Steel factor enhances supraoptimal antigen-induced IL-6 production from mast cells via activation of protein kinase C-beta.

Ag-triggered mast cell (MC) activation follows a bell-shaped dose-response curve. Reduced activation in response to supraoptimal Ag concentrations is thought to be due to preferential engagement of inhibitory-acting proteins like SHIP1, Lyn, and protein kinase C (PKC)-delta. We show in this study that short-term prestimulation with Steel factor (SF) prevents supraoptimal Ag inhibition, resulting in synergistic MC degranulation and IL-6 secretion. These events are preceded by synergistic phosphorylation/activation of numerous signaling proteins, e.g., Erk, p38, and LAT. However, these effects of prestimulation with SF appear not to be due to reduced engagement of the attenuator SHIP1. Pharmacological analyses suggest that the activation of conventional PKCs is important for this synergy. Specifically, although we found that the conventional PKC inhibitor, Gö6976, likely has some PKC-independent targets in MCs, it led us to further studies that established SF plus Ag-induced IL-6 secretion was severely impaired in PKC-beta(-/-) MCs, but not PKC-alpha(-/-) MCs. Thus, PKC-beta joins PI3K and Btk as important players in this synergistic MC activation.

Requirement for CD45 in fine-tuning mast cell responses mediated by different ligand-receptor systems.

The receptor-like protein tyrosine phosphatase CD45, the most abundant cell surface phosphatase on all nucleated hemopoietic cells, is a critical regulator of the activation status of Src family kinases (SFKs). To study the impact of CD45 on mast cell function, we compare bone marrow-derived mast cells (BMMCs) from CD45-deficient mice and from mice expressing an activating point mutation (E613R) in the juxtamembrane wedge of CD45. In response to Ag-triggered FcepsilonR1-mediated activation, CD45-deficient BMMCs exhibit increased inhibitory Lyn phosphorylation and drastically reduced effector functions (degranulation and cytokine secretion). In contrast, CD45 E613R BMMCs show stronger effector functions after Ag-triggering than wild-type (WT) BMMCs. Despite these dichotomous phenotypes, phosphorylation of the inhibitory tyrosine in the SFK Lyn of CD45 E613R BMMCs is comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fyn-regulated phosphatidylinositol-3-kinase pathway. Interestingly, depending on the receptor system addressed, CD45-deficient and CD45 E613R BMMCs show uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or SF is enhanced compared to WT BMMCs. Together, the data indicate that CD45 plays a complex and essential role in fine-tuning mast cell responses mediated by different ligand-receptor systems.

Stimulation of mast cells via FcvarepsilonR1 and TLR2: the type of ligand determines the outcome.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam(3)CSK(4) and MALP-2, on Ag (DNP-HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam(3)CSK(4), but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam(3)CSK(4), most probably by electrostatic forces, reduced the level of active DNP-HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE+Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam(3)CSK(4) indicate that one must be cautious when interpretating results obtained with "model" substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcepsilonR1) and TLR2 are examined.

Insulin and insulin-like growth factor-1 promote mast cell survival via activation of the phosphatidylinositol-3-kinase pathway.

OBJECTIVE: Mast cells (MCs) play central roles for the onset and development of immediate-type and inflammatory allergic reactions. Since the inverse relationship between atopic disorders and diabetes mellitus has been observed in animals and humans, we investigated the effects of insulin (Ins) on MC signaling and biological function. METHODS: In bone marrow-derived MCs (BMMCs) from wild-type as well as SHIP-deficient mice Ins as well as insulin-like growth factor-1 (IGF-I)-triggered intracellular signaling events and MC effector functions were studied. RESULTS: We found that the addition of either Ins or IGF-1 to BMMCs triggers the phosphorylation of protein kinase B (PKB) and p38 kinase but not extracellular signal-regulated kinase (Erk). We also found that Ins/IGF-1 stimulates the tyrosine phosphorylation of SHIP1 and, in keeping with this, Ins/IGF-1-induced PKB phosphorylation is higher in SHIP1-/- BMMCs and is inhibited in SHIP+/+ as well as SHIP1-/- BMMCs with inhibitors of phosphatidylinositol-3-kinase (PI3K). Ins/IGF-1, like antigen (Ag), also stimulates the Rac-dependent activation of PAK as well as the production of hydrogen peroxide (H2O2). To elucidate the role of Ins and IGF-1 in MC biology, we studied their effects on Ag-mediated degranulation and MC survival. Although both only slightly enhanced Ag-mediated degranulation, they significantly promoted MC survival in the absence of IL-3 in a PI3K-dependent manner. CONCLUSION: The promotion of BMMC survival by induction of Ins/IGF-1 signaling may, in part, be responsible for the inverse correlation observed between atopic disorders and diabetes mellitus.