Phagocytosis

1
Phagocytosis
Neutrophils and Macrophages are professional
phagocytes.
Recognition of target
Direct recognition
bacteria
Mannose, fucose
Lipoteichoic acids (Gram) LPS (Gram-)
Lectin-like carbohydrate recognition domains
Scavenger receptor A (SR-A), MARCO (macrophage
receptor with collagenous structure)
Mannose Receptor (MR) and DEC205 (macrophages)
Phagocyte
2
Indirection Recognition Opsonin Receptors
Antigen binding site
Fab
Antibody
Papain protease
Fc
Bacteria opsonized with antibodies
IgG
Opsonin
FC?RIIIB
FC?RI
FC?RIIA
Fc Receptors
?
?
?
?2
?2
Phagocyte
3
Opsonin receptor
Complement Receptor
Bacteria opsonized with C3b
Opsonin
Factor I
C3b
iC3b
CR1
CR1 binds C3b-opsonized target, but does
not induce phagocytosis.
?X?2
?M?2
CRIg
CR4
CR3
Phagocyte
4
Opsonin Receptor
Receptors for collectins (MBL, SPA)
Bacteria opsonized with collectins
LPS
Mannose
Lung surfactant protein A (SPA)
Mannose binding lectin (MBL)
C1q receptor
SPR210
phagocyte
5
Phagocyte
Bacteria
Mannose
Mannose receptor
Scavenger receptor
LPS, LTA
Fc receptor
Ag
Ab
Surface protein or carbohydrate
C3b
Complement receptor
C1q receptor
MBL
Mannose
SPR
SPA
LPS
Opsonin
6
Engulfment
IgG-opsonized particle
FcR
target
Phagocyte
pseudopods
FcR activation induces actin polymerization
30s
target
3 minutes
Phagocyte
Extension of pseudopods around the target
7
Engulfment
Complement Receptor
Phagocytosis by complement receptor requires
activation with inflammatory cytokines. FcR does
not require activation by other signals.
Complement-opsonized particle
target
Activation
No activation
phagocyte
Phase contrast
target
No pseudopods
Sinking of target into phagocyte
Staining for CR
CR surrounds the phagocytosed particle.
8
FcR activates the production of
inflammatory cytokines.
IgG-opsonized particle
Proinflammatory cytokines (e.g. TNF-?)
FcR signaling
Actin polymerization
Activation of genes for inflammatory cytokines
Activate components of TLR signaling pathway
Recruit TLR to Phagosomes
CR does not activate inflammation.
TLR2 is recruited to phagosomes.
9
Maturation of Phagosomes
phagocytosis
receptor
Endocytosis
receptor
Actin polymerization
Clathrin-coated pit
phagosome
endosomes
Fusion with endosomes and lysosomes
Gogi
Hydrolytic enzymes
lysosomes
Phagolysosome
10
5 minutes
Phagocytosis of latax beads by macrophage
Endocytosed HRP marker protein in endosomes
10 minutes
endosomes
Latax bead in phagosome, not fused with endosome.
30 minutes
Phagosomes and endosomes fused Phagosomes contain
HRP from endosomes.
60 minutes
11
Fusion of phagosomes with lysosomes
Macrophages lysosomes are labeled with red and
green marker proteins. Red and Green merge to
five yellow.
Preferential delivery of red marker to phagosomes
Green markers are delivered into phagosomes at
later time Points.
12
Neutrophil granules fuse with phagosomes.
Primary granule (azurphil granule)
Hydrolytic enzymes
Lysozyme
Proteases (cathepsin, elastase, proteinase 3)
Anti-microbial peptides
Defensins and BPI (bacerial permeability increasin
g protein)
Synthesis of reactive oxygen products
Myeloperoxidase
Secondary granule
lysozyme
Lactoferrin sequester iron
NADPH oxidase synthesis of reactive oxygen
products
13
Production of reactive oxygen products
HClO-
Myeloperoxidase
Cl-
Kill bacteria
Hydrogen peroxide
H2O2
Superoxide dismutase (SOD)
phagosome
Respiratory burst
O2-
superoxide
p22
gp91
O2
e-
NADPH oxidase complex
rap1A
rap1A
NADP
Rac-GDP
Rac-GTP
NADPH
GEF
Cytoplasm
FcR activation
kinase
p47
p40
p67
14
Chronic Granulomatous Disease
HClO-
Myeloperoxidase
Cl-
Kill bacteria
Hydrogen peroxide
H2O2
Superoxide dismutase (SOD)
O2-
superoxide
O2
gp91
e-
rap1A
NADPH oxidase complex
NADP
NADPH
Patients are deficient in components of NADPH
oxidase complex.
The most common form is deficiency of gp91. The
gene for gp91 is on X-chromosome. Most patients
are males.
Patients suffer bacterial and fungal infections.
15
Blood neutrophils
/
PMA
Activated neutrophils
/-
-/-
oxidation of NADPH
NBT
Reduced NBT (purple)
Detection of deficiency in NADPH oxidase
Multi-nucleated giant cells
Macrophages phagocytose bacteria. Lack of NADPH
oxidase prevents killing of bacteria
T cells
Granuloma
Chronic intracellular bacterial infection
16
The role of protease in neutrophil killing
activity
Mice deficient in elastase (a protease in
neutrophil granule) are more susceptible
to Bacterial infection
Survival of phagocytosed bacteria in elastase
deficient neutrophil.
17
FcR
CR
Recognition
Mannose receptor

Macrophage
neutrophil
Engulfment and formation of phagosome
Production of inflammatory mediators
Fusion with endosomes and lysosomes To form
phagolysosomes
Fusion with granules
Killing of phagocytosed bacteria through reactive
oxygen products and hydrolases.
18
phagocytosis
Effector
MAC
Neutrophils
Bacteria
Macrophages
Classical, lectin alternative
PAMPs-TLR
PAMPs-TLR
Macrophages
Complements
Sensor activation
C5a, C3a
Mast cells
Inflammation
Chemoattractants
Proinflammatory mediators
IL-1, TNF-?, prostaglandins, leukotrienes Histamin
es, C5a, C3a
CXCL8, C5a, leukotrienes, N-formyl-peptides
Recruitment
Neutrophils
Monocytes
19
Resolution of inflammation
Class switch of lipid mediators
Injection of TNF-? induces inflammation
Prostaglandin E2
Leukotriene B4
The level of neutrophil infiltration at various
time points after TNF-? injection
Lipoxin A4
The level of neutrophils and lipids in the
exudate of inflammation site
20
Lipoxin A4 (LXA4) reduces neutrophil
infiltration into inflammation site.
TNF
TNFLXA4
21
Lipoxin production is initiated during
inflammation.
Prostaglandins (PEG2) induces 15-lipoxygenase in
neutrophils. 15-lipoxygenase (15-LO) produces
lipoxin.
PEG2
15-LO
lipoxin
Lipoxins return the blood vessel to normal state
and stop the recruitment of neutrophils.
prostaglandins
leukotrienes
Transcellular synthesis
Leukotriene intermediates
lipoxin
platelet
12-lipoxygenase in platelet
Interaction between neutrophils and platelet
Leads to the synthesis of lipoxin.
lipoxins
22
Aspirin stimulates the production of lipoxins.
Arachidonic acid
Arachidonic acid
Aspirin
COX-2
Acetyl COX-2
Lipid intermediate (15R-HETE)
Prostaglandins
Inflammation
5-lipoxygenase
Lipoxins
Resolution
Resolvins and Protectins are lipid mediators for
resolution.
Polyunsaturated fatty acids
Resolvins and Protectins
23
Apoptosis (Programmed cell death)
24
Apoptosis Pathway
Cytochrome c
Death signal
mitochondria
Release of cytochrome c
Cystein protease Cleaves after Asp.
Caspase-9
ATP
Apaf-1
Activation of Caspase-9 by autocleavage
Activation by proteolytic cleavage
Caspase 3, 7
Degradation of cellular proteins (e.g.
Lamins) Activation of CAD to degrade chromosomes
(CAD caspase-activated DNase)
25
The regulation of apoptosis
mitochondria
Bax, Bad
IAP (Inhibitor of Apoptosis) inhibits caspases.
Release of cytochrome c
Bcl-2, Bcl-XL
Caspase-9
ATP
Apaf-1
Activation of Caspase-9 by autocleavage
IAP
Activation by proteolytic cleavage
Caspase 3, 7
IAP
Degradation of cellular proteins (e.g.
Lamins) Activation of CAD to degrade chromosomes
26
Apoptosis of neutrophils
Inflammatory mediators promote the survival of
neutrophils
Reduction of inflammatory mediators
Short-lived
Apoptosis
Phagocytosis of apoptotic neutrophils by
macrophages
27
Apoptotic cells are recognized by change in
membrane lipids and glycosylation patterns.
Recognized by receptors on macrophages
Phosphatidylserine flipped to the outer leaflet
outerleaflet
Inner leaflet
phosphatidylserine
Limited to inner leaflet
Apoptotic cell
Cytoplasm
Cytoplasm
28
Questions
How do antibodies and complements facilitate
phagocytosis?
What is the difference between FcR and CR in
mediating phagocytosis?
What is the connection between phagocytosis and
inflammation?
How do phagocytes destroy bacteria?
Relevant parts in textbook
P65-68, p182-184, p339, 344, p347-348, p26-28.