Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 micrograms/ml anti-IgE released 38 +/- 4% of their available histamine and 39 +/- 12 ng LTC4/10(6) basophils within 15-30 min. F-Met peptide (n = 8) caused the release of 54 +/- 8% histamine and 42 +/- 25 ng LTC4/10(6) basophils within a period of 2-5 min. C5a caused the release of 22 +/- 3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67 +/- 8 and 82 +/- 11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 microM [3H]AA incorporated 290 pmol/10(6) cells, or 32 +/- 5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73 +/- 5%), with 20 +/- 3% as neutral lipid, and only 5 +/- 2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24 +/- 2%), phosphatidylinositol, PI (22 +/- 1%), and phosphatidylethanolamine, PE (15 +/- 3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 micrograms/ml anti-IgE revealed [3H]LTC4 (2.4 +/- 1.0%), [3H]5HETE (1.0 +/- 0.1%), unmetabolized [3H]AA (91 +/- 2%), and an unidentified peak (3.4 +/- 1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.     

Effect of deuterium oxide on leukotriene C4 generation in immunologically stimulated human leukocytes

Addition of deuterium oxide (D2O), 6-36%, resulted in a dose-dependent increase in allergen- or anti-IgE-induced leukotriene C4 (LTC4) generation from human basophils. In the presence of 36% D2O, the enhancement was 260 +/- 135% for allergen stimulation and 480 +/- 152% for anti-IgE stimulation as compared with the control incubated in normal buffer. The increasing effect of D2O on LTC4 generation from basophils was completely reversed by washing the cells before incubation with allergen. Vinblastine as well as colchicine, at a concentration of 100 microM, counteracted the effect of D2O. The enhanced release of histamine and LTC4 from basophils challenged with allergen was suppressed by Dimaprit, a histamine H2 receptor agonist, at a concentration required to inhibit the release by 50% of 5 X 10(-5) M for histamine and 10(-5) M for LTC4. These observations suggest that microtubules may be involved in LTC4 generation from immunologically stimulated basophils.     

Inhibitability and enhanceability of basophil histamine release in asthmatic and normal subjects

Circulating human basophils contain histamine, a potent mediator of inflammation. Previous in vitro studies have shown that histamine 'releasability' in asthmatic subjects differs from normal subjects but have not evaluated possible differences in the immunopharmacological control of the release of this mediator which might account for these differences. The purpose of the present study was to examine the immunopharmacologic control of basophil histamine release in 14 asthmatics and 10 normal subjects who were characterized by pulmonary function tests, allergic status (skin tests and serum IgE levels) and nonspecific airways reactivity to methacholine and histamine. Basophils were stimulated with anti-IgE, and the inhibitory effects of the H2 agonist, dimaprit, and dibutyryl cyclic AMP (dbcAMP), as well as the enhancing properties of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and indomethacin on the modulation of histamine release, were investigated. Although no statistically significant differences were seen in the percent histamine release triggered by anti-IgE in these two groups, enhancement of histamine release by 5-HPETE was more consistent in the asthmatic subjects (10 of 10) than in control subjects (6 of 8). The percent increase in histamine release produced by 5-HPETE in asthmatic subjects averaged 3.9 +/- 1.3% using 0.03 micrograms anti-IgE/ml and 4.8 +/- 3.2% using 0.1 microgram anti-IgE/ml (p less than 0.002, Wilcoxon's signed rank test), and averaged 3.0 +/- 4.3 and 3.1 +/- 5.3%, respectively, in control subjects (p greater than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Density heterogeneity of human lung mast cells

Suspensions of enzymatically dispersed human lung parenchymal mast cells were fractionated according to density by flotation through discontinuous Percoll gradients and examined for their responsiveness to release stimulants and pharmacologic agonists. Mast cells localized to all six density fractions (I-VI) examined: densities varied from specific gravities of 1.053 gm/ml to 1.123 gm/ml. Most (67%) lung mast cells localized to fractions III and IV, corresponding to specific gravities of 1.077 to 1.088 gm/ml, respectively. Histamine content increased with density from 2.7 +/- 0.3 pg per cell in fraction 1 to 4.8 +/- 0.7 pg per cell in fraction VI (mean +/- SEM; n = 19). Fraction III was least responsive to high concentrations of anti-IgE than to any other fractions and, along with fraction IV, the most responsive to ionophore A23187. All fractions released the arachidonate mediators prostaglandin D2 and leukotriene C4 in response to anti-IgE. In four of eight lungs tested, formyl methionine peptide (10(-6) to 10(-4) mol/L) weakly elicited histamine release (3% to 6%) in fractions I and II cells. Compound 48/80 (0.1 to 10 micrograms/ml; n = 3) failed to induce histamine release in any fractions. The cyclic adenosine monophosphate-active drugs, isoproterenol (10(-4) mol/L), dibutyryl cyclic adenosine monophosphate (3 mmol/L), and isobutylmethylxanthine (3 X 10(-4) mol/L) inhibited anti-IgE-induced histamine release from all fractions equivalently. Dimaprit (3 X 10(-5) mol/L) and cromolyn sodium (10(-5) -3 x 10(-3) mol/L) failed to significantly inhibit any fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Release of leukotriene C4 (LTC4) from human eosinophils following adherence to IgE- and IgG-coated schistosomula of Schistosoma mansoni

The release of leukotriene C4 (LTC4) from human low-density eosinophils following adherence to live or formalin-fixed schistosomula of Schistosoma mansoni coated with parasite-specific IgE or IgG obtained from pooled human anti-S. mansoni serum has been studied. IgE-rich fractions were obtained after fractionation of pooled immune sera on fast-protein liquid chromatography (FPLC; polyanion SI-17 column) and were identified by parasite-specific RAST. Contaminating IgG was removed by adsorption on a Staphylococcus aureus-protein A affinity column. IgG-rich FPLC fractions were identified by a specific ELISA assay. IgG-dependent activities were confirmed by protein A adsorption. Low-density eosinophils adhered to live and formalin-fixed schistosomula coated with specific antisera and released 11.7 +/- 2.7 and 16.5 +/- 3.5 pmoles of LTC4/10(6) cells, respectively. LTC4 release induced by A23187 (5 x 10(-6) M) from the same cells was 80 +/- 24 pmoles/10(6) cells and 9.9 +/- 1 pmoles/10(6) cells in the presence of Sepharose particles (CNBr-activated 4B beads) covalently coated with normal human IgG. Fixed schistosomula coated with FPLC-purified IgE and IgG gave 7.6 +/- 0.4 and 6.0 +/- 0.1 pmoles of LTC4 per 10(6) low-density eosinophils, respectively. The same IgE- and IgG-rich fractions induced eosinophil-mediated cytotoxicity of live schistosomula in vitro. Removal of IgE by an anti-IgE affinity column abolished both the IgE-dependent release of LTC4 and the in vitro killing of larvae. Conversely, IgG-dependent activities were abolished by protein A, but not anti-IgE, adsorption. Normal density eosinophils generated undetectable amounts of LTC4 when incubated with IgE-coated schistosomula, whereas with IgG-coated larvae 4.6 pmoles/10(6) cells were obtained. Following preincubation with platelet-activating factor (PAF) (10(-7) M) and leukotriene B4 (LTB4) (10(-7) M), normal density eosinophils released LTC4 when in contact with larvae coated with antigen-specific IgE. Lyso-PAF had no effect in any of the systems tested. The synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) had no influence on IgE-dependent release of LTC4 from eosinophils. In contrast, FMLP (10(-7) M) enhanced the IgG-dependent LTC4 release, with PAF and LTB4 also showing a small enhancing effect. None of these agents substantially altered the release potential of low-density eosinophils in either IgE- or IgG-dependent events. Thus the results presented here indicate that in an IgE-dependent system, human low-density eosinophils can be induced to adhere to and kill IgE-coated helminthic targets and release biologically relevant amounts of LTC4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxatomide inhibits the release of chemical mediators from human lung tissues and from granulocytes

The effects of oxatomide on the release of histamine and leukotriene C4 (LTC4) from human lung fragments and granulocytes were examined and the findings compared with the effects of the antiallergic drugs ketotifen, azelastine and tranilast. Oxatomide inhibited the release of both histamine and LTC4 from human lung fragments in cases of passive sensitization with human IgE myeloma serum upon anti-epsilon stimulation. IC50 values for the release of LTC4 from human lung fragments were as follows: oxatomide, 2.35 x 10(-5) M; azelastine, 27.2 x 10(-5) M; ketotifen, 52.1 x 10(-5) M; tranilast, 62.9 x 10(-5) M. Oxatomide also inhibited the release of both histamine and LTC4 from human mixed leukocytes stimulated by the calcium ionophore A23187. IC50 values for the release of LTC4 from human mixed leukocytes were as follows: oxatomide, 1.67 x 10(-5) M; azelastine, 3.65 x 10(-5) M; ketotifen, 12.2 x 10(-5) M; tranilast, 15.1 x 10(-5) M. The effects of oxatomide on the release of LTC4 from purified human neutrophils and eosinophils were also given attention. Oxatomide inhibited the release of LTC4 from eosinophils more effectively than from neutrophils and mixed leukocytes. As there is evidence that eosinophils play an important role in the development of late asthmatic responses and/or in the aggravation of asthma, oxatomide is expected to be an effective treatment for such conditions.     

Mouse IL-3 induces histamine release from mouse peritoneal mast cells.

In this study, we have attempted to determine whether mouse peritoneal mast cells released histamine in response to IL-3. Recombinant mouse (m)IL-3 induced histamine release from mouse peritoneal mast cells in a dose-dependent fashion. Histamine release did not occur in the absence of phosphatidyl serine (PS), and was dependent on PS concentrations. The release was 14.3 +/- 3.8 and 43.5 +/- 11.5% (mean +/- SEM, n = 5) at 1 nM IL-3 in the presence of 10 and 20 micrograms/ml of PS. Calcium was required for the response, and in the absence of calcium, significant histamine release was not observed. The kinetics were slower than those of anti-IgE-induced response. IL-3-induced histamine release reached a peak within 15 min, while that by anti-IgE reached 80% of the maximum in 3 min. Lower concentrations of IL-3, which failed to directly induce histamine release, did not enhance anti-IgE-induced histamine release. Other cytokines, including mIL-4, mIL-5, m-granulocyte-macrophage colony-stimulating factor, human (h)IL-1 alpha, hIL-1 beta and hIL-8, neither induced histamine release nor enhanced anti-IgE induced histamine release. IL-4 had no capacity to enhance IL-3-induced histamine release. These results suggest that locally produced IL-3 might modulate mast cell-related inflammation through histamine release from mast cells.     

Immunological and non-immunological release of leukotrienes and histamine by guinea-pig heart

Fragments of sensitized guinea-pig heart (1 g wet weight) were incubated with 5 micrograms/ml of antigen for up to 30 min, and the incubation media were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) for the presence of leukotrienes LTB4, LTC4, LTD4 and LTE4. Maximum release of LTB4, LTC4 and LTD4 was observed after 15 min (32.8 +/- 4, 8 +/- 2 and 9.5 +/- 2.5 pmol/g tissue wet weight, respectively, mean +/- SEM). At the same time, histamine was also released, reaching a maximum at 5 min (290 +/- 63 pmol/g tissue) as determined by radioenzymatic assay. Similarly, the non-sensitized guinea-pig heart stimulated with the ionophore A23187 (4 microM) released LTB4, LTC4, LTD4 and LTE4, as well as the 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE as determined by RP-HPLC. The release of LTB4, LTC4 and LTD4 was at a maximum after 10-15 min of stimulation (63 +/- 8.4, 10.7 +/- 2 and 17.5 +/- 4 pmol/g tissue). The ionophore also stimulated the release of histamine in heart tissue, with a peak maximum after 5 min (325 +/- 77 pmol/g tissue). These data demonstrate that heart as well as pulmonary tissues release significant amounts of leukotrienes and histamine during immunological or non-immunological challenges.     

Role of protein kinase C in histamine release from human basophils

In this study, we investigated the role of calcium and phospholipid-dependent protein kinase (protein kinase C, PKC) in the modulation of histamine release from human basophils. A novel and potent inhibitor of PKC, K-252a, inhibited the release of histamine induced by anti-IgE in a dose-dependent manner with ID50 (the dose required for 50% inhibition of histamine release) of 2.2 x 10(-8) M. Histamine release stimulated with 12-0-tetradecanoyl-phorbol-13-acetate(TPA) was also suppressed by K-252a with maximal inhibition of 48.0 +/- 9.3% at 10(-7) M. In contrast, K-252a did not inhibit the release of histamine in response to FMLP and ionophore A23187. Another inhibitor of PKC, H-7, exhibited a dose-dependent inhibition of anti-IgE-induced histamine release with ID50 of 8.6 x 10(-4) M. H-8 and HA1004, which closely resemble H-7 in chemical structure but are less potent in inhibiting PKC, did not inhibit histamine release stimulated with anti-IgE, but rather enhanced the release at higher concentrations. These results strongly suggest that PKC activation plays a crucial role in the mediation of IgE-mediated histamine release from human basophils.     

Inhibitory effect of terfenadine on mediator release from human blood basophils and eosinophils

The effect of terfenadine on histamine release from human basophils and LTC4 production and release from human eosinophils was evaluated. Eosinophils and basophils were obtained by discontinuous gradient centrifugation of the peripheral blood of atopic asthma patients who were off medication. Anti-IgE-induced histamine release from human basophils was significantly inhibited by terfenadine. Maximum inhibition was obtained at 1 x 10(-5) M terfenadine (percentage inhibition = 57.0 +/- 20.1; P less than 0.05). However, only the highest dose of terfenadine used in this study, i.e. 2 x 10(-5) M, significantly inhibited calcium ionophore (A23187)-induced histamine release from human basophils (percentage inhibition = 40.0 +/- 14.6; P less than 0.05), and LTC4 production from human eosinophils (percentage inhibition = 59.8 +/- 9.9; P less than 0.05. These findings demonstrate that terfenadine, in addition to its known antihistamine property, also has an inhibitory effect on chemical mediator release.     

Immunologic release of histamine from dog lung: comparison of in vivo and in vitro responses in the same animal

The purpose of this study was to compare, for the first time, antigen-induced histamine release from the lung in the same natively allergic dogs both in vitro and in vivo. In six dogs, maximal antigen-induced histamine release from the lung correlated closely in vitro and in vivo (r = 0.94), although it varied widely between dogs (0% to 75.5% of total tissue histamine content); similarly, the antigen concentration to produce 50% of maximal histamine release varied sixfold between dogs (40 micrograms/ml to 250 micrograms/ml). In each of five other dogs, terbutaline sulfate administered intravenously caused a dose-dependent inhibition of antigen-induced histamine release from lung fragments in vitro: the maximal inhibition produced by 1 mg/kg was 60 +/- 4.5% (mean +/- SEM). In these same dogs, 10(-5)M terbutaline incubated with lung fragments in vitro caused inhibition of antigen-induced histamine release comparable to 1 mg/kg terbutaline in vivo. Increasing the dose of terbutaline in vitro produced maximal inhibition at 10(-4)M with no greater effect of the drug at 10(-3)M (71.4 +/- 3.8% inhibition). In both experimental situations propranolol caused a dose-dependent inhibition of beta-adrenergic modulation of Ascaris-induced release of histamine. This result supports the conclusion that terbutaline produced its effects by actions mediated by beta-adrenergic receptors on pulmonary mast cells. This experimental approach provides a suitable preparation in which to estimate the effective dose of agonists that modulate antigen-induced mast cell function in vivo.     

Leukocyte histamine release to suxamethonium in patients with adverse reactions to muscle relaxants

In an earlier study we confirmed the usefulness of intradermal skin tests and histamine release in diagnosis of patients reactive to muscle relaxants, and we suggested an IgE-mediated reaction rather than an idiosyncratic mechanism. In a later study, we studied the relationship between (Formula: see text) that is one of the muscle relaxants producing the most frequent adverse reactions under anesthesia. Histamine release was measured in five patients with increasing concentrations of suxamethonium in the presence or absence of human serum albumin in Tris buffer. Suxamethonium by itself without any carrier in the buffer could, in vitro, act as a true allergen on target leukocytes in the sensitized patients' group. Acetylcholine (20 and 200 micrograms/ml) did not induce significant histamine release in five patients with positive histamine release in the presence of suxamethonium. Preincubation of leukocytes from 11 patients for 30 min with 20 and 200 micrograms of acetylcholine in Tris albumin CA++ Mg++ buffer decreased the histamine release induced by suxamethonium (10 micrograms/ml); mean maximal histamine release of 46% +/- 4.2 was reduced to 31.4 +/- 5.8 and 7% +/- 4 (p less than 0.001), respectively. However, in eight control subjects similar concentrations of acetylcholine did not change the maximal histamine release induced by anti-IgE (0.2 micrograms/ml). In the same way acetylcholine did not modify histamine release induced by Dermatophagoides pteronyssinus extract (1/10,000 w/v) in six patients allergic to this allergen. This study suggests that suxamethonium acts as a true allergen and that acetylcholine or one of its metabolites may act as a hapten inhibitor in the model of histamine release induced by suxamethonium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of dexamethasone and its effect on histamine release from rat mast cells

Purified rat peritoneal mast cells were incubated for 20 h with or without dexamethasone (4 x 10(-6) M) and then passively sensitized with serum from Trichinella spiralis-infected rats. The release of histamine using various secretagogues (concanavalin A, crude antigen of T. spiralis and polymyxin B) was determined. Dexamethasone treatment markedly inhibited IgE-dependent release of histamine (from 33.9 +/- 5.0% to 12.4 +/- 5.1% and from 39.8 +/- 7.9% to 14.2 +/- 6.5% of total cellular histamine content, respectively) whereas histamine release stimulated by the nonimmunological stimulus, polymyxin B was unaffected by this steroid. This suggests that the effects of dexamethasone cannot be exclusively explained by inhibition of phospholipases. Specific binding of 3H-dexamethasone to purified mast cells displayed sigmoidal dependence on concentration which may be the result of either negative cooperativity or the presence of a different class of binding sites. Two saturation plateaux at 20-30 x 10(-9) M and 70-90 x 10(-9) M were observed. The equilibrium dissociation constant for the higher affinity binding sites was Kd1 = 1.9 x 10(-8) M and represented 25,290 sites/cell, whereas the apparent Kd2 for lower affinity sites amounted to 5.5 x 10(-8) M and represented about 120,000 sites/cell.     

Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells activated with peptides

Rat peritoneal mast cells co-cultured with mouse 3T3 fibroblasts (MC/3T3) are fully responsive to immunologic stimuli. To assess their nonimmunologic activation MC/3T3 were challenged with various peptides. Optimal concentrations of substance P (10(-4) M) and bradykinin (5 x 10(-5) M) induced histamine release of 58.2 +/- 9.3 and 66.8 +/- 6.6%, respectively, while neurotensin (10(-4) M) released only 16.6 +/- 3.7% histamine. Freshly isolated mast cells (F-MC) challenged with the same concentrations of peptides released lower percentages of histamine (substance P 45.6 +/- 5.1%, bradykinin 32.5 +/- 5.3%, neurotensin 11.3 +/- 6.0%). In both MC/3T3 and F-MC, only minute amounts of prostaglandin D2 (PGD2) were produced. In contrast, activation with anti-IgE antibodies and compound 48/80 caused both histamine release and PGD2 generation. Compound 48/80-stimulated MC/3T3 and F-MC released 80.2 +/- 3.4 and 51.8 +/- 6.2% histamine, respectively, and produced 15.4 +/- 2.8 ng/10(6) mast cells and 3.9 +/- 1.4 ng/10(6) mast cells PGD2, respectively. These findings indicate that peptides and bradykinin induce selective release of histamine with no PGD2 production in both F-MC and MC/3T3. Moreover, MC/3T3 preserve their functional characteristics of connective tissue mast cells since they are fully responsive to these peptides as F-MC.     

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.     

Generation and Metabolism of Leukotrienes and Release of Histamine from Human Dispersed Tonsillar Cells

We studied the generation and metabolism of leukotrienes (LT) and the release of histamine by human tonsillar cell suspensions. Human tonsils were dissected and mechanically dispersed. This procedure yielded a single cell suspension with 1.6 +/- 0.5 X 10(8) cells/g tissue consisting of 97.3 +/- 0.4% lymphocytes, 1.4 +/- 0.3% granulocytes, 1.3 +/- 0.3% macrophages/monocytes, and 0.03 +/- 0.02% mast cells/basophils. The cells were stimulated either with Ca-ionophore A 23187, melittin, or anti-human IgE. Determination of the 5-lipoxygenase products LTB4 and LTC4 was performed with specific radioimmunoassays (RIA), and histamine release was measured by the fluorophotometric technique. A time- and dose-dependent release of the mediators was monitored. LTB4 exceeded the amount of LTC4 in the supernatants. The concentration of leukotrienes ranged between 0.8 and 5.4 ng LTB4/1 X 10(8) cells or 0.5 and 1.5 ng LTC4/1 X 10(8) cells, depending on the stimulus. Histamine release after stimulation ranged between 25 and 35% of the total histamine content, whereas buffer controls amounted to 17%. The incubation of the cells (1 X 10(8) with exogenously added LTB4 resulted in the formation of omega-oxidated products (20-OH and 20-COOH-LTB4) and a novel unpolar metabolite, as identified by thin layer chromatography. This metabolite was not immunoreactive in the LTB4-RIA used. LTC4 and LTD4 were converted into LTE4 when added either to sonicated cells or to the cell-free supernatants of prestimulated tonsillar cells, indicating the release of gamma-glutamyltranspeptidase and dipeptidase, respectively. Our data clearly demonstrate the generation and metabolism of the 5-lipoxygenase products LTB4 and LTC4 as well as the release of histamine from human dispersed tonsillar cells, suggesting that they have a modulatory function with respect to the inflammatory potential at local sites.     

Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release.     

The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) is a potent inhibitor of mediator release from human dermal mast cells and peripheral blood basophils

BACKGROUND: The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) has recently been developed as a novel and cell-selective inhibitor of inflammatory cytokine secretion; it has fewer adverse effects than currently available drugs. OBJECTIVE: In this study, we investigated the capacity of pimecrolimus to directly inhibit in vitro mediator release from human skin mast cells and basophils. METHODS: Purified cutaneous mast cells or basophil-containing peripheral blood leukocytes were obtained from healthy human donors and preincubated with pimecrolimus (0.1 nmol/L to 1 micromol/L) in the absence or presence of its specific antagonist (rapamycin), cyclosporin A (100 nmol/L to 1 micromol/L), or dexamethasone (1 micromol/L) and then stimulated with anti-IgE or with calcium ionophore A23187 plus phorbol myristate acetate. Cell supernatants were kept for analysis of histamine, tryptase, LTC4, and TNF-alpha. RESULTS: Pimecrolimus caused a strong and dose-dependent inhibition of anti-IgE--induced release of histamine from mast cells and basophils (maximally 73% and 82%, respectively, at 500 nmol/L pimecrolimus) and of mast cell tryptase (maximally 75%) and a less pronounced inhibition of LTC4 (maximally 32%) and of calcium ionophore plus phorbol myristate acetate--induced mast cell TNF-alpha release (90% maximum at 100 nmol/L pimecrolimus). In contrast, inhibition achieved during mast cell histamine release was maximally 60% with cyclosporin A and only 28% with dexamethasone. CONCLUSION: These data demonstrate a marked inhibitory capacity of pimecrolimus on mediator release from human mast cells and basophils with a potency exceeding that of cyclosporin A and dexamethasone. Pimecrolimus might thus be expected to be effective in the treatment of mast cell-- and basophil-dependent diseases.     

Interleukin-8 and RANTES inhibit basophil histamine release induced with monocyte chemotactic and activating factor/monocyte chemoattractant peptide-1 and histamine releasing factor.

The objective of this study was to investigate the effect of interleukin-8 (IL-8) and RANTES on basophil histamine release induced with monocyte chemoattractant peptide-1 (MCP-1) and crude histamine releasing factor (HRF). IL-8 induced low levels of histamine release (8.5 +/- 0.5%) from basophils obtained from only six of 20 donors at high concentrations (10(-6) M). RANTES induced histamine release (16 +/- 2%) from basophils of four of 15 donors at 10(-7) M concentration. However, both IL-8 and RANTES inhibited MCP-1 and HRF-induced histamine release from basophils dose-dependently at concentrations of 10(-9) to 10(-7) M. Basophils from all donors showed a significant inhibitory response (greater than 15%). The maximal inhibition of MCP-1 and HRF by IL-8 was 28 +/- 4% and 48 +/- 8%, respectively. The maximal inhibition of MCP-1 and HRF by RANTES was 26 +/- 4% and 43 +/- 6%, respectively. Peripheral blood mononuclear cell-derived HRF was purified into three distinct peaks by reverse-phase high performance liquid chromatography. Peak I contained MCP-1 as judged by binding to an immunoaffinity column that was prepared with anti-MCP-1 antibody. IL-8 inhibited histamine release induced with all three peaks of HRF. The inhibition of histamine release by IL-8 was significantly higher in normal subjects than in allergic patients (59 +/- 9% versus 31 +/- 7%, P less than 0.05). Both IL-8 and RANTES inhibited cytokine-induced histamine release only and did not affect histamine release by anti-IgE, FMLP, and C5a.(ABSTRACT TRUNCATED AT 250 WORDS)