Evaluation of the anti-inflammatory effects of β-adrenoceptor agonists on human lung macrophages

The principal mechanism by which bronchodilator β-adrenoceptor agonists act is to relax airways smooth muscle although they may also be anti-inflammatory. However, the extent of anti-inflammatory activity and the cell types affected by these agonists are uncertain. The purpose of this study was to evaluate whether β-adrenoceptor agonists prevent pro-inflammatory cytokine generation from activated human lung macrophages. Macrophages were isolated and purified from human lung. The cells were pre-treated with both short-acting (isoprenaline, salbutamol, terbutaline) and long-acting (formoterol, salmeterol, indacaterol) β-agonists before activation with lipopolysaccharide (LPS) to induce cytokine (TNFα, IL-6, IL-8 and IL-10) generation. The experiments showed that short-acting β-agonists were poor inhibitors of cytokine generation. Of the long-acting β-agonists studied, formoterol was also a weak inhibitor of cytokine generation whereas only indacaterol and salmeterol showed moderate inhibitory activity. Further experiments using the β2-adrenoceptor antagonist ICI-118,551 suggested that the effects of indacaterol were likely to be mediated by β2-adrenoceptors whereas those of salmeterol were not. These findings were corroborated by functional desensitization studies in which the inhibitory effects of indacaterol appeared to be receptor-mediated whereas those of salmeterol were not. Taken together, the data indicate that the anti-inflammatory effects of β-adrenoceptor agonists on human lung macrophages are modest

The anti-inflammatory effects of prostaglandin E 2 on human lung macrophages are mediated by the EP 4 receptor

Background and purpose: Prostaglandin E2 (PGE2) has been shown to inhibit cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti-inflammatory effect of PGE2 has not been elucidated definitively. The aim of this study was to identify the EP receptor by which PGE2 inhibits cytokine generation from human lung macrophages. This was determined by using recently-developed EP receptor ligands. Experimental approach: The effects of PGE2 and EP-selective agonists on lipopolysaccharide (LPS) induced tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) generation from macrophages were evaluated. The effects of EP2-selective (PF-04852946, PF-04418948) and EP4-selective (L-161,982, CJ-042794) antagonists on PGE2 responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT-PCR. Key results: PGE2 inhibited LPS-induced and Streptococcus pneumoniae-induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP2 and EP4 receptors. L-902,688 (EP4-selective agonist) was considerably more potent than butaprost (EP2-selective agonist) as an inhibitor of TNFα generation from macrophages. EP2-selective antagonists had marginal effects on the PGE2 inhibition of TNFα generation whereas EP4-selective antagonists caused rightward shifts in the PGE2 concentration-response curves. Conclusions and implications: These studies demonstrate that the EP4 receptor is the principal receptor that mediates the anti-inflammatory effects of PGE2 on human lung macrophages. This suggests that EP4 agonists could be effective anti-inflammatory agents in human lung disease

Effects of fostriecin on β2-adrenoceptor-driven responses in human mast cells

As part of the intracellular processes leading to mast cell and basophil activation, phosphorylation of key substrates is likely to be important. These processes, mediated by phosphatases, are responsible for regulating phosphorylation. The aim of the present study was to determine effects fostriecin – a selective inhibitor of PP2A (protein phosphatase-2) – on β2-adrenoceptor-driven responses in human mast cells. Here, the effects of fostriecin (PP inhibitors) on the inhibition of histamine release from HLMC, on β-adrenoceptor-driven responses in mast cells and on desensitization were investigated. Long-term incubation (24 h) of mast cells with fostriecin (10−6 M) resulted in a significant (p < 0.001) reduction in the maximal response (from 41.2 [± 3.0] to 29.9 [± 4.2] %) to salbutamol following fostriecin treatment. The results showed that fostriecin pretreatment significantly attenuated the inhibitory effects of salbutamol. Overall, the present study suggested that PP2A has an important role in regulating mast cell β2-adrenoceptors

A standard, single dose of inhaled terbutaline attenuates hyperpnoea-induced bronchoconstriction and mast cell activation in athletes

Release of broncho-active mediators from mast cells during exercise hyperpnoea is a key factor in the pathophysiology of exercise-induced bronchoconstriction (EIB). Our aim was to investigate the effect of a standard, single dose of an inhaled β2-adrenoceptor agonist on mast cell activation in response to dry air hyperpnoea in athletes with EIB. Twenty-seven athletes with EIB completed a randomised, double blind, placebo-controlled, crossover study. Terbutaline (0.5 mg) or placebo was inhaled15 min prior to 8 min of eucapnic voluntary hyperpnoea (EVH) with dry air. Pre- and post-bronchial challenge, urine samples were analysed by enzyme immunoassay for 11β-prostaglandin(PG)F2α. The maximum fall in forced expiratory volume in 1 sec(FEV1) of 14 (12-20)% (median and interquartile range) following placebo was attenuated to 7 (5-9)% with the administration of terbutaline (P<0.001). EVH caused a significant increase in 11β-PGF2α from (27-57) ng·mmol creatinine-1 at baseline to (43-72) ng·mmol creatinine-1 at its peak post-EVH following placebo (P=0.002). The rise in 11β-PGF2α was inhibited with administration of terbutaline: 39 (28-44) ng·mmol creatinine-1 at baseline vs. 40 (33-58) ng·mmol creatinine-1 at its peak post-EVH (P=0.118). These data provide novel in vivo evidence of mast cell stabilisation following inhalation of a standard dose of terbutaline prior to bronchial provocation with EVH in athletes with EIB

Prostaglandin D2 generation from human lung mast cells is catalysed exclusively by cyclooxygenase-1

Mast cells are an exceptionally rich source of prostaglandin D2 (PGD2). PGD2 is pro-inflammatory and can cause bronchoconstriction. The enzyme cyclooxygenase (COX) is central to the generation of prostanoids such as PGD2. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX. COX exists as two isoforms, COX-1 and COX-2. The principal aim of this study was to establish whether COX-1 and/or COX-2 mediates PGD2 generation from human lung mast cells. Mast cells were isolated from human lung tissue and purified by flotation over Percoll and immunomagnetic bead separations. The cells were activated with anti-IgE or Stem Cell Factor (SCF). The generation of PGD2 was determined by ELISA. The effects of NSAIDs (aspirin, ibuprofen, diclofenac, naproxen, indomethacin), COX-1 selective (FR122047), and COX-2 selective (celecoxib) inhibitors on PGD2 generation were determined. The expression of COX-1 and COX-2 in mast cells was determined by Western blotting. All the NSAIDs tested abrogated stimulated PGD2 generation from mast cells except aspirin which was only weakly effective. FR122047 was an effective inhibitor of PGD2 generation (EC50 ~25 nM) from mast cells whereas celecoxib was ineffective. Immunoblotting indicated that COX-1 was strongly expressed in all mast cell preparations while COX-2 expression was weak. No induction of COX-2 was observed following activation of mast cells. These findings indicate that COX-1 is the principal isoform involved in generating PGD2 from human lung mast cells. These studies provide insight into the potential behaviour of NSAIDs in the context of respiratory diseases

Prenatal exposures and exposomics of asthma

This review examines the causal investigation of preclinical development of childhood asthma using exposomic tools. We examine the current state of knowledge regarding early-life exposure to non-biogenic indoor air pollution and the developmental modulation of the immune system. We examine how metabolomics technologies could aid not only in the biomarker identification of a particular asthma phenotype, but also the mechanisms underlying the immunopathologic process. Within such a framework, we propose alternate components of exposomic investigation of asthma in which, the exposome represents a reiterative investigative process of targeted biomarker identification, validation through computational systems biology and physical sampling of environmental medi

Adenosine closes the K+ channel KCa3.1 in human lung mast cells and inhibits their migration via the adenosine A2A receptor

Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2adrenoceptor through a Gαs-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10–5–10–3 M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Gαs-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease

IL-4 Amplifies the Pro-Inflammatory Effect of Adenosine in Human Mast Cells by Changing Expression Levels of Adenosine Receptors

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A2BsiRNA and selective A2B receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A2B and reduced expression of A2A adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine

Gs-Coupled Adenosine Receptors Differentially Limit Antigen-Induced Mast Cell Activation

Mast cell activation results in the immediate release of proinflammatory mediators prestored in cytoplasmic granules, as well as initiation of lipid mediator production and cytokine synthesis by these resident tissue leukocytes. Allergen-induced mast cell activation is central to the pathogenesis of asthma and other allergic diseases. Presently, most pharmacological agents for the treatment of allergic disease target receptors for inflammatory mediators. Many of these mediators, such as histamine, are released by mast cells. Targeting pathways that limit antigen-induced mast cell activation may have greater therapeutic efficacy by inhibiting the synthesis and release of many proinflammatory mediators produced in the mast cell. In vitro studies using cultured human and mouse mast cells, and studies of mice lacking A2B receptors, suggest that adenosine receptors, specifically the Gs-coupled A2A and A2B receptors, might provide such a target. Here, using a panel of mice lacking various combinations of adenosine receptors, and mast cells derived from these animals, we show that adenosine receptor agonists provide an effective means of inhibition of mast cell degranulation and induction of cytokine production both in vitro and in vivo. We identify A2B as the primary receptor limiting mast cell degranulation, whereas the combined activity of A2A and A2B is required for the inhibition of cytokine synthesis

Functional variability of the adenosine A3 receptor ( ADORA3

BACKGROUND: To improve understanding of aspirin hypersensitivity, this study focused on adenosine as a noncyclooxygenase target molecule of aspirin. Adenosine may affect the release of histamine from cutaneous mast cells through a mechanism mediated by the adenosine A3 receptor. OBJECTIVES: To investigate the genetic contribution of adenosine A3 receptor gene (ADORA3) polymorphisms in the pathogenesis of aspirin-induced urticaria (AIU) in a case-control association study in a Korean population. METHODS: A case-control association study was performed in 385 patients with AIU and 213 normal controls from a Korean population. The functional variability of genetic polymorphisms in the ADORA3 gene was analysed in in vitro studies that included a luciferase reporter assay and an electrophoretic mobility shift assay (EMSA), and ex vivo studies that included real-time polymerase chain reaction for mRNA expression in peripheral blood mononuclear cells and a histamine release assay. RESULTS: A significant association of ADORA3 promoter polymorphism at -1050G/T was found with the phenotype of AIU. Patients with AIU showed higher frequency of the haplotype, ht1 (T(-1050) C(-564) ), compared with normal healthy controls. Moreover, ht1 (TC) was found to be a high-transcript haplotype by the luciferase activity assay, and a -564C allele-specific DNA binding protein was found by EMSA. Increased basophil histamine release was noted in subjects who had the high-transcript haplotype, ht1 (TC). CONCLUSION: These results suggest that the high-transcript haplotype, ht1 (TC), of the ADORA3 gene may contribute to the development of cutaneous hyper-reactivity to aspirin, leading to the clinical presentation of AIU