FAGOCITOSIS 2011

8/12/2019 FAGOCITOSIS 2011

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FAGOCITOSIS:FISIOLOGIA Y PAPEL EN LA RESPUESTA

INMUNITARIA INNATA

Luis González Hernández

Instituto de Inmunología

Facultad de Medicina UCV

Febrero 2011



Reacciones defensivas mediadas por fagocitos

barreras defensivas iniciales

Sistema Fagocítico



Fagocitosis

Proceso por el cual los leucocitosreconocen y destruyen patógenos invasores.

Acción celular de comerincorporación de partículas grandes (bacterias)

Proceso por el cual los leucocitos y otras células ingieren ligandos particuladoscuyo tamaño excede 1mM

Proceso filogenéticamente ancestralElie Metcknikoff (Siglo XIX)



FUNCIÓNES INMUNITARIAS

Muerte microbiana

.- enzimas lisosomales

.- estallido respiratorioDirecccionamiento de péptidos a MHC-I y II

FAGOCITOSIS



Fagocitos

Sistema Fagocítico:Mononuclear y

Polimorfonuclear

FAGOCITOSIS



Anti-microbial function of neutrophils:first step = phagocytosis

12

3

4

5Within the tissue:Killing of pathogensis followed bytheir own death



Phagocytosis step by step



Regulation of the neutrophil-mediated inflammatoryresponse to infection

Kobayashi et al .

M icrobes & I nfect. 2003, 5, 1337

Effect of opsonization on the phagocytosis of S. aureus

by human PMNs. Unopsonized (circle) or serum-

opsonized (square) S. aureus strain was combined with

human PMNs at a 10 bacteria: 1 PMN ratio, as

indicated. Phagocytosis was measured with flowcytometry

Interaction of group A Streptococcus

(blue) with a human PMN

Opsonisation and phagocytosis



RECEPTORES PARA OPSONINAS Receptores Fc Receptores para complemento (específicos para C3b) Receptores de lectinas (glicoproteínas con residuos de fucosa, manosa

y N-acetilglucosamina)

Receptor Fc

Receptores Fc

Fagocitos:

FcγRI (IgG2a)

FCγRII (IgG1/IgG2b)

↑ Entrada de Na++ al interior celular ↑ Concentración de Ca++ intracelular Estallido respiratorio Cascada del ácido araquidónico

PLA2 Ácido Araquidónico

↑ AMPc intracelular



Receptores de Complemento

Activación de PKC

Fosforilación de Proteínas

CR1CR2

C3b

OLIGOSACÁRIDOSLECTINOFAGOCITOSIS

Lectina Azúcar

Azúcar Lectina

Azúcar AzúcarLectina



Hipótesis del “Modelo de laCremallera”



INGESTIÓN

1. Formación de Seudópodos2. Englobamiento de la partícula3. Formación de la vesícula fagocítica (FAGOSOMA)

Receptor Fc

Señales transmembrana

Cambios metabólicos

Liberación de agentes oxidantes

H2O2

N-cloraminas

Oxidación degrupos -SH

Puentes -S-S-



↑ CONSUMO DE OXÍGENO

↑ CONSUMO DE GLUCOSA

↑ ACTIVIDAD CICLO DE LAS PENTOSAS

↑ BIOSÍNTESIS DE LÍPIDOS

↑ PRODUCCIÓN DE H2O2 Y RADICALES TOXICOS DEL O2

↓ DEL pH (VACUOLA FAGOCÍTICA)

ACTIVACIÓN DE ENZIMAS HIDROLÍTICAS (PROTEASAS,GLUCOSIDASAS, LIPASAS, DNAsas, RNAsas..etc.)

FAGOCITOSIS

FASES DIGESTIÓN (DESTRUCCIÓN)

Generación de cambios bioquímicos



Anti-microbial function of neutrophils:the weapons for killing

Intracellular

granules containing multipleanti-microbialmolecules

Oxygen independant(degranulation)

and dependantMechanisms

(oxidative burst)



Oxygen-independant mechanisms

Release of peptides and proteins from the granules (degranulation)Bacterial permeability-increasing protein BP1

Defensins and chatelicidins/antimicrobial peptidesHighly cationic/positive charge mediated binding to microorganisms

whose surface are anionic



MECANISMOSMICROBICIDASOXIGENO-

INDEPENDIENTES

LIBERACIÓN DE ENZIMAS LÍTICAS Y PEPTIDOS ANTIMICROBIANOS

O i d d h i



Oxygen-independant mechanisms:first example

LysozymeDiscovered byA. Flemming

Degradation ofbacterialmembranePeptidoglycan



Oxygen-dependant mechanisms or oxidative burst:NADPH-oxydase

+ myeloperoxidase (MPO)

H2O2 (hydrogen peroxide)

HOCL (hypochlorous acid)

BactericidalOxidation of proeteins, nucleic acids..

ROS: reactive oxygen species

Phagolysosome membrane/assemblyof a large complex



Oxygen-dependant mechanisms:

1

2NO

RNI: reactive nitriteintermediates

Bacterial killingIn particular in macrophages

more than in neutrophils

Nitric oxide synthase



Netting pathogens: Neutroph i l extracel lu lar traps (NETs)

Neutrophil extracellular traps (NETs) were described as a novel defence mechanism forthe first time in 2004 [Science, Vol 303]. NETs are extracellular net-like structuresconsisting of a chromatin matrix to that specific proteins from the neutrophil granuls areattached These complex tree-dimensional structures form a physical barrier which trapspathogens and kills them by providing a high concentration of antimicrobial proteins. NETs

are produced by activated neutrophils.



New data: an extracellular killing mechanism in addition to theintracelular killing

NETs : Neutrophil Extracellular Traps

Yellow: neutrophil elastaseOrange: NET

Resting cells Activated cells

Smooth fibers (chains of nucleosomes from unfoldedchormatin) diam: 15-17nm

+ globular domains: diam 25nm



PROTEINS PRESENT IN NETs AND THEIR CELLULAR ORIGINS

SOURCE OF PROTEIN NAME

Nucleus Histones h1,h2a,h2b,h3,h4

Azurophilic (primary) granules Neutrophil elastase

Cathepsin G

Myeloperoxidase (MPO)

BPI

Specific (secondary) granules Lactoferrin

Tertiary granules Gelatinase

Peptidoglycan recognitin proteins(PGRPs)



How and when NETs are formed?

Following their activation by e.g. PMA, LPS (lipopolysaccharide) or bacteria, neutrophilsundergo a specific form of programmed cell death which comprises the re-organization

of the nucleus and the granula and, ultimately, leads to NET formation. This process,which is to be distinguished from cell-death by apoptosis or necrosis, is called “NETosis” [JCB, Vol 176, No2; 2007].

Upon activation (IL-8, LPS, microorganisms)Neutrophils start a programme that leads to their death and formation of NETs

In vitro, about 1/3 neutrophils upon activation are forming NETs

Simultaneous stimulation of several receptorsRearrangement of the nuclear and granular architecture

Dependant on ROSDependant on ROS (H

20

2)

NETs are released when the cell membrane ruptures ant the cell dies

NETosis: NEW FORM OF CELL DEATH DIFFERENT FROM AP0PTOSIS ANDNECROSIS

(Caspase independent, no DNA fragmentation)



Degradation of bacterial proteins involved in virulenceEx: Shigella

Mechanisms for selective degradation of proteins ????

Zychlinsky et al. Nature 2002, Science 2004

Binding of microorganisms to NETs

NETs:bind microorganisms,and ensures high local concentrationof anti-microbial agents



FAGOCITOSISFASES

ELIMINACIÓN DE DESECHOS

Desechos

Intracelulares

Aprovechamiento de biomoléculas: aa, lípidos,azúcares, nucleótidos

Material indigerible

Defecación (exocitosis)

Acumulación en “cuerposde múltiples vesículas”

Muerte celular



Fagosoma

1. PROTECCIÓN INESPECÍFICA

2. INDUCCIÓN DE RESPUESTAS INMUNES

ESPECÍFICAS

Células presentadoras del antígeno

ACTIVIDADFAGOCITICA DELOSMACRÓFAGOS



GRACIAS POR SU ATENCIÓN

LUIS R GONZALEZ HDEZ UCV D A ll Ph li I tit t P t

FAGOCITOSIS 2011

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