MICR 304 Immunology & Serology

1. MICR 304 Immunology & Serology 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

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 GEMM-Progenitor • Important cytokines • IL-3 • GM-CSF • G-CSF 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. 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 Two Major Types of Neutrophil Granules

10. 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

11. Neutrophil Abundance in Pus

12. Monocytes and Macrophages 8957B 8957B

13. Monocyte Development

14. The Development of Monocytes and Macrophages GEMM-Progenitor • Important cytokines • IL-3 • GM-CSF • M-CSF • IFN-g from activated TH cells Myeloblast Monoblast Neutrophil Promonocyte Monocyte Macrophage

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

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

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 • Rolling adhesion • Firm adhesion • 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. Endothelial Cell Selectin ICAM CD31 (PECAM) Leukocyte Sialyl-LewisX Integrin CD31 (PECAM) Extravasation Requires Activation of Endothelium and Leukocyte

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 Fc-Receptor for Antibodies Complement Receptor for C3b

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_xh-bkiv_c

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

41. Killing and Digestion by Phagocytes • Reactive oxygen and reactive nitrogen intermediates • O2-, H2O2 • 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. Opsonophagocytosis of C. albicans and Generation of ROI Formazan Crystals C. albicans PMN C. albicans

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 Enzymatic attack Pore formation

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

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/IMAGES/MONOCYTE%20IN%20SMEAR.JPG