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.     

Effects of the new 5-lipoxygenase inhibitor E6080 on leukotriene release in vitro.

Fundamental studies were conducted to examine the release of histamine and leukotriene (LT) C4 from lung fragments of guinea pigs and the effects of E6080 on the release of LTB4 and LTC4 from lung fragments or inflammatory cells. The release of histamine and LTs showed large interindividual variations and a marked dependence on experimental conditions. Addition of 10 mM L-cysteine significantly increased LTC4 release compared with that in its absence (about 1.7 times, in terms of mean value). E6080 inhibited antigen-stimulated LTB4 and LTC4 release from passively sensitized human (IC50: LTB4 0.08 microM, LTC4 0.2 microM) and guinea-pig lung fragments (IC50: LTC4 1.1 microM). The LTB4 and LTC4 releases from healthy human polymorphonuclear leukocytes (calcium ionophore A23187) and from allergic patients' leukocytes (basophils, antigen) were inhibited by E6080 with IC50 values of below 1.0 microM. Furthermore, the LTC4 release from rat alveolar macrophages (silica particles) was inhibited by E6080 with an IC50 of 0.2 microM. The potent inhibition by E6080 might be a result of the inhibition of 5-lipoxygenase, since 5-lipoxygenase in rat basophilic leukemia cell was inhibited by E6080 with an IC50 of 0.2 microM. The results confirm the potent inhibitory effects of E6080 on the release of LTs.     

Human neutrophil-derived histamine-releasing activity (HRA-N) causes the release of serotonin but not arachidonic acid metabolites from rat basophilic leukemia cells.

The effects of neutrophil-derived histamine-releasing activity (HRA-N) on arachidonic acid (AA) metabolism is unknown. Human basophils exposed to HRA-N released 25% of total histamine but no leukotriene C4 (LTC4). To confirm this phenomenon, rat basophilic leukemia (RBL) cells were exposed to HRA-N as well as anti-IgE, or calcium ionophore A23187. RBL cells incubated with A23187 released 44% of available serotonin and 59 and 124 pmol/10(6) cells of prostaglandin D2 (PGD2) and LTC4, respectively. Anti-IgE stimulation resulted in 34% serotonin release and the generation of 34 pmol PGD2 per 10(6) cells and 72 pmol LTC4 per 10(6) cells. In contrast, HRA-N (2 U/ml) induced 20% serotonin release, 4 pmol PGD2 per 10(6) cells, and 0.6 pmol LTC4 per 10(6) cells. Neither increasing the dose nor the incubation time of HRA-N enhanced the generation of AA metabolite. Additionally, the spectrum of AA metabolites generated by RBL cells in response to those agents was examined by reverse-phase high-performance liquid chromatography. RBL cells stimulated with A23187 released PGD2, LTB4, and its isomers, LTC4, and 5-hydroxyeicosatetraenoic acid. In contrast, HRA-N stimulation resulted in only minimal PGD2 generation and no other discernable AA metabolites. Thus, HRA-N causes selective release of serotonin without inducing AA metabolites. These data suggest that HRA-N activates mast cells through a unique pathway.     

Nasal response to a single antigen challenge in patients with allergic rhinitis — inflammatory cell recruitment persists up to 48 hours

BACKGROUND: Allergen challenge in some patients with respiratory allergy is followed by an early and a late reaction. OBJECTIVE: To evaluate the duration of mediator release and inflammatory cell recruitment during the late antigen-induced nasal response. METHODS: Eight patients with seasonal allergic rhinitis due to grass pollen underwent local challenge with the relevant allergen, a non-relevant allergen (Parietaria judaica), and nebulized saline solution. Nasal lavages were performed at baseline and 6, 24, 48, 72 h after challenge. Eosinophil cationic protein (ECP), leukotriene C4 (LTC4), leukotriene B4 (LTB4) myeloperoxidase (MPO) and prostaglandin D2 (PGD2) levels were radioimmunoassayed and histamine concentration was measured by an automated fluorometric method. RESULTS: Nasal challenge with the relevant antigen induced a response 6 h after stimulation, which subsided within 24 h. Eosinophilia, observed in the nasal lavages collected from 6 to 24 h after this challenge, was accompanied by ECP release. Neutrophilia were found in the nasal lavages collected from 6 to 24 h after challenge. The increase in neutrophil number correlated with MPO levels and LTB4 concentrations, but not with the intensity of nasal obstruction. Antigen challenge also induced significant recruitment of mononuclear cells 48 h after provocation. The challenge significantly raised histamine, but not PGD2, levels in the nasal lavages collected 6 h after provocation. A trend towards an increase in LTC4 levels in the nasal lavages collected 6 h after specific antigen challenge was also found. Nasal challenge with a non-relevant allergen or with saline solution did not cause either inflammatory cell recruitment or mediator release. CONCLUSION: Nasal challenge with the relevant antigen can induce a late response characterized by local accumulation of eosinophils, neutrophils and mononuclear cells persisting for 48 h and accompanied by release of ECP, MPO, LTB4 and histamine. These results indicate that a single antigen challenge in patients with allergic rhinitis causes prolonged inflammatory alterations which may contribute to the development of airway hyperreactivity.     

Release of 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin E2 (PGE2) by cultured human bronchial epithelial cells

Human bronchial epithelial cells were isolated from macroscopically normal bronchi obtained from lobectomy specimens. Cells were grown in nutrient F12 medium, and after the third or fourth subculture they were stimulated with arachidonic acid, histamine, leukotrienes (LT) C4, D4, or E4, prostaglandin (PG) D2, anti-IgE, acetylcholine, bradykinin, or phorbol myristate acetate (PMA). Neither mast cell mediators (i.e., histamine, LTC4, LTD4, LTE4, or PGD2) nor anti-IgE stimulated the release of arachidonic acid metabolites from the epithelial cells. However, arachidonic acid, acetylcholine, bradykinin, and PMA stimulated the release of 15-hydroxyeicosatetraenoic acid (15-HETE) as major and prostaglandin E2 (PGE2) as minor products. The maximal release of 15-HETE and PGE2 occurred in 1 h with arachidonic acid stimulation and in 2 h with other stimuli. Arachidonic acid at 30 microM caused the release of 258 +/- 76 ng and 29 +/- 15 ng (n = 12) of 15-HETE and PGE2, respectively, from 10 x 10(6) epithelial cells, whereas acetylcholine, bradykinin, or PMA caused the release of approximately 2- to 10-fold less 15-HETE and PGE2. These results demonstrate that human bronchial epithelial cells selectively generate 15-HETE as the predominant arachidonic acid product and PGE2 as a minor metabolite. The role of bronchial epithelial cells and their mediators in the pathogenesis of bronchial hyperresponsiveness needs further study.     

Enhanced granulocyte cytotoxicity by mediators derived from anti-IgE-stimulated human leucocytes.

Basophil-containing leucocyte fractions stimulated with an anti-human IgE, F(ab')2, generated histamine and the leukotrienes LTB4 and LTC4 with significant correlations between LTB4 and histamine (P less than 0.01; n = 26) and LTC4 and histamine release (P less than 0.001; n = 29) in the cell-free supernatants (SN). SN from these anti-IgE-treated cells enhanced the cytotoxicity of eosinophils and neutrophils (against complement-coated schistosomula of Schistosoma mansoni) in vitro. When SN were fractionated by reverse phase-high performance liquid chromatography (RP-HPLC), the enhancing activity for neutrophils was almost totally confined to fractions having LTB4 immunoreactivity (co-eluting as a single peak with the synthetic LTB4 marker). In contrast, the LTB4-containing fraction had minimal effects on eosinophil cytotoxicity, whereas synthetic histamine gave comparable enhancement to the unfractionated SN. The generation of LTs (but not histamine), as well as enhanced neutrophil cytotoxicity from basophil-containing leucocytes by anti-IgE treatment, was maximally inhibited by the 5-lipoxygenase inhibitors U-60, 257 and BW755C. Conversely, the cyclooxygenase inhibitor indomethacin did not significantly affect LT release, nor did it affect the subsequent cytotoxicity enhancing activity of SN from such cells. These results indicate that LTB4 and LTC4 are released from basophils, together with histamine, by IgE-dependent mechanisms, LTB4 enhances the cytotoxicity of bystander neutrophils, and histamine (and to a lesser extent, LTB4) augments eosinophil cytotoxicity.     

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.     

Inhibition of human basophil leukotriene release by antiinflammatory steroids

We have previously shown that 24-hour culture of human basophils with the antiinflammatory steroid dexamethasone produces an inhibition of the IgE-dependent release of histamine. In contrast, similar treatment of purified human lung mast cells does not inhibit the subsequent release of either histamine, prostaglandin D2, or leukotriene (LT) C4. We now show that incubation of mixed leukocytes for 24 h with 10(-7) M dexamethasone produces an inhibition of anti-IgE-induced basophil LTC4 release as detected by radioimmunoassay. In three experiments, control (CON) and dexamethasone (10(-7) M; DEX)-treated cells were challenged with 0.01, 0.03 and 0.1 micrograms/ml of anti-IgE, and histamine and LTC4 were monitored. LTC4 release (ng LTC4/micrograms total cell histamine) from cells stimulated with anti-IgE was: 0.01 micrograms/ml anti-IgE, 6.9 +/- 4, 0.3 +/- 0.1 (CON, DEX); 0.03 micrograms/ml anti-IgE, 13.8 +/- 4.7, 0.9 +/- 0.5; 0.1 micrograms/ml anti-IgE, 19.5 +/- 2.3, 4.9 +/- 1.2. Histamine release was inhibited by 50-75% by treatment with DEX in these experiments. Dose-response studies (n = 4) indicate that the inhibitory actions of DEX on LTC4 release occur in the range of 10(-10) to 10(-7) M. The concentration of DEX at which LTC4 release was inhibited by 50% (IC50) was approximately 2 X 10(-9) M. Another glucocorticoid (betamethasone) inhibited LTC4 release, while the nonglucocorticoids tetrahydrocortisone and beta-estradiol were inactive. High performance liquid chromatography (HPLC) analysis (coupled with RIA) indicated that the relative proportions of LTC4, LTD4, and LTE4 did not differ in supernatants from CON- and DEX-treated, anti-IgE-challenged cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological and non-immunological release of leukotrienes and histamine from human nasal polyps

Nasal polyps were obtained from 22 patients undergoing polyp surgery. They were chopped into fragments of approximately 2 mm2, washed free of blood, and passively sensitized with serum from timothy allergen sensitive patients (RAST 30-40%), then, challenged with timothy allergen. Analysis of the incubation medium after a 30 min challenge as assessed by reversed phase high performance liquid chromatography and bioassay revealed the presence of leukotriene (LT) LTB4, LTC4, LTD4 and LTE4 (29 +/- 9, 6.6 +/- 3, 62 +/- 13 & 86 +/- 23 pmol/g tissue wet weight, respectively). The human nasal polyps also released histamine which reached a maximum after approximately 5 min (2.8 +/- 1.28 nmol/g tissue), as measured by radioenzymatic assay. A similar profile of leukotrienes and histamine release was observed when the nasal polyps were stimulated with ionophore A23187. However, the ionophore stimulated release was greater than that observed for the antigen challenge. When human polyps were stimulated with a mixture of the ionophore and arachidonic acid, all the above products as well as 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE were detected in the medium. These data, taken together, demonstrate that human nasal polyps are able to release significant amounts of leukotrienes and histamine upon both immunological and non-immunological stimulations and that these responses might significantly contribute to the symptoms of allergic rhinitis.     

Mizolastine provides effective symptom relief in patients suffering from perennial allergic rhinitis: a double-blind, placebo-controlled study versus loratadine.

BACKGROUND: Mizolastine is a nonsedating H1 histamine receptor antagonist with additional antiallergic properties currently marketed in Europe for the treatment of seasonal and perennial allergic rhinitis (PAR) and urticaria. OBJECTIVE: This multicenter, randomized, double-blind, parallel-group study was conducted to evaluate the efficacy and safety of mizolastine in PAR compared with loratadine and placebo. METHODS: After a 1-week placebo run-in period, 428 adult PAR patients received placebo (146 of 428), mizolastine 10 mg (141 of 428), or loratadine 10 mg (141 of 428) once daily for 28 days. Symptoms were evaluated by patients and physicians using a total nasal score, evaluating itching, rhinorrhea, nasal blockade, and sneezing severity. RESULTS: Mizolastine treatment resulted in a significantly greater decrease in patient-rated total nasal score than placebo after 2 weeks (D14; -42%, P < 0.001) and at the end of the treatment period (-46%, P = 0.01), and significantly greater than that observed with loratadine at D14 (P = 0.031). No significant difference in change in total nasal score was observed between loratadine and placebo at 2- and 4-week visits. The global safety was satisfactory and the incidence of adverse events was similar in the three treatment groups. CONCLUSIONS: Mizolastine provides effective symptom relief in PAR together with a satisfactory safety profile. Improvement with mizolastine was significantly greater than placebo throughout the study despite a large placebo effect. Also mizolastine's effects were greater those observed with loratadine after 2 weeks of treatment.     

Inhibition of mediator and cytokine release from dispersed nasal polyp cells by terfenadine

The mechanism of action of H₁-blockers requires elucidation because they may possess properties unrelated to the blockage of histamine at its receptor level. A study was performed with enzymatically dispersed cells obtained from nasal polyps to examine the effect of terfenadine (0.1–10 μmol) on the release of leukotrienes (LT) (LTC₄/D₄ and LTB₄) after stimulation by anti-IgE, and on the spontaneous release of cytokines (granulocyte/macrophage-colony stimulating factor [GM-CSF] and tumor necrosis factor-alpha [TNF-α]) released from cells cultured for 6 h. Terfenadine inhibited significantly, and in a dose-dependent manner, the release of LTC₄/D₄, LTB₄, TNF-α, and GM-CSF. IC₅₀ values were determined for LTC₄/D₄ (8 μmol), LTB₄ (9.9 μmol), TNF-α (6.1 μmol), and GM-CSF (4 μmol). Terfenadine was found to possess new antiallergic properties with a novel in vitro model which mimics more closely inflammatory cells of allergic rhinitis or asthma.     

Reduced release of leukotrienes B4 and C4 from alveolar macrophages of rats with silicosis.

Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-alpha (TNF-alpha) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-alpha production, which was higher in AM from silica-exposed than from control rats. To study the interrelation between leukotriene and TNF-alpha release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosan-induced TNF-alpha release from AM in a dose-dependent manner, and TNF-alpha production could be restored almost completely by addition of LTB4. These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-alpha production during silicosis.     

Leukotrienes and isocyanate-induced asthma: a pilot study

BACKGROUND: The role of leukotrienes (LTs) in the pathophysiology of isocyanate-induced asthma is not well known. OBJECTIVE: We sought to characterize the type of airway inflammation induced by exposure to isocyanates and to investigate whether exposure to isocyanates induced an increase in LT receptor cysteinyl leukotriene ((CysLT)(1), CysLT(2) and leukotriene B(4) receptor (BLT(1))) expression, as well as a release of LT (LTC(4) and leukotriene B(4) (LTB(4))) and IL-8 in both asthmatics with isocyanate-induced asthma and healthy subjects. METHODS: We investigated eight subjects with isocyanate-induced asthma and eight healthy subjects. Both groups underwent specific inhalation challenges to isocyanates in the laboratory. Induced sputum was collected before and after exposure to isocyanates. CysLT(1), CysLT(2) and BLT(1) expression was assessed by flow cytometry, whereas LTC(4), LTB(4) and IL-8 were measured in the sputum supernatants by enzyme immunoassay. RESULTS: Exposure to isocyanates induced an increase in sputum neutrophils only in subjects with occupational asthma. There was a significant increase in CysLT(1) and BLT(1) receptor expression, as well as a release of LTB(4) and IL-8 after exposure to isocyanates compared with the baseline, only in subjects with isocyanate-induced asthma, whereas there was no increase in LTC(4). Exposure to isocyanates did not induce any change in LT receptor expression nor in the levels of LTC(4), LTB(4) and IL-8, in healthy subjects. CONCLUSION: The neutrophilia observed after exposure to isocyanates is likely to be related to the release of LTB(4), probably enhanced by the increased expression of BLT(1) on neutrophils as well as by the release of IL-8. The significance of the increase of CysLT1 receptor expression on neutrophils is unknown and needs further investigation.     

Pharmacological modulation of activation-secretion of rat serosal mast cells by chymase, an endogenous secretory granule protease.

The action of pharmacologic agents on chymase-induced exocytosis of beta-hexosaminidase and arachidonic acid (AA) metabolism by rat serosal mast cells (RSMC) was determined and compared with their effects on anti-IgE induced activation. Indomethacin (INDO) (less than or equal to 10 microM), a cyclooxygenase inhibitor, did not affect chymase- or anti-IgE-mediated exocytosis, while completely inhibiting prostaglandin D2 (PGD2) release at 1.25 microM. Theophylline (THEO), mepacrine, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C), and diethylcarbamazine (DEC), inhibitors of adenosine binding and phosphodiesterases, phospholipases, AA metabolism, and vesicular transport as well as leukotriene A4 formation, respectively, inhibited exocytosis with ID50 values of 3.4, 0.22, 3.4 and 1.9 mM for chymase and 2.4, 0.17, 2.8 and 5.2 mM for anti-IgE. These agents inhibited net PGD2 release with ID50 values of 2.1, 0.04, less than 0.05, and 1.5 mM for chymase and of 0.5, 0.1, less than 0.05, and 4 mM for anti-IgE. 5,6-Dehydroarachidonic acid (DHA) and arachidonyl hydroxylamine (AH), 5-lipoxygenase inhibitors, did not affect chymase-mediated exocytosis; anti-IgE-mediated exocytosis was not altered by AH but was suppressed by DHA (ID50 = 20 microM). Nordihydroguaiaretic acid (NDGA), an antioxidant, inhibited chymase-mediated exocytosis dose-dependently (ID50 less than or equal to 13.3 microM) while decreasing anti-IgE-mediated exocytosis by only 30% at 2.5-20 microM; net PGD2 release induced by both stimuli was inhibited dose-dependently. 2',5'-Dideoxyadenosine (DDA) and 1,6-di(0-(carbamoyl)cyclohexanone oxime)hexane (RHC 80267) and inhibitors of adenylate cyclase and of di-triglyceride lipases, respectively, had little effect on exocytosis induced by chymase but inhibited that induced by anti-IgE with ID50 values of 0.4 mM and 37 microM, respectively. With DDA the inhibition of net PGD2 release occurred with anti-IgE but not chymase, whereas RHC 80267 inhibited both chymase and anti-IgE-mediated PGD2 release. Differential inhibition of activation-secretion suggests either that chymase provides a step inhibited in IgE-mediated exocytosis by DDA, RHC 80267 and DHA, or that the activating pathway initiated by chymase is distinct.     

(9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido [1,2-a] pyrimidin-4-one), AS-35, inhibits leukotriene synthesis

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.     

Pharmacological effect of KC-404 on leukotriene release from human leukocytes induced by ionophore A23187

Release of leukotriene (LT) B4, LTC4 and histamine from human peripheral mixed leukocytes stimulated with calcium ionophore A23187 was evaluated. The amounts of LTB4 and LTC4 in supernatants were analyzed by high performance liquid chromatography and histamine was measured by an automated fluorometric technique. A time-course study demonstrated the characteristic patterns of the release of these mediators. LTB4 and LTC4 were more slowly released than histamine and the release of these LTs was inhibited by KC-404, a novel anti-asthmatic drug which has no effect on histamine release.     

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.     

Humoral and cellular responses to histamine and pollen allergen in a skin chamber model: effect of mizolastine.

BACKGROUND: Mizolastine is a new non-sedative antihistamine and antiallergic drug proven to be effective and safe in the treatment of allergic rhinitis and urticaria. OBJECTIVE: To quantitatively explore the time course of mediator release and cell recruitment during allergen challenge and the effects of mizolastine on the event, using the skin chamber model. METHODS: Twelve pollen-sensitive patients (23+/-6 years) were included in a double-blind crossover study. Patients received 10 mg mizolastine or placebo once daily in the first 4-day period and, after a 3-week washout period, vice-versa in the crossover period. On day 4 of each period, a non-invasive in vivo skin chamber technique was used to determine the alteration of vascular permeability, mast cell mediator release, the release of soluble intercellular adhesion molecule -1(sICAM-1) in skin sites challenged with exogenous histamine or grass pollen allergen extract, over an 8-hour period. RESULTS: Challenge with allergen-induced significant mast cell activation, as indicated by the release of histamine, tryptase and LTC4, in chamber fluids 2 hours after initiation of the allergic reaction and during the following 6 hours. Both exogenous histamine and allergen induced significant vasodilatation, which was sustained during the 8-hour challenge, as indicated by the accumulation of protein in the chamber fluids. Likewise, both histamine and allergen induced the release of significant amounts of ICAM-1 throughout the 8-hour period. Mizolastine significantly inhibited the histamine- and allergen-induced extravasation (after 2 hours, P = .003; after 8 hours, P = .009; after 2 hours, P = .044; after 8 hours, P = .003 respectively) and the histamine- and allergen-induced--ICAM-1 release (after 2 hours, P = .004; after 8 hours, P = .05; after 2 hours, P = .03 respectively). CONCLUSION: Mizolastine strongly inhibited the local response to histamine in this skin chamber model with, of interest, inhibition of the release of the soluble adhesion-molecule ICAM-1.     

Development of a system for monitoring the production of leukotriene B4, C4, D4 and E4 in whole blood of pediatric asthmatic patients

We have developed a new method using a computerized photodiode-array spectrophotometer (CPAS), to characterize the biosynthesis and the inhibition of leukotriene (LT) B4, C4, D4 and E4 in calcium ionophore (Ca-I)-stimulated whole blood. The results obtained were as follows: 1. The use of CPAS enabled us to identify and measure LTs from 2 ml volume of Ca-I stimulated whole blood as well as to check the purity of LTs, without any use of radioimmunoassay or bioassay. 2. LTC4 produced in whole blood was almost completely converted to LTE4 after 80 min incubation, and at this time both the production of LTE4 and LTB4 reached a plateau and remained constant thereafter. 3. Azelastine added in vitro caused a dose-dependent inhibition of Ca-I stimulated LTB4 and LTE4 production with an IC50 of 10 microM.     

Cytokines modulate IgE-mediated histamine liberation from human basophils

The effect of recombinant human IL-1α, IL-1β, IL-2, IL-3, Il-4, IL-6, TNF, LT, IFNα, IFNγ, GM-CSF and M-CSF on human basophil histamine release induced by anti-IgE antibody was examined in seven healthy donors using washed whole blood and a glass microfibre assay. The blood was preincubated for 20 min with cytokines and incubated for another 60 min after addition of the antibody. IL-3, GM-CSF and M-CSF were found to enhance the IgE-mediated response by 60–80%, whereas the other cytokines did not affect the mediator release to any significant degree.