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.
The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo.
The beta adrenergic agonist isoprenaline inhibited the IgE-triggered release of the preformed mediator histamine from human lung mast cells (HLMC) in a dose-dependent fashion. After prolonged (> or = 4 h) preexposure of HLMC to isoprenaline, there was a subsequent diminution in the effectiveness of a second exposure of isoprenaline to inhibit the release of histamine from activated HLMC. This induced hyporesponsiveness to isoprenaline was both concentration and time dependent. Although maximal levels of desensitization were obtained after an initial prolonged (14-h) preincubation with a high (10(-5) M) concentration of isoprenaline, exposure of HLMC for a shorter (4-h) time period with a lower (3 x 10(-7) M) concentration of isoprenaline was also effective at inducing a functional desensitization to isoprenaline. The inhibitory activity of the beta 2 agonist fenoterol was attenuated after a prolonged (14-h) pretreatment step with isoprenaline (10(-5)M), whereas the inhibitory properties of other adenylate cyclase activators, prostaglandin E2 and forskolin, were not affected appreciably. Prolonged (12-h) exposure of HLMC to the beta agonists fenoterol, salbutamol, and terbutaline also induced hyporesponsive states of beta agonists, qualitatively similar to that obtained with isoprenaline. The beta receptor antagonist propranolol, if coincubated with isoprenaline during the prolonged pretreatment step, protected against the subsequent refractoriness of the HLMC to isoprenaline. The glucocorticoid dexamethasone failed to prevent the isoprenaline-induced functional desensitization. In total, these results indicate that prolonged exposure of HLMC to beta agonists induces a state of selective hyporesponsiveness to agonists that act at beta adrenoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)
The long‐acting β 2 ‐adrenoceptor agonist, salmeterol (10 −9 –10 −5 M ), inhibited the IgE‐mediated release of histamine from human lung mast cells (HLMC) in a dose‐dependent fashion. Additional β‐adrenoceptor agonists were studied and the rank order of potency for the inhibition of histamine release from HLMC was isoprenaline>salmeterol>salbutamol. Approximate EC 50 values for the inhibition of histamine release were 10 n M for isoprenaline and 100 n M for salbutamol. An EC 50 value for salmeterol could not be calculated because maximal responses to salmeterol were not observed over the concentration range employed. Both salmeterol and isoprenaline inhibited the generation of sulphopeptidoleukotrienes (sLT) more potently and more efficaciously than the release of histamine from immunologically‐activated HLMC. Salmeterol (EC 50 <0.1 n M ) was more potent than isoprenaline (EC 50 0.4 n M ) at attenuating sLT generation. The β‐adrenoceptor antagonist, propranolol (1 μ M ), and the selective β 2 ‐adrenoceptor antagonist, ICI 118,551 (0.1 μ M ), both caused rightward shifts in the dose‐response curve for the inhibition of histamine release by isoprenaline. The antagonism of salmeterol effects by propranolol and ICI 118,551 was more complex. At lower concentrations (<1 μ M ) of salmeterol, both antagonists shifted the dose‐reponse curve to salmeterol rightward. At a higher concentration (10 μ M ) of salmeterol, neither ICI 118,551 nor propranolol was an effective antagonist of the salmeterol‐mediated inhibition of histamine release. Prolonged exposure (4 h) of HLMC to isoprenaline (1 μ M ) caused an approximately 50% reduction in the effectiveness of a second exposure to isoprenaline (10 μ M ) at inhibiting the release of histamine, whereas this pretreatment did not affect the salmeterol (10 μ M ) inhibition of histamine release. Isoprenaline (10 −9 –10 −5 M ) caused a dose‐dependent increase in total cell cyclicAMP levels in purified HLMC which paralleled the inhibition of histamine release. Salmeterol (10 −9 –10 −5 M ) was considerably less potent than isoprenaline at increasing HLMC cyclicAMP levels. In summary, these data indicate that salmeterol is an effective inhibitor of the stimulated release of mediators from HLMC. The present data also suggest that salmeterol may act to inhibit mediator release from HLMC by β‐adrenoceptor‐dependent and independent mechanisms. British Journal of Pharmacology (1998) 123 , 1009–1015; doi: 10.1038/sj.bjp.0701703
The β-adrenoceptor agonist, isoprenaline, inhibited the immunoglobulin E-mediated release of histamine from human lung mast cells (HLMC). Long-term (24 h) exposure of HLMC to isoprenaline reduced the subsequent effectiveness of isoprenaline to inhibit histamine release. The extent of this functional desensitization was variable with some HLMC preparations resistant and others highly susceptible. We sought to determine whether the variability in the degree of functional desensitization was influenced by genetic polymorphisms in the β2-adrenoceptor. HLMC preparations were genotyped at two polymorphic loci, positions 16 (arg to gly) and 27 (gln to glu), and the effect of desensitizing conditions (24 h with 10−6 m isoprenaline) on the subsequent ability of isoprenaline (10−7 m) to inhibit histamine release from HLMC was determined (n = 72). In HLMC preparations expressing β2-adrenoceptors with arg (wild-type) or gly (mutant) at position 16, desensitization was 71 ± 5% (n = 18) or 43 ± 5% (n = 26), respectively, whereas the desensitization was 59 ± 6% (n = 28) for heterozygotes at this position. In HLMC preparations expressing β2-adrenoceptors with gln (wild-type) or glu (mutant) at position 27, desensitization was 65 ± 5% (n = 25) or 28 ± 7% (n = 17), respectively, whereas the desensitization was 61 ± 5% (n = 30) for heterozygotes at this position. These data suggest that mutant (gly16 and glu27) forms of the receptor are resistant to desensitization compared to wild-type (arg16 and gln27) forms. However, analyses to determine the relative contributions of positions 16 and 27 suggest that position 27 is more important in influencing the degree of functional desensitization.
Abstract A number of structurally diverse antigens preferentially stimulate the synthesis of IgE antibodies, but no unifying principle has been proposed that explains the nature of isotype selection. In the present study, we show that common allergens present in bee venom, house dust mite emanations and parasite proteins induce mast cell and basophil degranulation and stimulate interleukin‐4 synthesis, and secretion in the absence of antigen‐specific IgE. These data point to a linkage between the initial activation of cells of the innate immune system and subsequent adaptive immune responses. They suggest that IgE‐independent mast cell and basophil degranulation is predictive of potential allergenicity and can be evaluated by means of a cellular assay. Our study indicates that non‐immunological degranulation by prototypic allergens, such as bee venom phos‐pholipase A 2 or proteases associated with house dust mite emanations, is critically dependent on enzymatic activity. These findings have potentially important implications for vaccine design in allergic and parasitic disease.
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