Phagocytes
List the armamentarium generated from (i) NADPH oxidase, (ii) lysosomal fusion to phagosome membrane, (iii) iNOS.
armamentarium= "armor" Armamentarium produced by NADPH oxidase activation includes: 1. Superoxide 2. Hydrogen Peroxide 3. Hydroxyl Radical 4. Hydroxyl Ion 5. Hypochlorite (note role and source of myeloperoxidase) Lysosomal granules contain many enzymes (including lactoferrin, lysozyme, and defensins) that are cytostatic/cytotoxic to microorganisms. These molecules play a role in host immunity as follows: (i) lactoferrin binds iron, thereby removing an essential ingredient for microbial growth; (ii) lysozyme destroys muramic acid in bacterial cell walls; (iii) defensins permeabilize bacterial and fungal membranes. Lysosomal granules also release myeloperoxidase, an enzyme required to generate hypochlorite, a potent antimicrobial agent that mediates its function by halogenating bacterial cell walls (next section). Synthesis of NO requires the induction of inducible nitric oxide synthase, a cytosolic enzyme that catalyzes the conversion of L-Arginine to L-Citrulline and NO in the presence of oxygen.High levels of NO are not only toxic to microbes but may even be toxic to the phagocytes producing it, and to surrounding cells, because NO is both lipid and water-soluble. The short half-life of NO (seconds) confers some protection. However, host tissues may be damaged in a nonspecific manner.
phagocytes
monocytes, macrophages and neutrophils are phagocytes [Monocytes have limited phagocytic ability; macrophages are specialized phagocytes; neutrophils have exceptional phagocytic capability.]
Describe the effect of IFNγ, TNF, IL-4, IL-10 and TGFβ on macrophage activation and the source of these cytokines. Be specific. **
. In vitro studies show that the induction of iNOS occurs in response to phagocytosis of Mycobacteria, Leishmania, gram-negative organisms, or the cytokine IFNγ ( Secreted by activated cells: NK cells and Th1 cells).Other studies have reported that the induction of iNOS requires two signals, (i) a priming signal delivered by bacterial products or (ii) a second signal reportedly delivered by TNF, a product of activated macrophages and activated Th1 cells. Down regulation of NO synthesis occurs in response to the cytokines interleukin-10 (IL-10), IL- 4, or transforming growth factor beta (TGF β), products of Th2 cells, a subset of activated T cells. Of these, TGFβ is the most effective inhibitor of NO synthesis. TNF (source= macrophages and Th1 cells) and IFN-gamma (source= Th1, NK, ThO cells) can increase iNOS IL-4 (source= basophils, mast cells, and Th2 cells), IL-10 (source= Th2 cells) and TGFB (source= macrophage and Th2 cells) down regulate iNOS "10-4 Good Buddy we have killed the pathogen"
Neutrophils
1. Do not function as antigen presenting cells 2. Do not secrete cytokines or chemokines (for the purpose of this course). 3. Recognition (direct and indirect) is the same as that described for macrophages. 3. Armamentarium products and functions are the same as that described for macrophage
List names of macrophages as a function of tissue (Review from first term). **different locations of the tissue macrophages
Bone Marrow Monoblasts CNS Microglial Cells Liver Kupffer Cells Synovium Synoviocytes Lung Alveolar Macrophages Lymph & Spleen Macrophages
Explain the role of CCL2/MCP-1 on monocytes. Describe the effect of GM-CSF on dendritic cells.
CCL2/MCP-1 attracts monocytes into circulation and into tissues. Activated monocytes secrete cytokines that play a role in hematopoiesis. Effect of GM-CSF on dendritic cells-> Enhances dendritic cell maturation, proliferation, and migration
Describe the role of the following cytokines in Hematopoiesis (and thymus): IL-3, IL-5, IL-7, GM-CSF, G-CSF, M-CSF.
IL-3-> creates pluirpotent stem cell & both myeloid/ lymphoid progenitor IL-5-> creates eosinophils IL-7-> creates B, T and NK cells GM-CSF->Source: Monocyte Fibroblasts Endothelial cells T cells Target Cell: myeloid progenitor & dendritic cells Role: differentiation of a myeloid progenitor to a GM progenitor in hematopoiesis. Enhances dendritic cell maturation, proliferation, and migration M-CSF-> Source:  Monocyte Fibroblasts Endothelial cells Target: GM-progenitor Role: differentiation of a myeloid progenitor to a monocyte in hematopoiesis. G-CSF-> Source:  Monocyte Fibroblasts Endothelial cells Target: GM-progenitor Role: differentiation of a myeloid progenitor to a granulocyte in hematopoiesis
Describe the role of IL-1 and TNF on the vascular endothelium in an inflammatory response. Include in your response E-selectins, VCAM-1, ICAM-1, ICAM-2 as well as their relevant counter molecules on neutrophils, lymphocytes, monocytes, and eosinophils. Also, include the CD molecules that constitute VLA-4 ((CD49d & CD29) and LFA-1 (CD11a & CD18)
Il-1-> -Critical role is inflammation esp. with TNF. Il-1 and TNF combo-> Role in inflammation; - Induces vascular endothelium to secrete: IL-8 (CXCL8) and MCP-1 (CCL2) - Induces de novo expression of VCAM-1, ICAM-1 and E-selectin ; - Upregulates expression of ICAM-2 Note: VCAM-1 on vascular endothelium interactions with VLA-4 (CD49d & CD29) on monocytes, lymphocytes and eosinophils. ICAM-1 and ICAM-2 on vascular endothelium interacts with LFA-1 (CD11a & CD18) on neutrophils, monocytes, lymphocytes, and eosinophils.
List the two main roles of macrophages.
In addition to their role in phagocytosis, activated macrophages function as antigen presenting cells for CD4+ T cells (See Dendritic cells and T cell Section).
Explain why opsonin-mediated recognition is called "indirect recognition".**
Indirect recognition refers to interaction of an intermediary protein, which has bound to an antigen, with a receptor on the phagocyte. This is referred to as opsonin-mediated phagocytosis.
Describe macrophages with respect to: (a) differentiation from monocytes, (b) role of MCP-1/CCL2 in the absence of infection and during infection.
Macrophages are phagocytes that reside in every tissue of the body. They derive from monocytes, blood borne cells that arise from bone marrow progenitors. Under normal conditions, monocytes comprise only about four per cent of circulating leukocytes. However, enhanced release of monocytes and migration into tissues follows tissue damage or infection (triggers for inflammation).In particular, the chemokine CCL2/MCP-1 (monocyte chemotactic protein-1) (See table below - Chemokines Secreted by Macrophages ) enhances recruitment of monocytes into circulation and migration into tissues where they differentiate into macrophages.
List the chemokines and cytokines secreted by activated macrophages. Identify the target cells for the chemokines and cytokines as well as the role of each of these molecules. See cytokine chart.
Macrophages secrete several chemokines ((IL-8/CXCL8, MCP-1/CCL2 ), as well as cytokines (IL- 1, TNF, IL-6, IL-12, IL-15, IL-18, IL-23, TGFβ) See tables below.
Describe differentiation of monocytes in bone marrow.
Monocytes Monocytes are derived from progenitor cells; [see chart and flow graph]
Describe neutrophils with respect to : differentiation, % of circulating leukocytes, half-life in circulation, chemotactic factors that attract neutrophils to site of inflammation.
Neutrophils comprise some 60% of the circulating leukocytes in peripheral blood. Neutrophils mature from precursor cells in the bone marrow, with more than a billion cells being generated daily. Only a small percent of these cells are stored in the bone marrow, with the majority being released into circulation. Once neutrophils are released into circulation they have a half-life of some 8 hours. A number of chemotactic factors (interleukin-8 (IL-8)/CXCL8 and C5a), generated in the early phase of an inflammatory response (induced by tissue damage or infection) results in enhanced release of neutrophils from the bone marrow and attraction to the site of the inflammation.
Explain how the opsonins were generated. **
Opsonins are generated when components, other than phagocytes, are activated in response to the pathogen.
Re: Indirect/Opsonin Mediated Recognition of Pathogens—list three opsonins that may be deposited on a pathogen. List the receptors on the phagocyte that interact with each opsonin.**
Opsonins are the products of: (i) complement activation (e.g., C3b); (ii) B cell activation (e.g., IgG ); and (iii) cytokine mediated activation of hepatocytes (e.g., C-reactive protein, CRP). Interaction of any of these pathogen- bound opsonins triggers the process of phagocytosis. Receptor-Opsonin pairing are as follows: Fcγ :FcγR (See antibodies); CRP: CRP-Binding Site (See IL-6, Macrophage cytokine); and C3b:CR1 (CR-1 is complement receptor-1: See complement).
Recognition of antigens/pathogens is "Direct" or "Indirect" . Explain the term "direct recognition". Re: Direct Recognition of Pathogens: Explain the role of pattern recognition receptors on phagocytes. Name one family of proteins that are classed as PRRs. Explain the role of pathogen associated molecular patterns on pathogens. **
PPRs interact directly with pathogens via PAMPs, This is referred to as "direct recognition/interaction". However, macrophages may also be activated following "indirect recognition/interaction" with pathogens. The indirect interaction is so called because molecules derived from activation of other components of the immune system bind to pathogens; it is the bound molecule that is recognized by receptors on the macrophage. The bound molecules are also referred to as "opsonins" (See below). Direct recognition of pathogens occurs via PRRs, which distinguish pathogens from self. PRRs exist as membrane bound receptors, cytosolic receptors, or secreted receptors. These receptors trigger cascades of biochemical events that lead to secretion of inflammatory cytokines, and other cellular responses associated with the innate immune response. PRR ligands are referred to as PAMPs. Family of PPR: A group of membrane bound receptors that exist both on cytosolic vesicles and external cell membranes are the toll-like receptors (TLRs). TLRs are so named for their sequence similarity to the Drosphilia protein, Toll. The TLRs are expressed on a various cell types including macrophages and neutrophils. There are 10 mammalian toll-like receptors; each recognizes different molecular patterns and signaling leads to different signaling cascades.
Explain the consequence of CD200R interaction with CD200. **
Phagocyte responses are regulated by multiple factors as well as by cell surface receptors, including the inhibitory signals resulting from ligation CD200R (which is expressed primarily by myeloid cells and a subset of T cells) by the widely distributed membrane protein, CD200.The CD200Rs are expressed primarily on cells of the myeloid lineage. However, they are also expressed on T cells (See T cell section) .
Describe steps in phagocytosis & formation of phagosome & phagolysosome. **
Recognition and binding of the pathogen is followed by its' ingestion as a portion of the plasma membrane extends outward and surrounds the microbe, forming a phagocytic vacuole termed a phagosome. This is referred to as "phagocytosis". Phagosomes are vesicles that contain the engulfed pathogen. In general, this phagocytic vacuole serves as the "battlefield" where microorganisms are subsequently destroyed by phagocytic "weapons" including: (i) lysosomal enzymes; (ii) reactive oxygen intermediates; and (iii) reactive nitrogen intermediates. These products target antigens in the phagosome leading to their degradation. Lysosomes, present in the cytosol, fuse with the phagosome to form a fusion product, the phagolysosome, into which lysosomal granules are discharged. Lysosomal granules contain many enzymes (including lactoferrin, lysozyme, and defensins) that are cytostatic/cytotoxic to microorganisms. These molecules play a role in host immunity as follows: (i) lactoferrin binds iron, thereby removing an essential ingredient for microbial growth; (ii) lysozyme destroys muramic acid in bacterial cell walls; (iii) defensins permeabilize bacterial and fungal membranes. Lysosomal granules also release myeloperoxidase, an enzyme required to generate hypochlorite, a potent antimicrobial agent that mediates its function by halogenating bacterial cell walls (next section).
In general terms describe the inflammasome with respect to signal 1 and signal 2 as well as outcome of the combined effects of these two signals. Include in your description the following: PAMPs, PRRs, NFκB, DAMPS, pro-IL-1, pro-IL-18, procaspase-1, IL-1, IL-18 and caspase-1. List 5 examples of DAMPs.
The NALP3 inflammasome is a large multi-protein complex that plays a critical role in host defense by mediating the conversion of the zymogen, procaspase-1 to caspase-1. Caspase-1 converts pro-IL-1 and pro- IL-18 to biologically active forms, (IL-1 and IL-18) which are secreted from the macrophage. The release of IL-1 and IL-18 is a two signal process: (i) signal 1 is activation of NFκB following PAMP (pathogen associated molecular pattern) binding to pattern recognition receptors (PRR), which leads to transcription of pro-IL-1 and pro-IL-18; (ii) signal 2 is the conversion of pro-caspase-1 to caspase-1. Caspase-1 then cleaves the biologically inactive precursors (pro-IL-1 and pro-IL-18) to their active forms IL-1 and IL-18, which are then secreted from the cell. Signal 2 can be triggered by (a) numerous molecules released during trauma or cell death, (b) reactive oxygen species, (c) ATP, and (d) cholesterol crystals, (e) free fatty acids, and other endogenous molecules. Molecules that trigger signal 2 are referred to as "danger associated molecular patterns" (DAMPs). Therefore activation of the NALP3 inflammasome, leading to secretion of IL-1 and IL-18, requires the coordinated response mediated by two distinct signals.
Explain why macrophages are not as effective as antigen presenting cells as dendritic cells.
The role of macrophages in antigen presentation during secondary immune responses can be explained, in part, by their tissue distribution. Secondary immune responses generally occur at the site of infection. In contrast, primary immune responses occur in secondary lymphoid tissues, and not at the site of infection. This explains why dendritic cells are probably more important in primary immune responses than macrophages, since unlike macrophages, dendritic cells can capture antigen in the periphery and transport it to the appropriate lymphoid tissue. Like dendritic cells, macrophages also express CD4 and the chemokine receptors CCR5 and/or CXCR4. Consequently, macrophages can readily be infected with HIV-1. Furthermore the constitutive expression of Class II MHC and costimulatory molecules is rather low in these cells. Macrophage activation upregulates Class II MHC and costimulatory molecules. See Dendritic Cell Section.
