Jiro Kitaura

Regulation of Mast Cell Survival by IgE

Mast cells play critical roles in hypersensitivity and in defense against certain parasites. We provide evidence that mouse mast cell survival and growth are promoted by monomeric IgE binding to its high-affinity receptor, Fc epsilon RI. Monomeric IgE does not promote DNA synthesis but suppresses the apoptosis induced by growth factor deprivation. This antiapoptotic effect occurs in parallel with IgE-induced increases in Fc epsilon RI surface expression but requires the continuous presence of IgE. This process does not involve the FasL/Fas death pathway or several Bcl-2 family proteins and induces a distinctly different signal than Fc epsilon RI cross-linking. The ability of IgE to enhance mast cell survival and Fc epsilon RI expression may contribute to amplified allergic reactions.

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

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcεRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcεRI. 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 FcεRI 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 FcεRI 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.

Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcεRI). In this study, we have made the following observations on growth properties and FcεRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcεRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-γ1 and C-γ2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcεRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-α and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Cα and protein kinase CβII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc∊RI

Aggregation of the high affinity receptor for IgE (FcεRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcεRI 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 FcεRI β subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcεRI β. The latter induced much higher levels of FcεRI β 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 FcεRI β, 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 FcεRI β showed that FcεRI β subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcεRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcεRI β-ITAM.

Mast Cell Survival and Activation by IgE in the Absence of Antigen: A Consideration of the Biologic Mechanisms and Relevance

Mast cells are not only major effector cells in allergy and host defense against parasites and bacteria but also important cellular components in other immune responses. Recent studies on the effects of monomeric IgE on mast cell survival and activation have made an impact on our view of the IgE binding to its high-affinity receptors, FcεRI. Traditionally, IgE binding to FcεRI has been considered as a passive action of “sensitization” before receptor aggregation by Ag. However, recent studies indicate that at high concentrations some monoclonal IgEs have effects on mast cells similar to or identical to those induced by IgE+Ag stimulation. These effects may be due to induction of FcεRI aggregation by these IgEs in the absence of Ag. This review will synthesize recent findings of the heterogeneity of IgEs in their ability to induce survival and activation events, their mechanisms, the potential in vivo significance of IgE-FcεRI interactions, and the implications of the mouse studies to human diseases.

AML1 mutations induced MDS and MDS/AML in a mouse BMT model

Myelodysplastic syndrome (MDS) is a hematopoietic stem-cell disorder characterized by trilineage dysplasia and susceptibility to acute myelogenous leukemia (AML). Analysis of molecular basis of MDS has been hampered by the heterogeneity of the disease. Recently, mutations of the transcription factor AML1/RUNX1 have been identified in 15% to 40% of MDS-refractory anemia with excess of blasts (RAEB) and MDS/AML. We performed mouse bone marrow transplantation (BMT) using bone marrow cells transduced with the AML1 mutants. Most mice developed MDS and MDS/AML-like symptoms within 4 to 13 months after BMT. Interestingly, among integration sites identified, Evi1 seemed to collaborate with an AML1 mutant harboring a point mutation in the Runt homology domain (D171N) to induce MDS/AML with an identical phenotype characterized by marked hepatosplenomegaly, myeloid dysplasia, leukocytosis, and biphenotypic surface markers. Collaboration between AML1-D171N and Evi1 was confirmed by a BMT model where coexpression of AML1-D171N and Evi1 induced acute leukemia of the same phenotype with much shorter latencies. On the other hand, a C-terminal truncated AML1 mutant (S291fsX300) induced pancytopenia with erythroid dysplasia in transplanted mice, followed by progression to MDS-RAEB or MDS/AML. Thus, we have developed a useful mouse model of MDS/AML that should help in the understanding of the molecular basis of MDS and the progression of MDS to overt leukemia.

PKCβ modulates antigen receptor signaling via regulation of Btk membrane localization

Mutations in Brutons tyrosine kinase (Btk) result in X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C‐β (PKCβ) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCβ‐deficient B cells. We provide direct evidence that PKCβ acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCβ results in a dramatic increase in B‐cell receptor (BCR)‐mediated Ca2+ signaling. We identified a highly conserved PKCβ serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcϵRI‐mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

O-methylated catechins from tea leaves inhibit multiple protein kinases in mast cells.

Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton’s tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.

Early Divergence of Fcε 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 (FcεRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcεRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcεRI 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 FcεRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.

A Ras activation pathway dependent on Syk phosphorylation of protein kinase C.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcεRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCβI and PKCα, respectively, are phosphorylated by Syk in the membrane compartment of FcεRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcεRI-stimulated mast cells.