FcepsilonRI Aggregation Research Articles

Genome-based expression profiling study of Hunner’s ulcer type interstitial cystitis: an array of 40-gene model

The aim of this study was to explore potential molecular mechanisms contributing to the pathogenesis of Hunner's ulcer type interstitial cystitis (IC). Dataset acquisitions from Gene Expression Omnibus under platform accession no GSE 11783. We compared global gene expression profiles in bladder epithelial cells from IC patients with Hunner's ulcer corresponding to normal controls. We re-sampling and exploit the correlation structure presented in the dataset through the transcriptional response. For each patient, two bladder biopsies were studied, one from an ulcer area and one from a non-ulcer area. RNA was extracted, and all labeled samples were hybridized to Human Genome U133 Plus 2.0 Array (Affymetrix, CA, USA). The Mahalanobis distance in hierarchical cluster analysis revealed a model of 40 genes expression which is increased in IC and ulcerated IC. Our results can be summarized as follows: First, the expressions of major histocompatibility complex (MHC) class IF and II molecules, leukocyte immunoglobulin-like receptors, hepatitis A virus cellular receptor 2, and interleukin 32 were increased in bladder epithelial from IC and ulcerative IC area. Next, there is an indication of antigen-mediated aggregation of the high-affinity Fc epsilon and gamma RI leading to allergic inflammation through the disease status. Third, the high-affinity Fc gamma RI subunit facilitated T-cell-mediated immune response through the disease status. Such changes, jointly termed "bladder remodeling," can constitute an important long-term consequence of Hunner's ulcer type IC. Our results indicate that genome-based expression profiling can be used for the diagnostic tests of Hunner's ulcer type IC in clinical practice.

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and beta-carotene significantly inhibited the antigen-induced release of beta-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (Fc epsilonRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited Fc epsilonRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of Fc epsilonRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of Fc epsilonRI to lipid rafts, which are known as platforms of the aggregation of Fc epsilonRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of Fc epsilonRI to lipid rafts. This is the first report that focused on the aggregation of Fc epsilonRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Loss of SOCS7 in mice results in severe cutaneous disease and increased mast cell activation

The Suppressor of Cytokine Signaling (SOCS) protein family plays a central role in the negative regulation of cytokine action and has been implicated in the development of atopic diseases. Lack of SOCS7 is associated with severe skin disease in mice. We sought to explore the underlying mechanisms resulting in this phenotype. Skin samples were analyzed and serum immunoglobulin production was measured. Cytokine production by bone marrow derived mast cells was determined by ELISA. Mast cell thymic stromal lymphopoietin (TSLP) production was assessed by quantitative real-time PCR. Data obtained revealed that Socs7(-/-) mice have increased serum IgE and IgG(1) production and exhibit an increased mast cell infiltrate, as well as un-provoked mast cell degranulation in the dermis as compared to controls. In vitro, bone marrow derived mast cells from Socs7(-/-) mice are hyperactive to IgE-mediated stimuli, with elevated production of pro-inflammatory cytokines (IL-13, IL-6, TNF-alpha). Further, activated Socs7(-/-) bone marrow derived mast cells have increased IL-7Ralpha transcript, which is part of the heterodimeric receptor for TSLP. Finally, lack of SOCS7 was accompanied by an increase in TSLP mRNA and protein production by mast cells following FcepsilonRI aggregation. These data implicate SOCS7 in the modulation of allergic inflammation and demonstrate that SOCS7 is involved in the regulation of TSLP signaling in mast cells.

Fc RI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells

A significant increase of mRNA expression of thymic stromal lymphopoietin (TSLP) has been reported in the bronchial mast cells (MCs) of asthmatic subjects; however, the mechanism underlying the upregulation of TSLP mRNA and protein remains unknown. FcepsilonRI-mediated activation of human MCs upregulated TSLP mRNA expression by 5.2+/-2.9-fold, while activation of the MCs using lipopolysaccharide and polyriboinosinic:polyribocytidylic acid failed to upregulate TSLP. Stimulation of MCs with interleukin (IL)-4 alone did not affect the TSLP mRNA expression, while pre-incubation of MCs with IL-4 for 48 h significantly enhanced the FcepsilonRI-mediated TSLP mRNA expression (by 53.7+/-15.9-fold; p<0.05) and the amount of TSLP in the cell pellets increased significantly from 23.4+/-4.3 pg mL(-1) to 121.5+/-3.7 pg mL(-1) (p<0.0001). However, the released TSLP was rapidly degraded by proteases that were released by MCs. We identified the population of cells expressing TSLP in the lungs of 16 asthmatic and 11 control subjects by immunohistochemistry. The percentage of TSLP-positive MCs in the total population of MCs was significantly increased in asthmatic airways (p<0.0001). Thus, MCs are able to store TSLP intracellularly and to produce TSLP following aggregation of FcepsilonRI in the presence of IL-4.

Osteopontin is produced by mast cells and affects IgE‐mediated degranulation and migration of mast cells

Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and FcepsilonRI aggregation in bone marrow-derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN-deficient (OPN(-/-)) and -sufficient (OPN(+/+)) mice without significant differences in yield, purity, granularity, and viability. Using OPN(-/-) FSMC, we found that recombinant OPN augmented IgE-mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin alphav). IgE-mediated passive cutaneous anaphylaxis was significantly reduced in OPN(-/-) mice compared with OPN(+/+) mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell-related pathological conditions.

A current understanding of FcɛRI-dependent mast cell activation

Mast cell activation via the high-affinity immunoglobulin (Ig) E receptor Fc epsilon RI is a topic of extensive investigation with therapeutic potential in allergic disease. The protein tyrosine kinases Fyn, Lyn, and Syk are intimately linked with the early events initiated by allergen-mediated aggregation of IgE-occupied Fc epsilon RI. Fyn and Lyn initiate signaling events that are organized by adaptor molecules, which compartmentalize and coordinate the activity of activated protein and lipid kinases and phospholipases to generate lipid products essential for signal amplification and mast cell function. Fyn and Lyn counter-regulate phosphatidylinositol 3-OH kinase (PI3K), controlling the produced amount of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a key regulator of mast cell degranulation. Fyn and Lyn also activate sphingosine kinases (SphK), which generate sphingosine-1-phosphate (S1P), thus contributing to mast cell chemotaxis and degranulation. Here, we summarize the current knowledge and future challenges and directions.

FcγRI-Mediated Activation of Human Mast Cells Promotes Survival and Induction of the Pro-survival Gene Bfl-1

Activation of mast cells through either FcepsilonRI or FcgammaRI leads to release of mediators contributing to the inflammatory response. One of the biologic characteristics of mast cells in allergic pathology is that these cells have the capacity to recover and regranulate after aggregation of FcepsilonRI. We have previously demonstrated that the pro-survival protein A1/Bfl-1 is required for mast cells to survive IgE-mediated activation. In the present study, we have investigated whether human mast cells show similar induction of bfl-1 and activation-induced survival after aggregation of FcgammaRI. Human cord blood-derived mast cells were activated by aggregation of either FcepsilonRI or FcgammaRI, and activation-induced survival and induction of bfl-1 was measured. We found that aggregation of FcgammaRI-induced expression of Bfl-1 and caused a comparable activation-induced mast cell survival as FcepsilonRI does. These data suggests that activation through Fc-receptors contribute to mast cell survival during antibody-dependent mast cell mediated inflammatory responses.

The mast cell and allergic diseases: role in pathogenesis and implications for therapy

Mast cells have long been recognized for their role in the genesis of allergic inflammation; and more recently for their participation in innate and acquired immune responses. Mast cells reside within tissues including the skin and mucosal membranes, which interface with the external environment; as well as being found within vascularized tissues next to nerves, blood vessels and glandular structures. Mast cells have the capability of reacting both within minutes and over hours to specific stimuli, with local and systemic effects. Mast cells express the high affinity IgE receptor (FcepsilonRI) and upon aggregation of FcepsilonRI by allergen-specific IgE, mast cells release and generate biologically active preformed and newly synthesized mediators which are involved in many aspects of allergic inflammation. While mast cells have been well documented to be essential for acute allergic reactions, more recently the importance of mast cells in reacting through pattern recognition receptors in innate immune responses has become recognized. Moreover, as our molecular understanding of the mast cell has evolved, novel targets for modulation have been identified with promising therapeutic potential.

Negative autoregulation of Src homology region 2-domain-containing phosphatase-1 in rat basophilic leukemia-2H3 cells

Src homology region 2-domain-containing phosphatase-1 (SHP-1) plays an important role in the regulation of signaling from various receptors in hematopoietic cells. In mast cells, SHP-1 has been shown to negatively regulate the initial signaling triggered by high-affinity receptor for IgE (FcepsilonRI) and positively regulate downstream outputs. To clarify the molecular mechanisms of SHP-1 in mast cells, we determined substrates for SHP-1 by using the substrate-trapping approach. When phosphatase-inactive SHP-1 was over-expressed in rat basophilic leukemia (RBL)-2H3 cells, tyrosine phosphorylation of a 68-kDa protein was enhanced before and after FcepsilonRI aggregation. Immunoprecipitation and western blot analyses revealed that this protein is SHP-1, either endogenous or ectopically expressed. FcepsilonRI-induced activation of Lyn and Syk was comparable between cells expressing wild-type (wt) and phosphatase-inactive SHP-1. In vitro phosphatase assay and combined transfection, immunoprecipitation and immunoblot analyses showed that tyrosine 536 of SHP-1 was potent phosphorylation site and that SHP-1 could dephosphorylate this site that had been phosphorylated by Lyn. Furthermore, the phosphatase activity of SHP-1 immunoprecipitated from cells expressing a phosphatase-inactive SHP-1 was increased compared with that from vector-transfected or wt SHP-1-expressing cells. Finally, expression of phosphatase-inactive SHP-1 resulted in decreased activation of mitogen-activated protein kinases and suppressed transcription of cytokine genes, whereas wt SHP-1 enhanced these processes. Taken collectively, these results suggest that SHP-1 may be a physiological substrate of SHP-1 in RBL-2H3 cells and that dephosphorylation of SHP-1 leads to a decrease in its catalytic activity and an enhancement of downstream signaling. A negative autoregulatory circuit of SHP-1 may contribute to mast cell regulation.

Negative signals from FcεRI engagement attenuate mast cell functions

Mast cells have long been recognized as the critical tissue-based effector cells in IgE-mediated allergic diseases. Ligation of the high-affinity receptor for IgE (FcepsilonRI), constitutively expressed on mast cells, promotes cell activation and immediate release and production of pro-inflammatory mediators. Besides these positive signals, FcepsilonRI aggregation has recently been understood to generate negative intracellular signals capable of limiting mast cell functional responses. This review is aimed at providing a summary of the mechanisms through which FcepsilonRI engagement can generate negative signals and regulate mast-cell function. Similar mechanisms are employed by other receptors expressed by immune cells, such as T cell and B cell receptors, pointing to a general concept in negative immunoreceptor signaling.

Targeting Kit Activation: A Potential Therapeutic Approach in the Treatment of Allergic Inflammation

The prevalence of allergic diseases is increasing worldwide. Hence, there is continued need for novel pharmacological therapies for the treatment of these disorders. As the mast cell is one of the essential cells that contributes to the inflammation associated with allergic diseases, this cell type remains an attractive target for such pharmacological intervention. Mast cells are major players in the early phase of the allergic response since they generate and release a variety of inflammatory mediators following antigen-dependent aggregation of IgE-bound FcepsilonRI (high affinity IgE-receptor) on the cell surface. These mediators also contribute to the late and chronic stages of allergic inflammation. Thus, the IgE/antigen response has been a major focus in the development of new drugs targeting mast cells. The essential role that stem cell factor (SCF) and its receptor, Kit, play in mast cell biology, however, may provide us with an alternative or adjunct therapy. SCF is necessary for mast cell development, proliferation and survival, but it is also known to play a role in homing and adhesion of mast cells. Furthermore, there is an increasing amount of literature demonstrating that SCF is necessary for optimal IgE/antigen-induced mast cell degranulation and cytokine production. Several drug candidates targeting SCF and/or Kit have been studied for their anti-allergic properties. These include anti-SCF antibodies, antisense oligonucleotides, Kit inhibitors, and inhibitors of downstream signaling molecules. In this review, we provide an overview of the role of SCF and Kit in mast cell activation and discuss potential drug candidates for targeting this response.

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcepsilonRI in mast cells triggers the influx of calcium (Ca(2+)) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca(2+) influx in mast cells. Trpm4(-/-) bone marrow-derived mast cells had more Ca(2+) entry than did TRPM4(+/+) cells after FcepsilonRI stimulation. Consequently, Trpm4(-/-) bone marrow-derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4(-/-) mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca(2+) entry in mast cells.

Decoding IgE Fc receptors

Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases by interacting with two membrane receptors: high-affinity FcepsilonRI and low-affinity FcepsilonRII (CD23). Allergeninduced IgE-occupied FcepsilonRI aggregation on the mast cell or basophil cell surface leads to the activation of intracellular signaling events and eventually the release of pre-formed and de novo synthesized inflammatory mediators. The role of FcepsilonRII in allergic diseases has been proposed to include regulation of IgE synthesis, enhanced histamine release from basophils, and a contribution to Ag-IgE complex presentation but the exact function of CD23 remains poorly understood. This review summarizes some new developments in IgE Fc-receptor studies with an emphasis on regulation of FcepsilonRI expression and signal transduction, including monomeric IgE, lipid raft segregation, and some recently identified negative regulators. A better understanding of signaling events following IgE FcR aggregation will shed new light on how allergy patients might be treated more safely and effectively.

Mast cells can promote the development of multiple features of chronic asthma in mice

Bronchial asthma, the most prevalent cause of significant respiratory morbidity in the developed world, typically is a chronic disorder associated with long-term changes in the airways. We developed a mouse model of chronic asthma that results in markedly increased numbers of airway mast cells, enhanced airway responses to methacholine or antigen, chronic inflammation including infiltration with eosinophils and lymphocytes, airway epithelial goblet cell hyperplasia, enhanced expression of the mucin genes Muc5ac and Muc5b, and increased levels of lung collagen. Using mast cell-deficient (Kit(W-sh/W-sh) and/or Kit(W/W-v)) mice engrafted with FcRgamma+/+ or FcRgamma-/- mast cells, we found that mast cells were required for the full development of each of these features of the model. However, some features also were expressed, although usually at less than wild-type levels, in mice whose mast cells lacked FcRgamma and therefore could not be activated by either antigen- and IgE-dependent aggregation of Fc epsilonRI or the binding of antigen-IgG1 immune complexes to Fc gammaRIII. These findings demonstrate that mast cells can contribute to the development of multiple features of chronic asthma in mice and identify both Fc Rgamma-dependent and Fc Rgamma-independent pathways of mast cell activation as important for the expression of key features of this asthma model.

FcεRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells

Topical application of a glucocorticoid is now widely recognized as the first-line therapy for bronchial asthma. However, glucocorticoid treatment is largely ineffective in relation to overproduction of sputum and lung tissue remodeling. The purpose of the current study was to identify human mast cell (MC) products that are related to goblet cell hyperplasia. The FcepsilonRI-mediated gene expression profile of MCs was examined by using high-density oligonucleotide probe arrays and RT-PCR. Secretion of a protein, amphiregulin, by the MCs was measured by ELISA. Upregulation of mucin genes in NCI-H292 cells by amphiregulin was evaluated by real-time RT-PCR. The expression levels of amphiregulin on histological sections obtained from 40 subjects with asthma and 6 healthy control subjects were estimated by immunohistochemical staining, and the correlation with the number of goblet cells was studied. Amphiregulin was secreted by human MCs after aggregation of FcepsilonRI, and its expression was not inhibited by a glucocorticoid (dexamethasone). Amphiregulin upregulated mucin gene expression in airway epithelial cells. Upregulation of amphiregulin expression was observed in MCs of patients with asthma, but not normal control subjects. Furthermore, upregulation of amphiregulin in MCs significantly correlated with the extent of goblet cell hyperplasia in the mucosa of patients with bronchial asthma. These results suggest that after exposure to antigens, human MCs may induce sputum production via release of amphiregulin. Therefore, amphiregulin may be a new target molecule for treatment of overproduction of sputum in bronchial asthma.

Early divergence of Fc epsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival.

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcepsilonRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcepsilonRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcepsilonRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcepsilonRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.

Signaling assemblies formed in mast cells activated via Fcepsilon receptor I dimers.

Although aggregation of the Fcepsilon receptor I (FcepsilonRI) is necessary for Ag-mediated mast cell triggering, the relationship between the extent of the FcepsilonRI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by FcepsilonRI dimers, elicited by binding of anti-FcepsilonRI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated FcepsilonRI, receptor dimers (1) induced a less extensive association of FcepsilonRI with detergent-resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross-linking mAb, and (5) failed to produce clusters of FcepsilonRI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of FcepsilonRI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that FcepsilonRI-activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized FcepsilonRI; formation of large FcepsilonRI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.

The Phospholipase Cγ1-dependent Pathway of FcϵRI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.

Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI.

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcepsilonRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcepsilonRI. Highly cytokinergic IgEs can efficiently induce production of cytokines and render mast cells resistant to apoptosis in an autocrine fashion, whereas poorly cytokinergic IgEs induce these effects inefficiently. Highly cytokinergic IgEs seem to induce more extensive FcepsilonRI aggregation than do poorly cytokinergic IgEs, which leads to stronger mast cell activation and survival effects. These effects of both types of IgEs require Syk tyrosine kinase and can be inhibited by FcepsilonRI disaggregation with monovalent hapten. In hybridoma-transplanted mice, mucosal mast cell numbers correlate with serum IgE levels. Therefore, survival effects of IgE could contribute to the pathogenesis of allergic disease.

Investigation of early events in Fc epsilon RI-mediated signaling using a detailed mathematical model.

Aggregation of Fc epsilon RI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the beta and gamma subunits of Fc epsilon RI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving Fc epsilon RI, Lyn, Syk, and a bivalent ligand that aggregates Fc(epsilon)RI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the beta- and gamma-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the beta ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the beta ITAM will act as an inhibitor.