fagocitosis 2011
TRANSCRIPT
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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
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Reacciones defensivas mediadas por fagocitos
barreras defensivas iniciales
Sistema Fagocítico
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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)
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FUNCIÓNES INMUNITARIAS
Muerte microbiana
.- enzimas lisosomales
.- estallido respiratorioDirecccionamiento de péptidos a MHC-I y II
FAGOCITOSIS
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Fagocitos
Sistema Fagocítico:Mononuclear y
Polimorfonuclear
FAGOCITOSIS
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Anti-microbial function of neutrophils:first step = phagocytosis
12
3
4
5Within the tissue:Killing of pathogensis followed bytheir own death
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Phagocytosis step by step
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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
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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
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Receptores de Complemento
Activación de PKC
Fosforilación de Proteínas
CR1CR2
C3b
OLIGOSACÁRIDOSLECTINOFAGOCITOSIS
Lectina Azúcar
Azúcar Lectina
Azúcar AzúcarLectina
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Hipótesis del “Modelo de laCremallera”
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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-
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↑ 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
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Anti-microbial function of neutrophils:the weapons for killing
Intracellular
granules containing multipleanti-microbialmolecules
Oxygen independant(degranulation)
and dependantMechanisms
(oxidative burst)
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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
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MECANISMOSMICROBICIDASOXIGENO-
INDEPENDIENTES
LIBERACIÓN DE ENZIMAS LÍTICAS Y PEPTIDOS ANTIMICROBIANOS
O i d d h i
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Oxygen-independant mechanisms:first example
LysozymeDiscovered byA. Flemming
Degradation ofbacterialmembranePeptidoglycan
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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
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Oxygen-dependant mechanisms:
1
2NO
RNI: reactive nitriteintermediates
Bacterial killingIn particular in macrophages
more than in neutrophils
Nitric oxide synthase
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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.
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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
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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)
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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)
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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
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FAGOCITOSISFASES
ELIMINACIÓN DE DESECHOS
Desechos
Intracelulares
Aprovechamiento de biomoléculas: aa, lípidos,azúcares, nucleótidos
Material indigerible
Defecación (exocitosis)
Acumulación en “cuerposde múltiples vesículas”
Muerte celular
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Fagosoma
1. PROTECCIÓN INESPECÍFICA
2. INDUCCIÓN DE RESPUESTAS INMUNES
ESPECÍFICAS
Células presentadoras del antígeno
ACTIVIDADFAGOCITICA DELOSMACRÓFAGOS
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GRACIAS POR SU ATENCIÓN
LUIS R GONZALEZ HDEZ UCV D A ll Ph li I tit t P t