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Anti-IgE antibodies act as an analogue of antigen and directly activate mast cells that have bound IgE on their surface. This use of anti-IgE to activate mast cells is similar to the use of anti-IgM antibodies as analogues of antigen to activate B cells, except that in the case of mast cells, secreted IgE, made by B cells, is bound to high-affinity Fc receptors on the cell surface rather than being synthesized as membrane IgE. Anti-IgE antibodies activate mast cells even in normal (nonatopic) individuals because, as mentioned earlier, mast cells are normally coated with IgE that can be cross-linked by the anti-IgE. In contrast, an antigen will activate mast cells only in individuals who are allergic to that antigen because only these individuals will produce enough specific IgE to be cross-linked by the antigen. Immediate hypersensitivity reactions can also be mimicked by the injection of other agents that directly activate mast cells, such as the complement fragments C5a, C4a, and C3a, called anaphyla-toxins, or by local trauma, which also causes degranulation of mast cells. Conversely, these reactions can be inhibited by agents that prevent mast cell activation.

Activation of mast cells results in three types of biologic response: secretion of the preformed granule contents by exocytosis (degranulation), synthesis and secretion of lipid mediators, and synthesis and secretion of cytokines. The signaling cascades initiated by allergen-mediated FceRI cross-linking are similar to the proximal signaling events initiated by antigen binding to lymphocytes (Fig. 19-5 and see Chapter 7). The Lyn tyrosine kinase is con-stitutively associated with the cytoplasmic tail of the FceRI P chain. On cross-linking of FceRI molecules by antigen, Lyn tyrosine kinase phosphorylates the ITAMs

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Phosphatidylcholine

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Phosphatidylcholine

FIGURE 19-5 Biochemical events of mast cell activation. Cross-linking of bound IgE by antigen is thought to activate protein tyrosine kinases (Syk and Lyn), which in turn cause activation of a MAP kinase cascade and a phosphatidylinositol-specific phospholipase C (PI-PLCy). PI-PLCy catalyzes the release of IP3 and DAG from membrane PIP2. IP3 causes release of intracellular calcium from the endoplasmic reticulum. Calcium and DAG activate PKC, which phosphorylates substrates such as myosin light chain protein and thereby leads to the degradation and release of preformed mediators. Calcium and MAP kinases combine to activate the enzyme cytosolic phospholipase A2 (PLA2), which initiates the synthesis of lipid mediators, including prostaglandin D2 (PGD2) and leukotriene C4 (LTC4).

FIGURE 19-5 Biochemical events of mast cell activation. Cross-linking of bound IgE by antigen is thought to activate protein tyrosine kinases (Syk and Lyn), which in turn cause activation of a MAP kinase cascade and a phosphatidylinositol-specific phospholipase C (PI-PLCy). PI-PLCy catalyzes the release of IP3 and DAG from membrane PIP2. IP3 causes release of intracellular calcium from the endoplasmic reticulum. Calcium and DAG activate PKC, which phosphorylates substrates such as myosin light chain protein and thereby leads to the degradation and release of preformed mediators. Calcium and MAP kinases combine to activate the enzyme cytosolic phospholipase A2 (PLA2), which initiates the synthesis of lipid mediators, including prostaglandin D2 (PGD2) and leukotriene C4 (LTC4).

in the cytoplasmic domains of FceRI P and y chains. The Syk tyrosine kinase is then recruited to the ITAMs of the Y chain, becomes activated, and phosphorylates and activates other proteins in the signaling cascade, including several adaptor molecules and enzymes that participate in the formation of multicomponent signaling complexes, as described in T cells. Linker for activation of T cells (LAT) is one of the essential adaptor proteins involved in mast cell activation, and one of the enzymes recruited to LAT is the y isoform of a phosphatidylinositol-specific phospholipase C (PLCy). Once bound to LAT, PLCy is phosphorylated and then catalyzes phosphatidylinositol bisphosphate breakdown to yield inositol trisphosphate (IP3) and diacylglycerol (DAG) (see Chapter 7). IP3 causes elevation of cytoplasmic calcium levels, and DAG activates protein kinase C (PKC). Another pathway of PKC activation in mast cells involves the tyrosine kinase Fyn, which phosphorylates the adaptor protein Grb-2-associated binder-like protein 2 (Gab2), which in turn binds phosphoinositide 3-kinase, leading to activation of PKC. Phosphorylation of the myosin light chains by activated PKC leads to disassembly of the actin-myosin complexes beneath the plasma membrane, thereby allowing granules to come in contact with the plasma membrane.

The mast cell granule membrane then fuses with the plasma membrane, a process that is mediated by members of the SNARE protein family, which are involved in many other membrane fusion events. Different SNARE proteins present on the granule and plasma membranes interact to form a multimeric complex that catalyzes fusion. The formation of SNARE complexes is regulated by several accessory molecules, including Rab3 guano-sine triphosphatases and Rab-associated kinases and phosphatases. In resting mast cells, these regulatory molecules inhibit mast cell granule membrane fusion with the plasma membrane. On FceRI cross-linking, the resulting increased cytoplasmic calcium concentrations and the activation of PKC block the regulatory functions of the accessory molecules. In addition, calcium sensor proteins, called synaptotagmins, respond to the elevated calcium concentrations by promoting SNARE complex formation and membrane fusion.

Following membrane fusion, the contents of the mast cell granules are released into the extracellular environment. This process can occur within seconds of FceRI cross-linking, and can be visualized morphologically by loss of the dense granules of mast cells (see Fig. 19-4). The biologic actions of the mediators released upon mast cell degranulation are described later.

Synthesis of lipid mediators is controlled by activation of the cytosolic enzyme phospholipase A2 (PLA2) (see Fig. 19-5). This enzyme is activated by two signals: elevated cytoplasmic calcium and phosphorylation catalyzed by a mitogen-activated protein (MAP) kinase such as extracellular receptor-activated kinase (ERK). ERK is activated as a consequence of a kinase cascade initiated through the receptor ITAMs, probably using the same intermediates as in T cells (see Chapter 7). Once activated, PLA2 hydro-lyzes membrane phospholipids to release substrates that are converted by enzyme cascades into the ultimate mediators. The major substrate is arachidonic acid, which is converted by cyclooxygenase or lipoxygenase into different mediators (discussed later).

Cytokine production by activated mast cells is a consequence of newly induced cytokine gene transcription. The biochemical events that regulate cytokine gene transcription in mast cells appear to be similar to the events that occur in T cells. Recruitment and activation of various adaptor molecules and kinases in response to FceRI cross-linking lead to nuclear translocation of nuclear factor of activated T cells (NFAT) and nuclear factor kB (NF-kB) as well as activation of activation protein 1 (AP-1) by protein kinases such as c-Jun N-terminal kinase. These transcription factors stimulate transcription of several cytokines (IL-4, IL-5, IL-6, IL-13, and tumor necrosis factor [TNF], among others) but, in contrast to T cells, not IL-2.

Mast cell activation through the FceRI pathway is regulated by various inhibitory receptors, which contain an immunoreceptor tyrosine-based inhibition motif (ITIM) within their cytoplasmic tails (see Chapter 7). One such inhibitory receptor is FcyRIIB, which coaggregates with FceRI during mast cell activation. The ITIM of FcyRIIB is phosphorylated by Lyn, and this leads to recruitment of the phosphatase called SH2 domain-containing inositol 5-phosphatase (SHIP) and inhibition of FceRI signaling. Experiments in mice indicate that FcyRIIB regulates mast cell degranulation in vivo. Several other inhibitory receptors are also expressed on mast cells, but their importance in vivo is not yet known.

Mast cells can be directly activated by a variety of biologic substances independent of allergen-mediated cross-linking of FceRI, including polybasic compounds, peptides, chemokines, and complement-derived ana-phylatoxins. These additional modes of mast cell activation may be important in non-immune-mediated immediate hypersensitivity reactions, or they may amplify IgE-mediated reactions. Certain types of mast cells or basophils may respond to macrophage-derived chemokines, such as macrophage inflammatory protein 1a (MIP-1a), produced as part of innate immunity, and to T cell-derived chemokines, produced as part of adaptive cell-mediated immunity. The complement-derived anaphylatoxins, especially C5a, bind to specific receptors on mast cells and stimulate degranulation. These che-mokines and complement fragments that activate mast cells are likely to be produced at sites of inflammation. Therefore, mast cell activation and release of mediators may amplify IgE-independent inflammatory reactions. Polybasic compounds, such as compound 48/40 and mas-toparan, are used experimentally as pharmacologic triggers for mast cells. These agents contain a cationic region adjacent to a hydrophobic moiety, and they work by activating G proteins.

Many neuropeptides, including substance P, soma-tostatin, and vasoactive intestinal peptide, induce mast cell histamine release and may mediate neuroendocrine-linked mast cell activation. The nervous system is known to modulate immediate hypersensitivity reactions, and neuropeptides may be involved in this effect. The flare produced at the edge of the wheal in elicited immediate hypersensitivity reactions is in part mediated by the nervous system, as shown by the observation that it is markedly diminished in skin sites lacking innervation. Cold temperatures and intense exercise may also trigger mast cell degranulation, but the mechanisms involved are not known.

Mast cells also express Fc receptors for IgG heavy chains, and the cells can be activated by cross-linking bound IgG. This IgG-mediated reaction is the likely explanation for the finding that Ig e chain knockout mice are not completely resistant to antigen-induced mast cell-mediated anaphylaxis. However, IgE is the major antibody isotype involved in most immediate hypersen-sitivity reactions.

Mast cell activation is not an all-or-nothing phenomenon, and different types or levels of stimuli may elicit partial responses, with production of some mediators but not others. Such variations in activation and mediator release may account for variable clinical presentations.

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