Recovery from desensitization of IgE-dependent responses in human lung mast cells.

BACKGROUND: Clinical desensitization and oral food immunotherapy are therapeutic interventions that allow individuals who react adversely to an allergen (drug or food) to be made tolerant to the allergen. However, tolerance is brief, and allergen hypersensitivity can recur within days following allergen withdrawal. OBJECTIVE: We hypothesize that the reason these treatments are temporary reflects rapid recovery of mast cells from a desensitized state. We sought to test this. METHODS: Desensitization of IgE-mediated histamine release from human lung mast cells was explored by methods that partially replicate the pattern of treatment during clinical desensitization. Specific and non-specific desensitization and changes in surface IgE were examined following desensitization. Recovery from desensitization was also studied. RESULTS: Desensitization of mast cell responses was readily induced with concentrations of antigen or anti-IgE that were suboptimal for secretion. There was little or no non-specific desensitization when lung mast cells were exposed to antigens. There was no loss of cell surface IgE following desensitization. Removing the desensitizing stimulus from the media following desensitization allowed the cells to recover with half-point of recovery of ~1.5 days and complete recovery after 5 days. Both the functional response and histamine content recovered within this time frame. The recovery appeared possible because both antigens and anti-IgE dissociated rapidly from cells after washing to remove excess stimulus. CONCLUSIONS AND CLINICAL RELEVANCE: Human lung mast cells readily recover from a desensitized state following removal of desensitizing antigen. This finding provides a potential explanation for the ephemeral nature of clinical desensitization.

Regulation of human skin mast cell histamine release by PDE inhibitors.

BACKGROUND: Mast cell and basophiles are thought to be central to inflammation that has an allergic basis as allergens activate these cells in an IgE-dependent manner to generate mediators such as histamine, eicosanoids and cytokines. Phosphodiesterase (PDE) is known to exist as multiple molecular forms of enzyme that metabolise the second messengers. Studies of our own have shown that, of a variety of isoform-selective drugs, the PDE4-selective inhibitors, such as rolipram, attenuate the IgE-mediated release of histamine from human basophiles but not from human lung mast cells (HLMC). The main aim of the present study was to characterise the type and role of PDEs regulating human skin mast cells by using selective and non-selective PDE inhibitors. METHODS: Cells were pre-treated for 15 min with these agents and then challenged with an optimal releasing concentration of anti IgE (1:300) for a further 25 min for the release of histamine. RESULTS: The data show that all the selective PDE-inhibitor compounds (10(-5)M) were ineffective whereas the non-selective PDE inhibitor, theophylline (10(-3)M), inhibited histamine release from HSMC (74 ± 4% inhibition; p<0.05). None of the selective PDE inhibitors had any effect on histamine release from HLMC whereas, in basophiles, compounds with activity at PDE 4 (rolipram, denbufylline, Ro-2017, Org 30029) were effective inhibitors of histamine release. CONCLUSION: The data suggest that unlike most inflammatory cells, PDE-selective inhibitors are ineffective stabilisers of HSMC activity which is similar to HLMC.

Characterization of the EP receptor subtype that mediates the inhibitory effects of prostaglandin E2 on IgE-dependent secretion from human lung mast cells.

BACKGROUND: Prostaglandin E2 (PGE2 ) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2 -selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. OBJECTIVE: The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. METHODS: The effects of non-selective (PGE2 , misoprostol), EP2 -selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4 -selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2 -selective (PF-04418948, PF-04852946) and EP4 -selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. RESULTS: Prostaglandin E2 , EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4 -selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2 -selective antagonists caused rightward shifts in the PGE2 concentration-response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.

Heterogeneity in the responses of human lung mast cells to stem cell factor.

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 D(2) (PGD(2) ) 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 PGD(2) 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 PGD(2), than hitherto appreciated and this could be important in the context of respiratory diseases.

Characterization of syk expression in human lung mast cells: relationship with function.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION AND CLINICAL RELEVANCE: 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.

Influence of beta2-adrenoceptor gene polymorphisms on beta2-adrenoceptor-mediated responses in human lung mast cells.

BACKGROUND AND PURPOSE: Previous studies have shown that beta(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 beta (2)-adrenoceptor gene (ADRB2) influence this variability in (a) beta(2)-adrenoceptor-mediated inhibition and (b) desensitization of beta(2)-adrenoceptor-mediated responses in human lung mast cells. EXPERIMENTAL APPROACH: Mast cells were isolated from human lung tissue. The inhibitory effects of the beta-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 beta (2)-adrenoceptor-mediated responses in mast cells. CONCLUSIONS AND IMPLICATIONS: These data indicate that certain polymorphisms in ADRB2 influence beta(2)-adrenoceptor-mediated responses in human lung mast cells.

Role of calcineurin in the regulation of human lung mast cell and basophil function by cyclosporine and FK506.

BACKGROUND AND PURPOSE: Cyclosporine and FK506 are thought to act by targeting the Ca2+-dependent protein phosphatase, calcineurin. The aim of the present study was to determine whether cyclosporine and FK506 stabilize mast cells and basophils by interacting with calcineurin. EXPERIMENTAL APPROACH: The effects of cyclosporine and FK506 on the IgE-mediated release of histamine from mast cells and basophils were evaluated. The presence of calcineurin in cells was determined by Western blotting. Ca2+-dependent protein phosphatase activities were assessed in cell extracts using a synthetic phosphorylated peptide that is known to serve as a substrate for calcineurin. KEY RESULTS: FK506 was about 100-fold more potent than cyclosporine as an inhibitor of IgE-dependent histamine release from mast cells and basophils. Immunoblotting of solubilized preparations of purified cells demonstrated the presence of calcineurin in mast cells and basophils. In enzyme assays, mast cells expressed approximately 7-fold higher Ca2+-dependent protein phosphatase activity than basophils. Whereas cyclosporine effectively inhibited Ca2+-dependent protein phosphatase activity in cell extracts, FK506 was considerably less effective. CONCLUSIONS AND IMPLICATIONS: FK506 and cyclosporine inhibit the stimulated release of histamine from mast cells and basophils. However, the ability of cyclosporine, but not FK506, to inhibit Ca2+-dependent protein phosphatase activity questions whether FK506 stabilizes mast cells and basophils by interacting with calcineurin.

Regulation of immunoglobulin E-mediated secretion by protein phosphatases in human basophils and mast cells of skin and lung.

A wide range of serine/threonine protein phosphatase (PP) inhibitors were studied for effects on the immunoglobulin E (IgE)-mediated release of histamine from human lung mast cells, human skin mast cells and basophils. Okadaic acid (OA) inhibited the release of histamine from all three cell types in a concentration-dependent manner. Two structural analogues of okadaic acid, okadaol and okadaone, known to be less active than the parent molecule as inhibitors of PP, were less active than okadaic acid as inhibitors of histamine release in these three cell types. A number of PP inhibitors, showing differences in selectivity for PP1 and PP2A, were also evaluated. Calyculin, which is roughly equipotent as a PP1 and PP2A inhibitor, attenuated the release of histamine from all three cell types. Similarly, tautomycin (TAU), which shows greater selectivity for PP1 over PP2A, was also effective at inhibiting histamine release in all three cell types. In contrast, fostriecin, which is very much more potent as an inhibitor of PP2A over PP1, was ineffective as an inhibitor in all three cell types. These data indicate that the regulation of mediator release by PPs is similar in lung mast cells, skin mast cells and basophils. Moreover, the data suggest that PP1 is important in the control of cellular activity.

Influence of genetic polymorphisms in the beta2-adrenoceptor on desensitization in human lung mast cells.

The beta-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 beta2-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 beta2-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 beta2-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.

Role of protein phosphatases in the regulation of human mast cell and basophil function.

Many extracellular stimuli mediate physiological change in target cells by altering the phosphorylation state of proteins. These alterations result from the dynamic interplay of protein kinases, which mediate phosphorylations, and protein phosphatases, which catalyse dephosphorylations. The antigen-mediated aggregation of high-affinity receptors for IgE on mast cells and basophils triggers rapid changes in the phosphorylation of many proteins and culminates in the generation of inflammatory mediators involved in allergic inflammatory diseases such as asthma. Although protein kinases have an established role in this process, less is known about the involvement of protein phosphatases. This imbalance has been redressed in recent years by the availability of phosphatase inhibitors, such as okadaic acid, that facilitate investigations of the role of protein phosphatases in intact cells. Here we review a number of studies in which inhibitors of protein phosphatases have been used to shed light on the potential importance of these enzymes in the regulation of human mast cell and human basophil function.

Beta-adrenoceptor reserve in human lung: a comparison between airway smooth muscle and mast cells.

The effects of several beta-adrenoceptor agonists on the relaxation of precontracted human bronchial rings and the inhibition of IgE-mediated histamine release from human lung mast cells (HLMC) were studied. For the relaxation of bronchial rings, isoprenaline, fenoterol and terbutaline were full agonists whereas salbutamol was a full agonist in some (two out of six) experiments and a partial agonist in the remainder. For the inhibition of histamine release, relative to isoprenaline, neither fenoterol, terbutaline nor salbutamol was a full agonist. Studies with the irreversible beta-adrenoceptor antagonist, bromoacetylalprenolol menthane, indicated that there was a larger beta-adrenoceptor reserve for the relaxation of precontracted bronchial rings than for the inhibition of histamine release from HLMC. Further studies indicated that the isoprenaline inhibition of histamine release was considerably more susceptible to desensitizing treatments than the isoprenaline relaxation of bronchial rings. Collectively, these data suggest that a larger beta-adrenoceptor reserve exists for the relaxation of smooth muscle than the inhibition of histamine release from HLMC and that differences in receptor reserve may contribute to the relative susceptibilities of the two systems to desensitization.

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils.

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid. 2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0.02-0.1 nM) > or = microcystin (0.1 nM) > okadaic acid (70 nM). 3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71+/-3% and 49+/-13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1). 4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates. 5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

Regulation of human mast cell and basophil function by cAMP.

1. Mast cells and basophils are important in mediating allergic disorders such as asthma. Activation of these cells results in the release of a wide variety of mediators that can promote inflammatory responses. 2. Receptor-mediated activators of adenylate cyclase such as the beta-adrenoceptor agonist, isoprenaline, and prostaglandin E2 (PGE2) are effective at inhibiting mediator release from human lung mast cells (HLMC) but not basophils. In HLMC, both isoprenaline and PGE2 elevate and sustain increases in cyclic adenosine 3',5'-monophosphate (cAMP) whereas in basophils, both compounds cause transient increases in cAMP. 3. Non-selective inhibitors of phosphodiesterase (PDE) such as theophylline and 3-isobutyl-1-methylxanthine are effective inhibitors of mediator release from both HLMC and basophils and both compounds cause elevations of cAMP that are sustained in both cell types. 4. Studies with selective inhibitors of PDE indicate that the cAMP-specific PDE, PDE 4, regulates the activity of basophils but not HLMC. The nature of the PDE regulating HLMC responses is uncertain. 5. These data indicate that agents that induce and sustain elevations in intracellular cAMP attenuate the stimulated release of mediators from mast cells and basophils. However, the responsiveness of HLMC and basophils to selected cAMP-active agents differs.

Influence of receptor reserve on beta-adrenoceptor-mediated responses in human lung mast cells.

1. The effects of the beta-adrenoceptor agonists isoprenaline and salbutamol on IgE-mediated histamine release from human lung mast cells (HLMC) were evaluated. Both agonists (10(-10)-10(-5) M) inhibited histamine release in a dose-dependent manner and isoprenaline (pD2, 8.3+/-0.1, mean+/-s.e.mean) was more potent than salbutamol (7.3+/-0.1). Moreover, the mean data indicated that salbutamol was a partial agonist when compared with isoprenaline. However, there was a large degree of interexperimental variability because, in 11 of 32 experiments, salbutamol was a full agonist and, in 21 of 32 experiments, a partial agonist relative to isoprenaline. These data suggest that different HLMC preparations possess variable receptor reserves. 2. The effect of the irreversible beta-adrenoceptor antagonist, bromoacetylalprenolol menthane (BAAM), on the inhibition of IgE-mediated histamine release by both isoprenaline and prostaglandin E2 (PGE2) was assessed. Whereas BAAM (100 nM) antagonized the isoprenaline inhibition of histamine release from activated HLMC, BAAM had no effect on the PGE2 inhibition. Pretreatment of HLMC with the beta2-selective competitive antagonist, ICI 118551 (100 nM), protected against the loss in responsiveness to isoprenaline following treatment with BAAM. 3. Concentrations of 1, 10 and 100 nM of BAAM caused dose-dependent rightward shifts in the dose-response curve for the isoprenaline inhibition of histamine release. Furthermore, there was a dose-dependent reduction in the maximal inhibitory response obtained with isoprenaline following treatments with increasing concentrations of BAAM. Although the rightward shifts in the isoprenaline dose-response curves, with a given concentration of BAAM, were similar in all experiments, there was some variability in the depression of the maximal response in individual experiments. Thus, in 6 of 16 experiments, BAAM (1 nM) did not depress the maximal response to isoprenaline, whereas in 10 of 16 experiments there was a depression (7 to 49% reduction) in the maximal response. These data suggest that different HLMC preparations possess variable receptor reserves. 4. Isoprenaline was more potent as an inhibitor in those HLMC preparations in which there was a larger receptor reserve (i.e. preparations in which the maximal inhibitory response to isoprenaline was unaffected by pretreatment with 1 nM BAAM). 5. The influence of receptor reserve on the inhibition by salbutamol of histamine release from HLMC was evaluated. There was a good correlation (r=0.77) between receptor reserve and the maximal response (relative to isoprenaline) obtained with salbutamol. Thus, HLMC preparations with larger receptor reserves were more responsive to salbutamol. 6. Receptor reserve influenced the desensitization of beta-adrenoceptor-mediated responses in HLMC. Cells were incubated (24 h) with isoprenaline (1 microM), washed and then the ability of a second isoprenaline (10(-10)-10(-5) M) exposure to inhibit histamine release was assessed. The pretreatment caused a reduction in the isoprenaline inhibition of histamine release although the extent of desensitization was highly variable, ranging from essentially negligible levels in some preparations to substantial reductions (93% desensitization) in the ability of isoprenaline to inhibit histamine release. There was a reasonable correlation (r=0.59) between receptor reserve and desensitization. Preparations that possessed a larger receptor reserve were more resistant to desensitization. 7. Collectively, these data suggest that a receptor reserve exists for the beta-adrenoceptor-mediated inhibition of histamine release from HLMC but that the size of this reserve varies between HLMC preparations. Moreover, the size of this receptor reserve may influence the sensitivity of HLMC to beta-adrenoceptor agonists and the susceptibility of individual HLMC preparations to desensitization.

Effect of radiographic contrast media on histamine release from human mast cells and basophils.

Radiographic contrast media (RCM) cause histamine-dependent allergic-like reactions. The direct effects of diatrizoate (high osmolar ionic monomer), ioxaglate (low osmolar ionic dimer), iopromide (low osmolar non ionic monomer) and iotrolan (iso-osmolar non ionic dimer) at the concentration of 200 mgI ml-1 (60 min exposure) on the release of histamine from human basophils, human lung mast cells (HLMC), and human skin mast cells (HSMC) were investigated. Diatrizoate induced 48 +/- 4% histamine release in basophils, 15 +/- 3% in HLMC and 25 +/- 6% in HSMC. The remaining RCM were relatively ineffective activators of histamine release in both HLMC and HSMC (ioxaglate 4 +/- 1% and 4 +/- 1%, iopromide 5 +/- 1% and 7 +/- 2%, iotrolan 7 +/- 2% and 10 +/- 3%, respectively). Both iotrolan and ioxaglate were effective in basophils inducing 21 +/- 3% and 24 +/- 6% histamine release, respectively, whereas iopromide was relatively ineffective (7 +/- 4%). Diatrizoate induced histamine release from all three cell types with optimal levels of histamine release after a 2-4 h incubation although significant levels occurred within 15 min. Dose-dependent histamine release from HLMC occurred in all four types of RCM, the largest response (37 +/- 3%) being produced by diatrizoate. The effect of osmolality on histamine release was investigated using different concentrations of mannitol solutions (0.25, 0.5 and 1 M). Histamine release from HLMC, HSMC and basophils after 90 min exposure to mannitol (1 M) was 24 +/- 2% (p < 0.05), 9 +/- 3% (p = 0.06) and 49 +/- 1% (p < 0.05), respectively, suggesting that hyperosmolality per se can induce histamine release from basophils and mast cells. Diatrizoate-induced histamine release in all three cell types was significantly reduced by lowering the temperature to 0 degree C and partially attenuated by the metabolic inhibitors antimycin A (1 microM) and 2-deoxyglucose (5 mM), and by the omission of glucose from the buffer solution. Diatrizoate-induced histamine release was not dependent on extracellular calcium. These data suggest that diatrizoate induces histamine release at least in part by non-cytotoxic mechanisms.

Salmeterol inhibition of mediator release from human lung mast cells by beta-adrenoceptor-dependent and independent mechanisms.

1. The long-acting beta2-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 beta-adrenoceptor agonists were studied and the rank order of potency for the inhibition of histamine release from HLMC was isoprenaline > salmeterol > salbutamol. Approximate EC50 values for the inhibition of histamine release were 10 nM for isoprenaline and 100 nM for salbutamol. An EC50 value for salmeterol could not be calculated because maximal responses to salmeterol were not observed over the concentration range employed. 2. 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 (EC50 < 0.1 nM) was more potent than isoprenaline (EC50 0.4 nM) at attenuating sLT generation. 3. The beta-adrenoceptor antagonist, propranolol (1 microM), and the selective beta2-adrenoceptor antagonist, ICI 118,551 (0.1 microM), 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 microM) of salmeterol, both antagonists shifted the dose-reponse curve to salmeterol rightward. At a higher concentration (10 microM) of salmeterol, neither ICI 118,551 nor propranolol was an effective antagonist of the salmeterol-mediated inhibition of histamine release. 4. Prolonged exposure (4 h) of HLMC to isoprenaline (1 microM) caused an approximately 50% reduction in the effectiveness of a second exposure to isoprenaline (10 microM) at inhibiting the release of histamine. whereas this pretreatment did not affect the salmeterol (10 microM) inhibition of histamine release. 5. 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. 6. 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 beta-adrenoceptor-dependent and independent mechanisms.

Protection by dexamethasone of the functional desensitization to beta 2-adrenoceptor-mediated responses in human lung mast cells.

1. The beta-adrenoceptor agonist, isoprenaline, inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent manner. Maximal inhibitory effects were obtained with 0.1 microM isoprenaline. However, the inhibition of histamine release from HLMC by isoprenaline (0.1 microM) was highly variable ranging from 33 to 97% inhibition (mean, 59 +/- 3%, n = 27). 2. Long-term (24 h) incubation of HLMC with isoprenaline led to a subsequent reduction in the ability of a second exposure of isoprenaline to inhibit IgE-mediated histamine release from HLMC. The impairment in the ability of isoprenaline (0.1 microM) to inhibit histamine release following desensitizing conditions (1 microM isoprenaline for 24 h) was highly variable amongst HLMC preparations ranging from essentially negligible levels of desensitization in some preparations to complete abrogation of the inhibitory response in others (mean, 65 +/- 6% desensitization, n = 27). 3. The ability of HLMC to recover from desensitization was investigated. Following desensitizing conditions (1 microM isoprenaline for 24 h), HLMC were washed and incubated for 24 h in buffer and the effectiveness of isoprenaline (0.1 microM) to inhibit IgE-mediated histamine release from HLMC was assessed. The extent of recovery was highly variable with some HLMC preparations failing to recover and others displaying a complete restoration of responsiveness to isoprenaline (mean, 40 +/- 6% recovery, n = 23). 4. The effects of the glucocorticoid, dexamethasone, were also investigated. Long-term (24-72 h) treatments with dexamethasone (0.1 microM) had no effect on IgE-mediated histamine release from HLMC. Additionally, long-term (24-72 h) treatments with dexamethasone (0.1 microM) had no effect on the effectiveness of isoprenaline to inhibit histamine release. However, long-term (24-72 h) treatments with dexamethasone (0.1 microM) protected against the functional desensitization induced by incubation (24 h) of HLMC with isoprenaline (1 microM). The protective effect was time-dependent and pretreatment of HLMC with dexamethasone for either 24, 48 or 72 h prevented desensitization by either 15 +/- 7, 19 +/- 5 or 51 +/- 10%, respectively (n = 5-7). 5. HLMC preparations which were relatively refractory to isoprenaline even after withdrawal (24 h) from desensitizing conditions responded more effectively to isoprenaline (0.1 microM) if dexamethasone (0.1 microM) was also included during the recovery period (19 +/- 9% recovery after 24 h in buffer; 50 +/- 8% recovery after 24 h with dexamethasone, n = 5). 6. These data indicate that the responses of different HLMC preparations to isoprenaline, the susceptibility of HLMC to desensitization and the ability of HLMC to recover from desensitizing conditions varies markedly. Dexamethasone, which itself has no direct effects on IgE-mediated histamine release from HLMC, protected HLMC from the functional desensitization to beta-adrenoceptor agonists. Because beta 2-adrenoceptor agonists and glucocorticoids are important in the therapeutic management of asthma and as the HLMC is probably important in certain types of asthma, these findings may have wider clinical implications.

Effects of phosphodiesterase inhibitors on human lung mast cell and basophil function.

1. The non-hydrolysable cyclic AMP analogue, dibutyryl (Bu2)-cyclic AMP, inhibited the stimulated release of histamine from both basophils and human lung mast cells (HLMC) in a dose-dependent manner. The concentrations required to inhibit histamine release by 50% (IC50) were 0.8 and 0.7 mM in basophils and HLMC, respectively. The cyclic GMP analogue, Bu2-cyclic GMP, was ineffective as an inhibitor of histamine release in basophils and HLMC. 2. The non-selective phosphodiesterase (PDE) inhibitors, theophylline and isobutyl-methylxanthine (IBMX) inhibited the IgE-mediated release of histamine from both human basophils and HLMC in a dose-dependent fashion. IBMX and theophylline were more potent inhibitors in basophils than HLMC. IC50 values for the inhibition of histamine release were, 0.05 and 0.2 mM for IBMX and theophylline, respectively, in basophils and 0.25 and 1.2 mM for IBMX and theophylline in HLMC. 3. The PDE 4 inhibitor, rolipram, attenuated the release of both histamine and the generation of sulphopeptidoleukotrienes (sLT) from activated basophils at sub-micromolar concentrations but was ineffective at inhibiting the release of histamine and the generation of both sLT and prostaglandin D2 (PGD2) in HLMC. Additional PDE 4 inhibitors, denbufylline, Ro 20-1724, RP 73401 and nitraquazone, were all found to be effective inhibitors of mediator release in basophils but were ineffective in HLMC unless high concentrations (1 mM) were employed. 4. Neither 8-methoxymethyl IBMX (PDE 1 inhibitor), zaprinast (PDE 5 inhibitor) nor a range of PDE 3 inhibitors (siguazodan, SKF 94120, SKF 95654) were effective inhibitors of mediator release from either basophils or HLMC. 5. In basophils, rolipram acted to potentiate the inhibitory effects of the adenylate cyclase activator, forskolin, whereas in HLMC, rolipram failed to potentiate the inhibitory effects of forskolin. 6. Extracts of purified HLMC and basophils hydrolysed cyclic AMP. IBMX (100 microM) inhibited the PDE activity in basophil extracts by 67 +/- 7% (P < 0.0001) and in HLMC extracts by 63 +/- 9% (P < 0.0005). The hydrolysis of cyclic AMP by basophil extracts was inhibited by the selective PDE inhibitors (all at 10 microM), rolipram (56 +/- 8%, P < 0.0001) and the mixed PDE 3/4 inhibitor, Org 30029 (47 +/- 9%, P < 0.01), whereas 8-methoxymethyl IBMX, siguazodan and zaprinast were ineffective. In HLMC, rolipram, Org 30029, 8-methoxymethyl IBMX, siguazodan and zaprinast all inhibited the hydrolysis of cyclic AMP by extracts to a significant (P < 0.05) and similar extent (approximately 25% inhibition at 10 microM). 7. In total, these data suggest that modulation of the PDE 4 isoform can regulate basophil responses whereas an association of the PDE 4 isoform with the regulation of HLMC function remains uncertain.

Preliminary characterization of the role of protein serine/threonine phosphatases in the regulation of human lung mast cell function.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE-dependent release of histamine was 0.2 microM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D2 (PGD2) and sulphopeptidoleukotrienes (sLT) with IC50 values of 0.2 microM and 0.6 microM respectively. 2. The IgE-mediated generation of histamine, PGD2 and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid > okadaol > okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3. Extracts of HLMC liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 microM), okadaic acid (73 +/- 4% inhibition, P < 0.0005), okadaol (26 +/- 7% inhibition, P < 0.05) and okadaone (8 +/- 7% inhibition, P = 0.52). The rank order of activity of okadaic acid > okadaol > okadaone parallels the activity of these compounds as inhibitors of isolated PPs. 4. Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15 +/- 3% (P < 0.001) by a low (2 nM) concentration of okadaic acid and by 88 +/- 4% (P < 0.0005) by a higher (5 microM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 microM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5. Inhibitor 2, a PP1-selective inhibitor, attenuated (71 +/- 3% inhibition, P < 0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6. Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated by extracts of purified HLMC and this activity was inhibited (81 +/- 8% inhibition, P < 0.005) by 2 nM okadaic acid suggesting that PP2A is resident in HLMC. 7. Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.

Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen-specific IgE.

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 phospholipase A2 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.