MICR 304 S2010 Lecture 4_Phago.ppt
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Transcript of MICR 304 S2010 Lecture 4_Phago.ppt
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MICR 304 Immunology &
Serology
MICR 304 Immunology &
Serology
Lecture 4Phagocytes
Chapter 2.4, Primary Literature
Lecture 4Phagocytes
Chapter 2.4, Primary Literature
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Overview of Today’s Lecture
• Definition of phagocytes• Phagocyte development• A closer look at neutrophils,
monocytes and macrophages• Phagocyte activation and
phagocytosis
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Key Players in Immunology
Innate Adaptive
Cells PhagocytesEpithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Effector Molecules
ComplementAntimicrobial (Poly)Peptides
Antibodies
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Phagocytes• Cells that take up microbes to kill and
digest them• The professionals
– Neutrophil granulocytes (not present in healthy tissue)
– Monocytes, macrophages (present in healthy tissue)
• Cells with phagocytic activities– Dendritic cells (specialized in antigen
presentation)– Basophil granulocytes, mast cells
(specialized mediators of inflammation)
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Neutrophils
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Granulocyte Development
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The Development of Granulocytes
• Important cytokines– IL-3– GM-CSF– G-CSF
GEMM-Progenitor
Myeloblast
MonocytePromyelocyte
Myelocyte(granules appear)
Metamyelocyte
Band Neutrophil
PolymorphonuclearNeutrophil (segmented)
(Eosinophils and Basophils mature similarly)
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Granulocytes
• Polymorphonuclear• Granule rich
– Neutrophil– Eosinophil– Basophil
Neutrophil granulocytes = polymorphonuclear cells = PMNs = Polys
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Two Major Types of Neutrophil Granules
• Primary (Azurophil)– Appear first during
mitotic development– Stain blue– Elastase, cathepsin
G, myeloperoxidase, defensins,LL37, lysozyme, glucuronidase
– Fuse with phagosome
– pH optimum 4-5
• Secondary (Specific)– Appear after mitotic
development– Outnumber the
primary granules– Lactoferrin,
lysozyme, C3/C5 proteases, receptors for fMLP, complement, Cytochrome b 558
– Secreted
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The Fate of Neutrophils
• Short lived cells (days)• Half Life in circulation 6 – 8 h• High turn over rate (1011/per day)• If unstimulated: migrate to
respiratory and digestive mucosal surfaces, apoptotic death
• If activated: will ultimately become necrotic pus
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Neutrophil Abundance in Pus
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Monocytes and Macrophages
8957B
8957B
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Monocyte Development
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The Development of Monocytes and Macrophages
• Important cytokines– IL-3– GM-CSF– M-CSF
– IFN-from activated TH cells
GEMM-Progenitor
Monocyte
Macrophage
Neutrophil
Myeloblast
Monoblast
Promonocyte
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Monocytes and Macrophages
• Mononuclear cells• Longer living (weeks – months)• Monocytes: in blood, exit into tissue to
differentiate into macrophages• Functions:
– Phagocytosis– Antigen-presentation– Primary activation of T-lymphocytes– Pivotal role in initiating an inflammatory
response
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Maturation and Differentiation of
Monocytes• Monocytes only found in bone marrow,
peripheral blood• Immature Cells• Monocytes are limited in receptor
expression, phagocytosis and cytokine production
• Main stimulators of maturation and differentiation to macrophages:– Interferon-gamma (T-Helper cells, NK cells)– GM-CSF (T-cells, macrophages)
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Macrophages : Interface to Adaptive Immunity
• Lymphocyte attraction and activation
• Antigen presentation through MHC II
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Specialized Macrophages
• Dendritic Cells: subepithelial, in solid organs, lymph nodes and lymphatic tissue
• Langerhans cells: in skin• Kupffer cells: in liver• Alveolar macrophages: in lung• Microglia cells: in brain
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Dendritic Cells
• Lymphoid and myeloid progenitor cells– Plasmocytoid DC: interferon producing
in response to viral infections– Conventional DC: antigen presentation
and activation of naïve T cells• Recognize common structures on
pathogens• Macropinocytosis : Receptor
independent• Highly specialized in antigen
presentation• After contact with antigen
migration to lymph nodes• Interact with T-lymphocytes in
lymph nodes
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Cytokines Secreted by Macrophages and Dendritic
CellsCytokine Target Cell Effect
IL-1 Lymphocytes Enhances responses
IL-6 Liver (Hepatocytes)
Induces acute phase protein secretion
CXCL8 (IL-8)
Neutrophils Chemoattractant
IL-12 NK cellsNaïve T cells
Activation of NK
TNF Vascular Endothelium
Cell adhesion and PermeabilityBlood clotting
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From Microbial Invasion to Successful Pathogen
Removal • Phagocytes are attracted to site of
invasion– Chemotaxis– Transmigration from blood vessel into tissue
• Physical contact between microbe and phagocyte– Opsonization
• Microbial uptake– Phagocytosis
• Killing
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Chemotaxis• Directed movement of phagocytes
towards the source of infection• Induced by chemoattractants:
– Bacterial products• Formylated peptides like fMLP
– Complement fragment• C5a
– Host derived lipid metabolites• LTB4 (arachidonic acid metabolite, produced upon
stress)
– Chemokines• CXCL-8 (formerly IL-8, acts on neutrophils)• CCL-2 (MCP-1, acts on monocytes)
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Classification of Chemokines
• Depending on amino acid structure• Number and spacing of cysteine
residues at N-terminus (C: cysteine; X: any amino acid)
• Chemokine families include– CXC (e.g. CXCL8, CXCL7)– CC (CCL2, CCL11)
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Chemokines Acting on Phagocytes
Chemokine
Producer Cells attracted
Major Effects
CXCL8(IL8)
MonocytesMacrophagesFibroblastsKeratinocytesEndothelial cells
NeutrophilsNaïve T-cells
MobilizationNeutrophil activation and degranulation
CXCL7(NAP-2)
Platelets Neutrophils Activates neutrophilsClot resorptionAngiogenesis
CCL2(MCP-1)
MonocytesMacrophagesFibroblastsKeratinocytes
MonocytesNK and T-cellsBasophilsDendritic cells
Activates macrophagesHistamin release by basophilsPromotes TH2 immunity
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Change of Cell Shape in Response to Chemokines
PMNs before and 5 sec after stimulation with chemokine
(Olsen et al, 2002)
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Phagocyte Movement: Cytoskeleton
Rearrangement
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Extravasation
1.Rolling adhesion2.Firm adhesion3.Transmigration
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Endothelial cells
Leukocytes
Endothelial cellsLeukocytes
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• Leukocyte:– Integrins
• Mac-1• LFA-1
• Endothelial Cell– Intercellular
adhesion molecules• ICAMs
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http://www.youtube.com/watch?v=I9zSe0qmXGw&NR=1
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Extravasation Requires Activation of Endothelium and
Leukocyte• Endothelial Cell
1. Selectin2. ICAM3. CD31 (PECAM)
• Leukocyte
1. Sialyl-LewisX
2. Integrin3. CD31
(PECAM)
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Opsonophagocytosis
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Opsonization
• Covering microbial surfaces with molecules recognizable by phagocytes:– Complement factors (C3b)– Immunoglobulins (IgG)– C-reactive protein– Mannose-binding protein and other
collectins– Surfactant
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Selected Opsonin Receptors
Complement Receptor for C3b
Fc-Receptor for Antibodies
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Opsonization and Engulfment
• Engagement of receptors trigger cytoskeletal movement
• Process continues until pseudopods make contact and seal
• A phagosome has been created
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• http://www.youtube.com/watch?v=I_xh-bkiv_c
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Phagolysosome Formation and Killing
• Engagement of receptors
• Triggering of killing mechanisms– Oxidative burst – Release of
lysosomal contents into phagosome • Antimicrobial
peptides
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Killing and Digestion by Phagocytes
• Reactive oxygen and reactive nitrogen intermediates– O2
-, H2O2
– NO
• Antimicrobial peptides• Low pH• Hydrolases, proteases,
phospholipases
Oxygendependent
Oxygenindependent
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Oxidative Burst
• Generation of oxygen radicals under consumption of molecular oxygen
• Initiated by NADPH oxidase– multi component membrane
enzyme complex including cytochrome b 558
• Delivered into phagolysosome and extracellular space
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C. albicans
FormazanCrystals
PMN
C. albicans
Opsonophagocytosis of C. albicans and Generation of
ROI
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A Second System to Produce Radicals Exist
• Nitric oxide synthase is the key enzyme
• Generates from L-arginine radicals like nitric oxide (reactive nitrogen metabolites or RNI)
• Readily detectable in murine macrophages
• Role in human PMN killing unclear
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Principal targets of ROI and RNI
• DNA• Hemes• Thioesters• Alkenes• Sulfhydryls
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Phagocytic Killing Mechanisms
Radical attack
Pore formation
Enzymaticattack
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Summary of Major Steps in Opsonophagocytosis
1. Opsonization2. Attachment3. Receptor clustering
and engulfment4. Phagosome
formation5. Phagolysosome
formation6. Killing and digestion
1.
2./3.
4. 5.
6.
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Today’s Take Home Message
• Chemotaxis is directed movement in response to a stimulus
• Chemokines bind to a seven membrane span receptor and have multiple effects
• Leukocyte extravasation is a three-step process: 1. rolling adhesion, 2. firm adhesion, and 3. transmigration mediated by 1. selectin:sialyl Lewis, 2. ICAM:integrin, and 3. PECAM:PECAM interaction between endothelial cell and leukocyte
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Additional Resources
• http://education.vetmed.vt.edu/curriculum/vm8054/Labs/Lab6/IMAGES/MONOCYTE%20IN%20SMEAR.JPG