1. MICR 304 Immunology & Serology Lecture 4 Phagocytes Chapter 2.4, Primary Literature Lecture 4 Phagocytes Chapter 2.4, Primary Literature

2. Overview of Today’s Lecture Definition of phagocytes Phagocyte development A closer look at neutrophils, monocytes and macrophages Phagocyte activation and phagocytosis

3. Key Players in Immunology InnateAdaptive Cells Phagocytes Epithelial Cells NK Cells Lymphocytes (B-Ly, T-Ly) Effector Molecules Complement Antimicrobial (Poly)Peptides Antibodies

4. 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)

5. Neutrophils

6. Granulocyte Development

7. The Development of Granulocytes Important cytokines –IL-3 –GM-CSF –G-CSF GEMM-Progenitor Myeloblast Monocyte Promyelocyte Myelocyte (granules appear) Metamyelocyte Band Neutrophil Polymorphonuclear Neutrophil (segmented) (Eosinophils and Basophils mature similarly)

8. Granulocytes Polymorphonuclear Granule rich –Neutrophil –Eosinophil –Basophil Neutrophil granulocytes = polymorphonuclear cells = PMNs = Polys

9. 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

10. The Fate of Neutrophils Short lived cells (days) Half Life in circulation 6 – 8 h High turn over rate (10 11 /per day) If unstimulated: migrate to respiratory and digestive mucosal surfaces, apoptotic death If activated: will ultimately become necrotic  pus

11. Neutrophil Abundance in Pus

12. Monocytes and Macrophages 8957B

13. Monocyte Development

14. 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

15. 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

16. 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)

17. Macrophages : Interface to Adaptive Immunity Lymphocyte attraction and activation Antigen presentation through MHC II

18. 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

19. 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

20. Cytokines Secreted by Macrophages and Dendritic Cells CytokineTarget CellEffect IL-1LymphocytesEnhances responses IL-6Liver (Hepatocytes) Induces acute phase protein secretion CXCL8 (IL-8)NeutrophilsChemoattractant IL-12NK cells Naïve T cells Activation of NK TNF  Vascular EndotheliumCell adhesion and Permeability  Blood clotting

21. 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

22. 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)

23. 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)

24. Chemokines Acting on Phagocytes ChemokineProducerCells attracted Major Effects CXCL8 (IL8) Monocytes Macrophages Fibroblasts Keratinocytes Endothelial cells Neutrophils Naïve T-cells Mobilization Neutrophil activation and degranulation CXCL7 (NAP-2) PlateletsNeutrophilsActivates neutrophils Clot resorption Angiogenesis CCL2 (MCP-1) Monocytes Macrophages Fibroblasts Keratinocytes Monocytes NK and T-cells Basophils Dendritic cells Activates macrophages Histamin release by basophils Promotes TH2 immunity

25. Change of Cell Shape in Response to Chemokines PMNs before and 5 sec after stimulation with chemokine (Olsen et al, 2002)

26. Phagocyte Movement: Cytoskeleton Rearrangement

27. Extravasation 1.Rolling adhesion 2.Firm adhesion 3.Transmigration

28. Endothelial cells Leukocytes Endothelial cells Leukocytes

31. Leukocyte: –Integrins Mac-1 LFA-1 Endothelial Cell –Intercellular adhesion molecules ICAMs

32. http://www.youtube.com/watch?v=I9zSe0qmXGw&NR=1

33. Extravasation Requires Activation of Endothelium and Leukocyte Endothelial Cell 1.Selectin 2.ICAM 3.CD31 (PECAM) Leukocyte 1.Sialyl-Lewis X 2.Integrin 3.CD31 (PECAM)

35. Opsonophagocytosis

36. Opsonization Covering microbial surfaces with molecules recognizable by phagocytes: –Complement factors (C3b) –Immunoglobulins (IgG) – C-reactive protein – Mannose-binding protein and other collectins – Surfactant

37. Selected Opsonin Receptors Complement Receptor for C3b Fc-Receptor for Antibodies

38. Opsonization and Engulfment Engagement of receptors trigger cytoskeletal movement Process continues until pseudopods make contact and seal A phagosome has been created

39. http://www.youtube.com/watch?v=I_x h-bkiv_c

40. Phagolysosome Formation and Killing Engagement of receptors Triggering of killing mechanisms –Oxidative burst –Release of lysosomal contents into phagosome Antimicrobial peptides

41. Killing and Digestion by Phagocytes Reactive oxygen and reactive nitrogen intermediates –O 2 -, H 2 O 2 –NO Antimicrobial peptides Low pH Hydrolases, proteases, phospholipases Oxygen dependent Oxygen independent

42. 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

43. C. albicans Formazan Crystals PMN C. albicans Opsonophagocytosis of C. albicans and Generation of ROI

44. 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

45. Principal targets of ROI and RNI DNA Hemes Thioesters Alkenes Sulfhydryls

46. Phagocytic Killing Mechanisms Radical attack Pore formation Enzymatic attack

47. Summary of Major Steps in Opsonophagocytosis 1.Opsonization 2.Attachment 3.Receptor clustering and engulfment 4.Phagosome formation 5.Phagolysosome formation 6.Killing and digestion 1. 2./3. 4. 5. 6.

48. 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

49. Additional Resources http://education.vetmed.vt.edu/curriculum/vm8054/Labs/Lab6/I MAGES/MONOCYTE%20IN%20SMEAR.JPG