Long term maintenance of IgE-mediated memory in mast cells in the absence of detectable serum IgE

Mast cells and basophils involved in allergic responses do not have clonotypic Ag receptors. However, they can acquire Ag specificity through binding of Ag-specific IgE to FcepsilonRI expressed on their surface. Previous studies demonstrated that IgE binding induced the stabilization and accumulation of FcepsilonRI on the cell surface and resulted in up-regulation of FcepsilonRI. In this study we have further analyzed the maintenance of IgE-mediated memory in mast cells and basophils in vivo by comparing kinetics of serum IgE levels, FcepsilonRI expression, and ability to induce systemic anaphylaxis. A single i.v. injection of trinitrophenyl-specific IgE induced 8-fold up-regulation of FcepsilonRI expression on peritoneal mast cells in B cell-deficient (micro m(-/-)) mice. Serum IgE levels became undetectable by day 6, but the treatment of mice with anti-IgE mAb induced a significant drop in body temperature on days 14, 28, and 42. The administration of trinitrophenyl -BSA, but not BSA, in place of anti-IgE mAb gave similar results, indicating the Ag specificity of the allergic response. This long term maintenance of Ag-specific reactivity in the allergic response was also observed in normal mice passively sensitized with IgE even though the duration was shorter than that in B cell-deficient mice. The appearance of IgE with a different specificity did not interfere with the maintenance of IgE-mediated memory of mast cells and basophils. These results suggest that IgE-mediated stabilization and up-regulation of FcepsilonRI enables mast cells and basophils not only to acquire Ag specificity, but also to maintain memory in vivo for lengthy periods of time.     

Different stabilities of the structurally related receptors for IgE and IgG on the cell surface are determined by length of the stalk region in their alpha-chains

A variant of the high affinity IgE receptor FcepsilonRI, which is composed of alpha- and gamma-chains without the beta-chain, is expressed on human APC, such as dendritic cells, and has been suggested to facilitate Ag uptake through IgE and hence to facilitate Ag presentation to T cells. The level of FcepsilonRI on these cells is correlated with the serum IgE concentration, suggesting IgE mediates the up-regulation of the alphagamma2-type FcepsilonRI. The IgE-mediated FcepsilonRI up-regulation on mast cells and basophils has been shown to enhance the ability of these cells to release chemical mediators and cytokines that are responsible for allergic inflammatory reactions. Here, to elucidate the mechanism controlling FcepsilonRI expression, we compared two structurally related Ig receptors, human FcepsilonRI and FcgammaRIIIA, which carry different alpha-chains but the same gamma-chains. The half-life of FcepsilonRI on the cell surface was short unless it bound IgE, whereas FcgammaRIIIA was stably expressed without IgG binding. Shuffling of the non Ig-binding portions of the FcepsilonRIalpha and FcgammaRIIIAalpha chains revealed that the stalk region was critical in determining the difference in their stability and ligand-induced up-regulation. Unexpectedly, analyses with added or deleted amino acids in the stalk region strongly suggested that the length rather than the amino acid sequence of the stalk region was of major importance in determining the different stabilities of FcepsilonRI and FcgammaRIIIA on the cell surface. This finding provides new insights into the mechanism regulating surface FcepsilonRI expression.     

Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo

The cross-linking of IgE-bound FcepsilonRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcepsilonRIgamma signaling to IgE/FcepsilonRI-mediated mast cell responses has not yet been elucidated. In this study FcepsilonRIgamma(-/-) mast cells were reconstituted with either wild-type or mutant FcepsilonRIgamma in transgenic mice and transfected mast cells in vitro. We demonstrate that FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcepsilonRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcepsilonRI is independent of FcepsilonRIgamma signaling. These results indicate that FcepsilonRIgamma-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.     

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.     

Critical role of protein kinase C betaII in activation of mast cells by monomeric IgE

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as "monomeric IgE" responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the FcepsilonRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the FcepsilonRI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca(2+) influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCbetaII, although Ca(2+) influx induced by cross-linking of the FcepsilonRI was intact. Transfection of PKCbetaII cDNA was found to restore the Ca(2+) influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKCbetaII (PKCbetaII/T500V) significantly suppressed the Ca(2+) influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCbetaII/T500V was found not to affect the antigen-induced responses. These results suggest that PKCbetaII plays a critical role in monomeric IgE responses, but not in antigen responses.     

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.     

Human eosinophils and human high affinity IgE receptor transgenic mouse eosinophils express low levels of high affinity IgE receptor, but release IL-10 upon receptor activation.

FcepsilonRI expressed by human eosinophils is involved in IgE-mediated cytotoxicity reactions toward the parasite Schistosoma mansoni in vitro. However, because receptor expression is low on these cells, its functional role is still controversial. In this study, we have measured surface and intracellular expression of FcepsilonRI by blood eosinophils from hypereosinophilic patients and normal donors. The number of unoccupied receptors corresponded to approximately 4,500 Ab binding sites per cell, whereas 50,000 Ab binding sites per cell were detected intracellularly. Eosinophils from patients displayed significantly more unoccupied receptors than cells from normal donors. This number correlated to both serum IgE concentrations and to membrane-bound IgE. The lack of FcepsilonRI expression by mouse eosinophils has hampered further studies. To overcome this fact and experimentally confirm our findings on human eosinophils, we engineered IL-5 x hFcepsilonRIalpha double-transgenic mice, whose bone marrow, blood, spleen, and peritoneal eosinophils expressed FcepsilonRI levels similar to levels of human eosinophils, after 4 days culture with IgE in the presence of IL-5. Both human and mouse eosinophils were able to secrete IL-10 upon FcepsilonRI engagement. Thus, comparative analysis of cells from patients and from a relevant animal model allowed us to clearly demonstrate that FcepsilonRI-mediated eosinophil activation leads to IL-10 secretion. Through FcepsilonRI expression, these cells are able to contribute to both the regulation of the immune response and to its effector mechanisms.     

Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and FcepsilonRI

Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

IL-10 suppresses mast cell IgE receptor expression and signaling in vitro and in vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcepsilonRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcepsilonRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.     

p28, a novel IgE receptor-associated protein, is a sensor of receptor occupation by its ligand in mast cells

Mast cells express the high affinity receptor for IgE (FcepsilonRI). Aggregation of this receptor by IgE and antigen leads to a signaling cascade resulting in the secretion of histamine, in the synthesis of other pro-inflammatory mediators such as leukotrienes and prostaglandins, and in the production of various cytokines, all of which participate in the development of the allergic reaction. In the last years, growing evidence accumulated that binding of IgEs to FcepsilonRI in itself induces active signals leading to mast cell survival, increased expression of FcepsilonRI, transient induction of histidine decarboxylase synthesis, and increased cell adhesion. The mechanisms underlying monomeric IgE signaling in the absence of receptor aggregation are still poorly understood. Here, we show that a protein of 28 kDa (p28) is physically and constitutively associated with FcepsilonRI in mast cells. Coimmunoprecipitation studies from (125)I surface-labeled cells demonstrated that this association involves at least 50% of membrane-expressed FcepsilonRI. After the addition of monomeric IgE to the cells, the p28.FcepsilonRI complex dissociates almost completely in less than 2 min. This dissociation is temperature-sensitive and is not due to the recruitment of additional proteins to the complex. Stripping bound IgE from the cells by acidic treatment promotes a rapid reassociation between p28 and FcepsilonRI. Altogether, these data are consistent with a conformational regulation of the complex. Thus, p28 is a sensor for FcepsilonRI occupation by IgE on mast cells, and its dissociation from the receptor could represent an early step of monomeric IgE signaling.     

Interaction of a monoclonal IgE-specific antibody with cell-surface IgE-Fc epsilon RI: characterization of equilibrium binding and secretory response

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.     

IL-10 inhibits Fc epsilon RI expression in mouse mast cells

FcepsilonRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcepsilonRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcepsilonRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcepsilonRI up-regulation. IL-10 and IL-4 reduced FcepsilonRI beta protein expression without altering the alpha or gamma subunits. The ability of IL-4 and IL-10 to alter FcepsilonRI expression by targeting the beta-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.     

TGF-beta 1 inhibits mast cell Fc epsilon RI expression

Mast cell activation through the high affinity IgE receptor (FcepsilonRI) is a critical component of atopic inflammation. The cytokine TGF-beta1 has been shown to inhibit IgE-dependent mast cell activation, possibly serving to dampen mast cell-mediated inflammatory responses. We present proof that TGF-beta1 inhibits mast cell FcepsilonRI expression through a reversible pathway that diminishes protein, but not mRNA, expression of the FcepsilonRI subunit proteins alpha, beta, and gamma. The stability of the expressed proteins and the assembled cell surface complex was unaltered by TGF-beta1 treatment. However, TGF-beta1 decreased the rate of FcepsilonRI beta-chain synthesis, arguing that this inhibitory cytokine exerts its effects at the level of mRNA translation. TGF-beta1 consistently diminished FcepsilonRI expression on cultured human or mouse mast cells as well as freshly isolated peritoneal mast cells. The related cytokines, TGF-beta2 and TGF-beta3, had similar effects. We propose that TGF-beta1 acts as a negative regulator of mast cell function, in part by decreasing FcepsilonRI expression.     

Cutting edge: CD4 T cell-mast cell interactions alter IgE receptor expression and signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4(+)CD25(+) regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4(+) T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcepsilonRI expression. Despite the known inhibitory functions of IL-10 and TGFbeta1, FcepsilonRI suppression was independent of IL-10 and TGF-beta1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C(4) production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcepsilonRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.     

Distinct aggregation of beta- and gamma-chains of the high-affinity IgE receptor on cross-linking.

The high-affinity IgE receptor (FcepsilonRI) on mast cells and basophils consists of a ligand-binding alpha-chain and two kinds of signaling chains, a beta-chain and disulfide-linked homodimeric gamma-chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/alpha-chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling beta- and gamma-chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of FcepsilonRI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of beta- and gamma-chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only gamma- but not beta-chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor beta- and gamma-chains do not co-aggregate but for the most part form homogenous aggregates of beta-chains or gamma-chains after crosslinking.     

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products

Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcepsilonRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (> or =0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcepsilonRI stimulation increased the cytosolic Ca(2+) levels to a similar extent, whereas GA, but not FcepsilonRI stimulation, caused mitochondrial Ca(2+) overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcepsilonRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca(2+) homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca(2+) overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.     

Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI

Aggregation of the high affinity receptor for IgE (FcepsilonRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcepsilonRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcepsilonRI beta subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcepsilonRI beta. The latter induced much higher levels of FcepsilonRI beta phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcepsilonRI beta, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcepsilonRI beta showed that FcepsilonRI beta subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcepsilonRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcepsilonRI beta-ITAM.     

IgE crosslinkage of Fcepsilon receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells

Since mast cells play pivotal roles in allergic inflammations, we investigated how IgE-mediated stimulation modulated mast cell matrix metalloproteinase (MMP)-9 production, and its enzymatic activation. In this study, we clearly demonstrated that proMMP-9 released from murine bone marrow-derived cultured mast cells (BMCMC) was activated to its valid form after crosslinking of surface immunoglobulin (Ig)E. Serine protease inhibitors sensitive to chymases inhibited the phenomenon, indicating that certain chymases may be responsible for activation of proMMP-9. Although binding of IgE to its specific receptors did not alter MMP-9 production, the IgE crosslinkage increased both expression of mRNA, and production of MMP-9 in mast cells. Glucocorticoid declined extra cellular processing of proMMP-9 without affecting mRNA expression. These findings give rise to the possibility that production and activation of mast cell MMP-9 may be increased in the affected sites, thereby resulting in an exacerbation of tissue degradation in inflammatory conditions.