Mast cells are crucial effector cells for the initiation of immediate hypersensitivity and also play critical roles in innate and acquired immunity. We have been studying mast cell signal transduction activated by cross-linking of the high-affinity IgE receptor (FceRI). In addition to signal transduction, we have been interested in IgE-dependent mast cell biology. In this regard our recent study has demonstrated that IgE binding to FceRI without deliberate incubation with antigen renders mast cells resistant to growth factor depletion-induced apoptosis. Substantial progress in the following areas has been made for the past few years.

Regulation of Bruton's tyrosine kinase (Btk) in mast cells

We previously showed that Btk plays a critical role in FceRI-induced mast cell activation, particularly in cytokine production through the activation of JNK1/2, one of three major MAP kinase subfamilies activated by FceRI stimulation. More recently, we defined the signaling pathway upstream and downstream of Btk leading to JNK: Protein kinase C (PKC)bI is strongly activated upon FceRI cross-linking and specifically regulated by Lyn, Syk, and Btk. PKCbI in turn regulates JNK and its downstream events leading to the production and secretion of cytokines such as IL-2 and TNF-a. Following these studies (PNAS 97:7423, 2000), we have been focusing on how PKC and other serine/threonine kinases regulate the activity of Btk. We have so far identified two serine residues, Ser-51 and Ser-180, of Btk as candidate target sites of phosphorylation by Akt (a crucial survival kinase) and PKC, respectively. By expressing phosphorylation site mutants in btk null mast cells, we, in collaboration with David J. Rawlings, University of Washington, have confirmed the negative regulatory effects of phosphorylation of these residues on Btk activity (EMBO J 20:5697, 2001). Our collaborative study also demonstrated that PKCb is specifically required for BCR- and FceRI-mediated NF-kB activation. Defects in this Btk/PKCb/IKK/NF-kB pathway and the Btk/PKCbI/JNK pathway may be the molecular bases of the phenotypic similarities found in btk and PKCb gene knockout mice. B cells from PKCb-deficient mice failed to recruit the IKK complex into lipid rafts, activate IKK, degrade IkB or up-regulate NF-kB-dependent survival signals. Inhibition of PKCb promoted cell death in B lymphomas characterized by exaggerated NF-kB activity. Together, these data define an essential role for PKCb in BCR survival signaling and imply that PKCbis a key therapeutic target for B-lineage malignancies (Nature Immunol. 3:780, 2002).

Novel Ras activation pathway dependent on PKC and Syk

Recently we have found that PKCa and PKCbI are phosphorylated on Tyr-658 and Tyr-662, respectively, in the membrane compartment of FceRI-stimulated mast cells. These tyrosines are phosphorylated by Syk, a protein-tyrosine kinase with two tandem SH2 domains. These phosphorylations do not change the activity of these PKC isoforms. Importantly, however, they create the binding site for the SH2 domain of the adaptor protein Grb-2. Sos, a guanine nucleotide exchange factor for Ras, complexed with Grb-2 is thought to be then recruited to the plasma membrane to activate Ras. This hypothesis was supported by our results that levels of active, GTP-bound Ras and ERK activation, a major event downstream of Ras, are lower in PKCb null mast cells than in wild-type controls upon FceRI stimulation. Furthermore, this notion was also supported by experiments using G?6976 that inhibits conventional isoforms of PKC (a, bI, bII, and y) and chronic PMA treatment that downregulates PMA-sensitive isoforms (both conventional and novel isoforms). This PKC/Syk-dependent pathway for Ras activation has solved a long-term enigma of how Ras is activated by PKC.

Monomeric IgE-induced anti-apoptotic effect on mast cells

Two recent studies including ours demonstrated that mouse mast cell survival and growth are promoted by monomeric IgE binding to FceRI (Immunity, 14:791, 2001; Immunity 14:801, 2001). These observations, together with earlier studies on IgE-induced upregulation of cell surface expression of FceRI, indicate that IgE binding to FceRI is not simply a "sensitization" step prior to stimulation with antigen, but it is an active signaling process. However, the two studies differ in their potential anti-apoptotic mechanisms: Krystal?s group showed that IgE-bound mast cells produce and secrete a variety of cytokines and render the cells resistant to growth factor deprivation-induced cell death in an autocrine fashion. Consistent with robust cytokine production and prolonged survival, the IgE-bound mast cells exhibit enhanced Bcl-XL and activation of MAP kinases and Akt. By contrast, we did not detect the secretion of any cytokines or activation of MAP kinases and Akt. The anti-apoptotic process does not involve the FasL/Fas death pathway or several Bcl-2 family proteins. To resolve the differences, we have compared anti-apoptotic ability of various monoclonal IgE molecules. Our results demonstrate that all of the various IgE molecules tested show anti-apoptotic effects on mast cells, but that the different IgEs exhibit a wide spectrum in their ability to induce the production and secretion of cytokines by mast cells. At one extreme, the most "highly cytokinergic" (HC) IgEs induce strong anti-apoptotic effects by an autocrine mechanism. At the other end of the spectrum, "poorly cytokinergic" (PC) IgEs induce less robust survival effects, but without inducing detectable cytokine production. Importantly, several lines of evidence indicate that binding of both HC and PC IgEs leads to FceRI aggregation in the absence of specific antigen, with more extensive FceRI aggregation induced by HC than by PC IgEs. Thus, HC IgEs induce degranulation, leukotriene release, receptor internalization, and proliferation. These IgE activities require Syk and are inhibited by monovalent hapten. In IgE hybridoma-transplanted mice, mast cell numbers in some mucosal tissues are correlated with serum IgE levels. Therefore, two types of IgE may affect the pathogenetic process of allergy in a distinctly different manner (Kitaura et al., submitted).