Peter T. Peachell

Background and Purpose: PGE₂inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti‐inflammatory effect of PGE₂has not been defined. The aim of this study was to identify the EP receptor by which PGE₂inhibits cytokine generation from human lung macrophages. This was determined by using recently developed EP receptor ligands.Experimental Approach: The effects of PGE₂and EP‐selective agonists on LPS‐induced generation of TNF‐α and IL‐6 from macrophages were evaluated. The effects of EP₂‐selective (PF‐04852946, PF‐04418948) and EP₄‐selective (L‐161,982, CJ‐042794) receptor antagonists on PGE₂responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT‐PCR.Key Results: PGE₂inhibited LPS‐induced andStreptococcus pneumoniae‐induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP₂and EP₄receptors. L‐902,688 (EP₄receptor‐selective agonist) was considerably more potent than butaprost (EP₂receptor‐selective agonist) as an inhibitor of TNF‐α generation from macrophages. EP₂receptor‐selective antagonists had marginal effects on the PGE₂inhibition of TNF‐α generation, whereas EP₄receptor‐selective antagonists caused rightward shifts in the PGE₂concentration–response curves.Conclusions and Implications: These studies demonstrate that the EP₄receptor is the principal receptor that mediates the anti‐inflammatory effects of PGE₂on human lung macrophages. This suggests that EP₄receptor agonists could be effective anti‐inflammatory agents in human lung disease.

Summary: Background: Prostaglandin E₂(PGE₂) has been shown to inhibit IgE‐dependent histamine release from human lung mast cells. This effect ofPGE₂is believed to be mediated byEP₂receptors. However, definitive evidence that this is the case has been lacking in the absence ofEP₂‐selective antagonists. Moreover, recent evidence has suggested thatPGE₂activatesEP₄receptors to inhibit respiratory cell function.Objective: The aim of this study was to determine the receptor by whichPGE₂inhibits human lung mast cell responses by using recently developed potent and selectiveEP₂andEP₄receptor antagonists alongside other establishedEPreceptor ligands.Methods: The effects of non‐selective (PGE₂, misoprostol),EP₂‐selective (ONO‐AE1‐259,AH13205, butaprost‐free acid) andEP₄‐selective (L‐902,688,TCS251) agonists on IgE‐dependent histamine release and cyclic‐AMPgeneration in mast cells were determined. The effects ofEP₂‐selective (PF‐04418948,PF‐04852946) andEP₄‐selective (CJ‐042794, L‐161,982) antagonists onPGE₂responses of mast cells were studied. The expression ofEPreceptor subtypes was determined byRT‐PCR.Results: Prostaglandin E₂,EP₂agonists andEP₄agonists inhibited IgE‐dependent histamine release from mast cells.PGE₂andEP₂agonists, but notEP₄agonists, increased cyclic‐AMPlevels in mast cells.EP₄‐selective antagonists did not affect thePGE₂inhibition of histamine release, whereasEP₂‐selective antagonists caused rightward shifts in thePGE₂concentration–response curves.RT‐PCRstudies indicated that mast cells expressedEP₂andEP₄receptors.Conclusions and Clinical Relevance: Although human lung mast cells may express bothEP₂andEP₄receptors, the principal mechanism by whichPGE₂inhibits mediator release in mast cells is by activatingEP₂receptors.

Summary: Background: Stem cell factor (SCF) is a growth factor that is involved in mast cell differentiation and proliferation. SCF primes human lung mast cells for enhanced responses to IgE‐directed activation but is not generally recognized as a direct activator. SCF mediates its effects through c‐kit.Objective: The aim of this study was to reappraise the effects of SCF on human lung mast cells.Methods: Mast cells were isolated from human lung. Mast cells were challenged with anti‐IgE or SCF and the generation of histamine, cysteinyl‐leukotrienes (cys‐LTs) and prostaglandin D₂ (PGD₂) was assessed as was expression of the activation marker, CD63. The effects of c‐kit inhibitors on mediator release were evaluated.Results: Stem cell factor (10 ng/mL) alone was unable to induce mediator release but primed mast cells for enhanced IgE‐dependent secretion. At higher concentrations (≥ 30 ng/mL), SCF had more varied effects and even when used alone was able to drive substantial levels of histamine release in about a third of all preparations studied. Similarly, SCF (100 ng/mL) alone was effective in stimulating the generation of cys‐LTs in half of the preparations studied. SCF (100 ng/mL) was even more effective at stimulating PGD₂ generation as almost all preparations generated substantial quantities of the prostanoid. Mediator release induced by SCF was accompanied by the up‐regulation of the activation marker, CD63. There was a positive correlation between the extent of mediator release induced by SCF and c‐kit receptor expression. The effects of SCF on mediator release from mast cells were reversed by the c‐kit inhibitor imatinib.Conclusions and clinical relevance: These data demonstrate that the responses of mast cells to SCF are heterogeneous. SCF can drive much greater levels of mediator release from mast cells, especially of PGD₂, than hitherto appreciated and this could be important in the context of respiratory diseases.

Previous studies indicate that the protein tyrosine kinase, syk, is critical in transducing FcɛRI-mediated signals. In human basophils, 'releasability' has been linked to the extent of syk expression. Human lung mast cells, like basophils, are also found to be variably responsive to IgE-dependent activation. The aim of the present study was to determine whether the wide variability in human lung mast cell responses, following IgE-dependent activation, has a relationship with syk expression. Mast cells were isolated from human lung tissue and 'releasability' was determined by activating the cells with a maximal releasing concentration of anti-IgE. Syk levels in mast cells were determined by immunoblotting and flow cytometry. Histamine release from mast cells, challenged with a maximal releasing concentration of anti-IgE, ranged from 0% to 69% (mean±SEM, 24±2%, n=53). A proportion of these preparations (nine out of 53) released very low levels of histamine (5%) in response to anti-IgE. Flow cytometry of a subset of preparations indicated that a weak response to anti-IgE was not related to a lack of surface IgE. Immunoblotting and flow cytometry studies demonstrated that, compared with mononuclear cells, human lung mast cells express low and variable levels of syk. However, there was no correlation between syk expression and mast cell releasability. Nonetheless, a number of putative inhibitors of syk including NVP-QAB205 (EC₅₀, 0.2 μm) effectively attenuated the IgE-dependent release of histamine from mast cells. These studies indicate that although syk may play an important role in mediating degranulation, the relative level of syk expression does not govern human lung mast cell releasability. Identification of the mechanisms that govern IgE-dependent activation of human lung mast cells is likely to be of wider clinical significance, given the central role that mast cells play in the development of allergic asthma.

Background and purpose: The long‐acting β₂‐adrenoceptor agonist, indacaterol, has been developed as a bronchodilator for the therapeutic management of respiratory diseases. The aim of the present study was to determine whether indacaterol has any anti‐inflammatory activity. To this end, the effects of indacaterol on human lung mast cell responses were investigated.Experimental approach: The effects of indacaterol, and the alternative long‐acting β‐agonists formoterol and salmeterol, were investigated on the IgE‐dependent release and generation of histamine, cysteinyl‐leukotrienes and prostaglandin D₂from human lung mast cells. Moreover, the extent to which long‐term (24–72 h) incubation of mast cells with long‐acting β‐agonists impaired the subsequent ability of β‐agonists to inhibit mast cell responses was assessed.Key results: Indacaterol was as potent and as efficacious as the full agonist, isoprenaline (EC₅₀, ∼4 nmol·L⁻¹), at inhibiting the IgE‐dependent release of histamine from mast cells. Formoterol was a full agonist whereas salmeterol was a partial agonist as inhibitors of histamine release. All three long‐acting β‐agonists were effective inhibitors of the IgE‐dependent generation of cysteinyl‐leukotrienes and prostaglandin D₂. Long‐term incubation of mast cells with long‐acting β‐agonists led to a reduction in the subsequent ability of β‐agonists to stabilize mast cell responses. This tendency to induce functional desensitization was least evident for indacaterol.Conclusions and implications: Indacaterol is an effective inhibitor of the release of mediators from human lung mast cells. This suggests that, as well as bronchodilation, mast cell stabilization may constitute an additional therapeutic benefit of indacaterol.

Background and purpose: Previous studies have shown that β ₂‐adrenoceptor‐mediated responses in human lung mast cells are highly variable. The aims of the present study were to establish whether polymorphisms of the β ₂‐adrenoceptor gene (ADRB2) influence this variability in (a) β ₂‐adrenoceptor‐mediated inhibition and (b) desensitization of β ₂‐adrenoceptor‐mediated responses in human lung mast cells.Experimental approach: Mast cells were isolated from human lung tissue. The inhibitory effects of the β‐adrenoceptor agonist, isoprenaline (10⁻¹⁰–10⁻⁵ M), on IgE‐mediated histamine release from mast cells were determined (n=92). Moreover, the inhibitory effects of isoprenaline were evaluated following a desensitizing treatment involving long‐term (24 h) incubation of mast cells with isoprenaline (10⁻⁶ M) (n=65). A potential influence of polymorphisms on these functional responses was determined by genotyping 11 positions, in the promoter and coding regions, of ADRB2 previously reported as polymorphic.Key results: There was no influence of any of the polymorphic positions of ADRB2 on the potency of isoprenaline to inhibit histamine release from mast cells with the exception of position 491C>T (Thr164Ile). There was no influence of any of the polymorphic positions of ADRB2 on the extent of desensitization of the isoprenaline‐mediated response following a desensitizing treatment except for position 46G>A (Gly16Arg). Analyses at the haplotype level indicated that there was no influence of haplotype on β ₂‐adrenoceptor‐mediated responses in mast cells.Conclusions and implications: These data indicate that certain polymorphisms in ADRB2 influence β ₂‐adrenoceptor‐mediated responses in human lung mast cells.British Journal of Pharmacology (2007) 152, 323–331; doi:10.1038/sj.bjp.0707400; published online 23 July 2007