Phagocytic Processes-Outline

1. Phagocytic Processes-Outline • The Defenders • Professional phagocytes • The strategy • The process • Extravasation and chemotaxis • Recognition and phagocytosis • Killing • Digestion • Role in resistance to microbes • Role in resistance to cancer • Role in wound healing/bone remodeling

2. Professional Phagocytes - (phagocytosis = ingestion of particles) • Monocytes-blood • Macrophages - Reside in tissues, kill microbes, and process and present antigen • Kupffer cells • Alveolar macrophages • Microglial cells • Osteoclasts • Neutrophils - Kill microbes, do not present antigen • Dendritic cells - Primary function is antigen processing and presentation

3. Macrophages in action Bacteria Lymphocyte

4. The Strategy • The anti-microbial strategy involving phagocytes could be described as a distributed (not a centralized) defense • Phagocytes are resident in some tissues and quickly move into others as infection, trauma, or other stimuli attract them and inflammation begins • This contrasts with the strategy for initiating acquired immune responses, which is centralized • Microbial antigens (either from lymph or ingested by dendritic cells accumulate in secondary lymphoid tissues, e.g., lymph nodes). Lymphocytes travel through, and those specific for the antigen respond

5. The process - extravasation • Endothelial cells in regions of infection, trauma, etc. become activated and express E- and P-selectin and ICAM-1, which bind to ligands on leukocytes causing them to stop and begin the process of diapedesis. They squeeze between endothelial cells, secrete proteases to break down the basement membrane, then enter the tissues. Fig. 8.11

6. The process - chemotaxis • Leukocytes in tissues move toward sites of infection by “crawling” toward higher concentrations of chemoattractants such as chemokines like IL-8 or bacterial products such as N-formyl peptides A macrophage extends pseudopods to ingest a yeast cell

7. Recognition • A variety of surface receptors on phagocytes bind to microbial components Fig. 8.8

8. Recognition • Professional phagocytes have receptors for complement components that greatly enhance recognition and binding of microbes Fig. 1.15

9. Recognition • Phagocytic cells have receptors that bind to antibody-coated particles. In some cases (e.g., bacteria with capsules), binding and phagocytosis are minimal without antibody, complement, or C-reactive protein Fig. 7.22

10. Phagocytosis • After recognition of microbial components by receptors on the surface of the phagocyte, the microbe is ingested by means of membrane invagination to produce a phagosome. This fuses with lysosomes which have a low pH and degradative enzymes to kill the microbe in a structure that is then termed a phagolysosome Fig. 1.13

11. Phagocytosis • Opsonization with antibody generally enhances the process of phagocytosis. However, some microbes (e.g., Salmonella, Listeria, Mycobacteria) can withstand the phagolysosome environment in unactivated macrophages Figure 7.23

12. Phagocytosis • Most bacteria are effectively retained in phagosomes and destroyed when these fuse with lysosomes. However, Listeria monocytogenes can escape into the cytoplasm in unactivated macrophages (left panel). However, activation by exposure to cytokines or LPS prevents this escape. From Higginbotham, J.N., Lin, T.L., and Pruett, S.B. Clin. Exp. Immunol. 88:492-498

13. Phagocytosis • The phagosome begins simply as a pinched off region of cytoplasmic membrane, but it quickly acquires many unique proteins that are involved in its functions. This is revealed in the 2-D gel shown; the proteins were identified by mass spectroscopy (this process is used to evaluate the cellular proteome). From Garin et al., J. Cell. Biol. 152:165, 2001

14. Phagocytosis • The Hollywood version

15. Killing • Mechanisms include agents that are resident in lysosomes of neutrophils and macrophages as well as processes that are triggered by phagocytosis (respiratory burst leading to generation of reactive oxygen species).

16. Killing • The respiratory burst generates large quantities of toxic oxygen metabolites that are potent anti-microbial agents. They are also often lethal to the phagocyte and may damage surrounding tissues as well NADPH Oxidase O2 + NADPH NADP + O2- + H+ O2- + H + O2 + H202 H202 + Cl- OCl- + H2O Superoxide Dismutase Myeloperoxidase

17. Killing • Neutrophils generate reactive oxygen species at near maximum levels when triggered, but macrophage production of reactive oxygen species can be increased substantially if the cells are exposed to certain T cell-derived cytokines such as IFN-g. Thus, some microbes are controlled but not completely cleared by the innate immune system, and an acquired cell-mediated immune response is required for clearance.

18. Killing • Reactive nitrogen intermediates, including nitric oxide (NO) are unequivocally involved in anti-microbial actions of macrophages in mice. There is considerable evidence that this is the case in humans as well, but the conditions required for macrophage activation for this function in humans are more rigorous and not yet well understood.

19. Digestion • Microbes are broken down by enzymes in the phagolysosome to basic constituents (amino acids, etc.) for reuse by the macrophage or excretion. In this process peptides are generated that associate with MHC class II proteins which are then transported to the cell surface for presentation to T cells. Dendritic cells are the most effective of the phagocytes in this regard. Macrophages can also perform this function, but they may require activation by cytokines for optimal performance.

20. Role in resistance to microbes Fig. 9.10 • Defects in phagocytes often are associated with profound immune deficits

21. Role in resistance to microbes • The effector mechanisms induced by antibody (neutralization and opsonization) ultimately depend on phagocytes (except at mucosal surfaces where inhibition of microbial attachment is critical).

22. Role in resistance to cancer • Tumor cells per se do not activate macrophages sufficiently to cause them to become tumoricidal. However, macrophages that are activated by other means can kill cancer cells by some of the same mechanisms used to kill bacteria as well as the production of TNF-a, which induces apoptosis in some cancer cells. • Thus, macrophages may or may not be important in normal immune surveillance for cancer, but they may be useful in immunotherapy, if properly activated.

23. Role in wound healing/bone remodeling/cellular homeostasis • Macrophages are largely responsible for recognizing, clearing, and recycling damaged or old leukocytes and red blood cells. • Macrophages are responsible for clearing apoptotic cells before the internal contents of the cells can be release and induce an inflammatory response. • Macrophages (or related cells called osteoclasts) are involved in tissue repair by clearing dead tissue and connective tissue components and in bone remodeling, healing, and growth, by degrading regions of bone so further growth can occur.