Abstract Inappropriate activation of mast cells via the Fc ε RI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to Fc ε RI ‐initiated mediator release. However, until now the role of TRPC calcium channels in Fc ε RI ‐mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC 1 and 6 in primary human lung mast cells and the LAD 2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to Fc ε RI ‐mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to Fc ε RI ‐mediated signaling in human mast cells. Although, the use of a TRPC 3/6 selective antagonist and agonist ( GSK ‐3503A and GSK ‐2934A, respectively) did not reveal evidence for TRPC 6 contribution to Fc ε RI ‐mediated calcium signaling in human mast cells. Similarly, inactivation of STIM 1‐regulated TRPC 1 in human mast cells (as tested by transfecting cells with STIM 1‐ KK 684‐685 EE ‐ TRPC 1 gating mutant) failed to alter Fc ε RI ‐mediated calcium signaling in LAD 2 human mast cells. Mediator release assays confirm that Fc ε RI ‐mediated calcium influx through Orai is necessary for histamine and TNF α release but is differentially involved in the generation of cytokines and eicosanoids.
Alveolar macrophages were treated with carbon blacks and adsorbates in order to evaluate the biologic effect of adsorbate, adsorbent and adsorbate-adsorbent complexes. Their capacity to phagocytize a subsequent challenge via the Fc-membrane receptor was quantified. Phagocytosis was suppressed in a dose-related manner with increasing concentrations of both carbon blacks and adsorbates. Carbon black N339 covered with 0.5 monolayers of the adsorbates suppressed phagocytosis more than N339 without the adsorbates. Increasing the adsorbate acrolein coverage from 0.5 to greater than 2.0 monolayers suppressed phagocytosis in a dose-related manner. Finally, samples of diesel particulate matter collected from an engine operated on a pure hydrocarbon fuel with various oxidizers, air (PSU #1) and an oxidizer free of nitrogen (N-free) were tested. Treatment of the macrophages with PSU #1 had a negligible effect on phagocytosis whereas the N-free sample suppressed phagocytosis in a dose-related manner. The data show that alveolar macrophage Fc-receptor-mediated phagocytosis is affected by: carbon black and adsorbate identity and concentration, coverage of the carbon black with adsorbates, and the oxidizer used in the generation of particles emitted by a diesel engine.
Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and therefore symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signalling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI- mediated human mast cell signalling has not been published. Here we show evidence for the expression of Orai 1,2 and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line; but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRImediated calcium entry. Calcium imaging experiments, utilising an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signalling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI- mediated calcium signalling in human mast cells. Similarly, inactivation of STIM1- regulated TRPC1 in human mast cells (as tested by transfecting LAD2 cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI- mediated calcium signalling in LAD2 human mast cells. Mediator release assays confirm that FcεRI- mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.
Summary Background Prostaglandin E 2 ( PGE 2 ) has been shown to inhibit IgE‐dependent histamine release from human lung mast cells. This effect of PGE 2 is believed to be mediated by EP 2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP 2 ‐selective antagonists. Moreover, recent evidence has suggested that PGE 2 activates EP 4 receptors to inhibit respiratory cell function. Objective The aim of this study was to determine the receptor by which PGE 2 inhibits human lung mast cell responses by using recently developed potent and selective EP 2 and EP 4 receptor antagonists alongside other established EP receptor ligands. Methods The effects of non‐selective ( PGE 2 , misoprostol), EP 2 ‐selective ( ONO ‐ AE 1‐259, AH 13205, butaprost‐free acid) and EP 4 ‐selective (L‐902,688, TCS 251) agonists on IgE‐dependent histamine release and cyclic‐ AMP generation in mast cells were determined. The effects of EP 2 ‐selective ( PF ‐04418948, PF ‐04852946) and EP 4 ‐selective ( CJ ‐042794, L‐161,982) antagonists on PGE 2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT ‐ PCR . Results Prostaglandin E 2 , EP 2 agonists and EP 4 agonists inhibited IgE‐dependent histamine release from mast cells. PGE 2 and EP 2 agonists, but not EP 4 agonists, increased cyclic‐ AMP levels in mast cells. EP 4 ‐selective antagonists did not affect the PGE 2 inhibition of histamine release, whereas EP 2 ‐selective antagonists caused rightward shifts in the PGE 2 concentration–response curves. RT ‐ PCR studies indicated that mast cells expressed EP 2 and EP 4 receptors. Conclusions and Clinical Relevance Although human lung mast cells may express both EP 2 and EP 4 receptors, the principal mechanism by which PGE 2 inhibits mediator release in mast cells is by activating EP 2 receptors.
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