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October 7, 2004 IMMUNITY ADAPTIVE CELL MEDIATED INNATE EFFECTOR SYSTEMS Fc Receptors Complement HUMORAL ANTIBODIES Antibodies are produced by B lymphocytes and plasma cells but perform their effector functions at sites distant from their production Many of the effector functions of antibodies are mediated by the heavy chain constant regions and different Ig heavy chain isotypes serve distinct effector functions Activation of the classical pathway of complement Effective in lysing bacteria Antigen receptor on naïve B lymphocytes First antibody made during the immune response Opsonization of Ags for phagocytosis by macrophages and neutrophils Activation of classical pathway of complement Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK and macrophages Neonatal immunity - transferred across the placenta and gut Secreted into the lumens of the GI and respiratory tracts Provides protection against pathogens that attack at the mucosal surfaces Antibody-dependent cell-mediated cytotoxicity involving eosinophils - parasite infection Mast cell degranulation - immediate hypersensitivity or allergy Antigen receptor of naïve B lymphocytes Although most effector functions are mediated by the heavy chain, the functions are triggered by the binding of antigen to the variable region Immunofluorescence Takes advantage of the fact that certain dyes such as fluorescein and rhodamine absorb light at one wavelenght and emit it at another characteristic wavelength Fluorescent dyes can be attached to Abs without altering their affinity. By using different filters and dyes it is possible to look simultaneously at multiple antigens. Direct and Indirect Detection of Surface Antigens Fluorescent activated cell sorter (FACS) Measures the fluorescent intensity of single cells The simplest form of the instrucment instrument counts each cell and records the level of fluorescence analysis or flow cytometry -FLOW Can also be used to sort cells FACS Analysis in one dimension Analysis in two dimensions uses two different fluorchromes attached to Abs specific for different antigens Flow cytometry can also be adapted to look at the presence of intracellular proteins such as cytokines. Non-ionic detergents at low concentrations are used to permeabilize the cells before the addition of antibody. Cells were first stained with an antibody specific for the cell surface protein CD4, then permeabilized and stained for the intracellular cytokine IFN-g Antibodies are effective in clearing pathogens and activating an inflammatory response in part because they bind to Fc receptors present on cells Fc There are three families of Fcg receptors FcgRI or CD64 FcgRII or CD32 FcgRIII or CD16 FcgRI or CD64 Expressed on macrophages and neutrophils Requires an associated g (or ) chain for expression and function; in g chain knock-out mice there is no expression of FcgRI. g chain has ITAM (YXXL) motifs for signaling. Can mediate ADCC and phagocytosis in response to X-linking Ligation also leads to macrophage activation and cytokine production Only receptor that binds monomeric IgG with high affinity FcgRII or CD32 Has signaling motifs in its cytoplasmic tail FcgRIIA the motif is an ITAM (activation) FcgRIIB the motif is an ITIM (inhibition) FcgRII or CD32 FcgRIIA Expressed on macrophages, neutrophils, eosinophils. Ligation leads to uptake. Present in the human, but not the mouse. FcgRII or CD32 FcgRIIB -- an inhibitory receptor Acts as an inhibitory receptor on B cells; crosslinking of surface IgM leads to proliferation only when F(ab)’2 is used. FcgRIIB bind the Fc of the intact Ab and provides an inhibitory signal. FcgRIIB also plays an inhibitory role in mast cell degranulation through FcRI. FcgRIII or CD16 FcgRIIIA, a transmembrane receptor is found on monocytes, macrophages, NK (the only Fc receptor) and T cells. g and/or chains are required for surface expression. FcgRIIIB is present on human neutrophils. It is secured in the membrane by a glycosyl phosphatidyl inositol (GPI) anchor. Antibody-Dependent Cell Mediated Cytotoxicity ADCC Leukocytes with Fc receptors bind to antibody-coated cells and destroy them Fc receptors are required for antibody-mediated tumor protection Mice were injected weekly with 2 ug/ml of therapeutic antibody Fcg-/- lack FcgRI and FcgRIII 4D5 - murine anti-HER2/neu Herceptin - humanized anti-HER2/neu HER2/neu is a protein present on breast and other cancers Rituxan - chimeric anti-CD20 CD20 is present on lymphoma cells Inhibitory FcgRIIB receptors also modulate the in vivo cytotoxicity of antibodies against tumors Mice were injected with a sub-therapeutic dose (0.2ug weekly) 4D5 is a murine anti-HER2/neu; Trastuzumab is the same as Herceptin, humanized anti-HER2/neu There is a balance between activating and inhibitory receptors From Ann. Rev. Immunol. 19:275, 2001. Uptake of antigen by macrophages (J. Immunol. 168:3697, 2002) Fc receptors influence the efficiency of antigen presentation Cell number Complement activation by antibodies also is important for effective clearing of pathogens and activation of an inflammatory response How was complement discovered? Lysis is dependent on two factors: Ag-spcific agent that is heat stable and a separate agent that is heat labile and complements Ab and causes lysis. Complement - an important effector system has important functions related to immune defense 1. Lysis of cells. This is the original function identified and causes hypotonic cell death by making hole. It is not effective against organisms with cell walls such as fungi and Gram positive bacteria 2. Opsonization. Macrophage and PMNs have FcRs and at least two different kinds of complement receptors that aid in phagocytosis. C3b, a cleavage product formed during activation is the major player. Antigen coated with C3b binds to cells bearing complement receptors and if the cell is a phagocyte the antigen will be phagocytosed. Complement - an important effector system has important functions related to immune defense 3. Inflammation. Peptides generated during activation play a role in inflammation. The anaphylatoxins of which C5a is the most potent bind receptors on mast cells and basophils and cause degranulation with the release of pharmacologically active mediators which induce smoothmuscle contraction and increases in vascular permeability. C3a, C5a and C5b67 act as chemoattractants and induce monocytes and neutrophils to adhere to vascular endothelial cells, extravasate through the endothelial lining of the capillaries and migrate to the site of complement activation in the tissue. Complement - an important effector system has important functions related to immune defense 4. Immune clearance. Removes immune complexes from the circulation and deposits them in the liver where they are degraded. C3b facilitates immune complex binding to CR1 on RBCs. In the liver and spleen the complexes are stripped from the RBC and phagocytosed. Complement also helps to solubilize immune complexes. 5. Enhanced immune response. CD21, part of the coreceptor on the B cell, binds cleaved C3. Recently it has also been shown that C3 is required for optimal expansion of T cells during a systemic viral infection. 6. Virus neutralization. Complement mediates viral neutralization by facilitating viral aggregation and by coating the viral surface. There are three pathways through which complement can be activated ADAPTIVE INNATE Classical pathway Requires Ab Mannose binding lectin pathway Alternative pathway The central player is C3 and all three pathways focus on generating activated C3 CLASSICAL PATHWAY Antigen-antibody C1qr2s2 (C1) Activated C1 C2 C4 C4b C2b C4b2a (C3 convertase) C4a C3 MBL Activated C1-like MASP complex LECTIN PATHWAY C3b C3a C4b2a3b (C5 convertase) C5 Ba B C3a C3 C3b C3bBb (C3 convertase) Microbial Surfaces Factor D ALTERNATIVE PATHWAY MAC C5b C6 C7 C8 C9 C5a C3bBb3b (C5 convertase) Cleavage of a labile thioester bond in C3, an abundant serum protein, creates an active group that allows C3 to attach to other proteins through NH2 or OH groups C Clq has 6 legs. When C1q binds the associated enzymes C1r and C1s are activated. The affinity of C1q for Fc is weak so that at least two Fcs must be bound to have activation. YY Activated proteases cleave C4 and C2. The cleavage products (C4b2a) form a new protease that cleaves C3 into C3a and C3b Association of C3b with the protease (C4b2aC3b) alters its specificity so that it now cleaves C5. C5b is the first protein in the membrane attack (MAC) complex IgM is a very rigid molecule Binding of Ag by IgM leads to a conformational change exposing the C1q binding sites. There are multiple sites in each IgM molecule so one IgM can bind C1q and activate the complement cascade IgG has only two sites per molecule. In order to achieve effective C1q binding and complement activation two IgG molecules must bind close to each other. YY Mannose Binding Protein Mannose Binding Protein Recognizes carbohydrate antigens common to pathogens Like C1q it has two associated proteases that become activated upon binding. These proteases cleave C4 and C2 and so the Lectin pathway is identical to the Classical pathway except for the first activation step C3 is always being spontaneously hydrolyzed. Usually it decays. However if it is in the proper environment such as a microbial surface it will complex with Factor B which will be cleaved by Factor D and will form a C3 convertase. The cleavage products produced during complement activation are very important effector molecules Anaphylotoxins: C3a, C4a, C5a C3a , C4a and C5a are anaphylatoxins Small peptides that causes smooth muscle contraction, increases vascular permeability and mast cell and basophil degranulation. C5a is also a chemoattractant and activator of WBC Anaphylatoxins Also amplify the inflammatory response by inducing the synthesis of pro-inflammatory cytokines. Their receptors are present on many cell types including leukocytes, mast cells, macrophages, endothelial cells, astrocytes and microglial cells Anaphylatoxins Anaphylatoxins trigger a cascade of events that contributes to the pathogenesis of a number of disease and conditions including hypersensitivity reactions, endotoxin shock, multiple organ failure and respiratory distress syndrome. Neutralization of the effects of anaphylatoxin can be of substantial clinical significance Cleavage products of C3b are opsonins They bind to receptors present on the surface of phagocytes Formation of the membrane attack (MAC) complex F Regulation of complement DAF: prevents assembly of and accelerates disassembly of C3 convertase displaces Bb from C3b displaces C2b from C4b Homologous restriction factor (HRF) and CD59: bind C5b678 preventing C9 binding and MAC formation Reactive species quickly decay Complement Receptors Type 1: CR1 or CD35 expressed on RBCs, m , neutrophils, eosinophils, B cell and som e T cells cells expressing CR1 can bind imm une complexes and particulate Ags to which C3b is attached, facilitating Agclearance Type 2: CR2 or CD21 expressed on B cells and some T cells bind degradation products of C3b potentiates response to Ags with C3b degradation products Type 3 and 4: CR3 and CR4 found on monocytes, m , neutrophils, NK and some T cells bind C3b degradation product binding of com plem ent coated particles triggers phagocytosis by phagocytic cells C3a/C4a and C5a present on mast cells, basophils and granulocytes and can be used for their degranulation Immune complex clearance Opsonization Enhances immune response Mediator Release Functions of complement Complement and Fc receptors synergize in promoting phagocytosis