari Angeles Munoz-Fernandez¶, Marıa Antonia Abad�, Jesus Jimenez-Barbero‡, Manuel Leal�, and Angel L. Corbı‡3
From the ‡Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientıficas, Ramiro de Maeztu 9, Madrid 28040,§Laboratorio de Microbiologıa Molecular, Hospital Doce de Octubre, Madrid 28041, the ¶Servicio de Inmunologıa, HospitalGeneral Universitario Gregorio Maranon, Madrid 28007, and the �Servicio de Enfermedades Infecciosas,Hospital Universitario Virgen del Rocıo, Sevilla 41013, Spain
The myeloid C-type lectin dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) recognizes oligosac-charide ligands on clinically relevant pathogens (HIV,Mycobac-terium, and Aspergillus). Alternative splicing and genomicpolymorphism generate DC-SIGN mRNA variants, which havebeen detected at sites of pathogen entrance and transmission.We present evidence that DC-SIGN neck variants are expressedon dendritic and myeloid cells at the RNA and protein levels.Structural analysis revealed that multimerization of DC-SIGNwithin a cellular context depends on the lectin domain and thenumber and arrangement of the repeats within the neck region,whose glycosylation negatively affects oligomer formation. Nat-urally occurring DC-SIGN neck variants differ in multimeriza-tion competence in the cell membrane, exhibit altered sugarbinding ability, and retain pathogen-interacting capacity,implying that pathogen-induced cluster formation predomi-nates over the basal multimerization capability. Analysis of DC-SIGN neck polymorphisms indicated that the number of allelicvariants is higher thanpreviously thought and thatmultimeriza-tion of the prototypic molecule is modulated in the presence ofallelic variants with a different neck structure. Our results dem-onstrate that the presence of allelic variants or a high level ofexpression of neck domain splicing isoforms might influencethe presence and stability of DC-SIGN multimers on the cellsurface, thus providing a molecular explanation for the correla-tion betweenDC-SIGN polymorphisms and altered susceptibil-ity to HIV-1 and other pathogens.
Dendritic cells (DCs)4 link the innate and adaptive branchesof the immune response by virtue of their capacity to recognizepathogen-specific structures (1) via pathogen-associatedmolecular pattern receptors (2). Immature DCs express a num-ber of lectins and lectin-likemolecules, which endow themwitha broad capacity for pathogen recognition, as they mediate thespecific recognition of parasitic, bacterial, yeast, and viralpathogens (3, 4). Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN, CD209) is a type II membrane C-type lec-tin (5, 6) abundantly expressed in vivo onmyeloid DC andmac-rophage subpopulations (5–12), as well as on in vitro generatedmonocyte-derived dendritic cells (MDDCs) and alternativelyactivated macrophages (12–14). DC-SIGN binds a large arrayof pathogens, including HIV (15), Ebola (16), hepatitis C (17–19), and Dengue virus (20) and Leishmania amastigotes andpromastigotes (21, 22), Mycobacterium tuberculosis (23, 24),Aspergillus fumigatus (25), andCandida albicans (26) via man-nan- and Lewis oligosaccharides-dependent interactions (27,28). In addition, DC-SIGN appears to mediate DC contactswith naıve T lymphocytes through its recognition of ICAM-3(6), DC trafficking through interactions with endothelialICAM-2 (8), and DC-neutrophil interactions by interactingwith the CD11b/CD18 integrin (29).Structurally, DC-SIGN contains a carbohydrate-recognition
domain, a neck region composed of eight 23-residue repeats,and a transmembrane region followed by a cytoplasmic tailcontaining recycling and internalization motifs (5, 30–32).Analysis of recombinantmolecules has revealed that themono-meric lectin domain has low affinity for carbohydrates, whereasfull-length DC-SIGN molecules form tetramers through theirneck domain, thus allowing high affinity recognition of specificligands (33–35). In addition to this prototypical structure, alter-native splicing events generate DC-SIGN isoform transcriptswhose presence exhibits inter-individual variations (36). Thenumerous DC-SIGN isoform transcripts reported to dateinclude an alternative cytoplasmic tail, an absent transmem-brane region, truncated lectin domains, and a variable number
* This work was supported in part by the Ministerio de Educacion y Ciencia(Grants SAF2005-0021, AGL2004-02148-ALI, and GEN2003-20649-C06-01/NAC), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (Span-ish Network for the Research in Infectious Diseases, Grant REIPIRD06/0008), and Fundacion para la Investigacion y Prevencion del SIDA enEspana (Grant FIPSE 36422/03) (to A. L. C.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.
1 Both authors contributed equally to this work.2 Supported by a Formacion de Profesorado Universitario predoctoral grant
(AP2002-2151) from Ministerio de Educacion y Ciencia (Spain).3 To whom correspondence should be addressed. Tel.: 34-91-837-3112 (ext.
4376); Fax: 34-91-562-7518; E-mail: [email protected].
4 The abbreviations used are: DC, dendritic cell; MDDC, monocyte-deriveddendritic cell; DC-SIGN, dendritic cell-specific ICAM-3-grabbing non-inte-grin; DC-SIGNR, DC-SIGN-related; HIV, human immunodeficiency virus;FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; DTSSP,dithiobis(succinimidylpropionate); MFI, mean fluorescence intensity.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 7, pp. 3889 –3903, February 15, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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of repeats within the neck domain (36). Moreover, the 23-resi-due repeat region of DC-SIGN is polymorphic at the genomiclevel (37, 38). Five different alleles for the DC-SIGN neckdomain have been identified to date, whose presence correlateswith altered susceptibility to HIV-1 transmission (38). Thefunctional relevance of the DC-SIGN neck variants has beenfurther suggested by their detection at mucosal HIV transmis-sion sites (39). Given the involvement of the neck domain inrecombinant DC-SIGNmultimerization, we hypothesized thatthe existence of this large array of polymorphic variants mighthave an impact on the repertoire of pathogen recognition bydendritic cells, as well as on the establishment of interactionsbetween dendritic cells and other cell types. We have charac-terized naturally occurring alternative splicing isoforms, allelicvariants and mutant isoforms of DC-SIGN in terms of surfacereceptor multimerization and adhesive and pathogen-recogni-tion capabilities, and found that the lectin domain contributesto DC-SIGN multimerization on the cell surface, that glycosy-lation of the neck domain negatively regulates formation ofmultimers, and that a neck domain with a single 23-residuerepeat is sufficient to mediate DC-SIGN multimerization onthe cell surface. Functional comparison of the distinct con-structs revealed that the basal multimerization of DC-SIGNdoes not correlate with enhanced binding to endogenous orpathogenic ligands, indicating that pathogen-induced clusterformation predominates over the basal multimerization capa-bility of the DC-SIGNmolecule and is the driving force for theDC-SIGN-dependent pathogen capture and internalization.
EXPERIMENTAL PROCEDURES
Generation of MDDCs
Human peripheral blood mononuclear cells were isolatedfrom buffy coats from healthy donors over a Lymphoprep(Nycomed, Norway) gradient according to standard proce-dures. Monocytes were purified from peripheral blood mono-nuclear cells by magnetic cell sorting using CD14 microbeads(Miltenyi Biotech, Bergisch Gladbach, Germany), and immedi-ately subjected to the dendritic cell differentiation protocol(40). Monocytes were cultured for 5–7 days in completemedium with 1000 units/ml granulocyte macrophage-colonystimulating factor (Schering-Plough, Kenilworth, NJ) and 1000units/ml interleukin-4 (PreProtech, Rocky Hill, NJ) cytokineaddition every second day, to obtain a population of immatureMDDCs.
Cells
The acute monocytic leukemia cell line THP-1, and theerythroleukemic K562 were cultured in RPMI 1640 mediumsupplemented with 10% fetal calf serum (complete medium).COS-7 and HEK293T cells were grown in Dulbecco’s modi-fied Eagle’s medium 10% fetal calf serum. THP-1 differenti-ation was induced by treatment with phorbol 12-myristate13-acetate (10 ng/ml), Bryostatin (10 nM), either alone or incombination with interleukin-4 (1000 units/ml), asdescribed before (14).
Isolation and Structural Characterization of AlternativelySpliced DC-SIGN Isoforms
DC-SIGN isoforms were isolated by reverse transcription-PCR on RNA from MDDCs of a healthy donor. Reverse tran-scription-PCR was performed essentially as described previ-ously (41). DC-SIGN mRNA was optimally amplified after 35cycles of denaturation (95 °C, for 45 s), annealing (62 °C, for45 s), and extension (72 °C, for 1 min), followed by a 10-minextension step at 72 °C.Oligonucleotides used for amplificationof the coding region of the prototypical DC-SIGN isoform 1A(DC-SIGN 1A) mRNA were CD209s (5�-GGGAATTCAGAG-TGGGGTGACATGAGTGAC-3�) and CD209as (5�-CCCCA-AGCTTGTGAAGTTCTGCTACGCAGGAG-3�) (6, 36).Amplification of DC-SIGN isoforms was accomplishedusing the primer pairs CD209s/CD209as, CD209soluble/CD209as, and CD209Ib/CD209as. The oligonucleotideCD209soluble (5�-GATACAAGAGCTTAGCAGTGTCCA-3�) spans through the exon Ic/exon III junction previouslydescribed for potentially soluble transmembrane-lacking DC-SIGN isoforms. The oligonucleotide CD209Ib (5�-GGGAATT-CTGGCCAGCCATGGCCTCAGC-3�) includes the alterna-tive translation initiation site found in exon Ib, which originatesthe DC-SIGN 1B isoforms (Fig. 1A). PCR-generated fragmentswere resolved in agarose gels, purified, sequenced, and clonedinto pCDNA3.1(�) vector.
Identification of DC-SIGN Polymorphic Isoforms andGeneration of His- and FLAG-containing DC-SIGNExpression Vectors
Three DC-SIGN allelic variants (-D3, -D5, and -D7) wereidentified by PCR on genomic DNA from 300 independentdonors. Amplification of the DC-SIGN neck domain-encodingexon was carried out on 300 ng of genomic DNA using oligo-nucleotides CD209-4F, (5�-GGGATTAACCAAGACCTTGG-CTC-3�) and CD209-4R, (5�-CCCAACTTCTCCTAGTCTG-GAGG-3�). After 35 cycles of denaturation (95 °C, for 45 s),annealing (61 °C, for 30 s), and extension (72 °C, for 90 s), fol-lowed by a 10-min extension step at 72 °C, PCR-generated frag-ments were resolved in agarose gels, purified, cloned intopCR4-TOPO vector (Invitrogen), and sequenced.Swapping of the neck domains between the allelic variants
and the prototypic form of DC-SIGN was done after introduc-tion of silentmutations creating restriction sites at Val63 (KpnI)and Ala247/Ala248 (SacII) in pCDNA3.1-DC-SIGN 1A and theallelic variants in pCR4-TOPO. Oligonucleotides used formutagenesis included: DC-SIGN-Val63s (5�-TGTCCAAGTG-TCCAAGGTACCCAGCTCCATAAGTCAG-3�), DC-SIGN-Val63as (5�-CTGACTTATGGAGCTGGGTACCTTGGACA-CTTGGACA-3�), DC-SIGN-247/248s (5�-GCTGACCCA-GCTGAAGGCCGCGGTGGAACGCCTGTGCCAC-3�), andDC-SIGN-247/248as (5�-GTGGCACAGGCGTTCCACCGC-GGCCTTCAGCTGGGTCAGC-3�). The resulting plasmids(pcDNA3.1-DC-SIGN-D3, pcDNA3.1-DC-SIGN-D5, andpcDNA3.1-DC-SIGN-D7) were verified by sequencing.An expression vector for N-terminal-His epitope-containing
DC-SIGN 1A (pCDNA3.1-DC-SIGN 1A-His) was created byPCR on pCDNA3.1-DC-SIGN 1A using oligonucleotides
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CD209His (5�-GGGAATTCGCCACCATGCATCATCAT-CATCATCATAGTGACTCCAAGGAACCAAGAC-3�) andCD209as. Generation of expression vectors for DC-SIGN-D3,-D5, and -D7 with FLAG epitope at the N terminus(pCDNA3.1-DC-SIGN-D3-FLAG, pCDNA3.1-DC-SIGN-D5-FLAG, and pCDNA3.1-DC-SIGN-D7-FLAG) was done by PCRusing oligonucleotides CD209FLAG (5�-GGGAATTCGCCA-CCATGGACTACAAGGACGACGATGACAAGAGTGAC-TCCAAGGAACCAAGAC-3�) and CD209as.
Stable and Transient Transfection of DC-SIGN Mutants andIsoforms
For transient transfections, COS-7 or HEK293T cells weretransfected with SuperFect (Qiagen) using pCDNA3.1-basedexpression plasmids containing the distinct isoforms ormutants of the DC-SIGN cDNA. To generate stable transfec-tants, K562 cells were transfected with pCDNA3.1-based con-structs using SuperFect and cultured in complete medium sup-plemented with 300�g/ml G418 (Invitrogen). Stable DC-SIGNexpression of the selected population was verified using theanti-DC-SIGN MR1 monoclonal antibody (13). Isolation ofK562-DC-SIGN1Aexpressing different levels ofDC-SIGNwasaccomplished by cell sorting after stainingwith theMR1mono-clonal antibody (13).
Site-directed Mutagenesis and Generation of DC-SIGNChimeric Molecules
Site-directed mutagenesis was carried out using theQuikChange site-directedmutagenesis kit (Stratagene, La Jolla,CA) on the pCDNA3-DC-SIGN 1A expression plasmid (13)according to themanufacturer’s instructions. Oligonucleotidesused for mutagenesis included: DC-SIGN-C/Ss (5�-GAGCTT-AGCAGGGTCTCTTGGCCATGGTC-3�) and DC-SIGN-C/Sas (5�-GACCATGGCCAAGAGACCCTGCTAAGCTC-3�),for mutation of Cys37 to Ser, with the resulting plasmid termedpCDNA3.1-(DC-SIGN C/S); and DC-SIGN-N/Qs (5�-GAC-GCGATCTACCAGCAGCTGACCCAGCTTAAAG-3�) andDC-SIGN-N/Qas (5�-CTTTAAGCTGGGTCAGCTGCTGG-TAGATCGCGTC-3�), for mutation of Asn80 to Gln, with theresulting plasmid termed pCDNA3.1-(DC-SIGN N/Q). Eachmutant construct was verified by DNA sequencing.To generate DC-SIGN expression vectors lacking the lectin
domain, PCR was performed on the pCDNA3.1-DC-SIGN 1Ausing oligonucleotides CD209s and CD209�Lectin (5�-CCCC-AAGCTTGTCACAGGCGTTCCACTGCAGC-3�). PCR-gen-erated fragments were resolved in agarose gels, purified, andsequenced. Fragments containing either the full-length (8d�L)and a 7- and 6-repeat neck regions (repeats 1 through 7, 7d�L;repeats 1 through 6, 6d�L) were cloned into pCDNA3.1(�) toyield pCDNA3.1-DC-SIGN 8d�L, pCDNA3.1-DC-SIGN7d�L, and pCDNA3.1-DC-SIGN 6d�L plasmids.
Flow Cytometry and Antibodies
Cellular phenotypic analysis was carried out by indirectimmunofluorescence, using FITC-labeled goat anti-mouseantibody (Serotec, Oxford, UK). Monoclonal antibodies usedfor cell surface staining includedMR1 (directed against the lec-tin domain of DC-SIGN), and the supernatant from the mouse
myeloma P3-X63Ag8 (X63) was used as the control. All incu-bations were done in the presence of 50 �g/ml human IgG toprevent binding through the Fc portion of the antibodies. Flowcytometry analysis was performed with an EPICS-CS (CoulterCientıfica, Madrid, Spain) using log amplifiers.
Immunofluorescence
Cells were resuspended in PBS and allowed to adhere ontopoly-L-lysine-coated coverslips for 60min at 37 °C. After a briefwashing step with PBS, cells were fixed and permeabilized in a1:1 solution of acetone:methanol for 10min at�20 °C, washed,and stained with the MR1 monoclonal antibody (13) followedby an incubation with an FITC-labeled goat anti-mouse anti-body. Coverslips were mounted in fluorescent mountingmedium (DakoCytomation, Carpinteria, CA), and representa-tive fields were photographed through an oil immersion lens ona Nikon Eclipse E800microscope equipped for epifluorescenceor by confocal microscopy.
Cell Surface Protein Labeling and Precipitation
For labeling, immatureMDDCswere washed with PBS 1mMEDTA, resuspended in PBS, pH 8.0, and incubated in 0.5mg/mlbiotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide estersodium salt (Pierce) for 30 min at 4 °C. Cells were extensivelywashed in PBS and lysed using 10mMTris-HCl, pH 8.0, 150mMNaCl, 0.025% sodium azide, 1% Brij 58 (Sigma-Aldrich), 1 mMiodoacetamide, 2 mM Pefabloc (Alexis Biochemicals, Lausen,Switzerland), and 2 �g/ml aprotinin, antipain, leupeptin, andpepstatin. For precipitation of biotin-labeled proteins, Strepta-vidin-agarose (Sigma-Aldrich) was added to the lysates, and themixture incubated for 1 h at 4 °C. After centrifugation, beadswere extensively washed in 10 mM Tris-HCl, pH 8.0, 150 mMNaCl, 0.025% sodium azide, 0.1% Brij 58, resuspended in 3�Laemmli sample buffer (2% SDS, 6.25 mM Tris base, 10% glyc-erol), and boiled. Eluted material was resolved by SDS-PAGEunder reducing or non-reducing conditions and subsequentWestern blot with polyclonal antibodies specific for DC-SIGN.Coprecipitation of DC-SIGN 1A/DC-SIGN 8d�L or DC-SIGN1A-His/DC-SIGN-D3/-D5-FLAG hetero-oligomers was per-formed on lysates from transiently transfected COS-7 withMR1 antibody as previously described (42), and precipitatedmaterial was detected with specific polyclonal antibodies, orusing anti-His antibody for precipitation and anti-FLAG-HRPantibody for detection of precipitated material, respectively.
Cross-linking Experiments
Cross-linking experiments were performed using the water-soluble cross-linking agent dithiobis(succinimidylpropionate)(DTSSP) according to themanufacturer’s instructions (Pierce).Briefly, immature MDDC was washed with PBS 1 mM EDTA,resuspended in 1 ml of PBS, and incubated in the presence of100 �l of 10 mM DTSSP in sodium citrate 5 mM, pH 5.0, for 30min at room temperature. Stop solution (20 mM Tris-HCl, pH7.5) was added (15 min at room temperature), and cells werewashed twice with PBS. Total cell lysates were obtained in 10mMTris-HCl, pH 8.0, 150mMNaCl, 0.025% sodium azide, 0.5%Nonidet P-40, 1mM iodoacetamide, 2mM Pefabloc (Alexis Bio-chemicals), and 2 �g/ml aprotinin, antipain, leupeptin, and
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pepstatin (Nonidet P-40 lysis buffer). 10 �g of each lysate wassubjected to SDS-PAGE as described for Western blot experi-ments. For cleaving the cross-linker agent, lysates were incu-bated with 5% �-mercaptoethanol in Laemmli sample buffer.
Generation of Polyclonal Antisera against DC-SIGN StructuralDomains
Peptides based on the sequence of the sixth repeated domainof the DC-SIGN neck region (GELPEKSKQQEIYQELTRL-KAAV), and the region between residues 6 and 33 of the cyto-plasmic tail (EPRLQQLGLLEEEQLRGLGFRQTRGYKS), weresynthesized by the multiple antigen peptide system (13). NewZealandWhite rabbits were immunized by subcutaneous injec-tion of each peptide (for DSG-1 and DSG-2 antisera) or therecombinant DC-SIGN lectin domain (for DSG-4 antiserum)expressed in bacteria (0.5 ml of a 1 mg/ml solution in PBS) incomplete Freund’s adjuvant (1:1) on day 0 and in incompleteFreund’s adjuvant (1:1) on days 21 and 42. Rabbits were bled onday 49, and serum was assayed for DC-SIGN recognition inWestern blot experiments.
Functional Characterization of DC-SIGN Isoforms and Mutants
C. albicans and A. fumigatus Binding Assays—Conidia werelabeled with 0.1 mg/ml FITC for 1 h at room temperature andextensively washed. For conidia-binding assays (25), cells werewashed, resuspended in complete medium, and pretreated for20 min at room temperature with anti-DC-SIGN (MR1) or anisotype-matched irrelevant antibody (X63). Then cells wereincubated with FITC-labeled A. fumigatus or C. albicansconidia at the indicated ratios for 30 min at room temperature.After extensive washing, cells were fixed with 2% paraformal-dehyde in PBS for 1 h at 4 °C, washed, and analyzed by flowcytometry.DC-SIGN-dependent Adhesion Assays—DC-SIGN-dependent
adhesionwas evaluatedusing Saccharomyces cerevisiaemannanas specific ligand. 96-well microtiter EIA II-Linbro plates werecoated overnight with mannan at 50 �g/ml in PBS at 4 °C, andthe remaining sites were blocked with 0.5% bovine serum albu-min for 2 h at 37 °C. Cells were labeled in RPMI 0.5% bovineserum albumin with the fluorescent dye 2�,7�-bis-(2-carboxy-ethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester(Molecular Probes, The Netherlands) at 37 °C and then prein-cubated for 20 min with either the isotype-matched controlX63 or the function-blocking MR1 antibodies. Cells were thenallowed to adhere to each well for 15 min at 37 °C. Unboundcells were removed by three washes with RPMI 0.5% bovineserum albumin, and adherent cells were quantified using a flu-orescence analyzer. Where specified, results are presented as“DC-SIGN-dependent binding,” defined as: DC-SIGN-dependent binding � (% bound cells in the presence ofP3X63 � % bound cells in the presence of MR1).Leishmania Amastigote Binding Assays—Cells were washed
in PBS 1 mM EDTA, resuspended in complete medium and5,6-carboxyfluorescein succinimidyl ester-labeled parasiteswere added onto the cells at a 10:1 (amastigotes:cell) ratio, andincubated at room temperature for 30 min. Afterward, cellswere fixed and analyzed by flow cytometry. For inhibitionassays, cells were preincubated for 10min at room temperature
with either MR1 antibody or an irrelevant antibody (X63) incomplete medium before parasite addition.DC-SIGN Internalization—Cells were washed, resuspended
in complete medium, and incubated with MR1 antibody (13)for 1 h at 4 °C to prevent DC-SIGN internalization. After exten-sive washing, cells were placed at 37 °C to allow internalizationto occur. At the indicated time points, internalization wasstopped by adding of cold PBS, and cells were immediatelyplaced at 4 °C. To detect the remainingmembrane-boundMR1antibody, an FITC-labeled goat anti-mouse antibody wasadded, incubated for 30 min at 4 °C, and analyzed by flowcytometry. All incubations were done in the presence of 50�g/ml human IgG to prevent binding through the Fc portion ofthe antibodies.EbolaGP1-Fc Binding Assays—Cells werewashed in PBS and
1mM EDTA, resuspended in complete medium, and incubatedwith GP1-Fc either in the presence of a monoclonal antibodyagainst DC-SIGN (MR1) or an irrelevant antibody (X63) for 20min at 4 °C. Then, cells were incubated with a phycoerythrin-labeled polyclonal antiserum against human IgG Fc (BeckmanCoulter), and analyzed by flow cytometry.Sugar-coated Fluorescent Bead Binding to DC-SIGN—Syn-
thetic fluorescein-labeled fucose- or Lewisx-containing polyac-rylamide beads (FITC-PAA-NAc-Gal, FITC-PAA-Fuc, andFITC-PAA-Lex) were obtained from Lectinity (Moscow, Rus-sia). After washingwith PBS and 1mMEDTA, transiently trans-fected HEK293T cells were resuspended in complete medium,and sugar-PAA-FLU beads were added to a final concentrationof 20 �g/ml and incubated at 37 °C for 30 min. After extensivewashing, cells were fixed for 1 h at room temperature, and ana-lyzed by flow cytometry. For inhibition assays, cells were prein-cubated for 10 min at room temperature with either MR1 anti-body or an irrelevant antibody (X63) in complete mediumbefore beads addition. Results from binding assays wereexpressed as “Binding Index,” which represents the DC-SIGN-dependent binding relative to DC-SIGN expression levelsaccording to the formula: Binding index � (mean fluorescentintensity (MFI) of cells plus beads � MFI of cells plus beads inthe presence ofMR1)/(MFI afterMR1 staining/MFI after stain-ing with X63).
NMR Experiments
Binding of soluble glucomannan from Candida utilis (IF) toDC-SIGN transfectants was done by basic Saturation TransferDifference, as previously described (43).
Western Blot
Total cell lysates were obtained in Nonidet P-40 lysis buffer,and 10 �g of each lysate was subjected to SDS-PAGE underreducing or non-reducing conditions and transferred onto anImmobilon polyvinylidene difluoride membrane (Millipore,Bedford, MA). After blocking of the unoccupied sites with 5%nonfat dry milk in 50 mMTris-HCl, pH 7.6, 150 mMNaCl, 0.1%Tween 20, protein detection was performed using the Super-SignalWest Pico chemiluminescent system (Pierce). DetectionofDC-SIGNwas carried out using polyclonal antiserumagainstthe C-terminal 20-residue peptide of DC-SIGN (C-20,sc-11038, Santa Cruz Biotechnology, Santa Cruz, CA), amino
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acids 61–200 (H-200, sc-20081, Santa Cruz Biotechnology), orpolyclonal antisera raised against peptides based on the sixth23-residue repeats within the DC-SIGN neck region (DSG-1),against a 28-residue peptide from the DC-SIGN cytoplasmictail (DSG-2), or against the whole lectin domain (DSG-4).
Carbohydrate Affinity Precipitations
For precipitation of mannan- and N-acetylgalactosamine-binding proteins, transiently transfected HEK293T or COS-7cells (3� 106)were lysed inNonidet P-40 lysis buffer. Then, 200�l of each lysatewas taken to 1mlwithNonidet P-40 lysis bufferand incubated with 50 �l of mannan- or N-acetylgalac-tosamine-agarose (Sigma-Aldrich) for 12 h at 4 °C. After exten-sive washing in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.025%sodium azide, 0.05%Nonidet P-40, bound proteins were elutedby boiling the agarose beads in 3� Laemmli sample buffer.
SDS-eluted and non-bound materials were resolved by SDS-PAGE and DC-SIGN detection accomplished with specificpolyclonal antibodies.
RESULTS
Range of DC-SIGN Alternatively Spliced Isoforms—MDDCsexpress a high number of alternatively spliced DC-SIGNmRNAspecies (36), which are also found atmucosalHIV trans-mission sites (39). To determine the range of DC-SIGNmRNAspecies found inMDDC from a single donor, three different setof primers were designed to specifically amplify the prototypi-cal DC-SIGN mRNA (DC-SIGN 1A), or species encodingeither an alternative cytoplasmic domain (DC-SIGN 1B) orlacking the transmembrane domain (DC-SIGN�TM) (Fig. 1A).Sequencing of the amplified fragments resulted in the identifi-cation of DC-SIGN mRNA species encoding for variants with
FIGURE 1. Detection of DC-SIGN isoforms on monocyte-derived dendritic cells. A, schematic representation of the DC-SIGN mRNA and the position ofoligonucleotides used to amplify DC-SIGN 1A isoforms (sense plus antisense), DC-SIGN 1B isoforms (Ib plus antisense), or DC-SIGN �TM isoforms without thetransmembrane region (soluble plus antisense). (Exons I–VI, genomic organization; ATG, translational start sites; CYT, cytoplasmic domain; TM, transmembraneregion). B, schematic structure of the major PCR fragments obtained from RNA of immature MDDCs from a single donor. C–F, lysates from COS-7 cellstransiently transfected with expression vectors for DC-SIGN 1A, a chimeric construct lacking the lectin domain (8d�L) or an empty vector (Mock) (C), precipi-tated material from surface (biotin-labeled) immature MDDCs (D), lysates from THP-1 cells differentiated with Bryostatin (Bryo) in the presence or absence ofinterleukin-4 (E), or lysates from immature MDDCs either untreated or incubated with the cross-linking agent DTSSP (F) were resolved by SDS-PAGE undernon-reducing or reducing conditions (in the presence of �-mercaptoethanol, �-MSH). The gels were then subjected to Western blot using polyclonal antiseraagainst the neck domain (DSG-1 in C and F; H-200 in E), against the cytoplasmic tail of DC-SIGN (DSG-2) (C, D, and F) or against the C-terminal 20-amino acidpeptide of DC-SIGN (C-20) (C). The specificity of the distinct antisera is indicated in each panel. Thin lines indicate the position of bands with higher mobility thanthe full-length DC-SIGN isoform. In D, the biotin-labeled proteins precipitated with streptavidin-agarose (SP) were analyzed in parallel with whole cell extracts(WE) and proteins in the supernatant or non-precipitated (SN).
Expression and Function of DC-SIGN Variants
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alternative cytoplasmic tails and potentially soluble isoforms,with each group including transcripts differing in the neckdomain or the carbohydrate-binding region (Fig. 1B). There-fore, and in agreement with previous reports (14, 36, 39), theDC-SIGN gene gives rise to a large number of alternativelyspliced mRNA species, most of which differ in the number of23-residue repeats within the neck domain, previously demon-strated to mediate multimerization of recombinant DC-SIGN(34).Next we generated polyclonal antisera specific for either the
neck domain (DSG-1) or the prototypic cytoplasmic tail of DC-SIGN 1A (DSG-2). Both DSG-1 and DSG-2 specificallydetected the 44-kDa band of the prototypic full-length isoformDC-SIGN 1A, as well as a deletion mutant lacking the lectindomain (8d�L), whereas a polyclonal antiserum against the 20C-terminal residues of the lectin domain (C-20) only detectedthe full-length molecule (Fig. 1C).To determine the degree of DC-SIGN multimerization on
MDDC, cell surface proteins were biotin-labeled, and strepta-vidin pulled-down material was analyzed for the presence ofDC-SIGN. Under non-reducing conditions, the DSG-2 anti-serum detected distinct several bands corresponding to DC-SIGN monomers, dimers, trimers, tetramers, and high ordermultimers either in the whole extracts and the pull-down (Fig.1D, left panel, lanes WE and SP, respectively), which suggeststhat DC-SIGN multimers are found on the cell surface ofMDDCs. Analysis of cell surface DC-SIGN molecules fromMDDCunder reducing conditions also revealed the presence ofadditional higher mobility bands that were also recognized bythe DSG-2 antiserum (Fig. 1D, right panel, lane SP). The samepattern was detected in total lysates of dendritic-like THP-1cells (14) using a polyclonal antiserum against the whole neckregion of the molecule (Fig. 1E), and similar bands could bedetected in MDDC lysates with both DSG-1 and DSG-2 anti-sera (Fig. 1F). Therefore, DC-SIGN isoforms can be detected onthe cell surface ofmonocyte-derived dendritic cells, although toa lower extent than the full-length DC-SIGN 1A.Contribution of the LectinDomain toDC-SIGNMultimeriza-
tion on the Cell Membrane—DC-SIGN multimer formation inMDDC could be readily identified by SDS-PAGE (Fig. 1D) (33,44). In fact, although treatment with the membrane-imperme-able cross-linker DTSSP enhanced the formation of high ordermultimers, DC-SIGN monomers, dimers, and multimers werereadily detected by C-20, DSG-1, and DSG-2 antisera undernon-reducing conditions (Fig. 1F). The detection of DC-SIGNmultimers was almost completely prevented in the presence ofreducing agents (Fig. 1, D and F), indicating that disulfidebridges contribute tomultimerization. Because all Cys residueswithin the lectin domain are engaged in intramolecular disul-fide bridges (27), we determined the effect of mutating Cys37,the onlyDC-SIGNcysteine residue outside of the lectin domainand located within the cytoplasmic tail. Mutation of Cys37 hadno effect on the degree of formation of DC-SIGN multimers(Fig. 2B, left panel), suggesting that multimerization could bedependent on cysteine residues within the lectin domain. Thelectin domainwas then removed fromeither theDC-SIGNpro-totypic isoform (8d�L) or from an isoformwith only six repeats(6d�L) (Fig. 2A). Both 8d�L and 6d�L constructs displayed
greatly reduced multimerization ability (Fig. 2B, right panel),which indicates that, although DC-SIGN multimerizationmight bemediated by the neck region (34, 35, 45), it requires oris stabilized by the lectin domain of the molecule.Along this line, the presence of lectin domain-lacking con-
structs (8d�L, 7d�L, and 6d�L) had a negative impact on thedegree of multimerization of DC-SIGN 1A, as we observed alower level of DC-SIGN 1A multimers in the presence of thesedeletion constructs (Fig. 2C). This could be explained by anincreased formation of heteromultimers (formed by DC-SIGN1A and constructs lacking the lectin domain), which mightexhibit lower stability in the presence of denaturing detergent,thus precluding its detection. If so, the existence of heteromul-timers could be demonstrated by coprecipitation experimentson lysates from cells cotransfected with DC-SIGN 1A and8d�L. The fact that the 8d�L isoform was pulled down afterimmunoprecipitation of lectin domain-containing moleculeswith theMR1monoclonal antibody (Fig. 2D) confirms that lec-tin domain-lacking constructs associate with the prototypicDC-SIGN 1A isoform, suggests that heteromultimers of DC-SIGN 1A and 8d�L are more sensitive to the presence of dena-turing agents than DC-SIGN 1A homomultimers and confirmsa role for the lectin domain of DC-SIGN in the formation ofstable oligomers.Structural Requirements of the Neck Domain for DC-SIGN
Multimerization—Although the neck domain is absolutelyrequired for the formation of multimers of recombinant non-glycosylated DC-SIGN and DC-SIGNR (34, 35, 45), its role inDC-SIGN multimerization on the cell membrane remainsunclear. To address this issue, we analyzed the pattern of mul-timerization of the prototypic full-length molecule (1A), natu-rally occurring (4d, 4d�, 2d, and 1d) or in vitro generated (3d)isoforms differing in the number and order of the neck regionrepeats, and constructs mutated at the N-linked glycosylationsite (1AN/Q and 1dN/Q) (Fig. 3A). Transient transfectionrevealed that the distinct DC-SIGN isoforms differed in theirability to form oligomers. A high proportion of full-length DC-SIGN 1A appeared as multimers, whereas deletion of half theneck region (4d) resulted in a considerable reduction of highorder multimers (Fig. 3A). By contrast, isoforms 3d and 2d,whose neck regions are composed of three and two repeats,exhibited an oligomerization ability roughly similar to that ofthe full-lengthmolecule, whereas isoform 1d showed the weak-est oligomerization (Fig. 3A). These results indicate that thepresence of at least two repeats within the neck region is suffi-cient for DC-SIGN multimerization. On the other hand, thelower multimerization of 4d suggests that there is no directcorrelation between the length of the neck region and oli-gomerization, and that the distinct repeats within the neckregion might not be functionally equivalent. This hypothesiswas confirmed when comparing the lowmultimerization capa-bility of 4d (composed of neck repeats 1, 6, 7, and 8) with thenormal (similar to 1A) oligomerization pattern of 4d� isoform,whose neck region is composed of repeats 1, 2, 3, and 8 (Fig. 3A),thus confirming that multimerization capability of DC-SIGNon the cell membrane is dependent not only on the number ofneck repeats but also on their arrangement, and that the repeats
Expression and Function of DC-SIGN Variants
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within the neck region of DC-SIGN are not functionallyinterchangeable.In agreement with the results obtained after transient trans-
fection, the 4d isoform also exhibited a greatly reduced propor-tion of DC-SIGN multimers when stably expressed in K562cells, whereasmultimerization of 2d isoformwas similar to thatof DC-SIGN 1A (Fig. 3B), a finding also observed after transfec-tion in T lymphoblastoid Jurkat cell (data not shown). Func-tional analysis of the three isoforms in K562 transfectantsrevealed that 1A, 4d, and 2d bound soluble C. utilis glucoman-nan (IF), as determined by one-dimensional saturation transferdifference (43, 46) (Fig. 3C), and were internalized after MR1-mediated engagement (Fig. 3D). Therefore, it can be concludedthat the degree of multimerization of functional DC-SIGN iso-forms on the cell surface is cell-type independent and does notinfluence the ligand-induced internalization of the molecule.
The inability of the first repeat to mediate multimerization(1d in Fig. 3A), and the fact that it contains the only potentialN-glycosylation site of DC-SIGN, prompted us to determinethe contribution of glycosylation to DC-SIGN oligomerization.Replacement of Asn80 for Gln in the context of the full-lengthmolecule (1AN/Q) greatly increased the proportion ofDC-SIGN multimers (compare 1A and 1AN/Q in Fig. 3A) andsuggests that glycosylation of the first neck repeat negativelyaffects DC-SIGN multimerization. The negative influence ofglycosylation on multimerization was even more evident uponanalysis of the 1dN/Q mutant, whose neck domain is formedonly by the first repeat with the Asn80/Gln replacement. Unlikethe 1d isoform, oligomers (and even high order multimers) ofthe 1dN/Q mutant could be easily detected (Fig. 3A). In fact,and like in the case of 1AN/Q, no 1dN/Q monomers wereobserved under non-reducing conditions (Fig. 3A). Therefore,
FIGURE 2. Determination of structural requirements for DC-SIGN multimerization. A, schematic representation of the DC-SIGN alternatively splicedisoforms (1A, 4d, 4d�, 3d, 2d, and 1d), mutants (1AC/S, 1AN/Q, and 1dN/Q) and chimeric molecules (8d�L, 7d�L, and 6d�L) used throughout the study. TheCys37 residue is indicated by a cross (†) on the transmembrane region, and the presence of potential N-glycosylation sequence is indicated by a black dot on thefirst repeat of the neck domain. B and C, COS-7 cells transiently transfected with the indicated DC-SIGN constructs were lysed, resolved by SDS-PAGE, andsubjected to Western blot using DSG-2 (B), or DSG-1 and DSG-4 (C) polyclonal antisera. D, lysates from COS-7 cells transiently transfected with the indicatedDC-SIGN constructs were immunoprecipitated with a monoclonal antibody against the DC-SIGN lectin domain (MR1) or a control antibody (CNT), andimmunoprecipitated proteins were resolved by SDS-PAGE and subjected to Western blot using DSG-1 polyclonal antiserum. Immunoprecipitated proteinswere analyzed in parallel with whole cell lysates (WE).
Expression and Function of DC-SIGN Variants
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glycosylation of the first repeat in the neck region impairs mul-timerization of DC-SIGN molecules.Influence of Multimerization on DC-SIGN Pathogen
Recognition—Despite the differences in their ability to multim-erize, transient transfection of the whole range of constructspreviously assayed revealed that all of them are capable ofbinding Candida yeasts and Leishmania amastigotes to asimilar extent (supplemental Fig. S1A). To rule out the subtledifferences in pathogen binding among the distinct constructswe evaluateCandida andL. pifanoi amastigotes binding by cellsexpressing decreasing levels of three naturally occurring iso-forms (1A, 4d, and 2d), and no significant difference wasobserved when comparing binding by cells expressing similarlevels of the three constructs (supplemental Fig. S1B and notshown). However, the results showed that the binding ability ofthe distinct DC-SIGN isoforms correlate with their expression
level (supplemental Fig. S1B). Therefore, because the multim-erization ability of the 4d isoform is considerably lower thanthat of 1A and 2d (see Fig. 3A), these results indicate that therecognition of C. albicans yeasts or L. infantum amastigotes bydistinct DC-SIGN isoform/mutants does not correlate withtheir multimerization degree. Consequently, the multimeriza-tion degree of an isoform does not predict its pathogen-bindingability.Influence of Multimerization on Sugar Recognition by
DC-SIGN—The lack of correlation betweenmultimerizationdegree and pathogen-binding ability of DC-SIGN isoformscould be explained by the large amount of DC-SIGN ligandsimmobilized on the pathogen surface, which would drive theformation of DC-SIGN-containing clusters (47, 48) andmight obscure the contribution of the affinity/avidity ofindividual molecules/oligomers to the whole interaction. To
FIGURE 3. Multimerization capacity of DC-SIGN isoforms and constructs. A and B, lysates from transiently transfected HEK293T cells (A) or K562 cellsstably transfected (B) with the indicated DC-SIGN constructs (see upper drawing) or a mock construct were analyzed by SDS-PAGE and subjected toWestern blot with the DSG-2 polyclonal antiserum. In B, two different clones of K562-DC-SIGN 1A were analyzed, whose relative level of DC-SIGNexpression is indicated by a dark triangle. C, binding of C. utilis glucomannan (IF) to K562 cells stably transfected with the indicated DC-SIGN isoforms bymeans of one-dimensional saturation transfer difference NMR. The lower profile represents the 1H NMR spectrum of IF in PBS at 298 K. For comparativepurposes, two subpopulations of K562-DC-SIGN 1A, which differ in their DC-SIGN cell surface expression level, were assayed. The graph illustrates thesignal intensity yielded by each transfectant (y-axis) and the chemical shift (�) in parts per million (ppm). D, monoclonal antibody-induced internaliza-tion of DC-SIGN isoforms in K562 cells stably transfected with the 1A, 4d, or 2d isoforms. DC-SIGN expression at different time points is shown relativeto the initial cell surface expression (100%, upper panel), and was determined by flow cytometry. For the three stable transfectants, the MFI (lowernumber) and the percentage of positive cells (upper number) at time zero are shown.
Expression and Function of DC-SIGN Variants
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avoid pathogen-induced clustering effects on the mem-brane, we assess the ability of the distinct DC-SIGN con-structs to be retained by sugars after membrane solubiliza-tion. As shown in Fig. 4A (upper panels), except 1d, allDC-SIGN constructs were specifically retained by mannan(a polysaccharide that blocks most DC-SIGN interaction).However, analysis of molecules not retained by mannan(supernatant) revealed that constructs 1A, 1AN/Q, 4d, and1dN/Q are retained with higher efficiency than the 4d�, 3d,and 2d constructs (Fig. 4A, lower panels). Monomers werepreferentially retained by mannan within the strong man-nan-binding and N-glycosylation-containing constructs (1Aand 4d) (lanes 1A and 4d in the left panels of Fig. 4A). Bycontrast, those exhibiting lower binding to mannan (4d�, 3d,and 2d) were preferentially retained as multimers, as mono-mers were almost exclusively detected in the supernatant(lanes 4d�, 3d, and 2d in left panels of Fig. 4A). Furthermore,and in agreement with the negative effect of N-glycosylationon DC-SIGN multimerization, the 1AN/Q and 1dN/Q con-structs were preferentially retained as multimers. Therefore,functional analysis of detergent-solubilized cellular DC-SIGN
demonstrates that multimer formation compensates for thelower mannan-binding affinity of certain DC-SIGN con-structs after membrane solubilization, an effect thatbecomes even more evident when less-than-optimal sugarligands (NAc-Gal) were used, which only retained lectinmultimers (Fig. 4B). Therefore, this set of data indicates thatthe number and arrangement of the repeats within the neckdomain directly influences the specificity and the sugar-binding ability of the DC-SIGN lectin domain.To further evaluate the relevance of DC-SIGN cell surface
multimerization on ligand binding, cell surface expressed 1dand 1dN/Q constructs were compared in their ability to bindFITC-PAA-Fucose and Lewisx beads. 1dN/Q, which appearsalmost exclusively as multimers, displayed a stronger bead-binding activity than 1d, whose multimers can barely bedetected, and the same finding was observed at three distinctcell surface expression levels (Fig. 4C). These results furthersupport the involvement of cell surface DC-SIGNmultimeriza-tion in ligand binding, and establishN-linked-glycosylation as acritical parameter for the DC-SIGN ligand-binding activity onthe cell surface.
FIGURE 4. Sugar recognition by DC-SIGN isoforms. A and B, lysates of HEK293T cells transiently transfected with the indicated DC-SIGN variants wereprecipitated with mannan- (A) or N-acetylgalactosamine (NAc-Gal)-agarose (B). Eluted proteins (upper panels) and non-bound proteins (supernatant, lowerpanels) were resolved by SDS-PAGE under reducing (right panels in A) or non-reducing (left panels in A, both panels in B) conditions, and subjected to Westernblot with DSG-2 antiserum. C, HEK293T cells transiently transfected with the indicated DC-SIGN variants (1A, 1d, and 1dN/Q) were incubated with eitherFITC-PAA-fucose or FITC-PAA-Lewisx beads (20 �g/ml) in the presence of MR1 blocking antibody or an irrelevant antibody, and the percentage of cells withbound beads was determined by flow cytometry. The percentage (upper number) and MFI (lower number) of cells stained with either a MR1 (black text) or anisotype-matched antibody (gray text) are indicated in each case. The results from three independent experiments on cells with different DC-SIGN cell surfacelevels (high, left panel; middle, middle panel; and low, right panel) are shown.
Expression and Function of DC-SIGN Variants
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Structural and Functional Characterization of PolymorphicVariants of DC-SIGN—The above results demonstrate that theneck region is an important determinant in the ligand-bindingactivity of DC-SIGN on the cell surface. It has been reportedthat, among the polymorphisms in the DC-SIGN gene (38,49–52), those affecting the length of the neck domain correlatewith altered susceptibility to HIV-1 infection (38). In fact, sim-ilar findings have been reported in the case of the related DC-SIGNR and susceptibility toHIV-1 and severe acute respiratorysyndrome infection (53–55). To evaluate the functional signif-icance of polymorphic DC-SIGN neck domains, three distinctallelic variants, whose neck domains contain only seven repeats,were identified at the genomic DNA and RNA level (Fig. 5, AandB) and functionally characterized. These polymorphic vari-ants lack repeats 3, 5, or 7, but theirmultimerization ability (Fig.5B), cell surface expression (Fig. 5C), and ligand-induced inter-nalization capability (Fig. 5D) were found to be indistinguish-able from that of the prototypic molecule. Moreover, the threevariants displayed unaltered capacity for recognition of Leish-
mania and Aspergillus (supplemental Fig. S2A) and mediatedcellular binding to Ebola GP1-Fc and Mannan (supplementalFig. S2B) and were retained by agarose-bound mannan aftermembrane solubilization (supplemental Fig. S2C), and medi-ated binding of C. utilis glucomannan to cells as determined byone-dimensional saturation transfer difference NMR (supple-mental Fig. S2D). Therefore, DC-SIGN polymorphic variantslacking a single neck domain repeat (3, 5, or 7) exhibit func-tional activities that are similar to those exhibited by the proto-typic DC-SIGN molecule.Because altered susceptibility to infections has been mostly
observed in individuals with heterozygosity at theDC-SIGN (orDC-SIGNR) gene (38, 53–55), we next evaluated the influenceof DC-SIGN polymorphic variants (-D3, -D5, and -D7, Fig. 5B)on the expression,multimerization, and functional capability ofthe prototypic molecule when expressed on the same cell.Transient transfection experiments demonstrated that theexpression of any of the polymorphic variants had no influenceon the DC-SIGN 1A total or cell surface expression (Fig. 6, A
FIGURE 5. Structural and functional characterization of DC-SIGN polymorphic variants. A, schematic representation of the DC-SIGN gene and the positionof oligonucleotides used to amplify the DC-SIGN neck domain-polymorphic variants (upper panel). Examples of the amplification of genomic DNA (left lowerpanel) and RNA (right lower panel) are shown, indicating the genotype of each donor (1A/1A, homozygote for DC-SIGN, with two 8-neck repeat alleles; 1A/-D3,1A/-D7, heterozygotes, with a full-length neck domain in one allele and a second allele coding for a neck with 7 repeats, missing either repeat 3 (-D3) or repeat7 (-D7), respectively). B, upper panel: schematic structure of the prototypic DC-SIGN variant (1A) and the three polymorphic alleles identified (-D3, -D5, and -D7).Lower panel: lysates from K562 cells stably transfected with the indicated constructs were lysed, subjected to SDS-PAGE under reducing and non-reducingconditions, and analyzed by Western blot with the DSG-2 polyclonal antiserum. C, cell surface expression of DC-SIGN polymorphic variants on stable transfec-tants on K562 cells, as determined by flow cytometry (upper panels) and immunocytofluorescence (lower panels). The middle panels show the correspondingphase contrast images. The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGN MR1 antibody (black text and profile) orthe X63 control antibody (gray text and profile) are indicated. D, monoclonal antibody-induced internalization of DC-SIGN in K562 cells stably transfected withthe indicated polymorphic variants. Flow cytometry expression is expressed relative to the level of DC-SIGN in each transfectant maintained at 4 °C (arbitrarilyconsidered as 100).
Expression and Function of DC-SIGN Variants
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andB). Like in the case of the 1A/8d�L cotransfectants, hetero-oligomers might have an increased sensitivity to denaturingagents. However, coimmunoprecipitation experiments withepitope-tagged molecules demonstrated that the DC-SIGN 1Amolecules preferentially formed homo-oligomers, and associ-ate weakly to seven repeat-containing polymorphic variants(�1% of the prototypic DC-SIGN molecules are engaged inhetero-oligomer formation) (Fig. 6C). Therefore, the shorterpolymorphic variants can be expressed on the cell surface buttend to form homo-oligomers and associate very weakly withthe prototypic DC-SIGN 1A full-length isoform. This resultwould imply that cells heterozygous at the DC-SIGN genemight almost exclusively express homo-oligomers on the cellsurface. We tested this hypothesis by analyzing the DC-SIGNisoform expression andmultimerization state inMDDC from adonor previously identified as a CD209 heterozygote (see Fig.5A). The prototypic and shorter variant of DC-SIGN wereexpressed to a similar extent in heterozygous dendritic cells,but no evidence was found of hetero-oligomer formation (Fig.
6D), confirming that DC-SIGN multimerization takes placepreferentially among variants whose neck region has identicalstructure.The low percentage (or impaired stability) of lectin hetero-
oligomers might explain the reduced pathogen-binding capac-ity exhibited by cells coexpressing allelic variants of DC-SIGNR(53) and the correlation between DC-SIGN/DC-SIGNR neckregion heterozygosity and susceptibility to viral infection (38,53–55). Consequently, DC-SIGN-dependent activities of cellscoexpressing different DC-SIGN allelic variants were evaluatedon transiently transfected cells. As shown in Fig. 6B, coexpres-sion of theDC-SIGN-D7 isoform (which lacks the seventh neckdomain repeat) did not significantly affect the ability of theprototypic DC-SIGN 1A isoform to bind immobilizedmannan.Along the same line, capture of fucose- or Lewisx-coated poly-acrylamide beads byDC-SIGN1Awas not affected by the coex-pression of the DC-SIGN-D3 isoform (which lacks the thirdneck domain repeat) (Fig. 7A and not shown). These resultsindicated that expression of polymorphic variants with shorter
FIGURE 6. Influence of DC-SIGN polymorphic variants on the expression, multimerization, and functional capability of the prototypic molecule.A, lysates from COS-7 cells transiently transfected with the indicated DC-SIGN constructs were subjected to Western blot with the DSG-2 polyclonal antiserum.The mobility of monomers and trimers is indicated. The right panel shows the same experiment after a longer electrophoretic separation for increasedresolution of the DC-SIGN trimers. B, adhesion to immobilized mannan of HEK293T cells transiently transfected with the indicated DC-SIGN variants in thepresence of either a blocking antibody (MR1) or an irrelevant antibody (X63) (lower panel). The DC-SIGN expression levels of the distinct transfectants wasdetermined by flow cytometry and is indicated in the upper panel. The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGNMR1 antibody (black text and profile) or the X63 control antibody (gray text and profile) are indicated. C, lysates from COS-7 cells transiently transfected with theindicated constructs were immunoprecipitated with an anti-5xHis monoclonal antibody, and immunoprecipitates were subjected to Western blot using eitheran anti-FLAG monoclonal antibody (upper panel) or the DSG-2 polyclonal antiserum (lower panel). Whole cell lysates were also analyzed as a transfectioncontrol. D, lysates from MDDCs generated from donors characterized as homozygote for DC-SIGN with 8 neck repeats (1A/1A) or heterozygote, with alleles withneck domain of 8 and 7 repeats (1A/-D7), were separated by SDS-PAGE under reducing and non-reducing conditions, and then subjected to Western blot withthe DSG-2 polyclonal antiserum. For control purposes, lysates from K562 cells stably transfected and COS-7 cells transiently transfected with the indicatedconstructs were included in the experiment.
Expression and Function of DC-SIGN Variants
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neck domains does not significantly alter the pathogen recog-nition ability of cells expressing the prototypic DC-SIGN 1Aisoform.Finally, because sugar precipitation had previously allowed
the identification of functional differences among alternativelyspliced isoforms (Fig. 4), lysates from MDDCs coexpressingDC-SIGN 1A and DC-SIGN-D7 were subjected to precipita-tion with mannan-agarose. Whereas a single band (corre-sponding to DC-SIGN 1A) was specifically retained from the1A/1A dendritic cells, both DC-SIGN 1A and DC-SIGN-D7isoforms were equally retained by mannan-agarose when thedendritic cell lysate from the 1A/-D7 donor was used (Fig. 7B).Therefore, DC-SIGN 1A and DC-SIGN-D7 are retained bymannan to a similar extent, confirming that shorter neck poly-morphic variants of DC-SIGN retain their sugar-recognitionability, showing no differences from that of the prototypic full-length DC-SIGN 1A molecule.
DISCUSSION
DC-SIGN-dependent binding and uptake of clinically rel-evant pathogens by dendritic cells relies on the lectin abilityto bind mannose- and fucose-containing glycans (56). Stud-ies on recombinant molecules have demonstrated that theavidity of such interactions is mediated through multimer-ization of the lectin, which is accomplished through inter-molecular associations mediated by the neck domain of themolecule (27, 34). The neck region of DC-SIGN is composedof eight 23-amino acid repeats, which are encoded in a singleexon whose polymorphism has been already demonstrated(38, 51). In fact, DC-SIGN alleles with 4–9 repeats within theneck region-coding exon have been described (51), and het-erozygosity at this specific exon correlates with altered sus-ceptibility to HIV-1 infection (38). Besides, numerous DC-SIGN alternatively spliced isoforms have been described at
the mRNA level (14, 36). The combination of alternativesplicing and genomic polymorphism predicts that a largerepertoire of DC-SIGN protein isoforms might exist, most ofwhich would differ in the size of the neck domain (14, 36, 38,51). However, to date, the functional characterization of DC-SIGN isoforms and allelic variants on the cell membrane hadnot been addressed. In the present manuscript we presentevidences that 1) DC-SIGN alternatively spliced mRNA spe-cies give rise to proteins that are expressed at the cell mem-brane on monocyte-derived dendritic cells, cell lines, andtransfectants; 2) DC-SIGN alternatively spliced isoforms dif-fer in their multimerization capability and sugar-bindingability; 3) the presence of two repeats within the neckdomain is sufficient for DC-SIGN multimerization; 4) theneck domain repeats are not functionally interchangeably,because the number and arrangement of repeats within theneck domain critically determines the multimerization andligand-binding ability; 5) the lectin domain of DC-SIGN sta-bilizes or contributes to the neck region-dependent multim-erization of DC-SIGN, which is negatively influenced by theN-linked glycosylation of the first neck domain repeat; 6)basal multimerization of the molecule does not predict thepathogen-binding ability and does not correlate with ligand-induced internalization; and 7) polymorphic variants differ-ing in neck domain composition can self-associate, but mul-timerize very poorly with the prototypic full-lengthmolecule, suggesting that the DC-SIGN molecules on thecell surface predominantly appear as homo-multimers. Thedata here presented constitutes the first demonstration thatalternative splicing and polymorphic variants of DC-SIGNare expressed on monocyte-derived dendritic cells, wherethey exhibit altered multimerization and carbohydrate-binding abilities (splicing variants) and tend to segregate
FIGURE 7. Influence of DC-SIGN polymorphic variants on the functional capability of the prototypic molecule. A, binding of either FITC-PAA-NAc-Gal or FITC-PAA-fucose beads to HEK293T cells transiently transfected with the indicated DC-SIGN constructs. Expression levels were determined byflow cytometry (upper panels). The percentage (upper number) and MFI (lower number) of cells stained with the anti-DC-SIGN MR1 antibody (black textand profile) or the X63 control antibody (gray text and profile) are indicated. B, lysates from MDDCs from a homozygote (1A/1A) and a heterozygote(1A/-D7) donors were incubated with mannan-agarose. Bound proteins (eluted, right panels) or whole cell lysates (whole lysate, left panels) wereresolved by SDS-PAGE under reducing conditions, and subjected to Western blot with the DSG-2 polyclonal antiserum or a monoclonal antibody againstCD45 as control (upper panels).
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from the prototypic molecule forming homo-multimers(polymorphic variants with a shorter neck domain).Whether DC-SIGN and related lectins are bona fide patho-
gen-recognition receptors or antigen-binding receptors whosefunction is subverted by pathogens is still unclear (4, 57). Ourresults indicate that the various alternatively spliced isoformsdiffer in their ability to be retained by immobilized mannan,whereas all of them are equally efficient in terms of pathogenbinding. We hypothesize that the large amount of DC-SIGNligands on the surface of interacting pathogens compensate forthe distinct affinity andmultimerization ability of the isoforms.If this is the case, pathogen-induced formation of DC-SIGN-containing clusters on the cell surface would counterbalancefor the diminished multimerization ability of certain isoformsandwould justify the large range of pathogens bound and inter-nalized via DC-SIGN. Therefore, according to this hypothesis,isoforms would have a physiological role (increasing the rangeof soluble antigens bound and internalized by DC-SIGN), butwould not have a major impact on the range of pathogensbound by DC-SIGN. Further studies are needed to clarify theseissues, because it is currently unknown whether the basal mul-timerization of DC-SIGN on the cell surface (33, 44) is exclu-sively mediated by intermolecular interactions or is a solubleligand-induced event. In this regard, all the experiments per-formed in the present study were done after extensive washingof the cells with EDTA, to prevent any carbohydrate-DC-SIGNinteraction that might affect multimerization of the moleculeon the cell surface.Sequence analysis has allowed the definition of 23-residue
repeats within the neck region of DC-SIGN, which is some-times divided into 7.5 repeats to account for the presence of anunrelated and unique sequence at the N-terminal half of thefirst repeat (34). Ultracentrifugation and cross-linking ofrecombinant truncated DC-SIGN molecules have establishedthat removal of the two N-terminal repeats only partiallyaffected the tetramerization ability, whereas recombinant pro-teins containing only repeats 7–8 formed partially dissociatingdimers. This has led to the proposal that repeats close to thelectin domain mediate dimer formation while the membraneproximal repeats are required for tetramer formation (34). Ourresults with transient and stable transfectants of the naturallyoccurring DC-SIGN 4d and 2d isoforms, which include repeats1, 6, 7, and 8 and 1 and 2 (Fig. 3A), indicate that the two moreN-terminal domains are sufficient for multimerization in a cel-lular context, a fact further confirmed by the very differentmul-timerization capability of the 4d (1, 6, 7, and 8) and 4d� (1, 2, 3,and 8) isoforms. The importance of repeats 1 and 2 for theability of DC-SIGN tomultimerize in the cellmembrane is evenmore evident when considering that the DC-SIGN 1d isoform(containing only repeat 1) does not multimerize, and thatremoval of the N-glycosylation site (1dN/Q mutant) allowsmultimerization within a cellular context. In addition, the 3dconstruct, which includes the first repeat followed by theN-ter-minal half of repeat 2, the C-terminal half of repeat 7 and theentire repeat 8, also exhibits an efficient multimerization capa-bility within a cellular context. Therefore, essential residues formultimerization can be mapped to the sequence GELSE at thebeginning of the second repeat, which includes a serine residue
unique among the repeats and contributes to the multimeriza-tion ability of recombinantDC-SIGNR (58). These results dem-onstrate the critical role of repeats 1 and 2 for DC-SIGN mul-timerization, because repeat 1 is capable ofmediatingmultimerformation, and themere presence of repeat 2 appears sufficientto overcome the inhibitory effect of the N-glycosylation atrepeat 1. These results are compatible and extend previous dataon the multimerization capability of recombinant DC-SIGN/DC-SIGNR molecules, and establish neck glycosylation as animportant parameter to limit the degree of DC-SIGN multim-erization in the cell.The combination of genomic polymorphism and alternative
splicing at theDC-SIGNgene results in the generation of a largenumber of isoforms/allelic variants of the molecule. Consider-ing their variable multimerization capability, and the higheravidity displayed by multimers, it is tempting to speculate thatthe existence of all these variants might endow macrophagesand dendritic cells with a broader repertoire of ligand-bindingaffinity and/or specificity. In fact, the ability ofmannan-agaroseto differentially retain the various DC-SIGN splicing isoforms(Fig. 4) would support this hypothesis. On the other hand, analternative function for the numerous DC-SIGN isoformscould be the modulation of full-length DC-SIGN-dependentfunctions. In this regard, and like the lectin domain-lackingchimeric constructs (Fig. 2), isoforms with truncated lectindomainsmight reduce the effective concentration of full-lengthDC-SIGN molecules on the cell membrane, thus impairing itsmultimerization on the cell surface and, consequently, thebinding and uptake of pathogens/ligands containing limitingamounts of sugar ligands.Regarding polymorphic variants, our results indicate that the
number of DC-SIGN allelic variants is greater than previouslythought. The study of Barreiro and Liu (38, 51) has definedpolymorphisms within the neck region of DC-SIGN and classi-fied them according to the number of repeats. However, and atleast within the Spanish population, the allelic variants contain-ing only 7 neck repeats are not structurally identical, and threedistinct alleles have been identified which differ in the missingrepeatwithin the neck domain (D3,D5, andD7). Therefore, it islikely that most of the previously defined CD209 alleles arereally heterogeneous in terms of the arrangement of the neckdomain repeats they contain. On the other hand, and despitethe association found between neck domain heterozygosity atthe CD209 and CD209L genes and altered susceptibility toHIV-1 (38, 55), hepatitis C (59, 60), or severe acute respiratorysyndrome infection (53), the polymorphic variants that we havecharacterized exhibit similar homo-multimerization capabilityand pathogen- and carbohydrate-binding specificity as the full-length molecule. However, the polymorphic variants contain-ing seven repeats (-D3, -D5, and -D7) exhibit a very weak abilityto assemble into hetero-multimers with the full-lengthDC-SIGN1Aprototypicmolecule, as hetero-multimers cannotbe observed by Western blot and an extremely low percentageof the -D3 variant can be coprecipitatedwithDC-SIGN1A (Fig.6). This result is in contrast to the reported ability of recombi-nant polymorphic forms of DC-SIGNR to engage in stablehomo- and hetero-tetramers (58). However, we feel that this isonly an apparent discrepancy, because the N-linked glycosyla-
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tion of the N-terminal neck repeat limits the extent of multim-erization of the molecular within a cellular context (Fig. 2) and,therefore, recombinant molecules (which are devoid of glyco-sylation) might display an enhanced tendency to multimerize.Whether the reduced ability of DC-SIGNpolymorphic variantsto associate with the full-length molecule contributes to thealtered susceptibility of heterozygous individuals to variousinfections remains to be determined. However, the preferentialformation of homo-multimers in heterozygous individualsmust lead to a reduction (50%) in the number of multimerscontaining the full-length DC-SIGN 1A molecule, what mightaffect the recognition of pathogens with a limiting amount ofcarbohydrate ligands. The fact thatCD209 gene promoter poly-morphisms, thought to affect DC-SIGN cell surface levels, alsoassociate with altered susceptibility toHIV-1 (52), Dengue (37),and tuberculosis (49) is compatible with the above explanation.Consequently, although further studies are required, ourresults demonstrate that expression of neck domain splicingand allelic variants influence the presence and stability of DC-SIGNmultimers on the cell surface, and provide relevant cluesabout the underlyingmolecularmechanisms for the associationbetweenDC-SIGNpolymorphisms and altered susceptibility toclinically relevant pathogens.
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Supplementary Figures
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3 1.5 0.752d
A79.9%6.8
84.3%11.4
90.0%14.5
88.0%17.9
90.4%18.0
84.1%9.0
7.1%0.4
80.2%9.0
75.5%4.4
51015202530354045
C. albicans yeasts L. infantum amastigotes Pathogen
% c
ells
with
boun
dpa
thog
ens
Mock1A1AN/Q4d4d'3d2d1d1d-N/Q
B
3 2 1 0.51A
µgDNA transfected
81,8 (9,9)79,2 (8.0)74,4 (5,7)39,4 (1,2)
77,7(5,8)66,8 (3,0)32,0 (0,9)
79,3 (7,0)52,7 (2,2)29,2 (0,8)
510152025303540
% c
ells
with
boun
dsp
ores
45
Mock 1A 4d 4d’ 3d 2d 1d 1dN/Q1AN/Q
3 1.5 0.754d
3 1.5 0.752d
Supplementary Figure 1.- Pathogen-binding capacity of DC-SIGN isoforms and mutants.- A, B. HEK293T cells transiently expressing the indicated DC-SIGN isoforms/mutants (A) or decreasing levels of DC-SIGN 1A, 4d or 2d (B), were incubated with fluorescent C. albicans yeasts and L. infantum amastigotes and the percentage of cells with bound pathogens was determined by flow cytometry. Cell surface expression of each DC-SIGN construct is indicated (thick lines, DC-SIGN; thin lines, control antibody for (A) and one profile for each distinct amounts of transfected plasmids in (B) (3 µg, thick line; 2 or 1.5 µg, thin line; 1 µg, dashed line; 0.75 and 0.5 µg, dotted line). In each case, the Mean Fluorescence Intensity and the percentage of positive cells are shown. The experiment was done three times with similar results, and a representative experiment is shown.
97
Leishmania
Aspergillus
1.2 (0.3)9.9 (0.5)12.3 (0.6)
0.2 (0.3)72.8 (5.7)16.4 (0.6)
0.8 (0.3)60.2 (5.5)13.4 (0.9)
0.1 (0.3)73.8 (6.4)20.1 (0.8)
0.3 (0.3)57.5 (4.0)10.7 (0.6)
0.0 (0.3)3.9 (0.4)4.1 (0.5)
0.0 (0.3)29.6 (1.5)6.2 (0.6)
0.1 (0.3)38.9 (2.1)7.6 (0.9)
0.1 (0.3)44.5 (2.9)9.2 (1.0)
0.1 (0.3)44.4 (3.0)12.5 (1.0)
K562 mock 1A -D3 -D5 -D7
K562-DC-SIGN
MOI=0MOI=10 + X63MOI=10 + MR1
A
DSG-2
1A -D3 -D5 -D7Mock1A -D3 -D5 -D7MockC
Eluted Supernatant
B
X63 MR1
2
4
6
8
1012
GP1
-Fc
bind
ing
(MFI
)
K562 mock1A low1A-D3-D5-D7
K562DC-SIGN
% B
indi
ng to
Man
nan
1020304050607080
Mannan-agarose precipitation
25015010075
50
37
25
Non-reduced
kDa
DC-SIGN -D7
DC-SIGN -D5
DC-SIGN -D3DC-SIGN 1ADC-SIGN 1A (low)K562-Mock1H mock +IF
D
4 2 δ (ppm)Si
gnal
inte
nsity
Leishmania
Aspergillus
1.2 (0.3)9.9 (0.5)12.3 (0.6)
0.2 (0.3)72.8 (5.7)16.4 (0.6)
0.8 (0.3)60.2 (5.5)13.4 (0.9)
0.1 (0.3)73.8 (6.4)20.1 (0.8)
0.3 (0.3)57.5 (4.0)10.7 (0.6)
0.0 (0.3)3.9 (0.4)4.1 (0.5)
0.0 (0.3)29.6 (1.5)6.2 (0.6)
0.1 (0.3)38.9 (2.1)7.6 (0.9)
0.1 (0.3)44.5 (2.9)9.2 (1.0)
0.1 (0.3)44.4 (3.0)12.5 (1.0)
K562 mock 1A -D3 -D5 -D7
K562-DC-SIGN
MOI=0MOI=10 + X63MOI=10 + MR1
A
DSG-2
1A -D3 -D5 -D7Mock1A -D3 -D5 -D7MockC
Eluted Supernatant
B
X63 MR1
2
4
6
8
1012
GP1
-Fc
bind
ing
(MFI
)
K562 mock1A low1A-D3-D5-D7
K562DC-SIGN
% B
indi
ng to
Man
nan
1020304050607080
B
X63 MR1
2
4
6
8
1012
GP1
-Fc
bind
ing
(MFI
)
K562 mock1A low1A-D3-D5-D7
K562DC-SIGN
K562 mock1A low1A-D3-D5-D7
K562DC-SIGN
% B
indi
ng to
Man
nan
1020304050607080
Mannan-agarose precipitation
25015010075
50
37
25
Non-reduced
kDa
DC-SIGN -D7
DC-SIGN -D5
DC-SIGN -D3DC-SIGN 1ADC-SIGN 1A (low)K562-Mock1H mock +IF
D
4 2 δ (ppm)Si
gnal
inte
nsity
Supplementary Figure 2.- Pathogen and sugar-binding capacity of DC-SIGN polymorphic variants.- A. K562 cells stably transfected with the indicated DC-SIGN variants were incubated with fluorescent C. albicans yeasts and L. infantum amastigotes, in the presence of either X63 or MR1 antibodies as indicated, and the percentage of cells with bound pathogens was determined by flow cytometry. In each case, the first number indicates the percentage of cells with bound amastigotes or conidia, and the Mean Fluorescence Intensity of the whole cell population is indicated in parenthesis. B. Adhesion to immobilized mannan (upper panel) or binding of Ebola GP1-Fc (lower panel) of K562 cells stably expressing the indicated DC-SIGN variants in the presence of a blocking (MR1) or an irrelevant antibody (X63) antibody. For comparative purposes, two subpopulations of K562-DC-SIGN 1A with different DC-SIGN cell surface expression levels were assayed. C. Lysates from COS-7 cells transiently transfected with the indicated DC-SIGN polymorphic variants were incubated with mannan-agarose. After extensive washing, bound (Eluted, left panel) and non-bound proteins (Supernatant, right panel) were resolved by SDS-PAGE under non-reducing conditions and subjected to Western blot with DSG-2. D. Binding of Candida utilis glucomannan (IF) to K562 cells stably transfected with the indicated DC-SIGN polymorphic variants by means of 1D Saturation Transfer Difference NMR. The lower profile represents the 1H NMR spectrum of IF-S in PBS at 298 K. Graph illustrates the signal intensity yielded by each transfectant (y-axis) and the chemical shift (d) in parts per million (ppm).
98
Resultados
4. Identificación de epítopos en la molécula de unión a patógenos DC-SIGN
DC-SIGN (Dendritic cell-specific ICAM-3-grabbing non-integrin) es una lectina tipo C que
reconoce oligosacáridos que contienen fucosa y/o manosa, presentes en patógenos con relevancia
clínica. La señalización intracelular iniciada tras la unión de DC-SIGN con el ligando interfiere con
las señales iniciadas por los TLR, y modula la activación y polarización de células T inducida por las
células presentadoras de antígeno. El dominio de reconocimiento de carbohidratos C-terminal (CRD)
de DC-SIGN es precedido por un cuello integrado por ocho repeticiones de 23 residuos, que media
la multimerización de la molécula, y cuyos polimorfismos se correlacionan con una susceptibilidad
alterada a la infección por SARS y HIV. Con el fin de definir epítopos estructurales y funcionales de
DC-SIGN, hemos utilizado isoformas que ocurren de forma natural y moléculas recombinantes
quiméricas. De los tres epítopos identificados en el CRD, uno de ellos está expuesto solamente en
la forma monomérica de DC-SIGN, siendo dependiente de la multimerización de la molécula. Los
epítopos del dominio del cuello son independientes de la conformación e inalterados por la
multimerización de DC-SIGN, pero son diferencialmente afectados por la ausencia de repeticiones
de esta región. Aunque los anticuerpos específicos frente al cuello de DC-SIGN exhiben menor
capacidad de bloqueo funcional, son más eficientes en la inducción de internalización de la molécula.
Por otra parte, la unión de los diversos anticuerpos a sus epítopos correspondientes da lugar a
distintos grados de agrupación de moléculas de DC-SIGN en la superficie celular. La identificación
de epítopos independientes en DC-SIGN podría facilitar el diseño de reactivos que modulen la
capacidad de activación y polarización de células T por las células que expresan DC-SIGN, sin
alterar su capacidad de reconocimiento de antígenos y patógenos.
99
a ,
fac-allyithingainor-ms
Molecular Immunology 47 (2010) 840–848
Contents lists available at ScienceDirect
Molecular Immunology
journa l homepage: www.e lsev ier .com/ locate /mol imm
Epitope mapping on the dendritic cell-specific ICAM-3-grabbing non-integrin(DC-SIGN) pathogen-attachment factor
Elena Sierra-Filardia , Ana Estechab , Rafael Samaniegob , Elena Fernández-Ruizc , María ColmenaresPaloma Sánchez-Mateosb, Ralph M. Steinmand, Angela Granelli-Pipernod, Angel L. Corbía,∗
a Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spainb Unidad de Inmuno-Oncología, Hospital General Universitario Gregorio Maranón, Madrid, Spainc Unidad de Biología Molecular, Hospital Universitario de la Princesa, Madrid, Spaind Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY, USA
a r t i c l e i n f o
Article history:Received 7 February 2009Received in revised form20 September 2009Accepted 30 September 2009Available online 30 October 2009
a b s t r a c t
DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachmenttor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinicrelevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes wTLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presentcells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domcomposed of eight 23-residue repeats which mediate molecule multimerization, and whose polymphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isofor
Keywords:HumanDendritic cellsAdhesion moleculesPathogen recognitionDC-SIGN
and chimaeric molecules, in combination with established recognition properties, were used to defineseven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which ismultimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domainwere conformation-independent and unaltered upon molecule multimerization, but were differentiallyaffected by neck domain truncations. Although neck-specific antibodies exhibited lower function-blocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of
s resu ionpes o lateand p en-ition
ed.
tinsoadiralvan-3-ane
al.,al.,a-2),et
ndis
01;
the different epitopeof independent epitothe T cell activatingand pathogen-recogn
1. Introduction
Dendritic cells (DC) express a large array of cell surface lecand lectin-like molecules, which endow them with a brcapacity for recognition of parasitic, bacterial, yeast and vpathogens (Cambi and Figdor, 2003; Robinson et al., 2006;Kooyk and Geijtenbeek, 2003). Dendritic cell-specific ICAMgrabbing non-integrin (DC-SIGN, CD209) is a type II membr
C-type lectin (Curtis et al., 1992; Geijtenbeek et al., 2000c) abun-dantly expressed in vivo on myeloid DC, macrophages (Bleijs etal., 2001; Curtis et al., 1992; Geijtenbeek et al., 2000a,c, 2001;Lee et al., 2001; Soilleux et al., 2001, 2002), and in vitro gener-ated monocyte-derived dendritic cells (MDDC) and alternativelyactivated macrophages (Puig-Kroger et al., 2004; Relloso et al.,2002; Soilleux et al., 2002). DC-SIGN binds HIV (Geijtenbeek and∗ Corresponding author. Tel.: +34 91 8373112x4376; fax: +34 91 5627518.E-mail address: [email protected] (A.L. Corbí).
he-0c;nd
0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.molimm.2009.09.036
lted in distinct levels of microclustering on the cell surface. The identificatn the DC-SIGN molecule might facilitate the design of reagents that moduolarizing ability of DC-SIGN-expressing cells without preventing its antigcapacities.
© 2010 Elsevier Ltd. All rights reserv
van Kooyk, 2003), Ebola (Alvarez et al., 2002), SARS (Marzi et2004), Hepatitis C (Lozach et al., 2004, 2003; Pohlmann et2003) and Dengue virus (Tassaneetrithep et al., 2003), Leishmnia amastigotes and promastigotes (Colmenares et al., 2004, 200Mycobacterium tuberculosis (Geijtenbeek et al., 2003; Tailleuxal., 2003), Aspergillus fumigatus (Serrano-Gomez et al., 2004), aCandida albicans (Cambi et al., 2003), via mannan- and Lewoligosaccharides-dependent interactions (Feinberg et al., 20Frison et al., 2003). Besides, DC-SIGN mediates intercellular adsion through its recognition of ICAM-3 (Geijtenbeek et al., 200Gijzen et al., 2007), ICAM-2 (Geijtenbeek et al., 2000a), CEA a
CEACAM1 (van Gisbergen et al., 2005a), and the CD11b/CD18 inte-grin (van Gisbergen et al., 2005b). The extracellular part of DC-SIGNcontains a carbohydrate-recognition domain (CRD) and a neckregion composed of eight 23-residue repeats (Curtis et al., 1992;Engering et al., 2002; Geijtenbeek et al., 2000b; Kwon et al., 2002).Analysis of recombinant molecules indicates that the neck domainmediates the formation of DC-SIGN tetramers, possibly as a strat-egy to increase the avidity for ligand binding (Bernhard et al., 2004;101
unolo
Feinbeand getransctransmSakunto HIV
EngprevenPipernintern(Caparwith tSIGNboundgen prto humpromo2005)be usecells, bdescriRellosepitopto disttope mmonomechainterncell su
2. Ma
2.1. G
Hulated(NycoMonoCD14and imtocoland 10et al.,
2.2. Sisoform
StapreviofectionSuperof thethe diCOS-7plemeand pfromated uby seqeratioPCR wet al.,�lectigeneraand se
nd 7epeacoRI/CDNIGN 4
.3. Im
Forere c
BS p.025%loc (protifterl., 20f sepith
0% gr nonolyclC-SIGestectedansfillip50 m
etectines
arrieran
ntise
.4. F
Indescrit al.,s a pe P3
nti-mlonalsed ance oortioith
mpli
.5. Dggreg
96ith mereereye 2xym
reincotyp
E. Sierra-Filardi et al. / Molecular Imm
rg et al., 2005; Mitchell et al., 2001). Alternative splicingnetic polymorphisms generate numerous DC-SIGN isoformripts whose presence has been detected at mucosal HIVission sites (Liu et al., 2005, 2004; Mummidi et al., 2001;
tabhai et al., 2005) and correlates with altered susceptibility-1 transmission (Liu et al., 2004).agement of cell surface DC-SIGN by monoclonal antibodiests pathogen-attachment (Geijtenbeek et al., 2000c; Granelli-o et al., 2005; Relloso et al., 2002), and also leads to moleculealization (Engering et al., 2002) and intracellular signalingros et al., 2006; Gringhuis et al., 2007; Hodges et al., 2007),he latter being antibody-specific (Hodges et al., 2007). DC-functions as an antigen-capturing molecule which targetsmolecules to endosomal compartments for subsequent anti-esentation (Engering et al., 2002). In fact, targeting antigensan dendritic cells via a humanized anti-DC-SIGN antibody
tes effective naive and recall T cell responses (Tacken et al.,. Consequently, DC-SIGN-specific monoclonal antibodies canful not only preventing pathogen spreading into myeloidut also as therapeutic tools. Through the use of previously
bed monoclonal antibodies (Granelli-Piperno et al., 2005;o et al., 2002), we now describe the identification of sevenes within the DC-SIGN molecule, some of which can be usedinguish the multimerization state of the molecule. The epi-apping on the DC-SIGN molecule suggests that anti-DC-SIGN
clonal antibodies can block DC-SIGN functions by alternativenisms, including ligand-binding blockade, antibody-inducedalization and modulation of the multimerization state on therface.
terials and methods
eneration of monocyte-derived dendritic cells (MDDC)
man peripheral blood mononuclear cells (PBMC) were iso-from buffy coats from healthy donors over a Lymphoprepmed, Norway) gradient according to standard procedures.cytes were purified from PBMC by magnetic cell sorting usingmicrobeads (Miltenyi Biotech, Bergisch Gladbach, Germany),mediately subjected to the dendritic cell differentiation pro-
using 1000 U/ml GM-CSF (Schering-Plough, Kenilworth, NJ)00 U/ml IL-4 (PreProtech, Rocky Hill, NJ) (Dominguez-Soto
2007), with cytokine addition every second day.
table and transient transfection of DC-SIGN mutants ands
ble transfectants of DC-SIGN 1A in K562 cells have beenusly described (Relloso et al., 2002). For transient trans-s, 2 × 105 COS-7 or HEK293T cells were transfected with
fect (Qiagen, Hilden, Germany) in 6-well plates, using 2 �gdistinct pCDNA3.1(−)-based expression plasmids containingstinct isoforms, mutants and chimaeric forms of DC-SIGN.and HEK293T cells were routinely grown in DMEM sup-
nted with 10% Fetal Calf Serum (FCS). DC-SIGN isoformsolymorphic variants were isolated by RT-PCR on RNAMDDC or genomic DNA, and the constructs were gener-sing standard molecular biology techniques and verified
arEpS
2
wP0baAaow1opDWjetr(Mindmc(Ga
2
deathacuepwa
2a
wwwdtopis
uencing, as described (Serrano-Gomez et al., 2008). Gen-n of DC-SIGN expression vectors lacking the lectin domainas performed on the pCDNA3-DC-SIGN 1A construct (Relloso2002) using oligonucleotides CD209 sense and CD209-
n (5′-CCCCAAGCTTGTCACAGGCGTTCCACTGCAGC-3′). PCR-ted fragments were resolved in 1.5% agarose gels, purifiedquenced. Fragments containing either the full-length (8d)
bodiesto adhremovent ceweremanngation
102
gy 47 (2010) 840–848 841
-, 6-, and 4-repeat neck regions (repeats 1 through 7, 7d;ts 1 through 6, 6d; repeats 1 through 4, 4d) were cloned intoHindIII-digested pCDNA3.1- to yield pCDNA3.1-DC-SIGN 8d,A3.1-DC-SIGN 7d, pCDNA3.1-DC-SIGN 6d and pCDNA3.1-DC-d plasmids.
munoprecipitation and Western blot
immunoprecipitation, DC-SIGN-transfected HEK293T cellsollected, washed with PBS 1 mM EDTA, resuspended in 1 ml
H 8.0 and lysed in 10 mM Tris–HCl pH 8.0, 150 mM NaCl,sodium azide, 0.5% NP-40, 1 mM iodoacetamide, 2 mM Pefa-
Alexis Biochemicals, Lausen, Switzerland), and 2 �g/ml ofnin, antipain, leupeptin and pepstatin (NP-40 lysis buffer).preclearing, anti-DC-SIGN antibodies (Granelli-Piperno et05) were added onto the cell lysate, followed by additionharose-coupled protein G. Retained molecules were eluted3× Laemmli’s sample buffer (2% SDS, 6.25 mM Tris base,lycerol), and eluates resolved by SDS-PAGE under reducing-reducing conditions, and subjected to Western blot with
onal antiserum raised against a 28-residue peptide from theN cytoplasmic tail (DSG2) (Serrano-Gomez et al., 2008). For
rn blot, 10 �g of total cell lysate in NP-40 lysis buffer was sub-to SDS-PAGE under reducing or non-reducing conditions and
erred onto Immobilon polyvinylidene difluoride membraneore, Bedford, MA). After blocking with 5% non-fat dry milkM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20, protein
ion was performed using the Supersignal West Pico Chemilu-cent system (Pierce, Rockford, IL). Detection of DC-SIGN wasd out with the distinct anti-DC-SIGN monoclonal antibodieselli-Piperno et al., 2005), or the DSG2 anti-DC-SIGN polyclonalrum.
low cytometry and antibodies
irect immunofluorescence was done using the previouslybed anti-DC-SIGN monoclonal antibodies (Granelli-Piperno2005), MR1 (directed against the lectin domain of DC-SIGN)ositive control (Relloso et al., 2002), or the supernatant ofX63Ag8 myeloma as negative control, and FITC-labeled goatouse IgG as a secondary antibody. Where indicated, a poly-antiserum against a neck-derived peptide (DSG1) was alsos a positive control. All incubations were done in the pres-f 50 �g/ml of human IgG to prevent binding through the Fcn of the antibodies. Flow cytometry analysis was performedan EPICS-CS (Coulter Científica, Madrid, Spain) using logfiers.
C-SIGN-dependent adhesion to S. cerevisiae mannan andation
-well microtiter EIA II-Linbro plates were coated overnightannan at 50 �g/ml in PBS at 4 ◦C, and the remaining sites
blocked with 0.5% BSA for 2 h at 37 ◦C. Transfected cellslabeled in DMEM containing 0.5% BSA with the fluorescent′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein ace-ethyl ester (Molecular Probes, The Netherlands) at 37 ◦C andubated for 20 min in DMEM 0.5% BSA containing either thee-matched control P3X63 or the distinct anti-DC-SIGN anti-
(Granelli-Piperno et al., 2005). Cells were then allowedere to each well for 15 min at 37 ◦C. Unbound cells wereed by three washes with DMEM 0.5% BSA, and adher-lls were quantified using a fluorescence analyzer. Results
expressed as “% Binding”, which indicates the percentage ofan-bound cells relative to the total cellular input. For aggre-
assays, K562-DC-SIGN cells were washed with PBS 1 mM
olecul
atese or
DC-of
andich
Thesed
R1
tedForur-ml)lde-ree
was
842 E. Sierra-Filardi et al. / M
EDTA, resuspended in RPMI 10% FCS, and placed in 24-well plat 105 cells/ml for 20 min at 37 ◦C and either in the presencabsence of anti-DC-SIGN antibodies.
2.6. Specificity of antibody recognition by ELISA
The cDNA region encoding the extracellular portion ofSIGN was generated by PCR, cloned in-frame downstreamthe hexahistidine sequence of pET100/D-TOPO (Invitrogen),transformed into BL21 bacteria to generate HIS-DC-SIGN, whwas purified on Ni2+-nitrilotriacetic acid-agarose (Qiagen).DSG1 peptide sequence (GELPEKSKQQEIYQELTRLKAAV) was ba
on the sequence of the sixth repeated domain of the DC-SIGNneck region. DSG1 peptide was coated onto 96-well MaxisorpImmunoplates (Nunc) and binding of anti-neck monoclonal anti-bodies assessed by a standard direct ELISA procedure usingHRP-conjugated anti-mouse IgG rabbit polyclonal antiserum. Forcompetition experiments, 96-well HIS-DC-SIGN protein-coatedplates were incubated with anti-CRD antibodies, followed by incu-Fig. 1. Identification of CRD- and neck-specific DC-SIGN antibodies. (A) Schematic sstudy. (B) The indicated DC-SIGN constructs (DC-SIGN 1A, DC-SIGN 8d and DC-SIGreactivity of the distinct anti-DC-SIGN monoclonal antibodies, the anti-DC-SIGN podetermined by flow cytometry. MFI (lower number) and percentage of positive celwith the indicated expression vectors (DC-SIGN 1A and DC-SIGN 8d), lysed after 48 hWestern blot using the indicated anti-DC-SIGN monoclonal antibodies. (D) Lysates othe indicated anti-DC-SIGN antibodies. Immunoprecipitates were resolved by SDS-polyclonal antiserum DSG2. The position of high-order multimers is indicated by aDC-SIGN constructs (1A, 1AN/Q, 2d and 1d) or an empty vector (Mock), and the reactiantibody MR1 (positive control) or the negative control P3X63 (Control) was deternumber) are shown in each case. Each experiment was performed twice with simila
ar Immunology 47 (2010) 840–848
bation with a suboptimal concentration of biotin-labeled Mantibody and HRP-conjugated streptavidin.
2.7. Immunofluorescence and confocal microscopy
105 immature MDDC were layered over fibronectin-coaglass coverslips (5 �g/ml) and incubated for 30 min at 37 ◦C.staining of early endosomes, cells were subsequently pulsed ding 10 min with A633 conjugated biferric-transferrin (10 �g/(Molecular Probes). Cells were then fixed (4% paraformahyde in PBS, 15 min at room temperature) and washed thtimes with 25 mM Tris buffer saline. When permeabilization
required, samples were incubated with 0.3% Triton X-100 (5 minat room temperature). After a blocking step in Blocking Reagent(Boheringer Manheim) containing 0.5% sodium azide and 1 �g/mlof human immunoglobulins (10 min at room temperature), cellswere labeled with the indicated antibody (30 min at 37 ◦C) fol-lowed by FITC-labeled anti-mouse IgG. Cells were finally washedin PBS and water, and mounted with fluorescence mountingtructure of the naturally occuring and chimaeric DC-SIGN constructs used in the presentN 4d) or an empty vector (Mock) were transient transfected in HEK293T cells, and thelyclonal antiserum DSG1 (positive control) or the negative control P3X63 (Control) wasls (upper number) are shown in each case. (C) COS-7 cells were transiently transfected, and cell lysates resolved by SDS-PAGE under non-reducing conditions and subjected to
f HEK293T cells transiently transfected with DC-SIGN 1A were immunoprecipitated withPAGE under reducing conditions, and subjected to Western blot with the anti-DC-SIGNn arrow. (E) HEK293T cells were transfected with expression vectors for the indicatedvity of the indicated anti-DC-SIGN monoclonal antibodies, the anti-DC-SIGN monoclonalmined by flow cytometry. MFI (lower number) and percentage of positive cells (upperr results, and one of the experiments is shown.
103
unolo
mediuwith37 ◦C)ondar100 scells.
Lasconfoctems,PL-APresolu∼150 nwith tintenstion awhichthe sc
resherge
. Re
.1. Idonoc
Ens re
Fig. 2. F1A, DC-repeat (antibodare showthe numthe DC-PE labeMab). Rmatchethe reco
E. Sierra-Filardi et al. / Molecular Imm
m (DAKO). For induction of patching, cells were incubatedthe indicated anti-DC-SIGN antibody (1 �g/ml, 10 min at, fixed and subsequently incubated with a FITC-labeled sec-y antibody. For microclustering quantification, more thanpots were measured from at least 10 randomly chosen
er scanning confocal microscopy was performed with aal scanning inverted AOBS/SP2-microscope (Leica Microsys-Heidelberg, Germany). All images were acquired with a 63XO NA 1.3 glycerol immersion objective. The theoretical x, y-tion of this lens at Airy-1 and 488 nm excitation length is
thm
3
3m
ie
m. Assessment of fluorophore colocalization was performedhe Leica software, using a global statistic method to performity correlation analysis. Plots display the intensity distribu-nd degree of colocalization corresponding to the entire cell,
is shown next to the scatter plot. Co-localizing events inatter plot were gated (double positive pixels above the dual
2000cizationet al.,functiies (Gto recAll an
ine specificity of the neck-specific anti-DC-SIGN monoclonal antibodies. (A and B) HEK2SIGN chimaeric constructs (1A, 2d and 1d) (A), three different DC-SIGN polymorphic varia-D3, -D5 and -D7) (B), or an empty vector (Mock) and the reactivity of the neck-specificy (positive control) or the negative control P3X63 (Control) was determined by flow cyto
n in each case. Each experiment was performed twice, and one of the experiments is shober of the neck region repeats (8d, 7d and 6d) were transient transfected in COS7 cells, an
SIGN cytoplasmic domain (DSG2) or the indicated neck-specific monoclonal antibodies. (led (PE-Mab), and their binding to K562-DC-SIGN 1A stable transfectants assayed by flowesults are expressed as the percentage of binding in the presence of each competing antibd antibody. (E) Recognition of the DSG1 peptide by the indicated neck-specific anti-DC-SIgnition of rHIS-DC-SIGN by the MR1 monoclonal antibody, as determined by inhibition E
104
gy 47 (2010) 840–848 843
old) and visualized as a white overlay on the green and redd image.
sults and discussion
entification of anti-CRD and anti-neck DC-SIGN-specificlonal antibodies
gagement of cell surface DC-SIGN by monoclonal antibod-sults in pathogen recognition blockade (Geijtenbeek et al.,
; Granelli-Piperno et al., 2005; Relloso et al., 2002), internal-(Engering et al., 2002) and intracellular signaling (Caparros2006; Gringhuis et al., 2007; Hodges et al., 2007). To defineonal epitopes within the molecule, ten monoclonal antibod-ranelli-Piperno et al., 2005) were assayed for their abilityognize natural and chimaeric variants of DC-SIGN (Fig. 1A).tibodies recognized the prototypical DC-SIGN isoform (DC-
93T cells were transiently transfected with expression vectors for DC-SIGNnts which contain only seven repeats but differ in the identity of the absentmonoclonal antibodies (2, 4, 7, 10 and 11), the anti-CRD MR1 monoclonal
metry. MFI (lower number) and percentage of positive cells (upper number)wn. (C) DC-SIGN deletion mutants lacking the lectin domain but differing ind cell lysates subjected to Western blot with a polyclonal antiserum against
D) Cross-inhibition experiments. Antibodies 1, 7 and MR1 were purified andcytometry in the presence of the indicated competing antibodies (Comp.
ody relative to the binding observed in the presence of an irrelevant isotype-GN monoclonal antibodies by ELISA. (F) Effect of CRD-specific antibodies onLISA.
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844 E. Sierra-Filardi et al. / M
SIGN 1A) on HEK293T cells (Fig. 1B), but only five (antibo2, 4, 7, 10 and 11) (numbering according to (Granelli-Pipernal., 2005)) bound the CRD-lacking construct (DC-SIGN 8d)retained their reactivity against a construct which lacks halfneck domain (DC-SIGN 4d, Fig. 1B). The same pattern of reactiwas observed by Western blot on lysates from COS-7 cells ovepressing DC-SIGN 1A or DC-SIGN 8d: antibodies 2, 4, 7, 10 andrecognized DC-SIGN 8d, and bound to monomeric and multimforms of DC-SIGN (Fig. 1C), while antibodies 3, 6 and 13 didrecognize denatured DC-SIGN, and antibodies 1 and 9 exclusibound to the monomeric form of full-length DC-SIGN 1A (Fig.The differences between both sets of antibodies were also sin immunoprecipitation assays, as DC-SIGN high-order multimwere only brought down by antibodies 2, 4, 7, 10 and 11 (Fig.Therefore, five antibodies (2, 4, 7, 10 and 11) recognize the nregion of DC-SIGN (Neck-specific antibodies), whereas the spficity of the rest (1, 3, 6, 9 and 13) depends on the presence ofCRD region (CRD-dependent antibodies).
The above experiments also allowed the definition of threetinct specificity groups within the CRD-dependent antibodies. Fthe Western blot analysis in Fig. 1C, it is evident that antibo
1 and 9 bind only denatured DC-SIGN monomers, thus suggest-ing that, unlike 3, 6 and 13, they recognize linear CRD epitopesnot accessible in DC-SIGN multimers. On the other hand, antibod-ies 1, 3, 9 and 13 yielded flow cytometry profiles (almost bimodal)which differ from that of antibody 6 (Fig. 1B). Therefore, three typesof CRD-dependent DC-SIGN antibodies might be defined: Group 1(antibodies 1 and 9), which recognizes CRD sequential epitope(s)Fig. 3. Function-blocking ability of anti-DC-SIGN monoclonal antibodies. (A) HEK29or an empty vector (Mock), and expression levels determinated by flow cytometrycells were labeled with BCECF and adhesion to S. cerevisiae mannan was performed ian irrelevant antibody (X63, Control) as negative control. (C) K562-DC-SIGN 1A stabat 37 ◦C either in the absence (Untreated) or presence of the indicated anti-DC-SIGNassayed anti-DC-SIGN monoclonal antibodies.
ar Immunology 47 (2010) 840–848
probably masked in DC-SIGN multimers, Group 2 (antibodies 313) and Group 3 (antibody 6).
3.2. Comparison of the specificity of neck-specific antibodies
To further define the specificity of the DC-SIGN neck-specantibodies, their reactivity was assayed on DC-SIGN construlacking the N-glycosylation site in the first neck domain repeat (SIGN 1A N/Q), or whose neck domain included only two repe(DC-SIGN 2d) or a single repeat (DC-SIGN 1d). Disruption ofN-glycosylation site led to a reduction in the reactivity ofneck-specific antibodies (Fig. 1E and not shown), possibly refling the higher degree of multimerization of DC-SIGN glycosylamutants (Serrano-Gomez et al., 2008).
Truncation of the neck domain to a single repeat (DC-S1d) did not have any influence on the binding of CRD-dependantibodies (Fig. 1E and not shown), indicating that recognitionCRD-specific antibodies is independent on the length of the ndomain. By contrast, neck-specific antibodies displayed a varialevel of reactivity against the DC-SIGN 2d construct, and their biing was completely abolished when the neck region was exclusiv
composed of the first repeat (DC-SIGN 1d) (Fig. 2A). Antibodies 4,10 and 11 bound DC-SIGN 2d to a similar extent as the prototypicDC-SIGN 1A molecule, implying that their epitope(s) is retainedwithin the two N-terminal repeats of the neck domain (Fig. 2A).Conversely, antibody 2 recognized DC-SIGN 2d to a lower extent,and antibody 7 displayed no binding to DC-SIGN 2d (Fig. 2A). There-fore, the two N-terminal repeats of the DC-SIGN neck retain the3T cells were transiently transfected with the prototypic isoform DC-SIGN (DC-SIGN 1A)with the anti-DC-SIGN MR1 monoclonal antibody. (B) Transiently transfected HEK293Tn the presence of the distinct anti-DC-SIGN antibodies (MR1, DSG1, 1, 2, 4, 6, 7 and 9) orle transfectants were seeded in 24-well plates at 105 cells/ml and maintained for 20 min
antibodies (10 �g/ml). (D) Schematic representation of the epitopes recognized by the
105
unolo
epitopbodiestheir mspecifivarianthe ideshownrepeatsis ofwhichbodiesneck-sor 7 (Frecogntial reallowineck d
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E. Sierra-Filardi et al. / Molecular Imm
e(s) recognized by antibodies 4, 10 and 11, whereas anti-2 and 7 require the presence of additional repeats to exhibitaximal binding activity. On the other hand, all the neck-
c antibodies equally recognized three different polymorphicts of DC-SIGN which contain only seven repeats but differ inntity of the absent repeat (-D3, -D5, -D7) (Fig. 2B and data not), suggesting that they recognize epitopes shared by severals. A similar conclusion was drawn from Western blot analy-the DC-SIGN 8d, 7d and 6d neck region deletion constructs,were recognized to a similar extent by the neck-specific anti-(Fig. 2C), and from cross-competition experiments, as all thepecific antibodies could inhibit the binding of antibodies 4ig. 2D and not shown). Therefore, neck-specific antibodiesize epitopes shared by repeats 2-to-8, but exhibit differen-
cognition of the 2-repeat-containing DC-SIGN 2d construct,ng the definition of three distinct epitopes on the DC-SIGNomain.extend the above findings, neck-specific antibodies wereted for their ability to bind the DSG1 peptide, whose
thththainsw
3a
H(Glaathtob
nce is based on the DC-SIGN neck repeat 6. Only three anti-(4, 10 and 11) bound significantly to DSG1-coated plates
E), indicating that they recognize common or overlappinges which differ from those recognized by antibodies 2 andgreement with flow cytometry data. Regarding CRD-specificdies, and based on the cross-inhibition experiments (Fig. 2D),apparent that MR1 recognized an epitope different from
polycl(Fig. 3the ceSIGN–(50%)antibo2 had
Immunofluorescence staining and antibody-induced DC-SIGN microclustering of immatxed and permeabilized fibronectin-bound MDDC with the indicated anti-DC-SIGN anti
s the colocalization analysis. The scatter plot (most right panel) displays the intensity distranel) Non-permeabilized fibronectin-bound MDDC were fixed and stained with the indicamagnification. (Antibody-treated MDDC panel) Fibronectin-bound MDDC were incubatedto remove unbound antibodies, and fixed and stained with FITC-labeled anti-mouse Ig
. Microclustering was evaluated by measuring more than 100 spots per antibody in a-containing microcluster size in MDDC under basal conditions (Untreated) or exposed to
are the range, mean and SD of the measurement of 10 individual cells from each experiy-treated cells was only seen with antibodies 7, 10 and MR1.
106
gy 47 (2010) 840–848 845
defined by antibodies 1/9, 3/13 and 6. To further confirmssibility, inhibition ELISA experiments were performed using
nding of biotin-labeled MR1 to recombinant HIS-DC-SIGN as-out. As shown in Fig. 2F, none of the anti-CRD antibodiested MR1 binding to recombinant HIS-DC-SIGN, thus demon-g that MR1 defines a separate epitope on the DC-SIGN CRDis distinct from those defined by antibodies 1/9, 3/13 and 6.
unctional comparison of CRD- and neck-specific monoclonaldies
e analyzed antibodies have been demonstrated to inhibittransmission from Raji-DCSIGN transfectants to T cells
elli-Piperno et al., 2005). To determine whether a corre-exists between epitope recognition and function-blocking, the antibodies were evaluated for their ability to inhibitnding of DC-SIGN 1A-expressing HEK293T cells (Fig. 3A)nnan-coated surfaces. Whereas MR1 antibody completelyd cell binding to S. cerevisiae mannan, the neck-specific DSG1
onal antiserum had no effect (Serrano-Gomez et al., 2007)B). Regarding CRD-dependent antibodies, and in line withll-binding experiments, antibodies 1 and 9 abrogated DC-mannan interaction, whereas antibody 6 caused a moderateinhibition (Fig. 3B). In the case of neck-specific antibodies,dies 4 and 7 partially reduced the binding, and antibodyno effect on the DC-SIGN–mannan interaction (Fig. 3B).ure MDDC. (A) Immunolocalization of transferin A633-loaded (red fluores-bodies (green fluorescence). In the case of antibody 10 (two right panels),ibution of each fluorochrome and the degree of colocalization. (B) (Untreatedted anti-DC-SIGN antibodies to determine the pattern of membrane stainingfor 10 min at 37 ◦C with the indicated anti-DC-SIGN antibodies (1 �g/ml),
G polyclonal antiserum. Marked areas are shown below at higher magni-minimum of 10 randomly chosen cells. (C) Quantitation of the size of thethe indicated anti-DC-SIGN antibodies for 10 min at 37 ◦C (Antibody-treated).mental condition. Significant difference (p < 0.001) between untreated and
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846 E. Sierra-Filardi et al. / M
Similar results were seen on the Leishmania-binding ability ofSIGN, with neck-specific antibodies exhibiting the lowest inhibieffect (data not shown). Likewise, the DC-SIGN-dependent hotypic aggregation of K562-DC-SIGN 1A cells was strongly inhibby CRD-dependent antibodies (1, 3, 6), but was almost unaffecby the 7 and 10 neck-specific antibodies (Fig. 3C). Therefore, aCRD antibodies consistently display a stronger function-blockcapacities than those directed against the neck domain.
The combination of structural and functional assays sodescribed allowed the identification of different structuralfunctional epitopes on DC-SIGN (Fig. 3D). At least four epitocould be defined within the CRD according to their linear/sequenarchitecture and their accessibility on the cell surface, with twthem differentially involved in DC-SIGN-dependent adhesive futions. The rest of the epitopes were mapped within the neck domof the molecule, and their existence is inferred from the distreactivity of the antibodies against truncated forms of the moleca peptide based on the sequence of a single repeat, and their distfunctional effects in adhesion assays.
3.4. Clustering- and internalization-inducing ability of DC-SIGNantibodies
Regardless of their specificity, all antibodies equally recnized DC-SIGN on MDDC after cell permeabilization, as tyielded a spotted staining enriched at the lamellipodium and
labeled discrete cytoplasmic structures near the plasma membrane(Fig. 4A and not shown). The identity of these structures as endo-cytic/sorting compartments was determined after a 10 min pulsewith A633-labeled transferrin, which labels both early and sort-ing endosomes, as most DC-SIGN cytoplasmic spots co-localizedwith transferrin-loaded endosomes (Fig. 4A, two rightmost pan-els). To determine the influence that each antibody might haveFig. 5. Internalization-inducing ability of anti-DC-SIGN antibodies. MDDC cells wesubsequently fixed and incubated with a Cy3-labeled secondary antibody to detect Dan FITC-labeled secondary antibody to detect internalized molecules. Representativeare shown in the bottom panel.
ar Immunology 47 (2010) 840–848
on the cell surface distribution of DC-SIGN, fibronectin-bonon-permeabilized MDDC were either stained with the distantibodies, or incubated with antibodies at 37 ◦C for 10 min besubsequent fixation and staining with secondary antibodiesshown in Fig. 4B (upper panel, Untreated MDDCs), all antibodexcept for #6, yielded an equivalent spotted distribution onplasma membrane of intact MDDC, with a generally enriched sting at the lamellipodium. Preincubation with the distinct antiborevealed that the antibodies differed in their ability to modifysize of DC-SIGN-containing microclusters on MDDC (Fig. 4B, lopanels, Antibody-treated MDDC). Quantitation of the microclusize indicated that only antibodies 7, 10 and MR1 significa(p < 0.001) enhanced the size of the MDDC DC-SIGN-containmicroclusters when compared to the size of the clusters obserat 4 ◦C under basal conditions (Fig. 4C). This result suggests thatability to promote DC-SIGN redistribution on the MDDC cell suris independent on their recognition specificity, as enhanced miclustering was induced by anti-neck (7, 10) and CRD-depend(MR1) antibodies. Given the differential function-blocking abof the microclustering-enhancing antibodies (Fig. 3), these resalso suggest that the ability of the antibodies to inhibit DC-Srecognition functions is not related to their ability to induceSIGN cell surface redistribution. Whether the clustering-inducability might affect their ability to trigger intracellular signaremains to be determined.
The ability of anti-neck antibodies of partially inhibiting
SIGN adhesive functions could be explained by their ability to alterthe multimeric state of the lectin on the cell surface through dis-ruption of the neck–neck intercellular interactions which mediateDC-SIGN multimerization. Alternatively, neck-specific antibodiescould also alter DC-SIGN internalization, thus reducing the numberof available cell surface molecules. To determine whether this wasthe case, the ability of the different antibodies to promote DC-SIGNre either kept at 4 ◦C or treated with the indicated antibodies for 10 min at 37 ◦C, andC-SIGN molecules on the cell surface. Then, cells were permeabilized and incubated withexamples of cells exposed to the different antibodies under both incubation procedures
107
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DCare su(Engerfactorcell sulocalizfrom trestric(Cambal., 20turingintera2007)al., 200surfactriggeof greinvolv
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Thepreseninternpathothe DCof morate (DdetermSIGNlocatedomaiDC-SIGor toConveefficiecientlycells (not be
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alization in MDDC in suspension was evaluated by compar-e DC-SIGN cell surface expression on MDDC maintained at
subjected to a 30 min incubation at 37 ◦C with the distinctdies (Fig. 5, upper panel). Interestingly, maximal internal-
was promoted by antibodies 2, 4, 7 and 10, all of whichize neck-specific epitopes (Fig. 5, lower panel). Among the
pecific antibodies, MR1 exhibited the highest internalization-ng ability (Fig. 5). Therefore, it is tempting to speculate thatnction-blocking ability of neck-specific antibodies derivesheir capacity to diminish the amount of available DC-SIGNules on the cell surface.-SIGN is an antigen-capturing molecule whose ligandsbsequently taken into the antigen-presentation pathwaying et al., 2002), and functions as a pathogen-attachment(den Dunnen et al., 2009). DC-SIGN nanoscale clusters on therface are not distributed randomly, as they are preferentiallyed to the leading edge of MDDC lamellipod and excludedhe ventral plasma membrane (Neumann et al., 2008). Thisted localization and its clathrin-dependent internalizationi et al., 2009) appear to be tightly regulated (Neumann et08) as a possible strategy to maximize recognition and cap-functions. In fact, the DC-SIGN cytoplasmic tail is known to
ct with the F-actin binding phosphoprotein LSP1 (Smith et al.,, which also contributes to DC-SIGN signaling (Gringhuis et9). The identification of reagents that modulate DC-SIGN cell
e distribution, like the DC-SIGN neck-specific antibodies thatr maximal DC-SIGN internalization in MDDC, can therefore beat value to identify cytoskeletal or cytoplasmic componentsed in the regulated cell surface distribution of the molecule.ti-DC-SIGN antibodies have been valuable tools to put for-he pathogen-binding ability of the receptor and its capacityger intracellular signaling (Caparros et al., 2006; Gringhuis2007; Hodges et al., 2007), that interferes with the NF�B
tion route and results in increased production of IL-10. Ingard, the DC-SIGN-initiated signaling has been shown to
ferentially promoted by distinct antibodies (Hodges et al.,, and different intracellular signals appear to be triggeredecognition of mannose- or Lewis-containing ligands by DC-Gringhuis et al., 2009). However, most studies on DC-SIGN
ellular signaling make use of ligands which are not exclu-recognized by DC-SIGN and might be also sensed by otherand/or pathogen recognition receptors. Thus, the identifica-antibodies that detect independent epitopes on the DC-SIGN
ule might constitute a first step for the design of reagentsdissect the DC-SIGN-initiated intracellular signaling path-
and (2) target lectin-initiated intracellular signals as a waydulate the polarizing ability of DC-SIGN-expressing antigen-ting cells.
antibodies whose epitopes have been mapped in thet manuscript can be also helpful to potentiate the
alization ability of the molecule without preventing itsgen-recognition capacity. The search for reagents specific for-SIGN related molecule L-SIGN has led to the identification
noclonal antibodies that greatly enhance its internalizationakappagari et al., 2006). In the case of DC-SIGN, we haveined that the antibodies that promote the strongest DC-
internalization in MDDC are directed against an epitoped within the neck domain and out of the ligand-recognitionn (CRD), what suggests that their moderate inhibitory of
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N recognition functions is due to either steric hindranceenhanced removal of the molecule from the cell surface.rsely, the anti-L-SIGN antibodies that display the mostnt ligand-blocking effect are also internalized most effi-
in K562 transfectants and liver sinusoidal endothelialDakappagari et al., 2006). Since the L-SIGN antibodies haveen structurally mapped, it is not possible at this time to
280Feinber
tive216
Frison,amrecointe278
108
gy 47 (2010) 840–848 847
ine whether this difference is due to either cell-specificlecule-specific effects. Regardless of the explanation, thealization-inducing DC-SIGN neck-specific antibodies appearideally suited to potentiate the generation of immuneses against DC-SIGN-interacting ligands without compro-the pathogen-attachment function of the molecule.
wledgements
is work was supported by the Ministerio de Educacióncia (Grants BFU2008-0149-BMC), Ministerio de Sanidad
sumo, Instituto de Salud Carlos III (Spanish Network forsearch in Infectious Diseases, REIPI RD06/0008, and AIDS
rch Network, RIS RD06/0006), and Fundación para la Investi-y Prevención del SIDA en Espana (FIPSE 36663/07) to ALC.
atefully acknowledge Dr. M.A. Abengózar for performing theania-DC-SIGN interaction experiment.
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Discusión
La plasticidad del proceso de activación de macrófagos se refleja en la existencia de
subpoblaciones de este tipo celular con funciones diferentes e, incluso, opuestas [87]. Con el fin de
determinar las bases moleculares de estas diferencias funcionales, se realizaron estudios de
expresión génica diferencial entre distintas poblaciones de macrófagos generados in vitro a partir de
monocitos de sangre periférica en presencia de GM-CSF (M1), M-CSF (M2), IFNγ (CAMØ) o IL-4
(AAMØ) (Figura 11).
Figura 11.- Diferenciación in vitro de macrófagos. Esquema ilustrativo de la generación in vitro de las poblaciones de macrófagos utilizadas en los ensayos de expresión génica.
El perfil de expresión génica de cada población de macrófagos se conoce como su “firma genética”,
y permite determinar su presencia en un tejido sano o su implicación en procesos inflamatorios. A su
vez, la firma genética puede ser utilizada para predecir la evolución de procesos patológicos, debido
a que el diferente comportamiento de estas subpoblaciones celulares permite prognosticar el
desarrollo de ciertas enfermedades, como por ejemplo la progresión de tumores (actividad pro-/anti-
tumoral). Por otro lado, la comparación de los perfiles de expresión génica de diferentes poblaciones
celulares permite establecer diferencias y semejanzas fenotípicas entre ellas. De este modo, hemos
identificado genes diferencialmente expresados entre las poblaciones de macrófagos analizadas,
cuya expresión restringida ha sido validada mediante PCR cuantitativa, y que podrían ser utilizados
como “marcadores fenotípicos” específicos de cada una de ellas (Figura 12). En este sentido, se
han identificado 149 genes diferencialmente expresados entre macrófagos generados con GM-CSF
(M1) y M-CSF (M2) (>2 veces de diferencia, p<0.05), 94 de los cuales están significativamente
sobre-expresados en macrófagos M2. Por otro lado, entre AAMØ y CAMØ existen 283 genes
diferencialmente expresados (>2 veces de diferencia, p<0.05), de los cuales 112 están asociados a
la activación alternativa de macrófagos. Entre los genes sobre-expresados en macrófagos M2
Macrófago pro-inflamatorio (M1)
Macrófago anti-inflamatorio(M2)
M-CSF
GM-CSF IL-4
INFγ
Activación clásica (CAMØ)
Activación alternativa (AAMØ)
Monocito
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Discusión
Figura 12.- Representación gráfica de la expresión relativa de genes en las diferentes poblaciones de macrófagos. La escala de colores representa la expresión relativa de cada gen respecto a la media de intensidad de fluorescencia de las cuatro poblaciones (negro), desde los genes con menor expresión (verde) a los genes más expresados que la media (rojo). A la derecha se han seleccionado algunos de los genes con mayor expresión en macrófagos generados con IL-4 (cuadro verde), M-CSF (cuadro azul) o IFNγ (cuadro naranja).
114
Discusión
generados en presencia de M-CSF encontramos genes que justifican su fenotipo anti-inflamatorio.
Por ejemplo, genes inducidos o regulados por TGFβ como TGFB1 o SEPP1, genes que promueven
angiogénesis como IGF-1, o genes involucrados en reparación de heridas como F13A1, se
encuentran diferencialmente expresados en macrófagos M2. Del mismo modo, en estas células se
sobre-expresan genes relacionados con la producción de la citoquina anti-inflamatoria IL-10, como el
factor de transcripción MAF, del cual depende su expresión y la inhibición de IL-12p70 [213, 214], o
la enzima HMOX-1 y el receptor “scavenger” CD163, que son inducidos por IL-10 [215]. Por otro
lado, hemos evaluado la expresión de grupos funcionales de genes entre las subpoblaciones de
macrófagos para intentar explicar sus diferentes características. Por ejemplo, la expresión de genes
implicados en la regulación de los niveles de hierro intracelular podría justificar la función microbicida
de los macrófagos M1 (asociada con bajos niveles de hierro intracelular) y el papel permisivo frente
a patógenos de los macrófagos M2 (asociado con un elevado contenido de hierro intracelular)
(Sierra-Filardi et al., Immunobiology, en prensa, 2010). A su vez, los estudios de expresión génica
en las diferentes poblaciones de macrófagos también nos han permitido identificar genes cuya
expresión no se encuentra descrita en dichas células, lo que posibilita el estudio de la regulación de
su expresión y de sus posibles funciones en ellas.
El receptor de folato β es un marcador de macrófagos anti-inflamatorios M2 y TAM,
cuya expresión es regulada por activina A.
El receptor de folato β (FRβ) es uno de los genes que hemos identificado como marcador de
macrófagos anti-inflamatorios generados en presencia de M-CSF (M2). El folato o vitamina B9 es un
nutriente hidrosoluble esencial en las células, ya que se encuentra implicado en numerosas rutas
bioquímicas como el metabolismo de aminoácidos, la síntesis de purinas y pirimidinas, y la
metilación de ácidos nucleicos, proteínas y lípidos [216]. La deficiencia de esta vitamina conlleva a
una serie de anormalidades clínicas [217] como anemia megaloblástica, retraso del crecimiento,
desórdenes neurológicos, depresión [216] y enfermedad de Alzheimer [218]. Por el contrario, su
suplemento óptimo previene defectos en el tubo neural durante el embarazo y reduce el riesgo de
enfermedades cardiovasculares, desarrollo de tumores [219] y la predisposición a aterosclerosis
[220]. La forma biológicamente activa es el tetrahidrofolato (THF), cofactor esencial en reacciones
de metilación entre las que se incluye la formación de metionina a partir de homocisteína en el
conocido como ciclo de la homocisteína/metionina [221], y que se encuentra acoplado a un segundo
ciclo, conocido como ciclo del folato [222] (Figura 13).
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Discusión
THF
5-MTHF5,10-MTHF
Metionina
Homocisteína
SAH SAM
MTHFR
Cisteína
Cistationina
SHMT
CH3X
Metionina sintasa
CBS
XMetilación de DNA, proteínas, lípidos
Folato
DHFR
Biosíntesis de nucleótidos
THF
5-MTHF5,10-MTHF
Metionina
Homocisteína
SAH SAM
MTHFR
Cisteína
Cistationina
SHMT
CH3X
Metionina sintasa
CBS
XMetilación de DNA, proteínas, lípidos
Folato
DHFR
Biosíntesis de nucleótidos
Figura 13.- Metabolismo del folato y vías relacionadas. Esquema simplificado de la conexión entre los ciclos del folato y de la homocisteína. Los rectángulos muestran los sustratos y los óvalos las enzimas. DHFR, dihidrofolato reductasa; THF, tetrahidrofolato; SHMT, serinhidroximetil transferasa; 5,10-MTHF, 5,10-metilentetrahidrofolato; MTHFR, 5,10-metilentetrahidrofolato reductasa; 5-MTHF, 5-metiltetrahidrofolato; SAM, S-adenosilmetionina; SAH, S-adenosilhomocisteína; X, sustrato para metilación; CBS, cistationina-β-sintasa.
El ácido fólico y sus formas reducidas (folatos) son captados a través de la membrana celular
mediante dos principales sistemas de transporte: 1) transporte vía canales o proteínas
transportadoras de membrana con baja afinidad (Kd~μM); y 2) endocitosis vía receptor de folato (FR)
de alta afinidad (Kd~pM) [223]. Además, existe un flujo de transporte asociado al metabolismo
energético de la célula que permite el flujo de folato hacia el exterior celular mediante hidrólisis de
ATP [224]. La expresión de los FR se encuentra restringida a aquellos tejidos que utilizan la
segunda vía de captación de folato. Así, a excepción de riñón y placenta, el resto de tejidos sanos
expresan niveles bajos o no detectables [225], mientras que numerosos tejidos tumorales (ovario,
mama, bronquios, riñón, cerebro) expresan altos niveles de estos receptores [226]. La expresión de
los FR se modula de manera inversa por la concentración extracelular de folato: en cultivos
celulares, niveles bajos de folato en el medio incrementan la expresión del receptor en membrana,
mientras que la adición de ácido fólico al medio revierte este aumento [227]. En humanos, existen 4
isoformas del FR, FRα (FOLR1), FRβ (FOLR2), FRγ (FOLR3) y FRδ (FOLR4) [228], cuyos perfiles
de expresión en tejidos normales y patológicos son diferentes [229]. Así, el FRα es la isoforma más
expresada en tejidos adultos sanos, sobre todo en tejidos epiteliales, y en algunos tipos de tumores
como los ginecológicos (ovario, útero) [230]. A diferencia del resto de isoformas, el FRγ y su forma
truncada (FRγ’) no se encuentran anclados a la membrana plasmática y son secretados por células
hematopoyéticas de algunos tejidos sanos y tumorales, como médula ósea, bazo y timo [231, 232].
Por el contrario, ni la expresión en tejidos adultos ni embrionarios, ni la capacidad de unir folato del
116
Discusión
FRδ ha sido descrita [233]. En ratón se han identificado homólogos de FRα y FRβ [234], y un tercer
FR que se expresa en bazo y timo [233]. Debido al papel del ácido fólico en el desarrollo
embrionario, los ratones Folbp1-/- y Folbp2-/- presentan defectos en la formación del tubo neural [235,
236].
Respecto a la expresión del FRβ, éste se encuentra restringido al linaje mieloide [237], se
detecta en células hematopoyéticas de médula ósea CD34+ [238] y está incrementado durante la
maduración de neutrófilos y la activación de monocitos y macrófagos [239]. En tejidos sanos el FRβ
sólo se expresa significativamente en placenta, bazo y timo [240], mientras que lo hace en el 70%
de los casos de leucemia mieloide aguda (AML), donde se localiza también en células CD34+ [237,
241]. Además, el FRβ se expresa en macrófagos sinoviales de pacientes con artritis reumatoide
(RA) [239] y en un modelo inducido de artritis en rata [242]. Respecto a su capacidad funcional, el
FRβ expresado en células hematopoyéticas CD34+, neutrófilos y monocitos no es capaz de unir
folato [238], mientras que sí lo hace en condiciones patológicas como AML [231, 241] y en
macrófagos sinoviales en RA [243]. Como se ha comentado anteriormente, en nuestros estudios de
expresión génica hemos determinado que el FRβ se expresa en macrófagos anti-inflamatorios M2,
donde su expresión es inducida por M-CSF. Adicionalmente, hemos observado que otras citoquinas
de activación alternativa de macrófagos, como IL-4 e IL-13, mantienen o aumentan su expresión en
dichas células, a diferencia de estímulos pro-inflamatorios, como GM-CSF o LPS, que la inhiben. En
este sentido, el patrón de expresión del FRβ en ratón es similar al que hemos observado en
humano, ya que se expresa en macrófagos peritoneales activados F4/80+ CD68+ que presentan un
fenotipo M2 [244][245], y se induce con IL-4 [128]. Por tanto, el FRβ también podría considerarse un
marcador de macrófagos anti-inflamatorios y de activación alternativa en ratón. Por otro lado, hemos
demostrado que los macrófagos M2 son capaces de captar folato mediante endocitosis vía receptor,
y que es únicamente el FRβ el que media esta unión, ya que ni el FRα ni el FRγ se expresan en
estas células. A diferencia de nuestros resultados, en macrófagos peritoneales de ratón el FRβ sólo
es funcional tras la activación con estímulos inflamatorios [246]. En conclusión, el FRβ constituye un
marcador de macrófagos anti-inflamatorios/alternativos, ya que su expresión es inducida por M-CSF
y citoquinas Th2 in vitro en monocitos y macrófagos M2, y además es el responsable de la captación
de folato por estas células.
Además de la inhibición de la expresión del FRβ por estímulos inflamatorios como GM-CSF,
observamos que el medio condicionado de macrófagos M1 es también capaz de inhibir su
adquisición. Este hecho nos llevó a buscar factores secretados por estas células que pudiesen
regular la expresión de este receptor. Según nuestros estudios de expresión génica, la activina A es
el gen más diferencialmente expresado entre macrófagos M1 y M2, y además hemos determinado
que es un citoquina secretada al medio de cultivo de los macrófagos M1. Según nuestras
observaciones, la expresión y secreción de activina A es inducida por GM-CSF in vitro en la
diferenciación de macrófagos a partir de monocitos de sangre periférica y de células de médula ósea
117
Discusión
de ratón. Además, y de acuerdo con su rápida secreción en procesos inflamatorios [247], la activina
A es liberada al medio de cultivo desde las primeras horas de estimulación con GM-CSF. Por el
contrario, los macrófagos diferenciados en presencia de M-CSF no son capaces de secretar activina
A ni siquiera en presencia de un estímulo inflamatorio como LPS, a diferencia de lo descrito en
macrófagos de ratón [248]. Por tanto, la diferente regulación de la expresión de activina A y FRβ por
GM-CSF y M-CSF, respectivamente, nos hizo pensar que la activina A podría ser un factor regulador
de la expresión del FRβ y otros marcadores de macrófagos M2.
Las activinas son factores de crecimiento y diferenciación pertenecientes a la familia de TGFβ,
que estructuralmente están compuestas por dos subunidades β (activina A, βA-βA; activina B, βB-
βB; activina AB, βA-βB) unidas por un único enlace disulfuro [249]. La activina A (INHBA) se
identificó inicialmente por su papel en el control de la secreción de la hormona estimuladora del
folículo (FSH) [2], y en la actualidad es conocida por sus numerosas funciones en diferenciación,
proliferación y apoptosis celular, homeostasis, reparación de heridas, inflamación, además de su
función endocrina y su papel en la progresión de tumores [249-253]. Su actividad biológica es
neutralizada por folistatina, debido a la elevada afinidad de esta proteína por las activinas [254-256].
Respecto a la señalización de la activina A, como miembro de la familia de TGFβ, se une a los
receptores transmembrana ActRIB y ActRIIA y señaliza a través de las proteínas Smad2/3 [257,
258]. La activina A es sintetizada por una gran variedad de células, incluyendo monocitos [259, 260],
macrófagos [261], células dendríticas [262, 263], células endoteliales [264], células del estroma de la
médula ósea [265-267] y mastocitos [268], y algunas células linfoides como timocitos [269] y células
esplénicas T CD4+ de ratón [270]. A su vez, la activina A es secretada durante procesos
inflamatorios por lo que está considerada como un factor modulador de la respuesta inmunitaria.
A pesar de su secreción por macrófagos pro-inflamatorios M1, la activina A está descrita como
una citoquina secretada preferentemente por células Th2 y que promueve activación alternativa de
macrófagos en ratón, al inducir la expresión de Arg1 e inhibir la expresión de iNOS [270]. Además,
otra citoquina Th2 como IL-13 aumenta la concentración de activina A en el lavado broncoalveolar e
intraepitelial en ratones naive y durante inflamación alérgica [271], mientras que IFNγ (citoquina Th1)
no es capaz de inducirla [272]. Sin embargo, según nuestras observaciones, la activina A secretada
por macrófagos M1, al igual que la proteína recombinante, ejerce funciones pro-inflamatorias en la
diferenciación de monocitos, ya que previene la adquisición de la expresión del FRβ (FOLR2), y
otros marcadores de macrófagos M2 como SERPINB2 o MAF. Además, hemos determinado que la
activina A regula la función anti-inflamatoria de los macrófagos generados en presencia de M-CSF,
al inhibir la secreción de IL-10 por estas células tras ser estimuladas con LPS. De acuerdo con
nuestro resultado, la activina A inhibe la producción de IL-10 en cultivos de células epiteliales
prostáticas [273]. Por tanto, la activina A es capaz de regular el fenotipo y función de los MDM. Por
otro lado, a pesar de regular la función anti-inflamatoria de los macrófagos M2 en cuanto a la
producción de IL-10, hemos observado que la activina A no afecta a la capacidad presentadora de
118
Discusión
antígeno de estas células. Además, el medio condicionado de macrófagos M1 no induce la
maduración de DC, mientras que sí lo hace después de estimular dichas células con LPS. Es decir,
según nuestras observaciones y de acuerdo con un trabajo previo [263], la activina A secretada
constitutivamente por macrófagos M1 no induce maduración de DC, mientras que sí lo hacen otras
citoquinas pro-inflamatorias secretadas tras estimulación. En conclusión, en papel modulador en la
respuesta inflamatoria de la activina A podría deberse a su capacidad de regular el fenotipo y
función de los macrófagos, ya que inhibe la adquisición de marcadores anti-inflamatorios y la
producción de IL-10 por macrófagos M2, pero no influye en la capacidad de activación de linfocitos T
en estas células o en la maduración de DC.
IL-1β TNFα
IL-6 iNOS
Inflamación
Célula TCélula NK
Macrófago
DC
Célula B
Activina A
NFκBMAPK
TLRRespuesta inmunitaria
IL-1β TNFα
IL-6 iNOS
Inflamación
Célula TCélula NK
Macrófago
DC
Célula B
Activina A
NFκBMAPK
TLRRespuesta inmunitaria
Figura 14.- Funciones de la activina A en procesos inflamatorios. La activina A es capaz de inducir la producción de mediadores inflamatorios, como IL-1β, TNFα, IL-6 e iNOS, a la vez que, por su función anti-inflamatoria, puede ejercer un efecto inhibidor sobre la respuesta inmunitaria [274].
Respecto al papel modulador en la respuesta inmunitaria de la activina A, existe una dicotomía
entre sus acciones pro- y anti-inflamatorias [274] (Figura 14), a diferencia de otro miembro de su
misma familia, como el TGFβ, que presenta siempre funciones supresoras [275]. Por su acción
inflamatoria, la activina A estimula la secreción in vitro de citoquinas pro-inflamatorias como
TNFα, IL-1β e IL-6 [276-278], así como la expresión de iNOS y la producción de NO [277, 279].
Además, la activina A está implicada en la degradación de IκB, la translocación al núcleo de NFκB y
la fosforilación de ERK1/2 y la quinasa p38 [280-282]. Respecto a sus acciones anti-inflamatorias, la
activina A puede bloquear la producción y función de citoquinas inflamatorias como IL-1β e IL-6. En
monocitos humanos, la activina A inhibe la producción de IL-1β, bloqueando la conversión de su
precursor a su forma activa, e induce la expresión del antagonista del receptor IL-1 (IL-1RN), dando
119
Discusión
lugar a una reducción neta en la actividad de IL-1β [282]. Respecto a la IL-6, se ha demostrado que
la activina A inhibe su expresión en respuesta a LPS en microglía de ratón in vitro e in vivo [283] y
en células humanas epiteliales del endometrio [284]. Por otro lado, la activina A inhibe numerosas
funciones pro-inflamatorias de estas citoquinas, incluyendo proliferación de células T y B, o la
fagocitosis de monocitos [282, 285-289] (Figura 14). Esta dualidad en los efectos estimuladores o
inhibitorios de la activina A depende de su concentración. Así, en células amnióticas humanas la
producción IL-6 se estimula a dosis bajas, mientras que a mayores dosis su efecto es inhibitorio
[290]. Por tanto, la actividad pro- o anti-inflamatoria de la activina A depende de su concentración
local en tejidos.
Como ya mencionamos en el apartado de Introducción, los TAM son considerados
fundamentalmente macrófagos anti-inflamatorios/M2 [111, 127, 291], aunque su fenotipo y función
varía durante el desarrollo de los tumores [118]. Por ello, nos planteamos determinar la expresión
del FRβ en TAM como marcador fenotípico de macrófagos M2. Así, hemos observado que este
receptor se expresa en TAM de melanoma primario y metastático, mientras que no lo hace en el
resto de células tumorales. Adicionalmente, hemos determinado que el FRβ se expresa en células
CD14+ aisladas ex vivo de líquido pleural de melanoma y fluido ascítico de adenocarcinoma de
mama metastático, donde es funcional y tiene la capacidad de captar folato. Estos resultados están
de acuerdo con la expresión del receptor en células CD68+ CD163+ en glioblastoma humano y de
rata [244]. Por el contrario, en otro estudio afirman que los TAM de fibrosarcoma de ratón expresa
menores niveles de FRβ que los macrófagos peritoneales elicitados con tioglicolato [127]. Esta
discrepancia puede ser debida a la influencia del microambiente tumoral en la expresión de este
receptor, y que puede variar en función del tipo de tumor o de la fase de desarrollo en la que se
encuentre. En este sentido, hemos determinado que tanto el líquido ascítico de algunos carcinomas
gástricos o los medios condicionados de algunas líneas celulares tumorales, como las citoquinas
anti-inflamatorias liberadas por los tumores como M-CSF e IL-10, inducen la expresión in vitro del
FRβ en monocitos. Además, la exposición conjunta a varias de estas citoquinas, situación similar a
la que ocurriría in vivo, tiene un efecto sinérgico en la inducción de este receptor. Por otro lado, está
descrito que la expresión de activina A es mayor en tejidos tumorales que en tejidos sanos [292,
293], y disminuye con la progresión tumoral, siendo más elevada en tumores primarios que en
carcinomas metastáticos [294]. Según nuestras observaciones, los líquidos ascíticos de algunos
carcinomas gástricos inducen la expresión del FRβ e inhiben la expresión de activina A en
monocitos y macrófagos pro-inflamatorios M1. Por tanto, el microambiente tumoral condiciona el
fenotipo de los TAM y, en consecuencia, el desarrollo tumoral. Además, la activina A secretada en la
primera fase de iniciación del tumor contribuiría a la inhibición de las propiedades supresoras de los
TAM, por lo que la activina A sería un regulador el establecimiento y desarrollo tumoral.
El fenotipo de los TAM es un factor importante en la progresión de los tumores, debido a que los
macrófagos son las células inflamatorias que regulan el inicio y desarrollo tumoral [129, 295, 296].
120
Discusión
En los sitios de inflamación crónica donde se inicia la formación del tumor los macrófagos presentan
un fenotipo inflamatorio M1 con actividad anti-tumoral [297, 298] (Figura 15). Posteriormente cuando
el tumor se establece, los TAM dejan de secretar citoquinas inflamatorias como TNFα, IL-1β e IL-6, y
producen señales de supervivencia para las células tumorales, apoyando el crecimiento,
angiogénesis y metástasis del tumor [120, 299-301]. Este hecho se ha observado en modelos de
cáncer colorectal inducido por colitis y cáncer de hígado inducido químicamente en ratón, donde
existe una inhibición de la producción de citoquinas inflamatorias (IL-6, TNFα) [302-304]. Por tanto,
los macrófagos presentan una función dual en el contexto de la progresión tumoral: mientras que en
las primeras etapas de formación del tumor los macrófagos tienen el potencial de expresar
actividades anti-tumorales (fenotipo M1), en tumores establecidos juegan un papel pro-tumoral
(fenotipo M2) [126] (Figura 15). Una posible explicación a estas diferencias funcionales de los
macrófagos puede ser la eficacia de la respuesta inmunitaria en las distintas etapas de desarrollo
tumoral [305, 306]. Así, en estados tempranos de formación del tumor, la respuesta inmunitaria es
eficaz y los macrófagos ejercen su actividad citotóxica eliminando células tumorales. Una vez
estabilizado el tumor, cuando las células tumorales persistentes han escapado del “ataque”
inmunitario, se crea un ambiente inmunosupresor con una polarización anti-inflamatoria/M2
predominante en los macrófagos. Por ello, una posible estrategia terapéutica frente al avance
tumoral podría consistir en alterar el balance funcional de los macrófagos hacia un fenotipo
tumoricida [307].
CD8
INICIACIÓN EQUILIBRIO ESCAPE
Progresión del tumor
M1 M2
CD4CD4
NK CD8CD8
NKNK
CD8 CD4
ELIMINACIÓN
CD4CD8
INICIACIÓN EQUILIBRIO ESCAPE
Progresión del tumor
M1 M2
CD4CD4
NK CD8CD8
NKNK
CD8 CD4
ELIMINACIÓN
CD4
Figura 15.- Polarización de los macrófagos en el desarrollo tumoral. El fenotipo de los macrófagos sufre un cambio progresivo con el avance tumoral, desde un fenotipo tumoricida/M1 en las primeras etapas de iniciación hasta un fenotipo supresor/M2 cuando el tumor se establece [308].
Por otro lado, la función de los TAM está condicionada por su localización en respuesta a
señales locales [309]. Así, en áreas del tumor poco vascularizadas los TAM son altamente pro-
121
Discusión
angiogénicos en respuesta a hipoxia [310], y su presencia se ha correlacionado con el crecimiento
de tumores de mama [311]. Por el contrario, un elevado número de TAM en áreas vascularizadas
del tumor se correlaciona con buena prognosis en algunos tumores humanos, sugiriendo un posible
fenotipo anti-tumoral M1 en esos sitios [312]. Además, existe un fenotipo “mixto” debido a que los
TAM pueden presentar al mismo tiempo características pro-inflamatorias (M1) y anti-inflamatorias
(M2). Así, en un modelo tumoral en ratón los TAM son capaces de expresar a la vez iNOS y Arg1
[313, 314]. De forma similar, monocitos de pacientes con tumor gástrico avanzado expresan niveles
elevados y mayores de IL-12 e IL-10, citoquinas pro- y anti-inflamatorias respectivamente, que los
monocitos de individuos sanos [315]. En estos casos los TAM pueden exhibir actividades que
previenen el establecimiento y la extensión de las células tumorales y, simultáneamente, contribuir al
crecimiento y diseminación del tumor [316].
Al igual que ocurre en tumores, los macrófagos parecen ser los promotores principales de
enfermedades inflamatorias/autoinmunes como RA, donde existe una correlación directa entre la
actividad de estas células y la inflamación de las articulaciones y la destrucción del hueso [243].
Esto es debido a que los macrófagos del líquido sinovial de RA secretan múltiples mediadores de
inflamación y destrucción de tejido, incluidas citoquinas pro-inflamatorias (IL-1, IL-6, TNFα),
quimioquinas, prostaglandinas, metaloproteasas y ROS [317]. Además, estos macrófagos activados
participan en presentación de antígeno contribuyendo a la activación y proliferación de las células T
y su consecuente actividad destructiva [318]. Por todo ello, los macrófagos presentes en el líquido
sinovial de RA tendrían un fenotipo pro-inflamatorio M1. De acuerdo con esto, la expresión de
activina A en membranas sinoviales y su concentración en el líquido sinovial es elevada en
pacientes con RA [286, 288, 319]. Sin embargo, está descrita la expresión del FRβ en los
macrófagos presentes en el líquido sinovial de RA [243]. La expresión de este marcador de
macrófagos anti-inflamatorios estaría justificada por los elevados niveles de M-CSF existentes en el
líquido sinovial de RA, producidos por los fibroblastos sinoviales [57, 59, 320]. Por tanto, al igual que
ocurre en tumores, los factores presentes en el líquido sinovial de RA pueden condicionar el fenotipo
de los macrófagos, existiendo un cambio entre sus funciones inflamatorias (M1) y supresoras (M2).
Por otro lado, la activina A está involucrada en otras enfermedades inflamatorias como la
enfermedad inflamatoria intestinal, meningitis, asma, etc. En enfermedad inflamatoria intestinal, la
activina A se localiza en la mucosa de tejidos inflamados donde existe una elevada expresión de IL-
1β [287], y en intestino y plasma en tres modelos diferentes de la enfermedad en ratón [321]. Los
niveles de activina A son elevados en células T CD4+ y en suero en pacientes asmáticos [272, 322]
y en el de lavado broncoalveolar en modelos de asma en ratón [268, 271, 323]. Por tanto, la activina
A secretada en los sitios de inflamación, podría condicionar el fenotipo de los macrófagos allí
presentes, regulando así la respuesta inflamatoria.
La expresión y capacidad de endocitosis selectiva del FRβ en TAM o macrófagos sinoviales de
RA, posibilita el desarrollo de conjugados de folato como agentes terapéuticos frente a tumores y
122
Discusión
enfermedades inflamatorias e autoinmunes [324] (Figura 16). Estos conjugados irían dirigidos de
forma específica frente a las células implicadas en procesos patológicos, evitando el daño colateral
en las células sanas [325]. A su vez, el ácido fólico es un ligando óptimo como agente terapéutico
debido a su facilidad de conjugarse y a su elevada afinidad por su receptor incluso después de su
conjugación [324]. Un ejemplo del uso de terapéutico del FRβ se demuestra en dos modelos de
ratón de lupus eritematoso sistémico, enfermedad autoinmune crónica caracterizada por inflamación
y daño en el tejido conjuntivo, y en la que los macrófagos activados contribuyen a su desarrollo al
secretar mediadores inflamatorios y atraer otras células inflamatorias. En ellos se ha demostrado
que la depleción de macrófagos FRβ+ mediante inmunoterapia frente al receptor reduce los síntomas
de la enfermedad, sin provocar daño en tejidos sanos, y prolonga la supervivencia de los animales
[326]. Por otro lado, un estudio reciente muestra la posibilidad de reducir el crecimiento tumoral por
la unión selectiva a los TAM de una inmunotoxina usando un anticuerpo monoclonal frente al FRβ
[244].
Célula tumoral
H+
R-SH
H+
R-SH
H+H+
H+
H+
H+H+
H+
R-SH
FRβ
Conjugado de folato
Endosoma
Lisosoma
H+
R-SH
H+
H+ Célula tumoral
FcRAnticuerpo
Macrófago
Célula NK
A B
Célula tumoral
H+
R-SH
H+
R-SH
H+H+
H+
H+
H+H+
H+
R-SH
FRβ
Conjugado de folato
Endosoma
Lisosoma
H+
R-SH
H+
H+ Célula tumoral
FcRAnticuerpo
Macrófago
Célula NK
A B
Figura 16.- Estrategias terapéuticas frente al FRβ. A. Endocitosis de conjugados de folato. Los conjugados de folato se unen al FRβ con elevada afinidad, se internalizan por invaginación de la membrana, y posteriormente, se separan y reducen liberando el agente terapéutico. B. Inmunoterapia mediada por el FRβ. Los haptenos unidos a folato son capaces de estimular una respuesta inmunitaria, produciéndose la destrucción de células tumorales por macrófagos y células NK.
123
Discusión
125
DC-SIGN constituye un marcador de macrófagos anti-inflamatorios M2 y AAMØ [56, 151], ya
que su expresión en monocitos y macrófagos es inducida por M-CSF e IL-4 [92, 150], e inhibida por
estímulos inflamatorios como IFNγ [92]. Por ello, DC-SIGN se expresa en macrófagos implicados en
reparación de heridas y en macrófagos alternativos/reguladores M2. Por otro lado, un estudio
reciente realizado en nuestro grupo demuestra que DC-SIGN se expresa en TAM, y al igual que en
el caso del FRβ, su expresión podría contribuir a la adquisición fenotipo inmunosupresor en los
tumores (Domínguez-Soto et. al, J Immunol en revisión, 2010).
Requerimientos estructurales de DC-SIGN para su multimerización. Influencia de la presencia de variantes con menor tamaño en la región del cuello.
El reconocimiento de PAMP por las células dendríticas (DC) es el primer paso para
desencadenar la respuesta inmunitaria. DC-SIGN es un receptor de DC que interacciona con
proteínas glicosiladas presentes en patógenos, por lo que está implicado en etapas tempranas de
infecciones producidas por virus, hongos y bacterias [149, 193, 196]. Este reconocimiento a través
de DC-SIGN permite la internalización, procesamiento y posterior presentación antigénica a los
linfocitos T [150, 164, 187]. Estructuralmente DC-SIGN es una proteína de membrana cuya región
citoplásmica contiene motivos de reciclaje e internalización [149, 150], y su dominio extracelular
(ECD) consta de un cuello formado por 8 repeticiones de 23 aminoácidos seguido de un CRD [164,
165]. Procesos de “splicing” alternativo y polimorfismos a nivel genético dan lugar a variantes de DC-
SIGN tanto a nivel de RNA como a nivel proteico [168, 327].
Mummidi y colaboradores identificaron variantes de “splicing” de DC-SIGN en PBMC activados
con PHA, DC maduras derivadas de células hematopoyéticas CD34+ y células THP-1, que
presentan un tamaño variable en la región del cuello y en el CRD, así como isoformas solubles o
con una cola citoplásmica “alternativa” [168]. En nuestro estudio por primera vez se identificaron
isoformas de “splicing” de DC-SIGN en células dendríticas derivadas de monocitos (MDDC) que,
además, dan lugar a proteínas funcionales que se expresan en membrana. La generación de
construcciones de DC-SIGN con diferente tamaño y disposición de las repeticiones del cuello a partir
de estas isoformas, y su expresión en líneas celulares, ha sido de gran ayuda para determinar los
requerimientos estructurales de la multimerización y función de DC-SIGN. A su vez, para este
estudio hemos generado y utilizado mutantes de DC-SIGN sin CRD y con un número variable de
repeticiones en la región del cuello.
Aunque ya existían evidencias de la capacidad de oligomerización de la proteína recombinante
[166], se ha descrito que DC-SIGN aparece formando tetrámeros en la membrana de MDDC [164],
aumentando así la avidez por sus ligandos [165, 328]. La región del cuello es la responsable de esta
multimerización, mientras que existe controversia en el papel que desempeña el CRD [166, 329]. En
Discusión
este sentido, un estudio reciente afirma que el CRD no influye en la formación ni en la estabilidad de
los tetrámeros de DC-SIGN [328]. Sin embargo, y en un contexto celular, nosotros observamos que
el mutante que carece de CRD tiene disminuida su capacidad de multimerización. Por tanto, aunque
la oligomerización de DC-SIGN tiene lugar a través de la región del cuello, las interacciones entre
CRD contribuirían a su estabilización. Así, la ausencia de este dominio podría provocar una
disminución en las interacciones entre las regiones c-terminal de los cuellos, que llevaría a la
pérdida de estabilidad de los tetrámeros. Por otro lado, hemos determinado que la coexpresión de la
forma completa de DC-SIGN (1A) y el mutante sin CRD (8d) disminuye la formación de homo-
multímeros 1A-1A, debido posiblemente a la formación de hetero-multímeros 1A-8d, y que tendrían
menor estabilidad frente a agentes desnaturalizantes ya que no son detectables mediante Western
blot. El hecho de que la forma prototípica de DC-SIGN sea capaz de asociarse con mutantes sin
CRD confirma que la multimerización tiene lugar a través del cuello, y la menor estabilidad de los
hetero-multímeros 1A-8d vuelve a demostrar que la presencia de CRD estabiliza los oligómeros.
Por otro lado, el estado de oligomerización de DC-SIGN depende en gran medida del pH del
entorno [329]. Así, la elevada acidez existente en los endosomas provoca un cambio de
conformación de los tetrámeros con la consiguiente pérdida de afinidad y liberación de los ligandos
unidos a DC-SIGN [330]. De acuerdo con nuestros resultados, la estabilización de los multímeros de
DC-SIGN por el CRD se observa en ensayos con proteínas recombinantes donde al disminuir el pH,
en ausencia de CRD los tetrámeros se disocian totalmente, mientras que el ECD completo no lo
consigue. Al igual que el pH, otros factores como la concentración de sales estabilizan los
tetrámeros por aumento en las interacciones hidrofóbicas entre las regiones de cuello de DC-SIGN
[331].
Respecto al número de repeticiones de la región del cuello requeridas para la multimerización
de DC-SIGN, un estudio con proteínas recombinantes con el ECD truncado afirma que la presencia
de seis dominios mantiene la formación del tetrámero, mientras que la deleción de sucesivos
dominios (≤ 5.5 repeticiones) lo disocia parcialmente (dímeros) [165]. Además, proteínas de 3.5-2.5
repeticiones son difíciles de purificar por su baja estabilidad, lo que impide la determinación de su
estado de oligomerización; proteínas de menos de 2 repeticiones son más estables y presentan una
parcial disociación de los dímeros (monómeros); y el CRD sólo o con 0.5 repeticiones aparece como
monómero. Por ello y según estos autores, se requiere la presencia de al menos 3 dominios en el
cuello para que DC-SIGN pueda tetramerizar. En nuestro estudio, las construcciones de DC-SIGN
generadas a partir de las isoformas de “splicing” alternativo identificadas en MDDC y que contienen
4, 3, 2 ó 1 dominio repetido en el cuello, difieren en su capacidad de multimerización en líneas
celulares. Así, la isoforma de DC-SIGN con sólo la primera repetición en el cuello aparece como
monómero, mientras que las construcciones con mayor número de dominios (2, 3 y 4) lo hacen de
forma similar a la proteína completa con las 8 repeticiones de esta región. Por tanto, sería necesario
la presencia de al menos 2 repeticiones en el cuello para que DC-SIGN pueda multimerizar. De
126
Discusión
acuerdo con nuestras observaciones, un estudio realizado con su homólogo DC-SIGNR afirma que
la secuencia GELSE es necesaria para la multimerización de la molécula [332], por lo que la
presencia del segundo dominio de DC-SIGN sería necesaria para su multimerización (Figura 17).
DOMINIOV S K V P S S I S Q E Q S R Q D A I Y Q N L T Q L K A A V 1
G E L S E K S K L Q E I Y Q E L T Q L K A A V 2G E L P E K S K L Q E I Y Q E L T R L K A A V 3G E L P E K S K L Q E I Y Q E L T W L K A A V 4G E L P E K S K M Q E I Y Q E L T R L K A A V 5G E L P E K S K Q Q E I Y Q E L T R L K A A V 6G E L P E K S K Q Q E I Y Q E L T R L K A A V 7G E L P E K S K Q Q E I Y Q E L T Q L K A A V 8
DOMINIOV S K V P S S I S Q E Q S R Q D A I Y Q N L T Q L K A A V 1
G E L S E K S K L Q E I Y Q E L T Q L K A A V 2G E L P E K S K L Q E I Y Q E L T R L K A A V 3G E L P E K S K L Q E I Y Q E L T W L K A A V 4G E L P E K S K M Q E I Y Q E L T R L K A A V 5G E L P E K S K Q Q E I Y Q E L T R L K A A V 6G E L P E K S K Q Q E I Y Q E L T R L K A A V 7G E L P E K S K Q Q E I Y Q E L T Q L K A A V 8
Figura 17.- Secuencia de aminoácidos de las repeticiones de la región del cuello de DC-SIGN.
Como puede verse en la Figura 17, las repeticiones que forman parte de la región del cuello de
DC-SIGN difieren en su secuencia de aminoácidos. Los estudios realizados con las isoformas
identificadas en MDDC que presentan el mismo número pero distinta disposición de los dominios del
cuello (4d vs. 4d’), nos permiten afirmar que las repeticiones no son equivalentes funcionalmente.
Así observamos que estas isoformas difieren en su capacidad de multimerización y unión a ligandos.
De nuevo el residuo de serina (S) presente en el segundo dominio y en la isoforma 4d’ parece
contribuir en la multimerización de DC-SIGN. Por otro lado, las repulsiones entre los residuos de
arginina (R) con carga positiva y presentes en los dominios 6 y 7 de la isoforma 4d podrían
desestabilizar los tetrámeros. De acuerdo con esto, las diferencias funcionales existentes entre DC-
SIGN y DC-SIGNR, como la estabilidad de los oligómeros, la especificidad de unión de azúcares y la
señalización intracelular [188], podrían justificarse en parte por la diferente secuencia de
aminoácidos de las regiones del cuello de ambos receptores. Así, la presencia de un residuo de
leucina (L) en las repeticiones 6 y 7 de DC-SIGNR en lugar de la glutamina (Q) existente en DC-
SIGN, hace que este receptor sea más estable en estudios desnaturalización [328]. Además, la
sustitución del residuo de leucina (L) por glutamina (Q) en moléculas quiméricas de DC-SIGNR hace
que adquieran el comportamiento de DC-SIGN [328].
Por otro lado y como se ha comentado anteriormente, DC-SIGN presenta variaciones génicas
tanto en la región reguladora como en la región codificante, y cuya presencia se asocia a una
susceptibilidad alterada frente a infecciones por HIV-1 o M. tuberculosis [171, 172]. Los
polimorfismos en el exón 4 de DC-SIGN dan lugar principalmente a variantes con diferente número
de repeticiones en la región del cuello, y su frecuencia entre la población es baja (aprox. 1%) [333].
La presencia de estas variantes puede influir en la función de la molécula debido al papel del cuello
en la multimerización y soporte del CRD. Al igual que ocurría en el caso de los polimorfismos en la
región reguladora de DC-SIGN, existe controversia entre el posible papel protector de las variantes
127
Discusión
del cuello frente a determinadas infecciones. En este sentido, existen estudios que asocian la
presencia de variantes génicas del cuello de DC-SIGN a una resistencia a la infección por HIV-1 en
individuos de EEUU [327] o China [334], mientras que otros estudios no encuentran esta asociación
en la población del norte de Asia [335], norte de India [336, 337], sur de África, Tailandia [337] o
China [333]. Respecto a la susceptibilidad a la infección por M. tuberculosis no se ha encontrado un
papel protector de estas variantes en individuos de Colombia [178], sur de África [338] o Túnez
[179].
Esta discrepancia existente en la presencia de variantes de DC-SIGN con menor tamaño del
cuello y su asociación con susceptibilidad alterada frente a la infección por HIV-1, puede ser debida
al grupo étnico en el que se realiza el estudio, ya que tanto el tipo de polimorfismo como su
frecuencia es diferente entre ellos [180, 339, 340]. Así, las variantes de DC-SIGN son más
numerosas y frecuentes en individuos de China respecto a la población caucásica [333, 334].
Mientras que en individuos de China se han identificado alelos con 4, 5, 6, 7 y 9 repeticiones en el
cuello (genotipos 4/8, 5/8, 6/8, 7/8 y 8/9) [333, 334], en individuos de EEUU, sur de África, norte de
India y Tailandia sólo se han encontrado variantes con 7 y 9 repeticiones (genotipos 7/8 y 8/9) [327,
337], y en individuos de Colombia y Túnez únicamente se han encontrado variantes con 7
repeticiones (genotipos 7/7 y 7/8, y 7/8, respectivamente) [178, 179]. Por el contrario, otro estudio ha
identificado alelos con 5, 6, 7 y 9 dominios repetidos (genotipos 5/8, 6/8, 7/8 y 8/9) en individuos del
sur de África [338]. La discrepancia en las variantes génicas de DC-SIGN encontradas en los
diversos grupos étnicos puede ser debida a la generación de formas “resistentes” del receptor frente
a los patógenos presentes en las diferentes áreas geográficas, constituyendo un mecanismo de
“escape” del sistema inmunitario frente a dichos patógenos. Por ejemplo, un estudio realizado en la
población de un país en vías de desarrollo y un país industrializado, demuestra que la diversidad
genética de DC-SIGN en la población de Zimbawe es mayor (82%) que en individuos de Canadá
(33%) [171].
De acuerdo con los estudios mencionados anteriormente, las variantes de DC-SIGN que hemos
identificado en la población española corresponden a alelos con una repetición menos en el cuello,
encontrando individuos heterocigotos con genotipo 7/8 y un individuo homocigoto con genotipo 7/7.
Además, hemos determinado que estos alelos carecen de las repeticiones 3, 5 ó 7. Con la intención
de justificar su posible papel protector, caracterizamos estructural y funcionalmente estas variantes.
Las variantes génicas de DC-SIGN que carecen del dominio 3, 5 ó 7 expresadas en líneas celulares
de forma estable o transitoria, mantienen respecto a la molécula completa su expresión en
membrana y su capacidad de multimerización, internalización de ligando y unión a ligandos o
patógenos. Por otro lado, demostramos por primera vez que los polimorfismos de DC-SIGN se
expresan a nivel de proteína en MDDC de individuos heterocigotos, y lo hacen de manera similar a
la proteína prototípica y sin influir en su expresión. Además, en MDDC estas variantes son
funcionales y mantienen su capacidad de unión de ligandos, sin existir diferencias funcionales entre
128
Discusión
las MDDC de individuos que no presentan polimorfismos en el exón 4 de DC-SIGN e individuos con
un genotipo 7/8 en esa región. Respecto a la capacidad de asociación de estas variantes con la
forma completa de DC-SIGN, en líneas celulares la formación de homo-oligómeros está favorecida
respecto a la hetero-oligomerización, mientras que en MDDC de individuos heterocigotos no hay
evidencias de asociación entre ellas. Esto confirmaría la asociación preferente de moléculas con un
tamaño de cuello similar, debido posiblemente a que el mayor contacto entre los cuellos de igual
tamaño estabiliza las interacciones homotípicas, mientras que la disposición de CRD a diferente
altura no las favorece.
A pesar de los estudios que relacionan la presencia de variantes alélicas en la región del cuello
de DC-SIGN con una protección frente a la infección por HIV-1, no está muy claro el papel protector
de estas variantes. Según nuestro estudio, la formación preferencial de homo-multímeros y el hecho
de que las variantes con una repetición menos en el cuello mantienen su capacidad de unión de
ligandos, no justificaría su papel protector frente a la infección por HIV-1 en individuos heterocigotos.
Por el contrario, una posible causa de esta protección podría ser que la presencia de algunos
polimorfismos en el cuello de DC-SIGN provoque una menor expresión de moléculas en membrana.
En este sentido, en un estudio reciente identifican variantes de DC-SIGNR a partir de DNA
genómico de individuos polimórficos, las expresan en células Raji, y realizan ensayos de trans-
infección de HIV-1 [341]. Los polimorfismos estudiados consisten en cuellos de diferente tamaño (6,
8 y 10 dominios) y un cambio de nucleótido (A/G) en el exón 5, y afectan a la susceptibilidad frente a
la infección por HIV-1 [342-344]. Según estos autores, estas variantes de DC-SIGNR afectan a la
cantidad de moléculas expresadas en la superficie celular, lo que se correlaciona con la eficiencia de
trans-infección de DC-SIGNR independientemente del tamaño de la región del cuello o del
aminoácido codificado en el exón 5 (A/G). Por tanto, esta justificación basada en la disminución en
la expresión in vivo de DC-SIGN o DC-SIGNR en membrana estaría de acuerdo con nuestros
resultados donde las variantes de DC-SIGN en MDDC mantienen su expresión y, por tanto, su
funcionalidad. En este mismo sentido, la variante -336G de DC-SIGN que afecta al sitio de unión de
Sp1, modula su actividad transcripcional in vitro disminuyendo su expresión [182], por lo que su
presencia justificaría también una protección frente determinadas infecciones. De acuerdo con esto,
la presencia de isoformas de “splicing” de DC-SIGN también puede influir en el número de
moléculas expresadas en la superficie. En mucosas genitales o intestinales esta descrita la
presencia de isoformas solubles que pueden ser intracelulares o secretadas [168, 345] y que son
más abundantes que la forma prototípica [346], dando lugar a una menor avidez de DC-SIGN por
HIV-1 y, en consecuencia, una protección frente a la infección.
Por otro lado, la cantidad de moléculas de DC-SIGN expresada en la superficie celular se
correlaciona con los diferentes estados de oligomerización y se refleja en su estabilidad y avidez por
los ligandos. Así, la protección de las variantes del cuello de DC-SIGN frente a la infección por HIV-1
estaría también justificada porque el menor número de repeticiones en el cuello disminuye la
129
Discusión
formación y la estabilidad de tetrámeros [171, 330]. A su vez, estas variantes alterarían la posición
del CRD respecto la superficie celular, afectando a su accesibilidad y unión de patógenos [347, 348].
La señalización de DC-SIGN modula la respuesta inmune en función del patógeno involucrado
[207]. En el caso de HIV-1, la señalización de DC-SIGN conlleva la trans-infección del virus a células
T, lo que promueve infección del hospedador siendo beneficioso para el virus [212]. En este sentido,
existen estudios in vitro que dicen que HIV-1 y otros patógenos utilizan DC-SIGN para escapar de la
vigilancia inmunológica y promover su supervivencia [349, 350]. Por ello, el posible papel protector
de las variantes de DC-SIGN quedaría reflejado en la disminución de la unión e internalización del
virus. Por otro lado, la expresión reducida de DC-SIGN por la presencia de polimorfismos en DC y
macrófagos podría tener efectos perjudiciales en la eliminación de patógenos por la disminuida
capacidad de presentación de antígeno de estas células [171]. Así, un estudio reciente en ratones
transgénicos que expresan DC-SIGN humano exhiben un reducido daño tisular y una prolongada
supervivencia [351]. Por tanto, la presencia de polimorfismos de DC-SIGN también puede tener un
efecto indirecto en la modulación de respuestas inmunológicas frente a la infección por diferentes
patógenos.
Identificación epítopos en la molécula de DC-SIGN.
En un segundo estudio hemos empleado las construcciones de DC-SIGN generadas a partir de
las variantes de “splicing” y polimorfismos identificados en MDDC, así como los mutantes sin CRD y
con distinto número de repeticiones en el cuello de la molécula, para identificar siete epítopos
independientes en la molécula de DC-SIGN y confirmar algunas de las observaciones a las que
llegamos en el trabajo anterior. Para ello, empleamos anticuerpos monoclonales frente a DC-SIGN
que son capaces de inhibir trans-infección de HIV-1 por células Raji-DC-SIGN a linfocitos T [151]. Al
igual que ocurría con la unión a ligandos de DC-SIGN, el CRD es reconocido por los anticuerpos
independientemente del tamaño del cuello, y el reconocimiento de la molécula por los anticuerpos
dirigidos frente al cuello es dependiente de su tamaño, siendo necesaria la presencia de al menos
dos dominios para su reconocimiento. Aunque según nuestro anterior estudio el estado de
multimerización de DC-SIGN no predice su capacidad de unión a ligandos o patógenos, los
anticuerpos frente al CRD reconocen mejor las formas de DC-SIGN que se expresan en la
membrana como monómeros (1d) que como multímeros (2d), debido posiblemente a la disposición
espacial de los epítopos que reconocen. Por otro lado y de acuerdo con su capacidad de unión de
ligandos, las variantes polimórficas de DC-SIGN son reconocidas por igual por los anticuerpos
dirigidos frente al cuello y el CRD.
130
Discusión
131
Respecto a la capacidad de los anticuerpos de inhibir la unión de DC-SIGN a ligandos o
patógenos, los anticuerpos frente al CRD la bloquean total o parcialmente, mientras que los
anticuerpos frente al cuello lo hacen en menor extensión o incluso no lo consiguen. La diferencia en
la capacidad de inhibición por parte de los anticuerpos frente al CRD puede ser debida al epítopo
que reconocen y bloquean en cada caso. En este sentido, existen aminoácidos en el CRD de DC-
SIGN claves en la unión de ligandos (Glu347, Asn349, Glu354 y Asn365), ya que interactúan con el Ca2+
dictando el reconocimiento específico de carbohidratos, y cuya mutación conlleva a la pérdida total
de capacidad de unión de ligandos [352]. Mientras, la modificación de otros residuos como Val351,
sólo afecta a la interacción con determinados ligandos sin afectar a la unión de otros, lo que indica
que los ligandos de DC-SIGN pueden tener diferentes, pero solapados, sitios de unión [162]. Por
otro lado, el bloqueo funcional que producen los anticuerpos frente al cuello puede deberse a su
habilidad de modificar el estado de multimerización de la molécula, que altera su avidez por
ligandos, o bien a su capacidad de inducir su internalización, disminuyendo así el número de
moléculas en superficie.
En conclusión, la identificación de epítopos independientes en la molécula de DC-SIGN podría
facilitar el diseño de reactivos que modulen parte de las funciones de las células que expresan este
receptor. De este modo, se podría alterar el estado de multimerización de DC-SIGN en membrana,
inducir su internalización o bloquear su unión a patógenos de manera independiente. Por ejemplo,
en este estudio describimos anticuerpos capaces de potenciar la internalización de DC-SIGN en
MDDC, lo que generaría el desarrollo de una respuesta inmunológica, sin bloquear su capacidad de
unión. Debido a que la señalización de DC-SIGN depende del ligando que involucrado [207], el uso
de reactivos específicos frente a diferentes epítopos podrían ser una herramienta útil para el estudio
de las vías de señalización de la lectina, o incluso para modular la polarización de linfocitos T por
DC.
Conclusiones
Conclusiones
1. El receptor de folato β es un marcador de macrófagos anti-inflamatorios/M2 in vitro y de
macrófagos asociados a tumores (TAM), en los que media la captación de folato.
2. La expresión del receptor de folato β se induce por factores presentes en el microambiente
tumoral como M-CSF o IL-10, y citoquinas promotoras de la activación alternativa de macrófagos
como IL-4 o IL-13, y es inhibida por estímulos inflamatorios como LPS o IFNγ.
3. La activina A es secretada por macrófagos pro-inflamatorios/M1 y condiciona la adquisición
de los marcadores y las funciones efectoras características de los macrófagos M2, lo que sugiere su
implicación en la progresión tumoral.
4. La multimerización del receptor de patógenos DC-SIGN en la membrana celular depende de
la región del cuello y el dominio de reconocimiento de carbohidratos, y requiere la presencia de al
menos dos dominios en la región del cuello.
5. Las variantes polimórficas de DC-SIGN se expresan en la membrana de células dendríticas
derivadas de monocitos, y mantienen la capacidad de multimerización y unión de ligandos.
6. Al menos se pueden identificar siete epítopos independientes en la molécula de DC-SIGN,
tres de ellos localizados en la región del cuello y cuatro en el dominio de reconocimiento de
carbohidratos.
135
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Anexo
Anexo
Durante la realización de esta Tesis Doctoral he participado en diferentes proyectos que han dado lugar a las siguientes publicaciones:
1.- Caparrós E, Muñoz P, Sierra-Filardi E, Serrano-Gómez D, Puig-Kröger A, Rodríguez-
Fernández JL, Mellado M, Sancho J, Zubiaur M, Corbí AL. DC-SIGN ligation on dendritic cells results
in ERK and PI3K activation and modulates cytokine production. Blood, 2006 May 15;107(10):3950-8.
2.- Núñez C, Rueda B, Martínez A, Malvenda C, Polanco I, López-Nevot MA, Ortega E, Sierra-Filardi E, Gómez de la Concha E, Urcelay E, Martín J. A functional variant in the CD209 promoter is
associated with DQ2-negative celiac disease in the Spanish population. World J Gastroenterol, 2006 Jul 21;12(27):4397-400.
3.- Puig-Kröger A, Domínguez-Soto A, Martínez-Muñoz L, Serrano-Gómez D, Lopez-Bravo M,
Sierra-Filardi E, Fernández-Ruiz E, Ruiz-Velasco N, Ardavín C, Groner Y, Tandon N, Corbí AL,
Vega MA. RUNX3 negatively regulates CD36 expression in myeloid cell lines. J Immunol, 2006 Aug 15;177(4):2107-14.
4.- Gómez LM, Anaya JM, Sierra-Filardi E, Cadena J, Corbí A, Martín J. Analysis of DC-SIGN
(CD209) functional variants in patients with tuberculosis. Hum Immunol, 2006 Oct;67(10):808-11.
5.- Serrano-Gómez D, Martínez-Nuñez RT, Sierra-Filardi E, Izquierdo N, Colmenares M, Pla J,
Rivas L, Martinez-Picado J, Jimenez-Barbero J, Alonso-Lebrero JL, González S, Corbí AL. AM3
modulates dendritic cell pathogen recognition capabilities by targeting DC-SIGN. Antimicrob Agents Chemother, 2007 Jul;51(7):2313-23.
6.- Sierra-Filardi E, Serrano-Gómez D, Martínez-Nuñez RT, Caparrós E, Delgado R, Muñoz-
Fernández MA, Abad MA, Jimenez-Barbero J, Leal M, Corbí AL. Structural requirements for
multimerization of the pathogen receptor DC-SIGN (CD209) on the cell surface. J Biol Chem, 2008 Feb 15;283(7):3889-903.
7.- Fernández de Palencia P, Werning ML, Sierra-Filardi E, Dueñas MT, Irastorza A, Corbí AL,
López P. Probiotic properties of the 2-substituted (1,3)-β-D-glucan producing Pedioccus parvulus 2.6.
Appl Environ Microbiol, 2009 Jul;75(14):4887-91.
8.- Sierra-Filardi E, Puig-Kröger A, Domínguez-Soto A, Samaniego R, Corcuera MT, López-
Aguado F, Ratnam M, Sánchez-Mateos P, Corbí AL. Folate receptor beta is expressed by tumor-
associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages.
Cancer Res, 2009 Dec 15;69(24):9395-403.
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9.- Sierra-Filardi E, Estecha A, Samaniego R, Fernández-Ruiz E, Colmenares M, Sánchez-
Mateos P, Steinman RM, Granelli-Piperno A, Corbí AL. Epitope mapping on the dendritic cell-specific
ICAM3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor. Mol Immunol, 2010 Jan;47(4):840-848.
10.- Martin-Gayo E, Sierra-Filardi E, Corbí AL, Toribio ML. Plasmacytoid dendritic cells resident
in human thymus drive natural Treg cell development. Blood, en prensa, 2010.
11.- Sierra-Filardi E, Vega MA, Sánchez-Mateos P, Puig-Kröger A, Corbí AL. Heme
Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10
release. Immunobiology, en prensa, 2010.
12.- Garai-Ibabe G, Dueñas MT, Irastorza A, Sierra-Filardi E, Werning ML, López P, Corbí AL,
Fernández de Palencia P. Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus
suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods.
Bioresour Technol, en prensa, 2010.
13.- Domínguez-Soto A, Sierra-Filardi E, Puig-Kröger A, Blanca Pérez-Maceda B, Gómez-
Aguado F, Corcuera MT, Sánchez-Mateos P, Corbí AL. M-CSF promotes DC-SIGN expression in
M2-polarized and Tumor-Associated Macrophages. J Immunol, en revisión, 2010.
14.- Sierra-Filardi E, Puig-Kröger A, Sánchez-Mateos P, Vega MA, Corbí AL. Activin prevents
the acquisition of M2/anti-inflamatoy markers and skews the macrophage cytikine profile.
Manuscrito en preparación.
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