Critical Role of Protein Kinase C βII in Activation of Mast Cells by Monomeric IgE

Abstract

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 Fc⑀RI, 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 Fc⑀RI 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 PKC␤II, although Ca 2؉ influx induced by cross-linking of the Fc⑀RI was intact. Transfection of PKC␤II cDNA was found to restore the Ca 2؉ influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKC␤II (PKC␤II/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 PKC␤II/T500V was found not to affect the antigen-induced responses. These results suggest that PKC␤II plays a critical role in monomeric IgE responses, but not in antigen responses.Activation of mast cells triggers allergic and inflammatory responses through the release of a wide variety of mediators, such as histamine, arachidonic acid metabolites, and neutral proteases, and modulates immune responses through the production of cytokines and chemokines (1, 2). One of the prominent mechanisms for activation of mast cells is cross-linking of the Fc⑀RI, the high affinity receptor for IgE, by the multivalent antigen, and various signaling molecules have been identified in this pathway (3, 4). Furthermore, accumulating evidence has indicated that IgE-mediated activation of mast cells can occur even in the absence of the multivalent antigen (5). Sensitization of IL-3 2 -dependent mouse bone marrow-derived mast cells (BMMCs) with IgE induces an array of events, such as up-regulation of the Fc⑀RI (6, 7), resistance to apoptosis under IL-3 deprivation (8 -11), cytokine production (9, 10), histamine synthesis (12), and adhesion to fibronectin (13,14). These results have clearly indicated that sensitization with IgE (monomeric IgE) is able to activate mast cells in the absence of antigen. However, it remains to be clarified as to how monomeric IgE activates mast cells and what kinds of signaling molecules are involved in this pathway.Some preceding studies revealed that signaling molecules are shared between monomeric IgE responses and responses induced by crosslinking with multivalent antigen; weak but sustained tyrosine phosphorylation of several signaling components, which a...

Positive and negative regulatory mechanisms in high-affinity IgE receptor-mediated mast cell activation

“…Even though the precise signaling pathways have not yet been elucidated, a number of signaling molecules have been identified, including the FcεRI β-chain [38], MAPKs [38,74], PKC [60,74], PLC, PLD and the nuclear transcription factor NFAT [80]. It is possible that monomeric IgE activates the same signaling pathways as antigen-IgE, but further research is needed to confirm the precise signaling network that is activated by monomeric IgE.…”

Altered MicroRNA Expression Profiles in Activated Mast Cells Following IgE-FcεRI Cross-Linking with Antigen

“…Additionally, accumulating evidence has demonstrated that IgE can also induce a wide variety of biological responses of mast cells even in the absence of antigen, such as up-regulation of the FcεRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, which is referred to as "monomeric IgE" responses [8][9][10][11][12]. Several intercellular molecules, such as spleen tyrosine kinase (Sky) and phospholipase Cγ (PLCγ), are involved in the signaling cascade following IgE binding to FcεRI or FcεRI aggregation, and regulate a series of IgE-mediated biological processes in stimulated mast cells [9,[13][14][15]. However, in these processes, the regulatory mechanisms that are involved in the expression of these signal molecules have not been extensively studied.…”

Impaired activation of mast cells upon IgE-mediated antigen stimulation in a stroke-prone spontaneously hypertensive rat strain, SHRSP.Z

“…The lysate was centrifuged at 10,000 × g for 30 min at 4 • C. Activities of three different types of granule proteases and histamine content in the resultant supernatants were measured as previously described [8,9].…”

“…Immunoblot analysis was performed as described previously [9] using antibodies raised against phosphotyrosine (clone 4G10, Millipore, Billerica, MA), LAT, phospholipase C-␥1 (Millipore), Lyn, Fyn (Santa Cruz, CA), phosphorylated Syk (Tyr525/526), phsophorylated p44/42 MAP kinase (phosphorylated ERK, Thr202/Tyr204), phosphorylated LAT (Tyr171), Syk, p44/42 MAP kinase (Cell Signaling Technology, Danvers, MA), and actin (Chemicon, Temecula, CA). …”