CELULAS DE LA INMUNIDAD INNATA.

LEUCOCITOS POLIMORFONUCLEARES

MSc Angela Conesa MSc Luis Gonzlez IDI-Mayo de 2011

Contenido
Desarrollo ontognico Caractersticas Generales Marcadores de Superficie Funciones Efectoras Papel en la Regulacin de la Respuesta Inmune

Papel en Enfermedad

Clulas Polimorfonucleares.
Clulas maduras incapaces de diferenciarse o dividirse Vida media: corta No muestran especificidad por el antgeno

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Mielopoyesis

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GRANULOPOYESIS
Produccin de neutrfilos: 5 x1010 -10 x1010/clulas x da

Vida media: 6-8 horas

MEDULA SEA

Mieloblasto
PROLIFERACIN (Pool Mittico) (7,5 das)

Promielocito

Mielocitos
(grnulos especficos)

MADURACIN (6,5 das)

Metamielocitos Cayados o Bandas

ALMACENAMIENTO

Segmentados

Caractersticas Generales
NEUTRFILOS:
12-15m cromatina compacta 2-5 lbulos VN: 65-75% Leuc. Totales 90% PMN Mecanismo efector: Fagocitosis y activacin de mecanismos bactericidas Tamao: Ncleo:

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Marcadores de superficie
Neutrfilos Eosinfilos Basfilos Mastocitos

Ag. Leucocitario CD45 Ag. Mieloides CD13 CD17 CD31 CD35 Ag. Linfoides CD3,5,7,19,22 Ag. NK CD56, 57

+ + + + + -

+ + + + + -

+ + + + + -

+ -

Marcadores de superficie
Neutrfilos Molculas de Adhesin
PSGL-1 + + + -

Eosinfilos

Basfilos

Mastocitos

CD62L CD29 CD18/CD11a,b,c CD15 Receptores para Fc de Igs CD16b CD32 CD64 CD89 CD23 Fc eRI Mac 2 Receptores para componentes C C3a C5a CR1 CR3 CR4

+ + +

+ + + +

+ + + +

nd + -

+ + + + + -

+ + +* +/+

+ + nd nd nd + + +

nd + nd nd nd + +

+ + + + +

+ + + + +

Marcadores de superficie
Receptores para Citocinas
Neutrfilos IL-1 IL-2 () IL-2 () IL-2 () IL-3 IL-4 IL-5 IL-8 G-CSF GM-CSF INF TNF RANTES
CCR2 CCR3

Eosinfilos + +/+ + + + + + + + +

Basfilos nd + ? + f + + + + nd f + + nd + +

Mastocitos nd ? ? f + f nd nd f nd -

+ + + + + + +/+ + -

Mediadores
NEUTRFILOS: a) Grnulos primarios: Marcadores de memb Hidrolasas, catepsina G, elastasa, CD63 proteinasa 3, mieloperoxidasas (MPO), lisozima, CD68 -manosidasa, glicerofosfatasa. BPI b) Grnulos secundarios: Lactoferrina Colagenasa Histaminasa Sistema NADPH oxidasa c) Grnulos terciarios Gelatinasa, lisozima, acetiltransferasa d) Vesculas secretoras Fosfatasa alcalina Mediadores lipdicos: LTB4, PAF, TXA2 CD15 CD66 CD67 CD11b/CD18 CD11b/CD18 CD11b/CD18, CD10, CD45, CD35
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Receptores
TLRs

Receptores Fc
Complemento Scavenger Quimiocinas fMLP
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Anti-microbial function of neutrophils: first step = phagocytosis

3
4

5
Within the tissue: Killing of pathogens is followed by their own death

Anti-microbial function of neutrophils: the weapons for killing

Intracellular granules containing multiple anti-microbial molecules Oxygen independant (degranulation) and dependant Mechanisms (oxidative burst)

Oxygen-dependant mechanisms or oxidative burst: NADPH-oxydase

Phagolysosome membrane/assembly of a large complex

H2O2 (hydrogen peroxide)

+ myeloperoxidase (MPO)
ROS: reactive oxygen species HOCL (hypochlorous acid)

Bactericidal

MECANISMOS MICROBICIDAS OXIGENO-INDEPENDIENTES

LIBERACIN DE ENZIMAS LTICAS Y PEPTIDOS ANTIMICROBIANOS

Mecanismos microbicidas independientes del oxgeno

Release of peptides and proteins from the granules (degranulation) Bacterial permeability-increasing protein BP1 Defensins and chatelicidins/antimicrobial peptides Highly cationic/positive charge mediated binding to microorganisms whose surface are anionic

Papel en Respuesta Inmune

Defensa contra microorganismos invasores Mecanismos no-oxidativos degranulacin (defensinas, lisozima, enzimas hidrolticas, TNF) Mecanismos oxidativos radicales oxgeno radicales nitrgeno

Grnulos azurfilos: Elastasa, catepsina G, proteasa, mieloperoxidasa

Mieloperoxidasa

Redes que atrapan los microorganismos Grnulos especficos y terciarios: Lactoferrina, Colagenasa Gelatinasa, lisozima, acetiltransferasa
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Nature Rev IMMUNOL 2006;6:173-182

Netting pathogens: Neutrophil extracellular traps (NETs)

Neutrophil extracellular traps (NETs) were described as a novel defence mechanism for the first time in 2004 [Science, Vol 303]. NETs are extracellular net-like structures consisting of a chromatin matrix to that specific proteins from the neutrophil granuls are attached These complex tree-dimensional structures form a physical barrier which traps pathogens 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 the intracelular killing

NETs : Neutrophil Extracellular Traps

Resting cells

Activated cells

Smooth fibers (chains of nucleosomes from unfolded chormatin) diam: 15-17nm + globular domains: diam 25nm

Yellow: neutrophil elastase Orange: NET

PROTEINS PRESENT IN NETs AND THEIR CELLULAR ORIGINS

SOURCE OF PROTEIN
Nucleus Azurophilic (primary) granules

NAME
Histones h1,h2a,h2b,h3,h4 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, neutrophils undergo 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 receptors Rearrangement of the nuclear and granular architecture Dependant on ROS Dependant on ROS (H202) NETs are released when the cell membrane ruptures ant the cell dies NETosis: NEW FORM OF CELL DEATH DIFFERENT FROM AP0PTOSIS AND NECROSIS (Caspase independent, no DNA fragmentation)

Binding of microorganisms to NETs

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

Degradation of bacterial proteins involved in virulence Ex: Shigella

Mechanisms for selective degradation of proteins ????

Zychlinsky et al. Nature 2002, Science 2004

Funciones Efectoras Neutrophil extracellular traps (NETs)

S. Pneumonie Group A Streptococci Componentes de los granulos 1.- Elastasa, Cattepsina G, Mieloperoxidasa 2.- Lactoferrina 3.- Gelatinasa, Proteinas de reconocimineto de peptidoglican

Current Op in Microbiology 2007;10:52

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Interaccin Neutrfilos Clulas Dendrticas

Neutrfilos apoptticos

Neutrfilos activados

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Funciones efectoras de los neutrfilos

Papel en Respuesta Inmune

Interaccin con monocitos, clulas dendrticas, clulas T y clulas B de una manera bidireccional y multicompartimental.
Linfocito T INF Macrfago Neutrfilo apopttico Catepsina G Azurocidin Degranulacin Induce quimiotaxis TNF Tejido extravascular

Monocito

Catepsina G Azurocidin Proquemerina Quemerina

Induce quimiotaxis

TNF Linfocito B CD inmadura o CD plasmocitoide IL-23 rgano linfoide secundario G-CSF IL-17 Clula del estroma CXCL12 Mdula sea Precursor neutrfilo Neutrfilo retenido en la mdula sea

Clula T o Clula T o

Nature Reviews 2006; 6 (1), 174.

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Caractersticas Generales
EOSINFILOS: Tamao: Ncleo: VN: Totales Mecanismo efector: Funcin: 12-17m 2 lbulos 2-5% Leu. 7% PMN Degranulacin Enf. Parasitarias LPR reaccin alrgica

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Mediadores
EOSINFILOS:
Productos preformados: Grnulos primarios: Proteina Charcot-Leyden Grnulos secundarios: MBP (Major basic protein) Eosinophil cationic protein Eosinophil peroxidase ECP, EPO, EDN Citocinas, quimiocinas Eosinophil-derived neurotoxin Grnulos pequeos: Fosfatasa, arilsulfatasa, catalasas Mediadores lipdicos, (cuerpos lipdicos): Va Ciclooxigenasa: TXB2 PGE1, PGE2, PGE2, PGD2, PGF1 Va Lipooxigenasa: LTC4 PAF Neuropptidos: sustancia P
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PRINCIPALES PROTEINAS DE LOS GRANULOS EOSINFILOS

PROTEINA BASICA PRINCIPAL:

Citotxica para helmintos y protozoarios Estimula liberacin de histamina de clulas cebadas y basfilos

PROTEINA CATIONICA EOSINOFILICA

Citotxica para clulas de mamferos y no mamferos Estimula liberacin de histamina de clulas cebadas y basfilos Inhibe proliferacin de linfocitos T Pre-activa el plasmingeno Aumenta produccin de moco en bronquios Estimula produccin de GAG por los fibroblastos

NEUROTOXINA DERIVADA DEL EOSINOFILO

Capacidad de provocar disfuncin cerebral y cerebelar en animales Inhibidor de respuestas de clulas T

PEROXIDASA EOSINFILA
Citotoxica para clulas tumorales y normales Estimula liberacin de histamina y degranulacin celular Inactiva leucotrienos

Marcadores de superficie

J Allergy Clin Immunol 2004;113(1):3-8

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Trfico de Eosinfilos

Eo as APC IL-5, E0-1, RANTES, MIP-1 Eo-1

Th2 and endothelial cell products IL5, IL-4, and IL-13,

Eo-1

RANTES and the eotaxins

Sitio de la lessin

TGF-

Ann. Rev. immunol 2006;24:147 Advences Immunol 2009;101:81

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Papel en Respuesta Inmune

Estimulacin
Tejido daado Infecciones Alergenos Aloinjertos Tumores

EDN ligando para TLR2 - Migracin y maduracin mCD - TH2 cytokine expression

EPO

, ECP

ECP

Proteinas de los grnulos: EPO, MBP, ECP, EDN Citocinas: IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF/, GM-CSF, TNF , INF Quimiocinas: Eotaxina 1, RANTES, MIP-1 Mediadores lipdicos, Leucotrieno LTC4s, PAF. Neuromediadores sustancia P

Secrecin

Activacin de Mastocitos Liberacin de histamina

Ann. Rev. immunol 2006;24:147 Adv Immunol 2009;101:81
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Papel en la Enfermedad

MBP MBP

ECP, EDN

NET

Adv Immunol 2009;101:81

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Caractersticas Generales
BASFILOS:
Tamao: Ncleo: VN: Totales Mecanismo: Funcin: 5-7m 2 lbulos 0-1% Leu. 3% PMN Liberacin del contenido de los grnulos Reaccin hipersensibilidad inmediata

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Mediadores
BASFILOS:
Productos preformados: Histamina Triptasa Serinas-proteasas Heparina Proteoglicanos Proteasas Charcot Leyden Sintetizados Va Ciclooxigenasa: PGD2 Va Lipooxigenasa: LTC4, LTD4, LTE4, LTB4 Citocinas IL-4, IL13 IL-25, IL-3
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Papel en Respuesta Inmune

PAPEL EN RESPUESTA INMUNE ADQUIRIDA Funcin efectora dependiente de IgE

Current Opinion Immnunol 2000, 12: 624

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Respuesta de Basfilos por diferentes vas

Adv Immnunol 2009, 101:123

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Papel en Respuesta Inmune

TNF

Papel en Respuesta Inmune Natural y Adquirida Respuesta protectora: Bacterias, Parsitos Enfermedad: Ag Ambientales

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Current Opinion Immnunol 2000, 12: 624

Papel en Respuesta Inmune Anafilaxis mediada por IgG.

Ba unen la mayor cantidad de CI. mAb Ba103 CD200R3 de ratn Ba No afecta la anafilaxis mediada por IgE Generacin de anafilaxis Pen V-conjugada-OVA

Fig. 1 Classical and alternative pathways toward systemic anaphylaxis. The classical pathway utilizes mast cells, IgE, and histamine while the alter native pathway utilizes basophils and/or macrophages, IgG, and PAF.
Allergology International 2009;58:11

Mo

anti-histamina anti-PAF

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Fig. 2 Basophils are one of the major players in the IgGmediated systemic anaphylaxis. When allergens enter the blood stream, they form immune complexes with specific IgG antibodies that circulate in the blood. Circulating basophils efficiently capture the immune complexes through their receptors for IgG (IgG-R), and are activated to release PAF, that in turn stimulates vascular endothelial cells, resulting in the increased vascular permeability leading to hypotension.

Papel en Respuesta Inmune. Basfilos como iniciador de la Inflamacin Alrgica Crnica.

Fig. 4 A proposed mode of IgE-mediated chronic allergic inflammation. When IgE-bearing basophils are recruited into the peripheral tissue and encounter the relevant allergens, basophils are activated to secrete soluble factors including cytokines. These factors act on surrounding tissue-resident cells to induce the production of chemokines that in turn recruit large numbers of proinflammatory cells such as eosinophils and neutrophils, resulting in chronic allergic inflammation.

Allergology International 2009;58:11

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GRACIAS POR SU ATENCION