Claudia Gonzalez-Espinosa

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcεRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcεRI also uses a Fyn kinase–dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcεRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Transactivation of Sphingosine-1–Phosphate Receptors by FcεRI Triggering Is Required for Normal Mast Cell Degranulation and Chemotaxis

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcɛRI) in RBL-2H3 and bone marrow–derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1–phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcɛRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcɛRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcɛRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcɛRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

Negative Regulation of Immunoglobulin E–dependent Allergic Responses by Lyn Kinase

A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn − / − bone marrow–derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn − / − mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn − / − mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcεRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcεRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn − / − mice because both lyn − / − and lyn − / − fyn − / − mice showed high IgE levels. Thus, lyn − / − mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.

Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells.

ABSTRACT The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcɛRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcɛRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cγ1 (PLCγ1) and PLCγ2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCγ1 and PLCγ2 and calcium responses. Thus, Vav1 is essential to FcɛRI-mediated activation of PLCγ and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCγ-activated calcium signals.

Preferential Signaling and Induction of Allergy-promoting Lymphokines Upon Weak Stimulation of the High Affinity IgE Receptor on Mast Cells

Mast cell degranulation and de novo cytokine production is a consequence of antigen-aggregation of the immunoglobulin E (IgE)-occupied high affinity receptor for IgE (FcɛRI). Herein, we report that lymphokines that promote allergic inflammation, like MCP-1, were potently induced at low antigen (Ag) concentrations or at low receptor occupancy with IgE whereas some that down-regulate this response, like interleukin (IL)-10, required high receptor occupancy. Weak stimulation of mast cells caused minimal degranulation whereas a half-maximal secretory response was observed for chemokines and, with the exception of TNF-α, a weaker cytokine secretory response was observed. The medium from weakly stimulated mast cells elicited a monocyte/macrophage chemotactic response similar to that observed at high receptor occupancy. Weak stimulation also favored the phosphorylation of Gab2 and p38MAPK, while LAT and ERK2 phosphorylation was induced by a stronger stimulus. Gab2-deficient mast cells were severely impaired in chemokine mRNA induction whereas LAT-deficient mast cells showed a more pronounced defect in cytokines. These findings demonstrate that perturbation of small numbers of IgE receptors on mast cells favors certain signals that contribute to a lymphokine response that can mediate allergic inflammation.

Impaired FcRI-Dependent Gene Expression and Defective Eicosanoid and Cytokine Production as a Consequence of Fyn Deficiency in Mast Cells 1

Fyn kinase is a key contributor in coupling FcεRI to mast cell degranulation. A limited macroarray analysis of FcεRI-induced gene expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production. Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1β). FcεRI-induced generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies revealed that JNK activity was associated with generation of arachidonic acid. FcεRI-mediated activation of IκB kinase β and IκBα phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-κB DNA binding activity that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate a selective negative regulatory role for certain cytokines.

Macromolecular protein signaling complexes and mast cell responses: a view of the organization of IgE-dependent mast cell signaling

The generation of signals following engagement of cell surface receptors is an ordered process that requires tight regulation as spurious signals could result in unwanted, and possibly deleterious, cellular responses. Like other cell surface receptors, stimulation of a mast cell via the high affinity IgE receptor (FcepsilonRI) causes multiple biochemical events that ultimately result in cell activation and effector responses. Recently, our knowledge of how these events are ordered has increased. We now have identified some of the molecules involved, how they are organized into macromolecular complexes by FcepsilonRI stimulation, and the role of some of the constituents of these macromolecular signaling complexes in mast cell effector responses. In brief, we review the knowledge on macromolecular signaling complexes used by FcepsilonRI in mast cell activation and provide our view on the regulation of signal generation and its effect on mast cell activation.

A Perspective: Regulation of IgE Receptor-Mediated Mast Cell Responses by a LAT-Organized Plasma Membrane-Localized Signaling Complex

Background: To understand how the high-affinity IgE receptor (FcΕRI) communicates with downstream effectors, we focused on exploring the functional importance of the FcΕRI-mediated formation and localization of a signaling complex that contains the hematopoietic cell-specific scaffolding protein linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. Methods: Using the mast cell line RBL-2H3, we explored the localization of these proteins by confocal microscopy and cell fractionation. Additionally, the mechanism of function and the importance of LAT and Vav1 to mast cells was studied in genetically disrupted mice and in mast cells derived from their bone marrow. Results: We found that LAT, Vav1 and the adapter molecule SLP-76 associated in detergent-resistant microdomains (lipid rafts) found in the plasma membrane upon FcΕRI stimulation. In the absence of LAT, mast cells showed a remarkable loss of the secretory response and reduced cytokine responses. Vav1 deficiency also affected secretion, although not to the extent of LAT deficiency, and inhibited IL-2 and IFN-γ production. LAT- and Vav1-deficient mice showed reduced blood histamine levels after a systemic anaphylaxis challenge as compared to their normal counterparts. Conclusions: The results demonstrate that LAT is a central mediator in IgE receptor signaling by regulating multiple signaling pathways that affect mast cell degranulation and cytokine production. Vav1, a component of this LAT-containing signaling complex, regulates a specific subset of these responses.

Rethinking the role of Src family protein tyrosine kinases in the allergic response: new insights on the functional coupling of the high affinity IgE receptor.

Antigen-induced cross-linking of immunoglobulin E (IgE) antibodies bound to the high-affinity IgE receptor (Fc∈RI), on mast cells results in the release of mediators that initiate an inflammatory response. This normal immune response has been abducted by immunological adaptation, through the production of IgE antibodies to normally innocu-our substances, to cause allergic disease. Therefore, understanding the molecular requirements in IgE-dependent mast-cell activation holds promise for therapeutic intervention in disease. Recent investigation on the functional coupling of F∈RI to the intracellular signaling apparatus has provided paradigm-altering insights on the importance and function of Src family protein tyrosine kinases (Src PTK) in mast-cell activation. In this synopsis, we review the current knowledge on the role of the Src PTKs, Fyn and Lyn, in mast-cell activation and discuss the implications of our findings on allergic disease.