Ambroxol inhibits histamine release from human adenoidal mast cells

Inflammation Research -     

Neuropeptide-induced secretion from human skin mast cells.

Unlike human mast cells associated with mucosal surfaces such as lung, adenoids, tonsils and intestine, skin mast cells may be stimulated to release histamine by the neuropeptides substance P, vaso-active intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine and compound 48/80. Release of histamine by neuropeptides is rapid and accompanied by minimal generation of the eicosanoids prostaglandin D2 and leukotriene C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both immunological and non-immunological stimulation, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellularly. Similarly, elevations of intracellular cyclic AMP induced by anti-IgE occur only in the presence of extracellular calcium, whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope. From these results we suggest that IgE-dependent and neuropeptide stimulation of human skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule-associated mediators.     

人肥大细胞的IgE依赖性组胺和类胰蛋白酶分泌

目的　利用人结肠组织的肥大细胞和肥大细胞激活的体外研究系统评价人肥大细胞释放类胰蛋白酶和组胺的能力及其机制。方法　经酶悬浮的人结肠肥大细胞与抗IgE抗体共同培养后记录浓度相关曲线和时间关系曲线。类胰蛋白酶用酶联免疫吸附试验的方法测量 ,而组胺则由一种以玻璃纤维为基础的荧光比色法测量。结果　抗IgE抗体可引起浓度相关性的组胺和类胰蛋白酶释放 ,最大组胺和类胰蛋白酶分泌量分别比基础分泌量超出 2 .7和 2 .5倍以上。抗IgE抗体的作用从加样后 10s开始 ,6min后达高峰并至少持续 15min。百日咳毒素和代谢抑制剂能够分别抑制抗IgE抗体引起的组胺和类胰蛋白酶释放。百日咳毒素还能够减少自发性类胰蛋白酶释放。结论　人结肠肥大细胞在受到抗IgE抗体刺激时具有平行释放类胰蛋白酶和组胺的能力 ,这个过程与肥大细胞膜G蛋白偶联受体的激活有关 ,并消耗能量。肥大细胞自发性释放组胺和类胰蛋白酶的功能可能是通过不同的机制实现的。     

Interferon-alpha/beta inhibits IgE-dependent histamine release from rat mast cells.

Although mast cells and interferons are both involved in numerous immune and inflammatory responses, little is known about how microenvironmental factors such as interferons (IFNs) influence mast cell function. To study this question, sensitized peritoneal mast cells (greater than 98% purity) obtained from rats infected 4 weeks earlier with the parasite Nippostrongylus brasiliensis were preincubated for 24 hr with rat IFN-alpha/beta in RPMI-1640, then stimulated to degranulate with worm antigens. In the absence of antigen, IFN-alpha/beta had no noticeable effect on histamine release. However, in the presence of antigen, IFN-alpha/beta (150-1500 U/ml) inhibited histamine release in a dose-dependent manner (22.2 +/- 7.5% to 56.3 +/- 6.9%, n = 10). This inhibitory effect was neither heat (56 degrees for 1 hr) nor acid (pH 2 for 18 hr) labile, but was completely blocked by anti-IFN antibodies. In the presence of compound 48/80 (1 microgram/ml) or substance P (5 X 10(-5) M), IFN-alpha/beta was ineffective at modulating histamine release. Histamine release induced by antigen in the presence of the membrane phospholipid phosphatidyl-serine (30 micrograms/ml) was inhibited by IFN in a dose-dependent manner, but maximal inhibition (25.3 +/- 2.7%, n = 10) was reached at a lower concentration of IFN (750 U/ml) than when antigen was used alone. Therefore, rat IFN-alpha/beta appears to inhibit histamine release from rat mast cells in a dose- and stimulus-dependent manner and may do so by reducing the fluidity of the cell membrane.     

Ambroxol inhibits the release of histamine, leukotrienes and cytokines from human leukocytes and mast cells

OBJECTIVES AND DESIGN: The effects of the mucolytic agents ambroxol and N-acetylcystein (NAC) were studied on the release of histamine, leukotrienes, cytokines and superoxide anions from a variety of cells involved in the pathogenesis of allergic inflammation. SUBJECTS: Mast cells were isolated from human adenoids and skin (n = 5-6). Basophils, monocytes and granulocytes were obtained from Buffy-coat blood obtained from healthy blood donors (n = 4-7) and enriched by density centrifugation. TREATMENT AND METHODS: Ambroxol or NAC were added to the cells for different periods before stimulation with various immunological and non-immunological secretagogues. Histamine release from mast cells, basophils and monocytes was assayed either by radioimmunoassay or spectrofluorometrically. LTC4 (basophils), LTB4 (neutrophil/eosinophil granulocytes or monocytes), IL-4 and IL-13 (basophils) were measured by ELISA. RESULTS: Ambroxol inhibited histamine release by more than 50% from human adenoidal mast cells (1000 microM ambroxol) and skin mast cells (100 microM ambroxol) stimulated by Con A and compound 48/80, respectively. Ambroxol (100 microM) strikingly inhibited anti-IgE induced release of both histamine, LTC4, IL-4 and IL-13 from basophils and reduced both histamine and LTB4 release induced by C5a or Zymosan in monocytes. The drug also reduced LTB4 and superoxide anion production in granulocytes stimulated by zymosan or fMLP. In all cell types studied, ambroxol was more efficacious following a short preincubation (5-15 min) of the drug with the cells before stimulation. In contrast, NAC produced no clear effects on any of the different cell types studied, regardless of the preincubation period, the concentration or the stimulus employed. CONCLUSIONS: Unlike NAC, ambroxol is able to not only inhibit acute mediator release from mast cells and leukocytes but also reduce immunomodulatory cytokine generation from basophils and may have beneficial effects in the treatment of allergic respiratory diseases.     

Cyclosporin A inhibits histamine release from human peripheral basophils and human skin mast cells

Cyclosporin A (CSA) is a strong immunosuppressive substance which is widely used for prevention of transplant rejection. Moreover, this compound is clinically active in different cutaneous inflammatory reactions. In the present study the effects of CSA on mediator release from activated human basophils and isolated human skin mast cells, as important effector cells of such skin diseases, were investigated. CSA exterted a dose-dependent inhibition of the IgE-mediated histamine release from both cell populations. The maximum inhibition was 64.4±9.3% and 85.8±14.2% for basophils and skin mast cells, respectively. Non-immunological histamine release induced by calcium ionophore A23817 was also inhibited. The present data suggest, therefore, that the anti-allergic properties of CSA on basophils and skin mast cells may be partially responsible for the strong anti-inflammatory effects of CSAin vivo.

     

Mercury induces inflammatory mediator release from human mast cells

Background  

Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl₂) on human mast cell activation.     

Platelet augmentation of IgE-dependent histamine release from human basophils and mast cells

A factor(s) from human platelets enhances IgE-mediated histamine release from human basophils and mast cells. This effect is directly related to the platelet number; at physiological platelet/leukocyte ratios (40:1), the enhancement was 66 +/- 11%. Platelet stimulation by thrombin more than doubled the enhancement, to 172 +/- 10% at 40:1. Mast cell release was also enhanced by platelets although the magnitude was more limited (86 +/- 13% at 40:1 with thrombin). Direct basophil/platelet contact was unnecessary in that platelet supernatants were fully active; a direct platelet factor/basophil interaction is suggested, however, by the fact that basophils purified 100-fold with respect to other leukocytes were enhanced by the platelet factors. The appearance of platelet-enhancing activity is associated with the release of an alpha-granule marker (PF4) rather than with products of arachidonic acid metabolism (thromboxane B2). The platelet factor(s) responsible for these effects are not dialyzable, are heat stable and do not appear to be identical to PF4 or platelet-derived growth factor (PDGF). Since anti-IgE-stimulated basophils cause PF4 release and this correlates with the release of enhancing factor, we suggest that a pro-inflammatory feed forward relationship exists. Together with our previous data showing that platelets are activated in vivo during antigen challenge of allergic asthmatic subjects, these results suggest that platelets may be important in modulating IgE-mediated allergic reactions in man.     

Conditioned medium from concanavalin A-stimulated spleen cells inhibits the IgE-dependent sensitization of murine peritoneal mast cells in vitro.

Conditioned medium (CM) from concanavalin A (Con A)-stimulated murine spleen cells inhibited release of histamine and 5-HT from murine peritoneal mast cells sensitized with monoclonal IgE anti-DNP antibody and challenged with DNP-human serum albumin (HSA) antigen. Inhibition was seen when the CM was added to the mast cells either 24 hr before or simultaneous with, but not 24 hr subsequent to, the IgE, thus showing that inhibition was at the IgE-dependent stage of mast cell sensitization. Unconditioned medium, prepared in the same way as CM but not exposed to spleen cells was without activity, demonstrating that inhibition was due to a spleen cell-derived factor. CM from unstimulated spleen cells was likewise without activity. The sensitization inhibitory factor appears to be a protein, since it was retained upon dialysis, and destroyed by heating at 70 degrees and above. The factor does not appear to be IgE, since it was stable at 56 degrees, and is not IL-1 or IL-2, since recombinant human IL-1 alpha and IL-1 beta, and recombinant mouse IL-1 alpha and IL-2 were without inhibitory activity. The active CM and all recombinant IL-1 and IL-2 preparations did not release histamine or 5-HT directly from mast cells during 48 hr of culture, and did not modulate the histamine content of these cells, nor their capacity to incorporate [3H]5-HT.     

Dispersion and characterisation of mast cells from human skin

A technique is described for the enzymatic dispersion of mast cells in high yield from human infant foreskin. Dispersed mast cells exhibit high viability as assessed by light microscopy, low spontaneous histamine release, and survival in culture. Dispersed mast cells release histamine in response to immunological stimulation and synthetic secretagogues including ionophore A23187, compound 48/80 and poly-L-lysine. Reactivity to these stimuli indicates that cutaneous mast cells differ in their properties from human pulmonary mast cells.     

Effect of cytokines on mediator release from human dispersed lung mast cells

To evaluate the contribution of human lung mast cells (HLMC) to allergic inflammation, we investigated whether or not cytokines, including stem-cell factor (SCF), monocyte chemotactic and activating factor (MCAF), and RANTES, activate HLMC. SCF induced histamine release from dispersed HLMC in a dose-dependent fashion (P<0.01). The release was 7.8 ± 1.0% at 500 ng/ml SCF (n= 9). This response was also observed in chopped lung tissue. HLMC from which surface IgE molecules had been removed by treatment with lactic acid responded to SCF, while these cells lost their response to anti-IgE. The process was relatively rapid and reached a maximum in 5 min. This response required extracellular calcium, and it was observed at 37°C, but not at 4°C or 20°. A brief preincubation (10 min) with lower concentrations of SCF, which were ineffective in releasing histamine, enhanced anti-IgE-induced histamine release (P<0.05), while its enhancing effect was lost by the longer preincubation (30 min). SCF did not prime basophils to enhance stimulated-histamine release. Interleukin (IL)-1α, IL-1β, IL-3, IL-4, IL-5, granulocyte/macrophage-colony stimulating factor (GM-CSF), MCAF, and RANTES neither induced histamine release nor enhanced the release stimulated by anti-IgE after a 10- or 30-min preincubation. The combination of IL-3 and IL-4 showed no effect on histamine release from HLMC. Leukotriene (LT)C₄/D₄/E₄ production by SCF was negligible, as compared with anti-IgE-induced LT production. SCF at 1.5 ng/ml augmented anti-IgE-induced LT generation significantly (536+ 117 pg/10⁵ mast cells and 1569 ± 258 pg/10⁵ mast cells; P<0.01). These results provide further evidence that numerous aspects of the phenotype of mast cells and basophils are heterogeneous, including structure, relevant secretagogues, and pharmacologic control.     

Olopatadine inhibits TNFalpha release from human conjunctival mast cells.

BACKGROUND: Tumor necrosis factor-alpha (TNFalpha) release likely plays a crucial role in allergic ocular inflammation via increasing ICAM-1 on epithelial cells and triggering other proinflammatory events. The immediate and prolonged release of TNFalpha from human conjunctival mast cells in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined. Olopatadine (Patanol) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine, tryptase, and PGD2 release in vitro and promote decreased H1 receptor binding activity in vitro and functional H1 receptor antagonism in vivo. OBJECTIVE: To investigate the effect of olopatadine on TNFalpha release from anti-IgE antibody challenged purified human conjunctival mast cells. METHODS: Human conjunctival mast cells were purified (>95%) from cadaveric tissues using a procedure combining enzymatic digestion and Percoll gradient centrifugation. These cells were incubated with olopatadine for 30 minutes then challenged with anti-IgE antibody for 90 minutes. Supernatants were analyzed for TNFalpha. RESULTS: Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFalpha release in a concentration dependent manner (optimum concentration was 10 microg/mL). Olopatadine pre-incubation resulted in a dose-dependent decrease in anti-IgE antibody mediated TNFalpha release (IC50 = 13.1 microM). At a concentration of 3 mM olopatadine reduced TNFalpha release to the level of unchallenged controls. CONCLUSION: Olopatadine inhibited anti-IgE antibody-mediated release of TNFalpha from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.     

TRAIL mediated signaling in human mast cells: the influence of IgE-dependent activation

Background:  Mast cells activation through FcɛRI cross-linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE-dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction.
Methods:  Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti-human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro-apoptotic stimulus, activated, IgE-treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF).
Results:  In accordance with a previous study of ours, it was found that IgE-dependent activation increased TRAIL-induced caspase-8 and caspase-3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE-dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL-1) anti-apoptotic molecules as well as the pro-apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL-1 and of BIM.
Conclusions:  These findings suggest that IgE-dependent activation of human mast cells induces an increase in both pro-survival and pro-apoptotic molecules. We therefore hypothesized that IgE-dependent activation may regulate human mast cell apoptosis by fine-tuning anti-apoptotic and pro-apoptotic factors.     

Differential modulation of mediator release from human basophils and mast cells by mizolastine

BACKGROUND: Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti-inflammatory activities by modulating mediator release from basophils and mast cells. OBJECTIVE: To study the in vitro effects of mizolastine, an H1 receptor antagonist, on the release of eicosanoids, histamine and IL-4 from human basophils and lung mast cells. METHODS AND RESULTS: Mizolastine (10(-7)-10(-5) M) concentration-dependently inhibited the release of cysteinyl leukotriene C4 from anti-IgE-stimulated basophils (IC(50): 3.85+/-0.28 microM) and mast cells (IC(50): 3.92+/-0.41 microM). The same concentrations of mizolastine did not affect anti-IgE-induced prostaglandin D2 release from lung mast cells. In contrast, mizolastine enhanced up to 80% IgE-mediated histamine release (EC(50): 4.63+/-0.14 microM) from basophils, but not from mast cells and it significantly potentiated IL-4 release from basophils induced by anti-IgE. Mizolastine did not affect histamine release from basophils induced by formyl peptide, whereas it inhibited cysteinyl leukotriene C4 release (IC(50): 1.86+/-0.24 microM). Blockade of cytosolic phospholipase A2 and arachidonic acid mobilization by pyrrolidine-1 did not alter the effect of mizolastine on histamine release from basophils, thereby excluding accumulation of arachidonic acid metabolic intermediates as the cause of this effect. Mizolastine did not influence anti-IgE-induced activation of extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) in human basophils. CONCLUSIONS: Mizolastine efficiently inhibits LTC4 synthesis in human basophils and mast cells presumably by interfering with 5-lipoxygenase. In contrast, it enhances histamine and IL-4 release only from anti-IgE-stimulated basophils. Therefore, mizolastine differentially regulates the production of mediators from basophils and mast cells in a cell- and stimulus-specific fashion.     

Biological properties of human skin mast cells

Mast cells and basophils, although sharing many constitutive properties, are quite distinct in their development, functions and biological properties. Mast cell granules are composed of a macromolecular matrix of proteoglycan and neutral protease of which heparin and tryptase, respectively, are predominant. The distribution of the other major neutral protease, chymase, allows human mast cell subpopulations to be subdivided immunocytochemically. All human mast cells respond to IgE-dependent stimulation with the secretion of the preformed mediator, histamine, and the newly generated lipid-derived eicosanoids PGD₂ and LTC₄. Although amounts of these products vary between mast cells dispersed from different tissues, it is uncertain whether this reflects true heterogeneity. Mast cells of the human skin, but not those of other tissues, are sensitive to stimulation by substance P, compound 48/80 and other basic non-immunological stimuli. The mechanism of mediator secretion induced by these agents is distinct from that induced by IgE-dependent stimulation. However, the morphological characteristics of degranulation are similar, suggesting that the distinct biochemical pathways merge into a common pathway before effecting degranulation.     

Relationship between mediator release from human lung mast cells in vitro and in vivo

There is now compelling evidence to incriminate bronchial mast cells in the pathogenesis of bronchoconstriction of allergic asthma. Human mast cells isolated from lung tissue or bronchoalveolar lavage release histamine and generate eicosanoids upon IgE-dependent activation. In this paper we present data that raise doubts about the significance of phospholipid methylation in IgE-dependent activation-secretion coupling and provide evidence that drugs such as 3-deazaadenosine inhibit mediator secretion by inhibiting phosphodiesterase, in addition to inhibiting putative methylation pathways. Activation of human mast cells and basophils also stimulates adenylate cyclase to increase levels of cyclic AMP, which, on the basis of pharmacological manipulation with purine nucleosides, we believe is involved in the progression of the secretory response. Human lung cells also generate both cyclo- and lipoxygenase products of arachidonate upon Ca++-dependent stimulation with complex interactions occurring between these pathways in the presence of the leukotriene inhibitor, Piriprost. The role of mast cells in the immediate airway response to inhaled allergens in asthma was demonstrated by showing an interaction between nonspecific bronchial reactivity and mast cell reactivity in predicting the airway response upon antigen inhalation. Further confirmation of this concept was obtained by showing an inverse relationship between the release of histamine and neutrophil chemotactic factor (NCF) into the circulation induced by antigen challenge, and nonspecific airway reactivity. The identification of significant increases in circulating mediators following antigen provocation of patients with seasonal asthma enabled the effects of drugs used in the treatment of asthma to be compared on airway calibre and mast cell mediator release. Sodium cromoglycate partially inhibited the airway and plasma histamine responses with antigen, but totally inhibited the increases in NCF. Salbutamol completely inhibited all responses, while ipratropium bromide, which produced the same bronchoconstriction as achieved with salbutamol, had no effect. The potent H1-antagonist astemizole partially inhibited bronchoconstriction without affecting histamine release. Antigen provocation produced a significant increase in circulating levels of the 13,14-dihydro-15-keto metabolite of PGF2 alpha which could originate from mast cell-derived PGD2. In both retrospective and prospective studies, a close relationship was shown between nonspecific bronchial reactivity and resting airway calibre in asthma.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of purification-associated reduction in IgE-dependent histamine release from rat peritoneal mast cells

Histamine release from purified rat peritoneal mast cells (PMC) was examined and compared to that from a non-purified preparation (PEC). Both PEC and PMC released similar amounts of histamine upon stimulation with compound 48/80, calcium ionophore A23187 and substance P. In contrast, IgE-dependent histamine release from PMC caused by antigen, anti-IgE and concanavalin A was very low compared to that of PEC. The reduced IgE-dependent histamine release from PMC, however, was recovered when PMC was reconstituted with non-mast cells (NMC) present in the peritoneal cavity. The effect was time-dependent and reached a plateau in 30 min. NMC from both sensitized and non-sensitized rats recovered the reduced histamine release from PMC dose-dependently. The potentiating effect of NMC was observed even in the presence of excess amount of phosphatidylserine. Supernatants of NMC and a mixture of PMC and NMC incubated for 1 hr at 37°C, however, failed to potentiate the histamine release. These results demonstrate that IgE-dependent histamine release from rat peritoneal mast cells is upregulated by other cells present in the peritoneal cavity, and that the mechanism involved is distinct from that of phosphatidylserine.