Mast Cell Purification Protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue. It is for this reason that, in this chapter, we provide detailed methods for the isolation and purification of human lung mast cells. However, it should be noted that the methods that are described in this chapter are generally applicable to the isolation of mast cells from different tissues, and this will also be discussed.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

An Optimized Protocol for the Isolation and Functional Analysis of Human Lung Mast Cells.

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Histamine H4 receptor mediates chemotaxis of human lung mast cells.

The diverse effects of histamine are mediated by discrete histamine receptors. The principal repository of histamine in the body is the mast cell. However, the effects of histamine on mast cells, especially those of human origin, have not been fully elucidated. In this study, the expression of histamine receptors in human lung mast cells was evaluated. Moreover, the effects of histamine receptor engagement on both mediator release and chemotaxis were investigated. Mast cells were isolated and purified from human lung tissue. Histamine receptor expression was determined by RT-PCR and q-PCR. Both methods for the detection of histamine receptors were in accordance and human lung mast cells expressed mRNA for histamine H4 and histamine H1 receptors, variably expressed histamine H2 receptor but did not express histamine H3 receptor. The effects of selective histamine receptor agonists on the release of both pre-formed (histamine) and newly-synthesised (cysteinyl-leukotriene, prostaglandin D2) mediators were investigated. None of the agonists tested had any direct effects on mediator release. None of the agonists modulated release stimulated by anti-IgE. Further studies showed that histamine induced migration of mast cells. Chemotaxis appeared to be mediated by the histamine H4 receptor since JNJ28610244 (H4 agonist) was chemotactic for mast cells whereas 2-(2-pyridyl) ethylamine (H1 agonist) was not. Furthermore, the selective histamine H4 receptor antagonist, JNJ7777120, effectively reversed the chemotaxis of mast cells induced by JNJ28610244. Overall, these experiments identify the histamine H4 receptor as chemotactic for human lung mast cells. This mechanism might influence mast cell accumulation in the lung.

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 ~25nM) 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.

Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.

Inappropriate activation of mast cells via the FcεRI 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 FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling 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 FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling 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 signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling 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.

Effects of fostriecin on ß2-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 ß2-adrenoceptor-driven responses in human mast cells. Here, the effects of fostriecin (PP inhibitors) on the inhibition of histamine release from HLMC, on ß-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 ß2-adrenoceptors.

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

The principal mechanism by which bronchodilator ß-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 ß-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) ß-agonists before activation with lipopolysaccharide (LPS) to induce cytokine (TNFα, IL-6, IL-8 and IL-10) generation. The experiments showed that short-acting ß-agonists were poor inhibitors of cytokine generation. Of the long-acting ß-agonists studied, formoterol was also a weak inhibitor of cytokine generation whereas only indacaterol and salmeterol showed moderate inhibitory activity. Further experiments using the ß2-adrenoceptor antagonist ICI-118,551 suggested that the effects of indacaterol were likely to be mediated by ß2-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 ß-adrenoceptor agonists on human lung macrophages are modest.

The anti-inflammatory effects of PGE2 on human lung macrophages are mediated by the EP4 receptor.

BACKGROUND AND PURPOSE: PGE2 inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti-inflammatory effect of PGE2 has not been defined. 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 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 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 receptor-selective agonist) was considerably more potent than butaprost (EP2 receptor-selective agonist) as an inhibitor of TNF-α generation from macrophages. EP2 receptor-selective antagonists had marginal effects on the PGE2 inhibition of TNF-α generation, whereas EP4 receptor-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 receptor agonists could be effective anti-inflammatory agents in human lung disease.

Cyclic nucleotide phosphodiesterase isoforms in human basophils and mast cells.

Cyclic nucleotide phosphodiesterase (PDE) exists as multiple molecular forms. Of the 11 families of PDE identified so far, PDE4, a cAMP-specific PDE, has been identified as the major isoform regulating inflammatory activity. The principle aim of the present study was to determine whether human basophils and human lung mast cells express PDE4. Four sub-classes of PDE4 (A, B, C, and D) have been identified and expression of these was determined by RT-CPR and by western blotting. In basophils, prominent expression of mRNA for PDE4A and PDE4D was observed whereas little if any expression of PDE4B and PDE4C was detected. These findings were paralleled by immunoblotting experiments as human basophils were found to express PDE4A and PDE4D with little evidence for the presence of either PDE4B or PDE4C. By contrast, human lung mast cells expressed very little, if any, mRNA for PDE4 sub-classes although, in some preparations, some modest levels of mRNA for PDE4D were detected. However, there was no evidence, at the protein level, that mast cells express PE4. Overall, these data indicate that basophils express PDE4 (4A and 4D) whereas human lung mast cells do not.

The effect of cyclic nucleotide analog drugs on the mediators release from basophils.

BACKGROUND: The cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), are intracellular second messengers that play an important role in modulating inflammatory cells involved in allergic diseases. In general, cAMP suppresses the activity of immune and inflammatory cells. We aim to evaluate the roles of cAMP and cGMP in regulating basophil activity. MATERIALS AND METHODS: Basophil-enriched preparations were incubated with analogs and then challenged with anti-IgE or IL-3 (4 or 24 hours). Supernatants were assayed for histamine, IL-4, and IL-13 release. The effects of Sp-8-CPT-cAMPS and Sp-8-CPT-cGMPS on IL-3-dependent mediator release from basophils were determined. The cells were pre-incubated with an analog and then incubated with IL-3 for 24 hours. RESULTS: Sp-8-CPT-cAMPS was an effective (P < 0.05) inhibitor of IL-4, IL-13, and histamine release from basophils. However, paradoxically, Sp-8-CPT-cGMPS enhanced histamine release and IL-13 generation, but by contrast, had little effect on IL-4 generation. Sp-8-CPT-cGMPS inhibited cytokine generation, but enhanced the release of histamine release to a modest extent. CONCLUSION: This study shows that the cAMP/protein kinase A (PKA) pathway may be inhibitory to the IgE- and non-IgE-dependent release of mediators from basophils.

Role of protein phosphatases inhibitors on the histamine release and the functional desensitization in human lung mast cells.

BACKGROUND: The ß2-adrenoceptor agonist, isoprenaline, is an effective inhibitor of histamine release from human lung mast cells (HLMC). Since phosphorylations of the ß2-adrenoceptors are probably important in inducing desensitization, we sought to investigate the importance of phosphorylation events by targeting protein phosphatases (PPs) in mast cells. To this end, the effects of the inhibitor of on the functional desensitization of ß-adrenoceptor-mediated responses in mast cells were investigated. MATERIALS AND METHODS: In this study the effects of PP inhibitors on the inhibition of histamine release from HLMC, on ß-agonists in mast cells and on desensitization were investigated. RESULTS: Long-term exposure of mast cells to both isoprenaline and salbutamol substantially reduced the extent to which isoprenaline inhibited histamine release. Pretreatments of up to 24 h with inhibitors alone had no effect on immunoglobulin E-mediated histamine release. Shorter (≤4 h) pretreatments had little effect on the activity of isoprenaline and salbutamol to inhibit histamine release from mast cells. CONCLUSION: Collectively, these data suggest that PP has an important role in regulating mast cell ß2-adrenoceptors.

Mast cell purification protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue. It is for this reason that, in this chapter, we provide detailed methods for the isolation and purification of human lung mast cells. However, it should be noted that the methods that are described in this chapter are generally applicable to the isolation of mast cells from different tissues and this will also be discussed.

Evaluation of the correlation and reproducibility between histamine, IL-4, and IL-13 release from human basophils.

Human basophils play a key role in allergic diseases such as asthma and in a variety of immunological disorders. The generation of IL-4 and IL-13 can be induced from basophil by IgE-mediated and non-IgE-mediated mechanisms. Time and stimulus-dependent differences in the regulation of these cytokines could have relevance to their biological effects. The aim of the present study was activation of basophils in order to evaluate the extent of histamine, IL-4, and IL-13 generations. Basophil-enriched suspensions were prepared by Percoll gradients. The release of histamine and cytokines was assessed after activation with either anti-human IgE (1/1000 or 1/10000, 4 h or 24 h) or IL-3 (100 ng/ ml, 24 h). Results were analysed statistically, using ANOVA test. Using anti-IgE, there was no significant correlation between the extent of either IL-4 (r=0.24, p=0.35) or IL-13 (r=0.47, p=0.098) and histamine release. Using IL-3 as stimulator, results showed that the extent of IL-13 correlated with histamine release(r=0.44, p=0.036). There was no correlation between the extent of IL-4 and the degree of either histamine (r=0.077, p=0.72) or IL-13 (r=0.162, p=0.5). The reproducibility of cytokines isolated from the same donor (on different occasions) indicated that the ability of anti-IgE to induce cytokines was consistently similar for a given donor. Our data showed that the pathways leading to IL-3-triggering histamine release and IL-13 generation show similarity. Donor-dependent differences may be responsible for this wide range in the extent of releasibility. The ability of IL-3 to release cytokines from basophils showed a wider range.

Preclinical evaluation of an inhibitor of cytosolic phospholipase A2α for the treatment of asthma.

Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA(2)α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA(2)α (7 nM) and was able to inhibit prostaglandin (PG)D(2) and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B(4), thromboxane A(2), and PGD(2) (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE(2) release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD(2) release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA(2)α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.

Influence of beta2-adrenoceptor gene polymorphisms on beta2-adrenoceptor expression in human lung.

BACKGROUND: The aim of the present study was to establish whether polymorphisms, especially those within the promoter region, of the beta(2)-adrenoceptor gene (ADRB2) influence beta(2)-adrenoceptor expression in human lung. METHODS: The density of beta-adrenoceptors in human lung tissue (n=88) was determined by saturation binding using the radioligand, iodinated cyanopindolol. Discrimination of beta(1)- and beta(2)-adrenoceptors was determined using the highly selective beta(1)-adrenoceptor antagonist, CGP20712A. Genotype was determined at 5 positions of ADRB2 previously reported as polymorphic. Potential influences of single nucleotide polymorphisms (SNPs) within the promoter region (-367, -47) and coding block (46, 79, 491) of ADRB2 on beta(2)-adrenoceptor expression were investigated. RESULTS: The density of beta(2)-adrenoceptors was variable among the 88 lung preparations studied ranging from 17 to 177fmol/mg protein (mean+/-S.E.M., 72+/-4fmol/mg protein). There was no influence of genotype on beta(2)-adrenoceptor expression for any of the polymorphisms studied except at position 491. The polymorphism at position 491C>T, leading to a change from thr to ile at amino acid 164, is uncommon. Preparations genotyped as heterozygous (126+/-15fmol/mg protein; n=5) expressed significantly (P=0.0005) higher levels of beta(2)-adrenoceptor than those that were homozygous (69+/-4fmol/mg protein; n=83). CONCLUSION: With the exception of position 491, these data indicate that polymorphisms of ADRB2 do not influence beta(2)-adrenoceptor expression in human lung.

The long-acting beta-adrenoceptor agonist, indacaterol, inhibits IgE-dependent responses of human lung mast cells.

BACKGROUND AND PURPOSE: The long-acting beta(2)-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 beta-agonists formoterol and salmeterol, were investigated on the IgE-dependent release and generation of histamine, cysteinyl-leukotrienes and prostaglandin D(2) from human lung mast cells. Moreover, the extent to which long-term (24-72 h) incubation of mast cells with long-acting beta-agonists impaired the subsequent ability of beta-agonists to inhibit mast cell responses was assessed. KEY RESULTS: Indacaterol was as potent and as efficacious as the full agonist, isoprenaline (EC(50), approximately 4 nmol x 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 beta-agonists were effective inhibitors of the IgE-dependent generation of cysteinyl-leukotrienes and prostaglandin D(2). Long-term incubation of mast cells with long-acting beta-agonists led to a reduction in the subsequent ability of beta-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.

Regulation of mast cells by beta-agonists.

The human lung mast cell is known to be a critical effector cell in the mediation of asthma. Activation of the mast cell by allergens and other stimuli leads to the release and generation of a wide variety of autacoids that cause bronchoconstriction, promote inflammation, and may influence airway remodeling. Therefore, the stabilization of mast cells has obvious value in the prevention of asthma. Among the drugs used to treat asthma, only beta-agonists are effective stabilizers of mast cells. Both short- and long-acting beta-agonists are effective against mast cells, but there are differences between agonists regarding the extent of inhibitory activity attained. Consequently, the type of beta-agonist prescribed influences the degree of mast cell stabilization possible. Despite the potential value of attenuating mast cell activity with beta-agonists, this benefit may diminish with time because of the development of tolerance. Both short- and long-acting beta-agonists can induce tolerance to mast cell stabilization, and generally, higher efficacy agonists tend to induce greater levels of tolerance; however, weaker agonists induce greater levels of tolerance than might be expected. Tolerance to the mast-cell-stabilizing effects of beta- agonists may be an issue clinically, because this occurs more readily than tolerance to smooth muscle relaxation. This could lead to a situation in which beta-agonists fail to prevent the release of mediators from mast cells but can still effectively relax airway smooth muscle. The continued ability to bronchodilate could mask the unfavorable consequences of unchecked mediator release from mast cells.

Prostaglandin E2 activates EP2 receptors to inhibit human lung mast cell degranulation.

The prostanoid, PGE2, is known to inhibit human lung mast cell activity. The aim of the present study was to characterize the EP receptor that mediates this effect. PGE2 (pEC(50), 5.8+/-0.1) inhibited the IgE-mediated release of histamine from mast cells in a concentration-dependent manner. Alternative EP receptor agonists were studied. The EP2-selective agonist, butaprost (pEC50, 5.2+/-0.2), was an effective inhibitor of mediator release whereas the EP1/EP3 receptor agonist, sulprostone, and the EP1-selective agonist, 17-phenyl-trinor-PGE2, were ineffective. The DP agonist PGD2, the FP agonist PGF(2alpha), the IP agonist iloprost and the TP agonist U-46619 were ineffective inhibitors of IgE-mediated histamine release from mast cells. PGE2 induced a concentration-dependent increase in intracellular cAMP levels in mast cells. The effects of the EP1/EP2 receptor antagonist, AH6809, and the EP4 receptor antagonist, AH23848, on the PGE2-mediated inhibition of histamine release were determined. AH6809 (pK(B), 5.6+/-0.1) caused a modest rightward shift in the PGE2 concentration-response curve, whereas AH23848 was ineffective. Long-term (24 h) incubation of mast cells with either PGE2 or butaprost (EP2 agonist), but not sulprostone (EP1/EP3 agonist), caused a significant reduction in the subsequent ability of PGE2 to inhibit histamine release. Collectively, these data suggest that PGE2 mediates effects on human lung mast cells by interacting with EP2 receptors.

Mast cell beta2-adrenoceptors.

The human lung mast cell is a crucial effector cell in the mediation of asthma. Activation of mast cells by allergens, and other insults, leads to the elaboration of a wide variety of autacoids that cause bronchoconstriction and promote inflammation. Of the drugs that are used to treat asthma, only bronchodilator beta2-adrenoceptor agonists are effective at inhibiting the elaboration of mediators from mast cells. Both short- and long-acting beta2-adrenoceptor agonists are effective inhibitors of mast cells although there are differences in the degree of inhibitory activity attained with a given agonist. Human lung mast cells express a homogeneous population of beta2-adrenoceptors. However, the density of beta2-adrenoceptors differs from preparation to preparation and this may influence the extent to which agonists stabilise mast cell activity. Tolerance to the mast cell-stabilising activity of beta2-adrenoceptor agonists can be readily demonstrated. As a generalisation, agonists that are more effective inhibitors of mediator release also induce greater levels of tolerance although weaker agonists induce greater levels of tolerance than might be expected. However, the extent of tolerance does not correlate with the degree of beta2-adrenoceptor loss. The inhibitory activity of agonists and the extent of tolerance observed may be influenced by genetic polymorphisms in the gene for the beta2-adrenoceptor.