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
Overview of Today’s Lecture
• Definition of phagocytes• Phagocyte development• A closer look at neutrophils,
monocytes and macrophages• Phagocyte activation and
phagocytosis
Key Players in Immunology
Innate Adaptive
Cells PhagocytesEpithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Effector Molecules
ComplementAntimicrobial (Poly)Peptides
Antibodies
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)
Neutrophils
Granulocyte Development
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)
Granulocytes
• Polymorphonuclear• Granule rich
– Neutrophil– Eosinophil– Basophil
Neutrophil granulocytes = polymorphonuclear cells = PMNs = Polys
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
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
Neutrophil Abundance in Pus
Monocytes and Macrophages
8957B
8957B
Monocyte Development
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
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
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)
Macrophages : Interface to Adaptive Immunity
• Lymphocyte attraction and activation
• Antigen presentation through MHC II
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
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
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
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
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)
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)
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
Change of Cell Shape in Response to Chemokines
PMNs before and 5 sec after stimulation with chemokine
(Olsen et al, 2002)
Phagocyte Movement: Cytoskeleton
Rearrangement
Extravasation
1.Rolling adhesion2.Firm adhesion3.Transmigration
Endothelial cells
Leukocytes
Endothelial cellsLeukocytes
• Leukocyte:– Integrins
• Mac-1• LFA-1
• Endothelial Cell– Intercellular
adhesion molecules• ICAMs
http://www.youtube.com/watch?v=I9zSe0qmXGw&NR=1
Extravasation Requires Activation of Endothelium and
Leukocyte• Endothelial Cell
1. Selectin2. ICAM3. CD31 (PECAM)
• Leukocyte
1. Sialyl-LewisX
2. Integrin3. CD31
(PECAM)
Opsonophagocytosis
Opsonization
• Covering microbial surfaces with molecules recognizable by phagocytes:– Complement factors (C3b)– Immunoglobulins (IgG)– C-reactive protein– Mannose-binding protein and other
collectins– Surfactant
Selected Opsonin Receptors
Complement Receptor for C3b
Fc-Receptor for Antibodies
Opsonization and Engulfment
• Engagement of receptors trigger cytoskeletal movement
• Process continues until pseudopods make contact and seal
• A phagosome has been created
• http://www.youtube.com/watch?v=I_xh-bkiv_c
Phagolysosome Formation and Killing
• Engagement of receptors
• Triggering of killing mechanisms– Oxidative burst – Release of
lysosomal contents into phagosome • Antimicrobial
peptides
Killing and Digestion by Phagocytes
• Reactive oxygen and reactive nitrogen intermediates– O2
-, H2O2
– NO
• Antimicrobial peptides• Low pH• Hydrolases, proteases,
phospholipases
Oxygendependent
Oxygenindependent
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
C. albicans
FormazanCrystals
PMN
C. albicans
Opsonophagocytosis of C. albicans and Generation of
ROI
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
Principal targets of ROI and RNI
• DNA• Hemes• Thioesters• Alkenes• Sulfhydryls
Phagocytic Killing Mechanisms
Radical attack
Pore formation
Enzymaticattack
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.
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
Additional Resources
• http://education.vetmed.vt.edu/curriculum/vm8054/Labs/Lab6/IMAGES/MONOCYTE%20IN%20SMEAR.JPG