INTRODUCTION: Mast cells when stimulated by antigen via the high affinity receptor for IgE (FcepsilonRI) release a variety of inflammatory factors by three mechanisms, degranulation, rapid synthesis of inflammatory lipids (eicosanoids) derived from arachidonic acid, and the generation of cytokines via gene transcription. Our general objective is to delineate the signaling pathways for each of these mechanisms and determine how therapeutic agents interact with these pathways. We have shown that degranulation is regulated primarily by changes in cytosolic Ca2+ (the calcium signal), protein kinase (PK) C, and phospholipase (PL) D (see accompanying report HL000993-18 LMI) whereas the generation of eicosanoids is dependent on a calcium signal and activation of mitogen activated protein (MAP) kinases. Expression of PLA2, the enzyme responsible for the generation of arachidonic acid, and downstream enzymes that convert arachidonic acid to eicosanoids such as cyclooxygenase-2 (COX-2) and 5-lipoxygenase is regulated by p38 MAP kinase. The activation of PLA2 itself requires phosphorylation by the MAP kinase, ERK1/2, and a calcium signal to promote the association of PLA2 with membrane substrates. The generation of cytokines is driven by induction/activation of a variety of transcription factors that are regulated by Ca2+, PKC, MAP kinases, and other signaling pathways. Most of these signalling processes as well as release of inflammatory mediators are potently suppressed by glucocorticoids (see previous reports in this series). CURRENT OBJECTIVES: This year we have investigated in more detail the regulation of cytokine production particulary the synergistic interactions of antigen with other mast cell stimulants such as stem cell factor (SCF), a mast cell growth factor, and pathogenic ligands of Toll-like receptors (TLRs). We have continued studies with glucocorticoids and have identified two key signaling mechanisms that are disrupted by dexamethasone, our prototypic glucocorticoid. Studies were conducted not only with rodent mast cell lines but also with human mast cells that were derived from peripheral blood pluripotent CD34+ cells because the receptor for SCF, c-KIT (CD117), is constitutively active in tumor mast cell lines. The new findings are as follows. SYNERGISTIC INTERACTIONS BETWEEN ANTIGEN AND SCF. In human mast cells, SCF failed to induce degranulation but acted in synergy with antigen to markedly enhance degranulation, levels of cytokine gene transcripts, and production of cytokines. Further examination revealed that antigen and SCF activated common and unique signaling pathways to account for these varied responses. SCF, unlike antigen, failed to activate PKC but activated PLC-gamma and calcium mobilization and augmented these signals as well as degranulation when added together with antigen. Both SCF and antigen induced signals that are associated with cytokine production namely, phosphorylation of the MAP kinases, phosphatidylinositol (PI) 3-kinase-dependent phosphorylation of Akt, and activation of the transcription factors, NF-kappa-B and NFAT. However, only SCF stimulated phosphorylation of STAT5 and STAT6 while antigen weakly stimulated the PKC-dependent induction and phosphorylation of c-Jun and associated AP-1 components, an action that was markedly potentiated by co-stimulation with SCF. Interestingly, most signals were down-regulated on continuous exposure to SCF (the physiologic situation) but were reactivated along with cytokine gene transcription on addition of antigen. This down regulation could serve to limit constitutive production of inflammatory cytokines under normal physiologic conditions. The findings, in total, indicated that a combination of Fc-epsilon-RI and KIT mediated signals and transcriptional processes were required for optimal physiologic responses of human mast cells to antigen Fc-epsilon-RI and KIT. The studies show for the first timme that the pattern of signals and cytokines produced in response to antigen varies depending on the prior exposure of mast cells to SCF and possibility other growth factors. GENERATION OF CYTOKINES ON STIMULATION OF MAST CELLS VIA TLRs: In addition to mediating IgE-dependent allergic reactions via Fc-epsilon-RI, mast cells are believed to play a critical protective role against acute bacterial and parasitic infections by production of inflammatory cytokines through the activation of TLRs. Studies with MC/9 and other mast cell lines revealed that activators of TLR2 (tripaltmitoyl Cys-Ser-(Lys)4, Pam3Cys) and TLR4 (lipopolysacchide, LPS) stimulated production of inflammatory cytokines and potentiated suc responses to antigen. However, neither Pam3Cys nor LPS stimulated degranulation and release of arachidonic acid or potentiated such responses to antigen. Examination of various signaling events also revealed that, as with SCF, Fc-epsilon-RI and TLRs mediated signals in common as well as signals that are unique to TLRs or to Fc-epsilon-R1. Stimulation of cells through both receptors resulted in activation of a wider array of cytokine gene related transcription factors than that induced by stimulation through individual receptors. The ability of TLR ligands to augment release of cytokines in antigen-stimulated cells may account for the exacerbation of IgE-mediated allergic diseases by pathogenic agents. IDENTIFICATION OF PRIMARY SITES OF ACTIONS OF GLUCORTICOIDS: Two sites were identified. One was the disruption of the interaction of PI 3-kinase with the adaptor protein, Gab2, and as a consequence downstream events that lead to degranulation (see accompanying report HL000993-18 LMI) and pathways (as yet undefined) that lead to transcription of inducible cytokines. This action is of slow onset (12 hrs for maximum effect), of long duration (24 hrs or more), and apparent with clinically relevant concentrations of dexamethasone (1-10 nM). Studies with related and unrelated steroids indicated that disruption was mediated through the glucorticoid receptor. The second site was identified through gene chip analysis. Treatment of RBL-2H3 mast cells with dexamethasone revealed that although transcriptional activity of genes for numerous proteins were either up or down regulated none of the proteins that are commonly associated with mast cell signaling mechanisms showed significant changes in expression of their mRNA. Of note, however, was a substantial increase in mRNA for the the inhibitory adaptor protein, downstream of tyrosine kinase (Dok)-1, which was previously shown to inhibit Ras activation by inhibiting the Ras GTPase-activating protein (RasGAP)in B cells. The increase in Dok-1 mRNA was accompanied by a fourfold increase in Dok-1 protein. The upregulation was of slow onset and apparent with as little as 1 nM to 10 nM dexamethasone--features that are reminiscent of the inhibition of the Erk pathway by dexamethasone. Treatment with dexamethasone resulted in enhanced tyrosine phosphorylation of Dok-1, augmented recruitment of RasGAP by Dok-1, and as in previous studies suppressed activation of the Erk pathway and recruitment of Raf-1 by Ras in antigen-stimulated cells. The same effects were obtained by transient overexpression of Dok-1but not by overexpression of Dok-1 that was mutated in RasGAP-binding domain. The negative regulatory role of Dok-1 was further validated by expression of small interfering RNA directed against Dok-1 which enhanced activation of MAP kinase and subsequent release of arachidonic acid. These findings identify Dok-1 as mediator of the anti-allergic actions of dexamethasone and as a negative regulator of the MAP kinase pathway and downstream events such as the release of arachidonic acid.
Showing the most recent 10 out of 13 publications
