calcimycin and Asthma

Asthma is a multifactorial disease on genetic basis. Its development is influenced by maternal and environmental factors, i.e. allergens and adjuvants. Early identification of candidates at high risk for development of asthma will enable giving recommendations on preventive measures focussing on exposure to tobacco smoke and other pollutants, indoor and outdoor allergens and possibly viral infections during infancy.

Topics: Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Eicosanoids; Eosinophilia; Eosinophils; Histamine Release; Humans; Lymphocytes; Macrophage Activation; Macrophages, Alveolar; Mast Cells; Membrane Lipids; Oxygen; Superoxides

Topics: Animals; Antigens; Asthma; Bronchoconstriction; Calcimycin; Guinea Pigs; Humans; Leukotrienes; Lipoxygenase Inhibitors; Lung; Macrophages; Mice; Naphthaleneacetic Acids; Neutrophils; Quinolines; Rats; Receptors, Immunologic; Receptors, Leukotriene; SRS-A; Zymosan

Immunologic or calcium-dependent activation of proteolytically dispersed human lung cells containing 5% mast cells causes the release of large amounts of PGD2 and TxB2. In cell purification experiments, only those fractions containing mast cells had the capacity to generate PGD2 and release histamine with IgE-dependent activation. The cells of origin of T X B2 are likely to be cells of the monocyte-macrophage series, although additional eicosanoid release may occur from immunologically activated lymphocytes and eosinophils. In men who have asthma, inhalation of low concentrations of PGD2 results in bronchoconstriction, whereas higher concentrations of PGD2 are needed to produce bronchoconstriction in normal subjects. Subjects with asthma exhibited 3.5-fold greater responsiveness to inhaled PGD2 than to PGF2 alpha. These observations demonstrate that PGD2 is the most potent bronchoconstrictor prostanoid tested in man. In both normal subjects and subjects with asthma, a single inhalation of PGF2 alpha resulted in a doubling in plasma levels of 13,14-dihydro-15-keto-PGF2 alpha. Plasma levels of this metabolite did not change after PGD2 inhalation. These results indicate that the 11-keto reduction of PGD2 to PGF2 alpha with subsequent inactivation is not important in the initial metabolism of PGD2.

Topics: Airway Resistance; Animals; Antibodies, Anti-Idiotypic; Asthma; Calcimycin; Cells, Cultured; Epoprostenol; Histamine Release; Humans; Hypersensitivity; Immunoglobulin E; Lung; Prostaglandins; Thromboxane A2

Topics: Adaptation, Physiological; Adrenal Cortex Hormones; Adrenal Glands; Adrenergic beta-Agonists; Aerosols; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Asthma; Bronchi; Calcimycin; Calcium; Cell Differentiation; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy; Forecasting; Glucose; Humans; Ion Channels; Lung; Methylprednisolone; Receptors, Glucocorticoid; Respiratory Hypersensitivity; Respiratory Therapy; Status Asthmaticus; Time Factors

Leukotrienes have been implicated in the mediation of airway obstruction induced by hyperventilation of cold dry air in asthmatic subjects. The effect of a novel inhibitor of 5-lipoxygenase activating protein, BAYx 1005, on the bronchospastic response to cold dry air hyperventilation was investigated in asthmatic patients.. After a screening cold dry air hyperventilation challenge to document cold air responsiveness, 16 asthmatic subjects (baseline forced expiratory volume in one second (FEV1) > 60% of predicted) underwent cold air challenge three hours after receiving 750 mg of BAYx 1005 or placebo using a randomised, double blind, crossover design. Leukotriene synthesis inhibition was estimated by measuring the concentration of leukotriene B4 in whole blood stimulated with calcium ionophore A21387.. Treatment with BAYx 1005 produced a 34% (95% CI 11 to 63) increase in the amount of cold air minute ventilation required for a 10% decrease in FEV1 (PD10VE) compared with placebo (mean (SE) 37.6 (1.12) 1/min compared with 28.0 (1.13) 1/min, p < 0.006). The PD20VE increased 19% (95% CI 8 to 31) after treatment with BAYx 1005 compared with placebo (57.3(1.10)1/min versus 48.1 (1.10) 1/min, p < 0.002). Treatment with BAYx 1005 produced a 15.4% decrease in ionophore-stimulated LTB4 production, while treatment with placebo produced a 7.1% increase in ex vivo LTB4 (p < 0.02).. Treatment with BAYx 1005, a novel inhibitor of leukotriene synthesis, produced a significant blunting of cold dry air responsiveness consistent with the hypothesis that leukotrienes mediate part of the bronchoconstriction induced by hyperventilation of cold dry air.

Topics: Adolescent; Adult; Analysis of Variance; Asthma; Bronchial Provocation Tests; Calcimycin; Cold Temperature; Cross-Over Studies; Double-Blind Method; Humans; Ionophores; Leukotriene B4; Lipoxygenase Inhibitors; Middle Aged; Quinolines

IV magnesium (Mg2+) has been proposed as an emergent treatment for acute asthma exacerbations. Recent studies have focused on the effects of Mg2+ on bronchial smooth muscle, yet asthma is primarily an inflammatory disease.. To assess the effects of Mg2+ on the neutrophil respiratory burst of adult patients with asthma.. A prospective, blind study of volunteer adult asthmatic patients was performed. The patients' polymorphonuclear neutrophils (PMNs) were isolated, purified, and placed into phosphate-buffered saline with the following test conditions: concentrations of magnesium chloride (MgCl2) added: 0 mmol MgCl2, 1 mmol MgCl2 (low), and 10 mmol MgCl2 (high) both with and without the calcium (Ca2+) ionophore A23187 (0.1 mmol). PMNs were activated using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 mumol), and the production of superoxide (O2-) was measured by the spectrophotometric reduction of cytochrome c.. Mg2+ reduced activated PMN O2- production compared with that for no Mg2+ (1.0 +/- 0.1 nmol O2-/5 x 10(5) PMN/min) in both low (-0.52* +/- 0.3 nmol O2-/5 x 10(5) PMN/min) and high (-0.76* +/- 0.3 nmol O2-/5 x 10(5) PMN/min; *p < 0.05) concentrations. The addition of A23187 increased O2- production in both the high (0.53* +/- 0.02 nmol O2-/5 x 10(5) PMN/min) and the low (1.5* +/- 0.6 nmol O2-/5 x 10(5) x 10(5) PMN/min) Mg2+ groups, with no change in the control group (1.2 +/- 0.2 nmol O2-/10(5) PMN/min).. In clinically relevant concentrations, Mg2+ attenuates the neutrophil respiratory burst in adult asthmatic patients. Mg2+ appears to affect PMNs by interfering with extracellular Ca2+ influx. Mg2+ may have a beneficial anti-inflammatory effect in asthmatic individuals.

Topics: Acute Disease; Adult; Asthma; Calcimycin; Calcium Channels; Double-Blind Method; Drug Therapy, Combination; Female; Humans; Inflammation; Ionophores; Magnesium Chloride; Male; Middle Aged; Prospective Studies; Respiratory Burst

The effects of dietary supplementation with highly purified eicosapentaenoic acid ethyl ester (EPA-E) (MND-21) on asthma symptoms, fatty acids in serum, and generation of leukotriene (LT) C4, LTC5, LTB4 and LTB5 by leukocytes stimulated with calcium ionophore A23187 were studied in 10 patients with bronchial asthma. The patients received nine capsules of MND-21 (2.7 g EPA-E) each day for 12 weeks. Leukocytes obtained from 39 patients with asthma who did not receive EPA-E were used as the control. Fatty acid composition was evaluated by gas chromatography and LT generation was measured by reverse-phase high performance liquid chromatography. EPA-E increased EPA content more than threefold, without changing the quantities of arachidonic acid in serum lipids. Leukocytes obtained from patients given EPA-E for 4 weeks generated less LTC4 (53.5 +/- 23.3 ng/10(7) cells) and LTB4 (24.9 +/- 12.4 ng/10(7) cells) than did those from control patients (142.4 +/- 91.6 ng/10(7) cells and 58.3 +/- 34.8 ng/10(7) cells, respectively). Although significant levels of LTC5 and LTB5 were present after 4 weeks of EPA-E (6.5 +/- 1.9 ng/10(7) cells and 4.6 +/- 2.7 ng/10(7) cells, respectively), generation of total LTC (LTC4 + LTC5) and total LTB (LTB4 + LTB5) were substantially suppressed. Symptoms had improved after two months of EPA-E, but the effect was temporary. We conclude that in patients with asthma, treatment with EPA-E may attenuate leukocyte function without distinctly changing the severity of asthma.

Topics: Aged; Asthma; Calcimycin; Cells, Cultured; Eicosapentaenoic Acid; Female; Humans; Leukocytes; Leukotrienes; Male; Middle Aged

The cysteinyl leukotrienes may play a central part in the mechanisms of aspirin-sensitive asthma. Previous work has shown that individuals with aspirin-sensitive asthma have high basal urinary LTE4 levels which increase further upon aspirin ingestion, and that sulphidopeptide leukotriene receptor antagonists attenuate aspirin-induced airflow obstruction. If the cysteinyl leukotrienes cause aspirin-induced asthmatic reactions, inhibition of the 5-lipoxygenase pathway should prevent aspirin-induced bronchospasm. This hypothesis has been tested with ZD2138, a specific non-redox 5-lipoxygenase inhibitor.. Seven subjects (four men) with aspirin-sensitive asthma with baseline FEV1 values > 67% were studied. ZD2138 (350 mg) or placebo was given on two separate occasions two weeks apart in a randomised double blind fashion. A single dose of aspirin was administered four hours after dosing and FEV1 was measured for six hours. Inhibition of the 5-lipoxygenase pathway by ZD2138 was assessed by measurements of urinary LTE4 levels and ex vivo calcium ionophore stimulated LTB4 generation in whole blood, before administration of drug or placebo and at regular time intervals after dosing and aspirin administration.. ZD2138 protected against the aspirin-induced reduction in FEV1 with a 20.3 (4.9)% fall in FEV1 following placebo compared with 4.9 (2.9)% following ZD2138. This was associated with 72% inhibition of ex vivo LTB4 generation in whole blood at 12 hours and a 74% inhibition of the rise in urinary LTE4 excretion at six hours after aspirin ingestion.. In aspirin-sensitive asthma the 5-lipoxygenase inhibitor ZD2138 inhibits the fall in FEV1 induced by aspirin and this is associated with substantial inhibition of 5-lipoxygenase.

Topics: Adult; Aspirin; Asthma; Bronchial Provocation Tests; Calcimycin; Double-Blind Method; Female; Forced Expiratory Volume; Humans; In Vitro Techniques; Leukotriene E4; Lipoxygenase Inhibitors; Male; Middle Aged; Pyrans; Quinolones

The enzyme 5-lipoxygenase catalyzes the metabolism of arachidonic acid to form products that have been implicated in the airway obstruction of asthma. We hypothesized that if products of the 5-lipoxygenase pathway are important in mediating this obstruction, then prevention of their formation should decrease the severity of an induced asthmatic response.. In a randomized, double-blind, placebo-controlled, crossover study, we examined the effect of A-64077, a 5-lipoxygenase inhibitor, on the bronchoconstriction induced by hyperventilation of cold, dry air in 13 patients with asthma. The completeness of 5-lipoxygenase inhibition was confirmed by examining the profile of eicosanoids produced in whole blood ex vivo after activation with the calcium ionophore A-23187.. A-64077 decreased the mean (+/- SEM) ionophore-induced synthesis of leukotriene B4, a 5-lipoxygenase product, by 74 percent (from 265.3 +/- 30.3 to 69.5 +/- 21.5 ng per milliliter, P less than 0.001), but it did not affect the ionophore-induced synthesis of thromboxane B2, a cyclooxygenase metabolite of arachidonic acid (80.0 +/- 17.1 ng per milliliter before A-64077 vs. 75.8 +/- 14.3 ng per milliliter after A-64077). In concert with the selective inhibition of 5-lipoxygenase by A-64077, the amount of cold, dry air (expressed as respiratory heat exchange) required to reduce the forced expiratory volume in one second by 10 percent was increased by 47 percent after A-64077 (3.0 kJ per minute for placebo vs. 4.4 kJ per minute for A-64077, P less than 0.002). Similar results were obtained when minute ventilation was used as an indicator of outcome (27.5 liters per minute for placebo vs. 39.8 liters per minute for A-64077, P less than 0.005).. Selective inhibition of 5-lipoxygenase by A-64077 is associated with a significant amelioration of the asthmatic response to cold, dry air, suggesting that 5-lipoxygenase products are involved in this response. This approach may be useful in the treatment of asthma.

Topics: Asthma; Bronchial Provocation Tests; Bronchoconstriction; Calcimycin; Cold Temperature; Double-Blind Method; Eicosanoids; Humans; Humidity; Hydroxyurea; Lipoxygenase Inhibitors; Male

Dryopteris crassirhizoma (DC) has a wide range of pharmacological effects, including antibacterial, anti‑influenza virus, anti‑tumor, anti‑reverse transcriptase and antioxidant effects. However, the inhibitory effect of DC on allergic inflammatory response remains unclear; therefore, the current study used an experimental ovalbumin (OVA)‑induced allergic asthma mouse model and phorbol myristate acetate (PMA)‑ and A23187‑stimulated HMC‑1 cells to reveal the effects of DC in regulating airway inflammation and its possible mechanism. Allergic asthma was initiated in BALB/c mice via exposure to OVA emulsified in aluminum, on days 1 and 14. Thereafter, the mice were treated with DC or dexamethasone (Dex) orally, before being challenged, from days 15 to 26. Subsequently, the mice were challenged with OVA on days 27, 28 and 29. The results of histological analysis indicated that the administration of DC decreased the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and suppressed eosinophilic infiltration, mucus production and collagen deposition in the lung tissue. DC treatment increased the level of T helper type 1 (Th1) cytokines (IL‑10 and interferon (IFN)‑γ) and decreased the levels Th2 cytokines (IL‑4, IL‑5 and IL‑13) and proinflammatory cytokines (IL‑6 and TNF‑α). Furthermore, DC treatment inhibited the activation of NF‑κB signaling (NF‑κB, p‑NF‑κB, IκB and p‑IκB), both in BALF and lung homogenates. Serum levels of total IgE and OVA‑specific IgE and IgG1 were significantly lower after DC treatment compared with after OVA treatment. However, the anti‑inflammatory effect of OVA‑specific IgG2a was higher after DC treatment. In addition, DC treatment attenuated the production of proinflammatory cytokines, including IL‑6 and TNF‑α, and the activation of NF‑κB signaling (NF‑κB and p‑NF‑κB), in PMA and calcium ionophore A23187‑stimulated HMC‑1 cells. In summary, the current study demonstrated that DC acts a potent anti‑allergic and anti‑inflammatory drug by modulating the Th1 and Th2 response and reducing the allergic inflammatory reaction in PMA and A23187‑stimulated HMC‑1 cells via NF‑κB signaling in an OVA‑induced allergic asthma model.

Topics: Animals; Anti-Allergic Agents; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cell Line, Tumor; Cytokines; Disease Models, Animal; Dryopteris; Humans; Lung; Male; Mast Cells; Mice; Mice, Inbred BALB C; NF-kappa B; Ovalbumin; Phytotherapy; Plant Extracts; Signal Transduction; Tetradecanoylphorbol Acetate

5-Lipoxygenase is the key enzyme in the biosynthesis of leukotrienes, powerful lipid mediators involved in inflammation, cell-cell communication, and other important physiological and pathological conditions. Particularly, cysteinyl-leukotrienes have been recognized as playing a significant role in the pathophysiology of asthma and potent and effective Cys-LT1 receptor antagonists have been developed for the treatment of this illness. Here we report that montelukast, a structural Cys-LT1 receptor antagonist, also exerts a substantial and apparently direct inhibitory effect on 5-lipoxygenase activity in vitro, at concentrations in the lower micromolar range, which are of potential therapeutic relevance. Thus, when human mast cells HMC-1 were stimulated with the Ca ionophore A23187 in the presence of montelukast (up to 100 microM) a substantial decline in 5-lipoxygenase biosynthesis was observed. Similar results were obtained in the rat mast cell-like RBL-1 cell model (IC50 congruent with 2.5 microM) and in human polymorphonuclear leukocytes. Moreover, montelukast directly inhibited human recombinant 5-lipoxygenase. Kinetic experiments revealed that the inhibition was of the non-competitive type, suggesting that montelukast binds a yet undefined allosteric site on 5-lipoxygenase. 5-Lipoxygenase inhibition by montelukast appears to be highly selective since the drug had no effects on other enzymes of the leukotriene cascade, viz. LTC4 synthase and LTA hydrolase.

Topics: Acetates; Allosteric Site; Animals; Anti-Asthmatic Agents; Asthma; Calcimycin; Calcium; Catalysis; Cell Line; Cell Line, Tumor; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclopropanes; Dexamethasone; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Inhibitory Concentration 50; Kinetics; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Mast Cells; Neutrophils; Protein Binding; Quinolines; Rats; Recombinant Proteins; Sulfides

The aim of this study is to investigate the histamine-releasing ability of mast cells from asthmatic bronchoalveolar lavage fluid (BALF). Following the measurement of the forced expiratory volume at the first second (FEV1), 29 mild asthmatics were included in the study and were subjected to fibreoptic bronchoscopy. The cells recovered from the BALF were challenged with anti-IgE, calcium ionophore A23187 (CI) or adenosine, and the released histamine was measured with an enzyme-linked chromogenic assay. Enzymatically dispersed mast cells from human lung or colon tissues were employed as control groups. The results showed that mast cells from BALF were at least 100 fold more sensitive to anti-IgE than those from lung or colon tissues. However, there was little difference between mast cells from BALF, lung or colon tissues in response to CI. Adenosine failed to stimulate histamine release from BALF mast cells. In conclusion, asthmatic BALF mast cells are much more sensitive to IgE-dependent stimulation than the non-IgE-dependent ones, indicating that mast cells may play a role in the pathogenesis of asthma.

Topics: Adenosine; Adult; Antibodies, Anti-Idiotypic; Asthma; Bronchoalveolar Lavage Fluid; Bronchoscopes; Calcimycin; Colon; Dose-Response Relationship, Immunologic; Female; Forced Expiratory Volume; Histamine Release; Humans; Immunoglobulin E; Ionophores; Lung; Male; Mast Cells; Middle Aged; Sensitivity and Specificity; Severity of Illness Index; Vasodilator Agents

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.

Topics: Airway Resistance; Animals; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Chelating Agents; Disease Models, Animal; Ditiocarb; Guinea Pigs; Injections, Intraperitoneal; Ionophores; Luminescent Measurements; Lung; Male; Ovalbumin; Reactive Oxygen Species; Superoxide Dismutase

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Asthma; Benzene Derivatives; Calcimycin; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Furans; Humans; Indomethacin; Ionophores; Isoenzymes; Leukotrienes; Lipopolysaccharides; Lipoxygenase; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Sulfonamides; Thromboxane B2

Our previous work on linkage analysis showed that histamine release from basophils to anti-IgE stimuli was linked to the gene marker of chromosome 11q13, where the beta chain of the high-affinity receptor for IgE (FcepsilonRI-beta) is located.. To evaluate the association between FcepsilonRI-mediated histamine release from basophils and four bi-allelic single nucleotide polymorphisms of the FcepsilonRI-beta gene.. Phenotypes of asthma, such as maximal histamine release from basophils and atopy, were measured from 80 randomly recruited asthmatic children. Polymorphisms of the FcepsilonRI-beta gene were determined by PCR-based methods.. The polymorphism in exon 7, resulting in Glu to Gly substitution, was significantly associated with histamine release from basophils to anti-IgE stimuli, but not with total IgE levels and skin test responses to aeroallergens.. This study supports a role for the FcepsilonRI-beta gene in the expression of high affinity IgE receptor-mediated histamine release from basophils.

Topics: Adolescent; Alleles; Asthma; Basophils; Calcimycin; Child; Female; Histamine Release; Humans; Male; Polymorphism, Single Nucleotide; Random Allocation; Receptors, IgE

Human blood polymorphonuclear cells, which biosynthesize eicosanoids from the 5-lipoxygenase (5-LO) pathway, are likely to be involved in asthma, in which glucocorticoids represent the first line of therapy. Their effects on leukotriene release after a short course of treatment, which have been reported in several studies, are controversial.. We sought to investigate whether long-term oral glucocorticoids inhibit lipid mediators from the 5-LO pathway.. Twelve normal control subjects, 29 asthmatic subjects, and 50 glucocorticoid-dependent asthmatic subjects were included in the study. Polymorphonuclear cells were studied for endogenous and transcellular metabolism of eicosanoids.. Total leukotriene B(4) production was significantly lower in cells from glucocorticoid-dependent asthmatic subjects (mean +/- SD, 177 +/- 26 ng/10(7) cells) than in control subjects (406 +/- 27), untreated asthmatic subjects (421 +/- 34), and asthmatic subjects treated with inhaled glucocorticoids (290 +/- 56). When incubated with arachidonic acid, these polymorphonuclear cells released very low amounts of 5(S)- and 12(S)-hydroxy-eicosatetraenoic acid (HETE), whereas endogenous 15(S)-HETE was found in substantial amounts. The transformation of exogenous 15(S)-HETE into 5(S),15(S)-diHETE and lipoxins was significantly more important in untreated asthmatic subjects than in control subjects and glucocorticoid-dependent asthmatic subjects.. This study showed that long-term oral corticotherapy affects the 5-LO activity and leads to a decrease production of all metabolites in contrast to short-term or inhaled glucocorticoids. This study also questions the site of action of glucocorticoids in regulating the availability of arachidonic acid and potential eicosanoid regulation, as previously held in phospholipase A2 studies.

Topics: 5-Lipoxygenase-Activating Proteins; Administration, Inhalation; Administration, Oral; Adrenergic beta-Antagonists; Adult; Albuterol; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Beclomethasone; Calcimycin; Calcium; Carrier Proteins; Chromatography, High Pressure Liquid; Drug Therapy, Combination; Eicosanoids; Female; Humans; Ionophores; Male; Membrane Lipids; Membrane Proteins; Methylprednisolone; Neutrophils; Phospholipids; Prednisone; Salmeterol Xinafoate; Theophylline

It has been postulated that T lymphocytes orchestrate the chronic inflammation in bronchial asthma. In animal models, infiltration of CD8+ T lymphocytes into the bronchial mucosa prevented bronchial hyperresponsiveness and decreased early and late phase reaction. IFN-gamma antagonizes IL-4-dependent IgE production as well as IL-5-induced proliferation and activation of eosinophils. We therefore investigated the secretion of IFN-gamma of isolated CD8+ T lymphocytes from peripheral blood of patients with allergic asthma (n = 6) and from healthy controls (n = 7) in vitro. In this setting we compared the effect of stimulation with anti-CD3 antibodies with that of phorbol myristate acetate (PMA) and calcium-ionophore. As expected, CD8+ T lymphocytes from peripheral blood of healthy volunteers produced significantly more IFN-gamma in the presence of PMA and calcium-ionophore than after stimulation with anti-CD3 antibodies. However, in subjects with allergic asthma, IFN-gamma secretion of CD8+ T cells was significantly higher when incubated with anti-CD3 antibodies than after activation with PMA and calcium-ionophore. While IFN-gamma secretion of CD8+ T lymphocytes of patients with allergic asthma was lower than that of healthy controls in the presence of PMA/calcium-ionophore, it was significantly elevated when compared with normal controls after stimulation with anti-CD3 antibodies. Thus, potent activators of cytokine secretion, such as PMA and calcium-ionophore, induce a cytokine profile different from that induced by weaker stimulants, such as anti-CD3 antibodies. These findings have implications for further studies investigating cytokine production of inflammatory cells in vitro.

Topics: Adult; Animals; Asthma; Calcimycin; Case-Control Studies; CD8-Positive T-Lymphocytes; Cytokines; Female; Humans; In Vitro Techniques; Interferon-gamma; Ionophores; Leukocytes, Mononuclear; Male; Middle Aged; Muromonab-CD3; Tetradecanoylphorbol Acetate

Sensory neuropeptides have been suggested to play a role in the pathogenesis of a number of respiratory diseases including asthma and chronic non-productive cough.. To investigate the action of sensory neuropeptides on airway mast cells obtained by bronchoalveolar lavage (BAL).. BAL was performed on 23 nonasthmatic patients with cough (NAC), 11 patients with cough variant asthma (CVA) and 10 nonatopic controls. Washed lavage cells were stimulated (20 min, 37 degrees C) with calcitonin gene-related peptide (CGRP), neurokinin A (NKA) and substance P (25 and 50 micromol/L).. The neuropeptides tested induced histamine release in all groups studied. Only CGRP (50 micromol/L) induced significantly more histamine release from both NAC and CVA patients compared with control subjects (P = 0.038 and 0.045, respectively).. Regardless of aetiology, mast cells from patients with chronic cough appear to have an increased responsiveness to CGRP compared with controls. The results of the present study suggest that the role of CGRP in chronic cough should be further investigated.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Calcitonin Gene-Related Peptide; Cough; Female; Histamine Release; Humans; Male; Mast Cells; Middle Aged; Neuropeptides; Substance P

Topics: Arachidonic Acid; Asthma; Calcimycin; Cell Culture Techniques; Eicosanoids; Glucocorticoids; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Macrophages, Alveolar; Monocytes; Neutrophils

To compare the mediator releasability between atopic and nonatopic asthmatics, we measured basophil histamine releasability (BaHR) using a calcium-ionophore A23187 and anti-IgE in 137 subjects who were treated at Seoul National University Hospital. Subjects were categorized into atopic (group AA, n=77) or nonatopic asthmatics (group NA, n=32), or normal controls (group NC, n=28). Serum total IgE levels were determined and correlation with BaHR was assessed. Anti-IgE-induced maximal BaHR in groups AA, NA, and NC was 41.0+/-3.2, 23.1+/-4.5, and 16.8+/-3.8, respectively (mean+/-SE, %). Anti-IgE-induced BaHR in group AA was significantly higher than that in groups NA and NC (p<0.05). Calcium ionophore A23187-induced maximal BaHR was 43.1+/-2.8, 40.8+/-4.4, and 50.5+/-5.2, respectively (mean+/-SE, %), and there was no significant difference among the groups. Serum total IgE level correlated significantly with anti-IgE-induced maximal BaHR (r=0.281, p<0.01) but not with that induced by calcium ionophore A23187. In conclusion, IgE receptor-related BaHR is higher in atopic asthmatics than in nonatopic asthmatics, and this increased BaHR in atopics is significantly associated with increased serum total IgE level.

Topics: Asthma; Basophils; Calcimycin; Child; Histamine Release; Humans; Immunoglobulin E; Ionophores

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.

Topics: 5-Lipoxygenase-Activating Proteins; Adult; Arachidonate 5-Lipoxygenase; Asthma; Biological Transport; Blotting, Western; Calcimycin; Carrier Proteins; Cell Nucleus; Cells, Cultured; Cysteine; Eosinophils; Female; Humans; Immunohistochemistry; Interleukin-5; Leukocyte Count; Leukotrienes; Male; Membrane Proteins; Staining and Labeling; Subcellular Fractions

The cytokine leukemia inhibitory factor (LIF) is known to be produced by both inflamed peripheral autonomic nerves and several cell types involved in the regulation of the immune response. We have recently demonstrated that several structural cell types in human airways produce LIF in response to inflammatory stimuli and that LIF augments contractile responses to tachykinins in airway explants. Because the eosinophil is a major effector cell in asthma and often found adjacent to the nerves, we hypothesized that eosinophils produce LIF and that LIF primes and upregulates eosinophil recruitment and function, allowing bidirectional neuroimmune interactions and augmentation of eosinophil-mediated injury.. The purpose of this study was to demonstrate that human eosinophils synthesize and release LIF, to determine the effects of LIF on eosinophil functions (ie, chemotaxis, granule protein release, expression of the activation marker CD69, and apoptosis), and to compare serum LIF levels between atopic and nonatopic individuals.. Reverse-transcription PCR, ELISA, immunocytochemistry, chemotaxis assay, and flow cytometry were used.. Peripheral blood eosinophils express LIF and messenger RNA for LIF and LIF receptor. Serum LIF levels were higher in atopic patients with mild asthma than in nonatopic normal donors. Eosinophils from nonatopic donors were stimulated by calcium ionophore to release small amounts of LIF (from almost none to 5.3 +/- 1.8 pg/10(6) cells). Eosinophils from atopic donors showed a 10-fold increase (from 45.1 +/- 38.7 pg/106 cells to 414.5 +/- 189.9 pg/10(6) cells). Preincubation of eosinophils with LIF increased eosinophil peroxidase release 4-fold. LIF was not chemotactic for eosinophils but augmented chemotaxis mediated by substance P by 82% and by platelet-activating factor by 31%. LIF did not effect eosinophil apoptosis but increased CD69 expression.. LIF has proinflammatory roles in eosinophil-dependent airway disorders.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Apoptosis; Asthma; Calcimycin; Chemotaxis, Leukocyte; Eosinophil Peroxidase; Eosinophils; Growth Inhibitors; Humans; Hypersensitivity, Immediate; Interleukin-6; Lectins, C-Type; Leukemia Inhibitory Factor; Lymphocyte Activation; Lymphokines; Peroxidases; RNA, Messenger

The genetics of the regulation of the release of mediators involving the interaction of IgE with cells and their ability to release mediators have not been extensively investigated. With use of the candidate gene approach, it was reported that the gene regulating the beta chain of the high-affinity receptor for IgE is on chromosome 11q13.. To determine whether gene(s) in chromosome 11q13 may control the expression of maximal histamine release from basophil to anti-IgE stimuli, linkage analysis between this phenotype and the gene marker of chromosome 11q13 was performed.. Maximal histamine release to anti-IgE and calcium ionophore A23187 and genotyping chromosome 11q13 with use of microsatellite marker (D11S97) were performed in 56 probands with asthma and 59 of their siblings. The linkage was analyzed by affected sib-pair analysis and the quantitative trait locus approach.. Maximal histamine release (mean +/- SE) to anti-IgE and A23187 was 43.3% +/- 3.5% and 30.9 +/- 3.4% in probands and 29.5% +/- 2.6% and 22.2% +/- 2.7 in siblings, respectively. Of 20 sib-pairs with the maximal histamine release to anti-IgE more than 33% (mean plus 1 SD of nonasthmatic controls), 11 (55%) shared 2 D11S97 alleles, 9 (45%) shared 1 allele, and neither sib-pair shared identical alleles, which indicates a significant linkage of maximal histamine to anti-IgE and gene marker of chromosome 11q13 (P =.02). The difference (mean +/- SE) of the maximal histamine release to anti-IgE between each proband and sibling was smaller in sib-pairs with 2 identical alleles than in those with 1 identical allele and with no identical allele (14.1% +/- 2.6% vs 25.8% +/- 3.1% vs 41.0% +/- 4.9%). However, the difference (mean +/- SE) to A23187 between each proband and sibling was not different among the 3 groups (9.7% +/- 1.8% vs 17.9% +/- 3.6% vs 10.4% +/- 4.8%).. Maximal histamine release from basophils to anti-IgE stimuli was linked to the gene marker of chromosome 11q13.

Topics: Adolescent; Asthma; Basophils; Calcimycin; Cells, Cultured; Child; Child, Preschool; Chromosomes, Human, Pair 11; Female; Genetic Linkage; Genetic Markers; Histamine Release; Humans; Immunoglobulin E; Ionophores; Male; Microsatellite Repeats; Receptors, IgE

The formation of leukotriene B4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB4. The addition of human recombinant LTA4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (> 4-fold) the formation of LTB4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA4 released by neutrophils. The formation of LTB4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA4 generated in the extracellular milieu. Investigations on the fate of LTA4 in plasma revealed that LTA4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA4, but directly release LTC4.

Topics: Asthma; Calcimycin; Cell Separation; Eosinophils; Epoxide Hydrolases; Erythrocytes; Granulocytes; Humans; In Vitro Techniques; Leukotriene B4; Neutrophils; Plasma; Pulmonary Eosinophilia; Rhinitis

This study was designed to examine further the role of platelet-activating factor (PAF) in asthma, comparing leukotriene B4 (LTB4) release, 5-lipoxygenase activity and intracellular calcium levels ([Ca2+]i) in macrophages. LTB4 and other lipoxygenase metabolites in macrophages in bronchoalveolar lavage fluids obtained from 23 asthmatic patients and 20 control subjects were measured by reverse-phase high-performance liquid chromatography. [Ca2+]i was monitored using the fluorescent probe fura-2. The basal LTB4 release of resting macrophages was not different between groups (0.02+/-0.01 versus 0.05+/-0.02 ng x 10(-6) cells). When stimulated with calcium ionophore A23187 (2.5 microM), however, macrophages from asthmatic patients released more LTB4 than cells from control subjects (30.2+/-3.4 versus 13.7+/-2.1 ng x 10(-6) cells). Although PAF alone did not alter LTB4 release, it enhanced the response to subsequent A23187 stimulation. This effect was noted following short treatment (i.e., 5 min) at concentrations of > or =1.0 microM PAF, with the maximal effect noted after treatment with 5.0 microM PAF + 2.5 microM A23187 (105.1+/-6.7 versus 15.3+/-2.6 ng x 10(-6) cells). Treatment of macrophages with PAF also increased 5-lipoxygenase activity and [Ca2+]i more in cytosols from asthmatic patients than in cytosols from control subjects. These findings support a role of intracellular calcium in the activation of 5-lipoxygenase which, in turn, augments the release of leukotriene B4. Because levels of platelet-activating factor may be increased in the lung during asthma and can increase the subsequent release of a chemotactic mediator leukotriene B4, from macrophages, these findings suggest that platelet-activating factor may prime the constitutive cells of the lung to augment inflammatory effects important in the pathogenesis of asthma.

Topics: Adult; Arachidonate 5-Lipoxygenase; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Calcium; Chromatography, High Pressure Liquid; Cytosol; Female; Humans; Inflammation Mediators; Ionophores; Leukotriene B4; Macrophages, Alveolar; Male; Middle Aged; Osmolar Concentration; Platelet Activating Factor; Reference Values

The release of histamine and leukotriene C4 (LTC4) from bronchoalveolar lavage (BAL) cells and peripheral blood stimulated with Ca ionophore A23187 was compared between atopic and nonatopic asthma. The proportion of basophilic cells in BAL fluid was significantly higher in atopic than in nonatopic asthma (p < 0.01); however, no significant differences were present in the other BAL cells between the two asthma types. The concentration of histamine in BAL fluid was significantly higher in younger patients (20-59 years) with atopic than in nonatopic asthma (p < 0.01). In contrast, the concentration of LTC4 was significantly higher in nonatopic than in younger patients with atopic asthma (p < 0.01). The release of histamine from BAL cells (p < 0.001) and peripheral blood (p < 0.01) was significantly larger in younger patients with atopic than in nonatopic asthma. The generation of LTC4 by BAL cells was significantly larger in nonatopic than in younger (p < 0.01) and older patients with atopic asthma (60+ years) (p < 0.05). These results suggest that both histamine and LTC4 participate in the onset mechanism of atopic asthma, and only LTC4 participates in that of nonatopic asthma.

Topics: Adult; Aging; Asthma; Blood Cells; Bronchi; Bronchoalveolar Lavage Fluid; Calcimycin; Female; Histamine; Humans; Hypersensitivity; Ionophores; Leukotriene C4; Male; Middle Aged; Pulmonary Alveoli

Monoterpenes are prescribed to treat chronic obstructive airway disorders mainly because of their familiar secretolytic properties. The aim of this study was to investigate the effect of 1.8-cineole (Soledum) on arachidonic acid (AA) metabolism in blood monocytes of patients with bronchial asthma. Patients with bronchial asthma (n = 10) and healthy test subjects (n = 12) were included in the study. Production of the representative AA-metabolites LTB4 and PGE2 from isolated monocytes stimulated with the calcium ionophore A23187 were measured ex vivo before therapy with 1.8-cineole (3 x 200 mg/day), after three days of treatment (day 4) and four days after discontinuation of 1. 8-cineole (day 8). The production of LTB4 and PGE2 from monocytes ex vivo was significantly inhibited on day 4 in patients with bronchial asthma (-40.3%, n = 10 and -31.3%, p = 0.1, n = 3 respectively) as well as in healthy volunteers (-57.9%, n = 12 and -42.7%, n = 8 respectively). In conclusion, 1.8-cineole was shown to inhibit LTB4 and PGE2, both pathways of AA-metabolism. Further studies are needed to show that 1.8-cineole is suitable in the treatment of bronchial asthma.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Asthma; Calcimycin; Cyclohexanols; Eucalyptol; Forced Expiratory Volume; Humans; Inflammation Mediators; Interleukin-1; Leukotriene B4; Menthol; Middle Aged; Monocytes; Monoterpenes; Terpenes

RANTES (regulated on activation, normal T expressed and secreted) has been shown to possess chemotactic activity for eosinophils. Eosinophils have been considered to play a key role in the allergic inflammation through the release of inflammatory molecules such as radical oxygen products. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils.. Eosinophils were isolated from heparinized venous blood of patients with bronchial asthma by the modified CD16-negative depletion method. Radical oxygen products were examined in terms of lucigenin-dependent chemiluminescence. To a mixture of 50 microl of eosinophils (2x10(6)/ml) and 50 microl of lucigenin (5x10(-4)M), 50 microl of calcium ionophore A23187 (final concentration 10(-5)M) was added, and radical oxygen products were determined for 600 s.. RANTES treatment resulted in the enhancement of peak value (0.64+/-0.23 RLU) and integrated value (119.08+/-20.52 RLU) as compared to untreated cells (0.15+/-0.03 RLU, 29.48+/-8.92 RLU, respectively). CONCLUSIONS We could conclude that RANTES might play an important role in the pathogenesis of allergic inflammation through involvement in selective eosinophil infiltration and eosinophil activation by augmentation of eosinophil oxidative metabolism.

Topics: Acridines; Asthma; Calcimycin; Chemokine CCL5; Eosinophils; Free Radicals; Humans; In Vitro Techniques; Inflammation; Inflammation Mediators; Ionophores; Luminescent Measurements; Reactive Oxygen Species

Asthma is considered a Th2-like disease, characterized by locally increased levels of interleukin (IL) 4. The bronchial epithelium plays an important role in the initiation and perpetuation of inflammatory reactions within the airways. However, little is known about the presence of IL-4 receptors on human bronchial epithelial cells, or the effects of IL-4 on these cells. In this report, definitive evidence of IL-4 receptor expression on human bronchial epithelial cells using several methods is presented. IL-4 receptor expression on human bronchial epithelial cells in vivo was demonstrated using in situ hybridization and immunohistochemistry. No difference in IL-4 receptor protein expression was observed between bronchial biopsies of healthy subjects compared to allergic asthmatics. Cultured human bronchial epithelial cells also expressed IL-4 receptor mRNA and protein (as determined by RT-PCR analysis and flow cytometry, respectively). IL-4 receptor protein expression by bronchial epithelial cells could be increased by stimulation with PMA+calcium ionophore, whereas IL-1beta and IL-6 decreased IL-4 receptor expression. A cyclic AMP analogue and IL-4 had no effect. Finally, it is shown that the IL-4 receptor is functionally active as IL-4 stimulates the release of IL-8, monocyte chemoattractant protein 1, and particularly IL-1 receptor antagonist by human bronchial epithelial cells. It is concluded that human bronchial epithelial cells express IL-4 receptors both in vivo and in vitro. Stimulation of human bronchial epithelial cells by IL-4 may result in the release of both pro- and anti-inflammatory mediators known to be upregulated in asthmatic airways.

Topics: Asthma; Bronchi; Bucladesine; Calcimycin; Cells, Cultured; Chemokine CCL2; Epithelial Cells; Gene Expression Regulation; Humans; In Situ Hybridization; Intercellular Adhesion Molecule-1; Interleukin 1 Receptor Antagonist Protein; Interleukin-4; Interleukin-8; Interleukins; Receptors, Interleukin-1; Receptors, Interleukin-4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Tetradecanoylphorbol Acetate

During the pollen season, peripheral blood mononuclear cells (PBMC) from allergic patients produce increased levels of Th2 cytokines after stimulation with allergen in vitro. We have studied the effect of a single bronchial provocation test (BPT) of allergic patients to determine whether allergen challenge in vivo modulates cytokine production by PBMC, after subsequent stimulation with the same allergen in vitro.. Twelve atopic asthmatic patients were challenged with the relevant allergen, and their PBMC, isolated before (T0) or 6 (T6) or 24 h (T24) after BPT, respectively, were cultured for 120 h in the presence or absence of the same allergen, after which cytokine production was measured by ELISA.. Allergen-specific activation of the PBMC at T0 resulted in interleukin (IL)-5 and IL-13 production, but not in detectable levels of interferon-gamma and IL-4. BPT did not induce the secretion of the latter cytokines. However, IL-5 and IL-13 production was significantly decreased at T24, as compared to T0. No statistically significant differences were found between the production of IL-10 before and after BPT.. In contrast to the effects of natural challenge with allergen, a decrease in the production of some Th2 cytokines by peripheral blood T cells was observed 24 h after BPT, suggesting a concomitant decrease in the frequency of allergen-specific T cells in the circulation.

Topics: Adolescent; Adult; Allergens; Asthma; Bronchial Provocation Tests; Calcimycin; Carcinogens; Cytokines; Female; Humans; Interferon-gamma; Interleukin-10; Interleukin-13; Interleukin-4; Interleukin-5; Ionophores; Leukocytes, Mononuclear; Male; Tetradecanoylphorbol Acetate; Time Factors

Although acyclovir (9-(2-hydroxyethoxymethyl) guanine) is an antiviral drug that inhibits DNA polymerase of herpes virus, we have had the experience of an asthmatic patient's peak flow rate being improved by oral administration of acyclovir.. The aim of this experiment is whether acyclovir has anti-asthma effects using an asthma model in guinea-pigs.. The airway response was induced by a single inhalation of calcium ionophore A23187 (2 mg/mL). The airway obstruction was estimated by the ratio of expiration to inspiration time (E/I). The peribronchial eosinophil infiltration and eosinophil influx into bronchoalveolar lavage (BAL) fluid 7 h after the inhalation were also examined. To assess the effects of acyclovir (1, 10, and 100 mg/kg), aminophylline (20 mg/kg) and pemirolast potassium (TBX, 20 mg/kg) on A23187-induced asthmatic response, the drugs were intraperitoneally administered before the inhalation.. The immediate airway obstruction was significantly suppressed by acyclovir (10 mg/kg) and aminophylline, whereas different doses of acyclovir (1 and 100 mg/kg) and TBX showed only a small inhibitory effect on the airway obstruction. On the other hand, the peribronchial eosinophilia was most successfully inhibited by TBX. Acyclovir (10 mg/kg) and aminophylline also suppressed the eosinophilia significantly. Furthermore, acyclovir significantly suppressed eosinophil influx into BAL fluid, whereas aminophylline and TBX weakly suppressed the influx.. These results suggest that acyclovir exhibits not only antiviral but also antiasthma activity.

Topics: Acyclovir; Aminophylline; Animals; Anti-Asthmatic Agents; Antiviral Agents; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Calcimycin; Eosinophils; Guinea Pigs; Histamine Antagonists; Humans; Male; Middle Aged; Peak Expiratory Flow Rate; Pyridines; Pyrimidinones; Time Factors

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC4 and LTB4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-lipoxygenase (5-LO). LTC4 synthase, and LTA4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC4 and LTB4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC4 and LTB4 when LTA4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca2+ ([Ca2+]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA2 and 5-LO activities without affecting the mobilization of [Ca2+]i.

Topics: Analysis of Variance; Animals; Arachidonic Acid; Asthma; Bronchodilator Agents; Calcimycin; Calcium; Cell Membrane; Drugs, Chinese Herbal; Ephedrine; Immunoglobulin E; Ionophores; Isotope Labeling; Leukemia, Basophilic, Acute; Leukotriene A4; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Phospholipases A; Phospholipases A2; Radioimmunoassay; Rats; Tritium; Tumor Cells, Cultured

It has been suggested that phospholipase A2 (PLA2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (lysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% (p < 0.01) and LTB4 synthesis 88-fold (p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB4 synthesis to 229-fold above the control values (p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretory (s) PLA2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal- and Cal+PMA-stimulated LTB4 synthesis. AACOCF3, a cPLA2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAI-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAI-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA2 had a greater role in stimulated LTB4 synthesis. These data indicate that PLA2 plays a direct role in human AM LT synthesis; both the cytosolic and secretory forms contribute to LT synthesis; PLA2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA2; and CoAI-TA contributes to PAF synthesis during PLA2 inhibition. With the exception of the greater role for cPLA2 in stimulated LTB4 synthesis in the patients with asthma, the contributions of PLA2 and CoAI-TA to AM LT and PAF synthesis appear to be similar in normal subjects and patients with asthma.

Topics: Acetyltransferases; Acyl-Carrier Protein S-Acetyltransferase; Arachidonic Acids; Asthma; Benzenesulfonates; Bronchoalveolar Lavage Fluid; Calcimycin; Calcium; Cells, Cultured; Cytosol; Enzyme Inhibitors; Humans; Ionophores; Leukotriene B4; Leukotrienes; Macrophages, Alveolar; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Tetradecanoylphorbol Acetate; Urea

The role of platelet activating factor (PAF) in asthma remains controversial. The priming effect of PAF on leukotriene B4 (LTB4) release, 5-lipoxygenase activity, and intracellular calcium levels in asthmatic neutrophils was examined.. LTB4 and other lipoxygenase metabolites in neutrophils obtained from 17 asthmatic patients and 15 control subjects were measured by reverse phase-high performance liquid chromatography (RP-HPLC). Intracellular calcium levels were monitored using the fluorescent probe fura-2.. The mean (SD) basal LTB4, release from neutrophils was not significantly different between the two groups (0.05 (0.01) vs 0.03 (0.02) ng/10(6) cells); however, when stimulated with calcium ionophore A23187 (2.5 microM), neutrophils from asthma patients released more LTB4 than cells from control subjects (15.7 (1.2) vs 9.9 (1.6) ng/10(6) cells). Although PAF alone did not alter LTB4 release, it enhanced the response to subsequent A23187 stimulation. This effect was observed following treatment for five minutes with PAF at concentrations > 1.0 microM. The maximal effect was seen with 5.0 microM PAF + 2.5 microM A23187 (62.7 (2.2) vs 18.6 (2.3) ng/10(6) cells). Pretreatment with PAF also increased 5-lipoxygenase activity and intracellular calcium levels in neutrophils from asthmatic patients to a greater extent than in those from non-asthmatic patients.. These findings indicate that, in neutrophils from asthmatic patients, PAF enhances LTB4 release and increases 5-lipoxygenase activity and intracellular calcium to a greater extent than in neutrophils from non-asthmatic patients.

Topics: Adult; Arachidonate 5-Lipoxygenase; Asthma; Calcimycin; Calcium; Cells, Cultured; Cytosol; Female; Humans; Ionophores; Leukotriene B4; Male; Middle Aged; Neutrophils; Platelet Activating Factor; Stimulation, Chemical

We examined the release of leukotriene B4 from calcium ionophore A23187-stimulated neutrophils from asthmatic patients treated with or without intravenous prednisolone during an asthmatic attack. The mean level of LTB4 in the supernatant of stimulated neutrophils from patients treated with intravenous prednisolone was significantly lower than that in the supernatant of stimulated neutrophils from those without prednisolone treatment. Preincubation with prednisolone caused a dose-dependent inhibition of LTB4 release from calcium ionophore A23187-stimulated neutrophils. These findings suggest that intravenous prednisolone inhibits the release of LTB4 from neutrophils in vivo.

Topics: Adult; Asthma; Calcimycin; Calcium; Cells, Cultured; Humans; Ionophores; Leukocyte Count; Leukotriene B4; Middle Aged; Neutrophils; Prednisolone; Secretory Rate

5-Lipoxygenase activation of human blood polymorphonuclear cells (PMN) from asthmatic patients (asthmatics) was studied to investigate whether differences may exist with healthy subjects (controls). The respective cell capacities to produce lipoxins (LXs), leukotrienes, and 5(S), 15(S)-dihydroxyeicosatetraenoic acid [5(S),15(S)-diHETE] were compared under in vitro stimulation by ionophore A23187, with or without exogenous 15(S)-hydroxyeicosatetraenoic acid [15(S)-diHETE]. Eicosanoids were analyzed by elution with an isocratic reverse-phase high performance liquid chromatography system, and their profiles, detected by simultaneous monitoring at 302, 280, and 246 nm, were evaluated on the basis of chromatographic behavior: UV spectral characteristics and coelution with synthetic standards. In the presence of exogenous 15(S)-HETE, human PMN were able to produce LXs and 5(S),15(S)-diHETE, PMN from asthmatics were able to produce 5(S), 5(S),15(S)-diHETE, and LXs from endogenous sources, whereas in the same experimental conditions, no detectable amounts of these compounds were released by PMN from controls. The levels of 5(S),15(S)-diHETE, and LXs biosynthesized from endogenous arachidonic acid were highly correlated. Two different LX patterns were observed involving two possible metabolic pathways: (a) via the intermediate 5,6-epoxytetraene alone for LXs generation from exogenous 15(S)-HETE; and (b) via 5,6- and/or 14,15-epoxytetraenes leading to the formation of an enzyme-bound delocalized carbocation for LXs generation from endogenous arachidonate, respectively. The enhanced 5-lipoxygenase activation of blood PMN from asthmatics and the metabolism of exogenous 15(S)-HETE may reflect a priming induced by various mediators released from environmental cells, and could be considered as a model of transcellular signalization between PMN and endothelial cells.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Male; Models, Biological; Neutrophil Activation; Substrate Specificity

Eosinophils play a major role in the pathogenesis of bronchial asthma. In this study, we examined the density characteristics of blood eosinophils from 9 normal healthy individuals and 9 allergic asthmatic patients. Furthermore, the effect of platelet-activating factor, a potent mediator of inflammation, and calcium ionophore, A23187, on the density of normodense eosinophils (density > 1.085 g/ml) has also been examined. Initially, asthmatic patients had 27.0 +/- 1.1% eosinophils of lighter density (density < or = 1.081 g/ml), significantly greater than that in the normal individuals (7.5 +/- 0.5%). After exposure to platelet-activating factor (1 microM) or calcium ionophore (A23187, 1 microgram/ml), the normodense eosinophils switched to hypodense in both groups: 16.7 +/- 2.1% and 54.2 +/- 3.7%, respectively, in normal individuals, and 30.6 +/- 5.7% and 77.4 +/- 2.3%, respectively, in asthmatic patients. These data demonstrated that a certain percentage of normodense eosinophils from asthmatics and normal subjects switched to hypodense after activation with platelet-activating factor or calcium ionophore. Furthermore, eosinophils from asthmatics switched to a greater degree than in normal subjects, suggesting that the normodense eosinophils in asthmatics become primed probably by endogenously released mediators.

Topics: Adult; Asthma; Calcimycin; Centrifugation, Density Gradient; Eosinophils; Female; Humans; Inflammation Mediators; Ionophores; Leukocyte Count; Male; Platelet Activating Factor

Eosinophils from asthmatic patients are known to release greater amounts of leukotrienes than normal eosinophils when stimulated by the calcium ionophore A23187. The effect of platelet activating factor (PAF) in priming eosinophils was investigated.. Eosinophils were obtained from 18 asthmatic patients and 18 healthy donors. Cells separated by the Percoll gradients were incubated with PAF (C-18) for 30 minutes and then stimulated with the calcium ionophore A23187 (2.5 microM) for 15 minutes. The amount of leukotriene C4 (LTC4) in supernatants was measured using a combination of high pressure liquid chromatography and radioimmunoassay.. The mean (SD) amount of LTC4 released by eosinophils from asthmatic patients upon stimulation with the calcium ionophore A23187 alone was 27.9 (9.9) ng/10(6) cells (n = 6). The amount of LTC4 released following stimulation with the calcium ionophore A23187 after pretreatment with PAF (1, 5, and 10 microM) was 57.2 (8.9), 75.1 (14.3), and 52.6 (10.7) ng/10(6) cells (n = 6), respectively. Trace amounts of LTC4 (0.9 (0.02) ng/10(6) cells, n = 6) were detected in the supernatant of the cells after stimulation by PAF alone (5 microM). The amount of LTC4 released upon stimulation by calcium ionophore A23187 alone in eosinophils from healthy donors was 10.3 (3.7) ng/10(6) cells (n = 4). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with PAF at concentrations of 1, 5, and 10 microM were 11.9 (3.5), 17.8 (5.6), and 12.7 (5.1) ng/10(6) cells (n = 4), respectively. Trace amounts of LTC4 (0.6 (0.02) ng/10(6) cells, n = 4) were detected in the supernatant of the cells upon stimulation with PAF alone (5 microM). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with lyso-PAF at concentrations of 1, 5, and 10 microM (n = 4 or 6) were 30.8 (5.2), 22.9 (5.1), and 27.3 (4.3) ng/10(6) cells (n = 6) from the eosinophils of asthmatic patients and 13.7 (3.3), 15.2 (4.9), and 14.7 (3.8) ng/10(6) cells (n = 4) from the eosinophils of healthy donors.. The results indicated that PAF enhanced LTC4 formation by eosinophils obtained from asthmatic patients stimulated with the calcium ionophore A23187, but not those obtained from normal subjects.

Topics: Adult; Aged; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Eosinophils; Humans; In Vitro Techniques; Leukotriene C4; Male; Middle Aged; Platelet Activating Factor; Stimulation, Chemical

The production of platelet-activating factor (PAF) by inflammatory cells is regulated by lyso-PAF acetyltransferase, and the activity of this enzyme is increased in neutrophils of stable asthmatic patients. The aim of this investigation was to determine whether acetyltransferase activity is further upregulated in asthmatic patients experiencing acute symptoms. A radioenzymatic assay was used to measure the enzymatic affinity constant (Km) and maximal enzymatic activity (Vmax) for acetyltransferase from unstimulated and Ca2+ ionophore (A23187)-stimulated neutrophils from 16 patients with acute asthma, and the measurement was repeated at the time of discharge (n=9) and after recovery from the acute episode (n=13). During acute asthma, Km (median 93.8 (interquartile range 64.1-109.7) microM) was lower than that measured in nonasthmatic subjects in a previous study using identical methods (155.1 (122.2-179.9) microM; p=0.0001), and in 10 out of 13 acute patients Km for unstimulated neutrophils increased following recovery. In A23187-stimulated neutrophils, Km during acute asthma (84.3 (73.6-100.2) microM) and at discharge (83.9 (83.1-94.8) microM) were similar, but Km after recovery was increased (115.0 (95.6-119.5) microM; p=0.02). The change in Km following stimulation with A23187 was also significantly less during acute asthma than previously measured in nonasthmatic subjects (p=0.003). Although Vmax during acute asthma (12.9 (interquartile range 10.5-22.5) nmol x min(-1) x mg(-1) protein) did not differ significantly from that at discharge (14.4 (12.3-20.4) nmol x min(-1) x mg(-1)) or after recovery (17.3 (12.3-18.4) nmol x min(-1) x mg(-1)), both median Km and Vmax tended to be lowest during acute asthma and increase at discharge and after recovery. An increase in lyso-PAF acetyltransferase activity alone may not account for increased systemic PAF concentrations during acute asthma. However, the reduction in the enzymatic affinity constant and its smaller change following in vitro stimulation suggest that alterations in the affinity of acetyltransferase for acetylcoenzyme A (CoA) and in the regulation of enzyme activity may be occurring during acute asthma.

Topics: Acetyltransferases; Acute Disease; Adult; Aged; Asthma; Calcimycin; Female; Humans; In Vitro Techniques; Ionophores; Male; Middle Aged; Neutrophils; Up-Regulation

The effects of the macrolide antimicrobial agents azithromycin, clarithromycin, erythromycin and roxithromycin on the prooxidative activity of stimulated human neutrophils have been investigated in vitro. Superoxide generation by activated neutrophils was measured by lucigenin-enhanced chemiluminescence. At the concentrations used (2.5-80 micrograms/ml) none of the test agents was cytotoxic, nor did they possess superoxide-scavenging properties. Treatment of neutrophils with all 4 macrolides was accompanied by dose-related inhibition of superoxide production by cells activated with FMLP or the calcium ionophore (A23187), while the responses activated by phorbol myristate acetate (PMA) or opsonized zymosan were minimally affected. The anti-oxidative interactions of roxithromycin with FMLP-activated neutrophils were neutralized by pretreatment of the cells with low, non-cytotoxic concentrations (0.5 microgram/ml) of the prooxidative, proinflammatory bioactive phospholipids, lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and lyso-PAF (LPAF). Using an assay of membrane-stabilizing activity, the macrolides antagonized the membrane-disruptive effects of LPC, PAF and LPAF, without affecting enzymes involved in their synthesis. These membrane-stabilizing interactions of macrolides with neutrophils may counteract the proinflammatory, prooxidative activity of several bioactive lipids which have been implicated in the pathogenesis of bronchial asthma.

Topics: Adult; Anti-Asthmatic Agents; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Asthma; Azithromycin; Calcimycin; Cell Membrane; Clarithromycin; Erythromycin; Humans; Ionophores; Luminescent Measurements; Lysophosphatidylcholines; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Respiratory Burst; Roxithromycin; Superoxides; Tetradecanoylphorbol Acetate; Zymosan

Eosinophils generate and release leukotrienes C4 and B4 (LTC4, LTB4) and platelet activating factor (PAF), all of which have the capacity to cause inflammation and tissue injury in the airways. This study has examined the effects of a new 5-lipoxygenase inhibitor, 6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride (CAS 120164-49-0, E6080) on the release of LTC4, LTB4 and PAF by human eosinophils, Eosinophils stimulated by 1 mumol/l calcium ionophore A23187 for 15 min released 37.5 +/- 2.2 ng, 2.3 +/- 0.3 ng and 4.0 +/- 0.3 pmol per 10(6) cells of immunoreactive LTC4, LTB4 and PAF, respectively (mean +/- SEM, n = 4). LTC4 and LTB4 releases were inhibited dose-dependently by the addition of E6080 to the cell suspension. The IC50 values were 0.26 mumol/l for LTC4 and 0.23 mumol/l for LTB4. PAF release was not inhibited. These results suggest that E6080 is a potent inhibitor of LTC4 and LTB4 release from eosinophils and may provide a protective effect against bronchoconstriction during late-phase asthmatic responses.

Topics: Asthma; Calcimycin; Dose-Response Relationship, Drug; Eosinophils; Humans; In Vitro Techniques; Ionophores; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Platelet Activating Factor; Thiazoles

Adenylyl cyclase is a transmembrane signaling system involved in the inhibition of cellular responses. Recently, we showed that the activity of adenylyl cyclase may be potentiated by stimuli that induce an increase of cellular responses but that do not activate adenylyl cyclase. This is probably an important physiologic feedback mechanism that prevents cells from becoming "overstimulated.". Because increased cellular activities are frequently observed in persons with asthma, we hypothesized that a defect in potentiation of adenylyl cyclase might be involved.. Potentiation of isoprenaline-induced adenosine cyclic monophosphate (cAMP) production with the mitogen phytohemagglutin (PHA; 45 micrograms/ml) or the calcium ionophore A23187 (1 mumol/L) was studied in peripheral blood mononuclear cells taken from patients with asthma (n = 8) and healthy control subjects (n = 11).. Isoprenaline-induced cAMP production was potentiated significantly in the healthy control subjects (PHA, 110% +/- 15%; A23187, 92% +/- 25%). In contrast, potentiation was not seen with PHA or A23187 in the total group of patients with asthma. However, some patients showed weak potentiation, whereas in others PHA decreased isoprenaline-induced cAMP production. Moreover, the effect of PHA on isoprenaline-induced cAMP production correlated significantly with the degree of bronchial hyperreactivity in patients with asthma (r = 0.96; p = 0.0001).. The observed defect in signal transduction could play an important part in bronchial hyperresponsiveness.

Topics: Adenylyl Cyclases; Adult; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchodilator Agents; Calcimycin; Cyclic AMP; Drug Synergism; Female; Humans; Ionophores; Isoproterenol; Leukocytes, Mononuclear; Male; Methacholine Chloride; Phytohemagglutinins; Signal Transduction

To determine the role of neutrophil elastase in asthmatic responses, we studied the effect of ONO-5046, a specific neutrophil elastase inhibitor, on antigen-induced asthmatic responses in allergic sheep. Pulmonary resistance (RL) was measured for 8 h after antigen challenge. Measurements of airway responsiveness to methacholine and bronchoalveolar lavage fluid (BALF) were obtained 8 h after challenge. Antigen challenge caused early and late increases in RL, airway hyperresponsiveness (AHR), and recruitment of neutrophils and eosinophils along with increases in TXB2 and LTB4 in BALF. ONO-5046 treatment significantly reduced both early and late bronchoconstriction, neutrophil recruitment, increases in LTB4 in BALF, and AHR. ONO-5046 post-treatment significantly reduced the increase in RL 8 h after antigen challenge. Another neutrophil elastase inhibitor, FR 134043, significantly reduced both early and late bronchoconstriction. ONO-5046 had little effect on calcium ionophore-induced LTB4 release from isolated neutrophils and whole blood obtained from drug-treated sheep. These findings suggest that neutrophil elastase is involved in antigen-induced bronchoconstriction and AHR mediated by neutrophil accumulation and 5-lipoxygenase products in allergic sheep.

Topics: Airway Resistance; Animals; Antigens; Asthma; Benzoquinones; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Calcimycin; Glycine; Heterocyclic Compounds; Leukocyte Elastase; Leukocytes; Leukotriene B4; Masoprocol; Methacholine Chloride; Pancreatic Elastase; Polycyclic Compounds; Sheep; Sulfonamides; Thromboxane B2

Fibronectin, an extracellular matrix component, is a ligand for very late activation antigen (VLA)-4, which is one of the beta 1-integrin family of molecules expressed by eosinophils. This study examined the effect of adherence to fibronectin on radical oxygen products from eosinophils. Adhesion of eosinophils to fibronectin resulted in enhancement of eosinophil production of radical oxygen species, as determined by luminol-dependent chemiluminescence of eosinophils stimulated with calcium ionophore. It was concluded that eosinophil adhesion to extracellular matrix via adhesion molecules may be important in the pathogenesis of allergic inflammation through eosinophil activation.

Topics: Asthma; Calcimycin; Cell Adhesion; Eosinophils; Fibronectins; Humans; In Vitro Techniques; Ionophores; Reactive Oxygen Species

Exposure to ozone has been reported to cause increased immediate bronchial reactivity to inhaled allergen in asthmatics. The purpose of these studies was to determine whether ozone induces either spontaneous physiological degranulation or enhanced immunoglobulin E (IgE)-mediated degranulation of mast cells, thus accounting for the in vivo effects noted in asthmatics. A rat mast cell line (RBL-2H3) was exposed to different levels of ozone (0.1, 0.3, 0.5, and 1.0 ppm), covered by different amounts of buffer, and both cytotoxic and nontoxic exposure conditions were determined. In addition to cytotoxicity, spontaneous release of granule products and prostaglandin D2 (PGD2) associated with ozone exposure were assessed. RBL-2H3 cells were also exposed to ozone under noncytotoxic conditions followed by stimulation with alpha-IgE to cross-link membrane-bound IgE and A23187 so that the effect of ozone on stimulated degranulation could be examined. Only exposure conditions associated with cytotoxicity were associated with spontaneous release of mast cell serotonin, indicating no physiologic degranulation due to ozone exposure. Data presented herein also demonstrate that ozone substantially inhibited both IgE- and A23187-induced degranulation. Neither catalase nor superoxide dismutase protected cells from the inhibitory effect of ozone, indicating that ozone does not act through generation of H2O2 or superoxide. Additionally, ozone caused a modest increase in spontaneous PGD2 generation only under cytotoxic conditions. Thus ozone appears to inhibit mast cell degranulation after IgE- or A23187-mediated stimulation and causes direct release of mast cell granule products and PGD2 only under conditions associated with membrane cytotoxicity.

Topics: Animals; Asthma; Calcimycin; Catalase; Cell Line; Cell Survival; Cytoplasmic Granules; Dose-Response Relationship, Drug; Humans; Immunoglobulin E; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Ozone; Prostaglandin D2; Rats; Superoxide Dismutase; Tumor Cells, Cultured

Toluene diisocyanate (TDI) is a volatile, highly reactive chemical widely used as a polymerizing agent in the production of polyurethane foams, lacquers, adhesives, and other items. Repeated airway exposures in the workplace to TDI may cause a concentration-dependent risk of developing chronic airway disorders. Different pathomechanisms are involved. IgE-mediated sensitization and irritative effects were clearly demonstrated in exposed subjects as well as in animals. In this study we examined the cellular and mediator composition in bronchoalveolar lavage fluid (BALF) of guinea pigs (eight in each group) exposed to TDI (10, 20, or 30 ppb) on 5 consecutive days for 2 hours each. Increased numbers of eosinophils and significantly elevated levels of LTB4 and LTC4/LTD4/LTE4 were obtained in BALF of all exposed animals when compared to nonexposed control animals. PGD2 and TXB2 remained unaltered in BALF. Stimulation of BALF cells of exposed and control animals with Ca-ionophore A23187 and arachidonic acid induced an increased generation of LTB4. Furthermore, BALF cells of the exposed animal groups generated immunoreactive LTC4/LTD4/LTE4, whereas controls did not show peptido-leukotriene formation in the presence and absence of stimuli. Our data clearly demonstrate an influx of eosinophils into the airways associated with mediator release and higher cellular responsiveness after TDI exposure.

Topics: Animals; Arachidonic Acid; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Calcimycin; Dose-Response Relationship, Drug; Eosinophils; Female; Guinea Pigs; Inflammation Mediators; Leukocyte Count; Leukotrienes; Prostaglandin D2; Thromboxane B2; Toluene 2,4-Diisocyanate

5-Lipoxygenase (5-LO) activation of human blood polymorphonuclear cells (PMN) from healthy subjects (HS) and from asthmatic patients (AP) was investigated comparing their respective capacities to produce lipoxins, 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and leukotrienes, under in vitro stimulation by ionophore A23187. PMN from AP were able to generate higher leukotriene levels from endogenous sources than PMN from HS. Moreover they produced 5,15-diHETE (from 50 to 280ng/10(7) cells) and lipoxins (from 1 to 30ng/10(7) cells), in a linear manner, whereas in the same experimental conditions no detectable amounts of these compounds appeared in PMN from HS. The enhanced 5-LO activation of blood PMN may reflect transcellular signalisation priming indicating that lipoxins and 5,15-diHETE could be much more specific inflammatory state biomarkers than leukotriene B4.

Topics: Arachidonic Acid; Asthma; Biomarkers; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neutrophils; Reference Values

Leukotrienes are inflammatory mediators implicated in the pathogenesis of asthma. The capacity of inflammatory cells within the airways to generate leukotrienes may be altered in asthma. This hypothesis was tested using bronchoalveolar lavage (BAL) to sample cells within the airways from atopic asthmatic and normal subjects, and by measuring their capacity to generate leukotriene B4 (LTB4) and leukotriene C4 (LTC4) in response to A23187, a potent stimulus of leukotriene generation.. Bronchoalveolar lavage was performed in 12 mild asymptomatic atopic asthmatic patients and 12 normal subjects. Mixed BAL cell aliquots (approximately 80% alveolar macrophages) were incubated with 0-20 microM A23187 for 10 minutes and with 4 microM A23187 for 0-30 minutes, and leukotrienes were measured by radioimmunoassay and high performance liquid chromatography.. Mixed BAL cells from asthmatic subjects generated less LTB4 than cells from normal subjects in dose response and time course experiments (area under the curve 81.5 (0.0-228.5) ng.min.10(-6) cells in asthmatic subjects and 197.9 (13.9-935.6) ng.min.10(-6) cells in normal subjects. There were no differences in LTC4 generation between BAL cells from asthmatic and normal subjects.. Generation of LTB4 by BAL cells from atopic asthmatic subjects in response to A23187 was reduced. As the alveolar macrophage is the major source of LTB4 in BAL cells, these results probably reflect reduced generation of LTB4 by alveolar macrophages from asthmatic patients. This may be a consequence of monocyte migration into the lung, or altered alveolar macrophage function in asthma, or both.

Topics: Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Leukotriene B4; Leukotriene C4; Macrophages, Alveolar; Male; Radioimmunoassay

Enhanced activities of peripheral blood cells are a common characteristic of patients with asthma.. Here we tested whether this could be due to a dysfunction in one or more signal transduction systems.. The production of 1,2-diacylglycerol (1,2-DAG) and arachidonic acid was compared in mononuclear blood cells from patients with asthma (n = 10) and healthy controls (n = 12).. Using three different stimuli (concanavalin A, aluminium fluoride or the calcium ionophore A23187) no difference in the production of both 1,2-DAG and arachidonic acid could be found between patients and controls before allergen challenge. Concanavalin A-induced 1,2-DAG production could be inhibited completely in the presence of isoprenaline; concanavalin A-induced arachidonic acid production, partially. The inhibitory effect of adenylate cyclase activation on the production of 1,2-DAG and arachidonic acid was identical in patients and controls. Following allergen challenge, there was a tendency to an increased production of 1,2-DAG and arachidonic acid in controls, whereas in patients there was a tendency to a decreased production.. Enhanced cellular activities found in patients with asthma are not caused by an intrinsic dysfunction in production of 1,2-DAG and arachidonic acid.

Topics: Adult; Allergens; Animals; Arachidonic Acid; Asthma; Calcimycin; Concanavalin A; Diglycerides; Drug Interactions; Dust; Female; Fluorides; Humans; Isoproterenol; Leukocytes, Mononuclear; Male; Mites

The effects of three histamine H1 receptor antagonists, epinastine (CAS 80012-43-7, WAL-801 CL), ketotifen (CAS 34580-13-7) and oxatomide (CAS 60607-34-3), on mediator release have been studied in human peripheral leukocytes. When leukocytes from asthmatic patients sensitive to mite were stimulated with the allergen, epinastine inhibited histamine release with a concentration required for 50% inhibition (IC50) of 3 x 10(-5) mol/l and leukotriene C4 generation. On the other hand, ketotifen or oxatomide showed little inhibiting effect on histamine release elicited with the allergen. When the cells were stimulated with calcium ionophore A23187, epinastine failed to inhibit histamine release and leukotriene C4 generation. Oxatomide caused a concentration related inhibition of calcium ionophore-induced histamine release with the IC50 value of 5 x 10(-5) mol/l. Ketotifen or oxatomide also showed an inhibition of leukotriene C4 generation induced by calcium ionophore in a dose-dependent manner and the IC50 value was 6 x 10(-6) mol/l for oxatomide and 8 x 10(-5) mol/l for ketotifen, suggesting that oxatomide is a more potent inhibitor of leukotriene C4 generation than ketotifen. These results indicate that epinastine inhibits IgE-mediated histamine release and LTC4 generation, and oxatomide has a capacity to inhibit calcium ionophore-induced mediator release from human leukocytes. Additionally, when platelet activating factor was quantitated by radioimmunoassay in the supernatant and the cell pellet after ionophore stimulation, epinastine inhibited the formation and the secretion in a dose-dependent manner.

Topics: Asthma; Calcimycin; Dibenzazepines; Histamine H1 Antagonists; Humans; Hypersensitivity; Imidazoles; Immunoglobulin E; In Vitro Techniques; Ketotifen; Leukocytes; Leukotriene C4; Neutrophils; Piperazines; Platelet Activating Factor

In asthma, activation and recruitment of eosinophils to the bronchial mucosa amplifies many cellular functions. The blood eosinophil count and the number of hypodense eosinophils increase with asthma severity. Eosinophils produce numerous proinflammatory mediators in response to a variety of agonists, notably the peptido-leukotriene (LT) C4, a potent bronchoconstrictor. In this study, we have evaluated blood eosinophil LTC4 release and its modulation by cytokines in normal individuals and in subjects with asthma of various severities: mild (beta 2-agonist on demand); moderate (inhaled steroids on a regular basis); and severe (inhaled and oral steroids on a regular basis). Eosinophils were isolated using a modified Percoll gradient technique, which recovers both hypodense and normodense eosinophils in a global cell population. Eosinophils released detectable amounts of LTC4 only in the presence of the stimulus (calcium ionophore A23187, 2 microM). The ionophore-induced LTC4 release was greater in moderate asthmatics (mean +/- SEM 5.7 +/- 1.3 pg x 10(3)/250,000 eosinophils) than in normal individuals (1.6 +/- 0.4 pg x 10(3)/250,000 eosinophils), mild asthmatics (1.8 +/- 0.3 pg x 10(3)/250,000 eosinophils) and severe asthmatics (2.0 +/- 0.3 pg x 10(3)/250,000 eosinophils). Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) amplified the ionophore-induced LTC4 release in the four groups from 1.9 to 2.6 and 1.9 to 2.8 fold, respectively. Interleukin-3 (IL-3) did not increase LTC4 production except by the eosinophils of the severe asthmatics whose ionophore-induced LTC4 production was enhanced by 1.9 fold. These data demonstrate that the asthmatic bronchial inflammatory process may modify blood eosinophil LTC4 release and its modulation by cytokines according to asthma severity and treatment.

Topics: Adult; Analysis of Variance; Asthma; Calcimycin; Cells, Cultured; Cytokines; Eosinophils; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation Mediators; Interleukin-3; Interleukin-5; Ionophores; Leukotriene C4; Male; Middle Aged; Severity of Illness Index

The ex vivo release of leukotriene B4 (LTB4) and leukotriene C4 (LTC4) from leukocytes was evaluated after stimulation with both Ca-ionophore (Ca-I) and opsonized zymosan (OZ) in children with atopic asthma. Twenty-seven patients with asthma of varying severity were evaluated and divided into three groups: 1) moderate to severe asthma using inhaled steroids and symptom-free for the last 3 weeks (n = 8), 2) mild asthma with sporadic symptoms, only using inhaled beta 2-agonists < 3 times/week (n = 8), and 3) acute asthmatic attacks admitted to hospital (n = 11). A group of children without atopic disease or any other known disease served as controls (n = 15). Total serum IgE levels were significantly increased in the children with asthma compared with the control group. LTC4 production was only significantly increased in the group of children with moderate to severe asthma after stimulation with Ca-I, when compared with controls. In the same group, a trend towards increased LTC4 production after stimulation with OZ was found. LTB4 was not significantly increased in any patient group compared with the control group. A significant correlation between LTC4 production after stimulation with Ca-I, but not OZ, and the relative blood eosinophil count was found in all subjects. LTC4 generation per eosinophilic cell after stimulation with Ca-I or OZ was not statistically different in any patient group compared with the controls. We conclude that the increased leukotriene (LT) levels found after the stimulation of peripheral white blood cells sampled from atopic children with asthma are mainly the result of increased numbers of LT-producing cells, rather than due to increased releasability from these cells.

Topics: Adolescent; Asthma; Calcimycin; Child; Child, Preschool; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Leukocytes; Leukotriene B4; Leukotriene C4; Opsonin Proteins; Zymosan

The effect of a new anti-asthmatic drug, TYB-2285 (3,5-bis(acetoxyacetylamino)-4-chlorobenzonitrile), was investigated in ovalbumin-sensitized guinea-pigs. When guinea-pigs were pretreated with TYB-2285 (300 mg kg-1, p.o., single dose or consecutively for 7 days), the immediate asthmatic response was inhibited as demonstrated by diminished cyanosis, but not the bronchoconstriction. TYB-2285, given singly or consecutively, inhibited the appearance of late asthmatic response and the infiltration of inflammatory cells, such as eosinophils, into the airway. Additionally, airway hyper-responsiveness was also reversed by the single administration of TYB-2285. Luminol-dependent chemiluminescence of airway-infiltrated cells stimulated with A23187 was inhibited by TYB-2285 in a dose-dependent manner. The present study suggests that TYB-2285 inhibits late asthmatic response and airway hyperresponsiveness by inhibiting the accumulation of eosinophils and other inflammatory cells into the airway, and also by inhibiting the production of oxygen radicals from airway-infiltrated cells.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Dose-Response Relationship, Drug; Guinea Pigs; Histamine; Luminescent Measurements; Luminol; Male; Nitriles; Ovalbumin; Respiratory Function Tests; Respiratory Hypersensitivity

Second generation antihistamines have been reported to have anti-inflammatory properties in addition to their potency as H1 antagonists. In this in-vitro study, we evaluated the effect of terfenadine on platelet activating factor-(PAF)-induced or N-formyl-methionyl-leucyl-phenylamine-(FMLP)-induced human eosinophil and neutrophil chemotactic responses; and on superoxide anion generation from human eosinophils and neutrophils activated by either PAF, calcium ionophore (A23187) or phorbol myristate acetate. Since eosinophil degranulation is also associated with tissue inflammation, we further examined the effect of terfenadine on the PAF-induced release of eosinophil cationic protein. The peak concentration of terfenadine-related materials in serum of adult individuals after 60 mg of oral administration has been reported to be 351 +/- 0.4 ng/mL. We therefore used 100 to 1000 ng/mL concentrations of terfenadine. Purified normodense-eosinophils and neutrophils were obtained by discontinuous gradient from allergic subjects. We observed that terfenadine had greater inhibitory effects on eosinophils than neutrophils in both chemotactic response and superoxide generation. Terfenadine, at concentrations of 500 and 1000 ng/mL, significantly inhibited PAF-induced and FMLP-induced eosinophil chemotaxis, whereas 1000 ng/mL of terfenadine was necessary to suppress neutrophil chemotaxis. Terfenadine, at concentrations achievable at standard dosing regimens, has anti-inflammatory properties in vitro.

Topics: Adult; Asthma; Calcimycin; Cell Degranulation; Cell Separation; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Eosinophils; Female; Humans; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Rhinitis; Superoxides; Terfenadine

To determine whether pemirolast, a new antiallergic drug, inhibits the activation of eosinophils, we investigated the effect of pemirolast on the release of leukotriene C4 (LTC4) and eosinophil cationic protein (ECP) from human eosinophils. Calcium ionophore A23187 caused both LTC4 and ECP release from human eosinophils, whereas PAF and FMLP induced only ECP release from the eosinophils. Pemirolast (10(-6) to 10(-3) M) inhibited A23187-induced LTC4 release from the eosinophils in a dose-dependent fashion with 77% inhibition at 10(-3) M. Pemirolast (10(-5) to 10(-3) M) inhibited A23187-induced ECP release from the eosinophils in a dose-dependent fashion with 42% inhibition at 10(-3) M. Pemirolast (10(-4) and 10(-3) M) also inhibited PAF-induced and FMLP-induced ECP release from the eosinophils. We conclude that pemirolast prevents the activation of human eosinophils to inhibit LTC4 and ECP release. These results suggest that pemirolast might be useful in controlling allergic diseases by inhibiting eosinophil activation.

Topics: Asthma; Blood Proteins; Calcimycin; Eosinophil Granule Proteins; Eosinophils; Humans; In Vitro Techniques; Leukotriene C4; N-Formylmethionine Leucyl-Phenylalanine; Platelet Activating Factor; Pyridines; Pyrimidinones; Ribonucleases; Secretory Rate

A simple method for purification of basophils from a relatively small volume of blood has been developed, which enables us to collect basophils with a purity of over 80%. Basophils were partially purified from 10-20 ml of citrated whole blood using the discontinuous Percoll density gradient centrifugation technique and then contaminant cells were removed using monoclonal antibodies against CD2, CD19, CD14 and CD16. At the end of the procedure, basophils were > 80% pure with lymphocytes accounting for most of the contaminating cells. When stimulated with anti-IgE or fMLP, histamine release from purified basophils was similar to that from mixed leukocytes. When highly purified basophils were challenged with calcium ionophore A23187, generation of leukotriene C4 (LTC4) was not significantly different between asthmatic patients and normal subjects (45.6 +/- 22.6 vs 52.7 +/- 25.6 ng/10(6) cells). Basophils were capable of generating LTC4 in approximately the same quantities as eosinophils (46.5 +/- 11.7 ng/10(6) cells, n = 3). Furthermore, it has been shown that incubation of basophils and eosinophils with calcium ionophore generates only small quantities of thromboxane B2 (TXB2).

Topics: Antibodies, Anti-Idiotypic; Asthma; Basophils; Blood Cells; Calcimycin; Cell Separation; Centrifugation, Density Gradient; Histamine Release; Humans; Leukotriene C4; N-Formylmethionine Leucyl-Phenylalanine; Thromboxane B2

Bronchial epithelial cells may be involved in the pathogenesis of asthma releasing several inflammatory mediators such as interleukins and lipoxygenase products. In this study we evaluated the spontaneous and A23128-induced release of 15-HETE, PGE2 and fibronectin as well as the spontaneous expression of HLA-DR and ICAM-1 molecules by bronchial epithelial cells obtained by bronchial brushing from 35 asthmatic and 27 normal subjects. The release of fibronectin and 15-HETE was studied using the EIA and RIA techniques. The expression of HLA-DR and ICAM-1 molecules was studied using the APAAP and the immunofluorescence methods. Bronchial epithelial cells from asthmatics released higher amounts of 15-HETE and fibronectin both spontaneously (p < 0.002, p < 0.05, respectively) or after stimulation with calcium ionophore compared with normals. On the other hand, PGE2 release was significantly higher only after stimulation with calcium ionophore (p < 0.002). The spontaneous expression of HLA-DR and ICAM-1 (p < 0.001) was significantly higher on epithelial cells from asthmatics than in normal subjects. Finally, the severity of asthma significantly correlated with the release of 15-HETE (p < 0.02) and the expression of HLA-DR and ICAM-1 respectively (p < 0.001 and p < 0.002, respectively). This study indicates that bronchial epithelial cells are in an activated state in asthma and that the degree of activation is correlated to the severity of the disease.

Topics: Adult; Alkaline Phosphatase; Asthma; Bronchi; Calcimycin; Cell Adhesion Molecules; Dinoprostone; Epithelium; Fibronectins; Fluorescent Antibody Technique; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; Intercellular Adhesion Molecule-1

The density distribution pattern of eosinophils over discontinuous isotonic Percoll gradients from the blood of normal, asymptomatic allergic and non-allergic asthmatic individuals was investigated. There was a completely identical distribution pattern between the investigated groups. Analysis of the expression of surface markers for complement receptors CR1 and CR3 and immunoglobulin G receptor on eosinophils derived from the density bands 1.080, 1.085 and 1.090 g/ml supported this finding since they did not reveal differences in expression between the bands within one group but also not between the three groups. Eosinophils of the various density bands were further purified and stimulated in vitro to produce leukotriene C4 (LTC4) by the calcium ionophore A23187 or serum treated zymosan. Equal amounts of LTC4 were synthesized by the eosinophils of the various density bands within one group. However, it appeared that the eosinophils of all density bands of allergic and non-allergic asthmatics synthesized significantly more LTC4 than the eosinophils from normal individuals (five- to tenfold). Probably this indicates in vivo priming of the eosinophils in asthmatic individuals which is not reflected by a change in density. Control experiments, dealing with possible artifacts due to the isolation procedure or the patient selection, to find differences in distribution patterns over discontinuous Percoll density gradients of the eosinophils of asthmatic compared to normal individuals failed to show such a difference. Therefore, the density distribution pattern of eosinophils over these gradients does not reflect cell activation, whereas LTC4 formation clearly does. This could mean that LTC4 formation is a more sensitive parameter for cell activation than density distribution or cell surface marker expression.

Topics: Adolescent; Adult; Artifacts; Asthma; Calcimycin; Eosinophils; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukocyte Count; Male; Middle Aged; Receptors, Complement; Receptors, IgG; Recombinant Proteins; SRS-A

The effect of ibudilast on anaphylactic bronchoconstriction was studied in guinea pigs sensitized actively with ovalbumin (OA). Animals were treated with indomethacin, tripelennamine and propranolol prior to the antigen challenge. Anaphylactic bronchoconstriction was prevented by ibudilast (1-4 mg/kg i.v. and 5-20 mg/kg p.o.) dose-dependently. FPL55712 and phenidone were also effective. Even when administered at the maximum development of bronchoconstriction, ibudilast (0.5 and 2 mg/kg i.v.) and FPL 55712 caused significant reduction of the increased airway tone, while phenidone did not. Ibudilast (1-4 mg/kg i.v.) and FPL55712 inhibited leukotriene D4-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol. Ibudilast (1.6 and 4 mg/kg i.v.) inhibited platelet-activating-factor (PAF)-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol, however, FPL 55712 inhibited PAF-induced airway responses only at a high dose such as 10 mg/kg i.v. Ibudilast (4 mg/kg i.v.) did not inhibit acetylcholine-induced airway response. Ibudilast showed inhibition of the release of slow-reacting substance of anaphylaxis (SRS-A) from guinea pig chopped lung sensitized with OA, which was significantly diminished by indomethacin. The drug little affected the activity of phospholipase A2 and 5-lipoxygenase in guinea pig polymorphonuclear leukocytes. These results indicate that ibudilast inhibits anaphylactic bronchoconstriction which is considered to be largely mediated by endogenously released SRS-A. The inhibitory effect of ibudilast on anaphylactic bronchoconstriction in the presence of indomethacin is considered to be exerted through its antagonism to SRS-A.

Topics: Acetylcholine; Anaphylaxis; Animals; Arachidonate 5-Lipoxygenase; Asthma; Bronchoconstriction; Bronchodilator Agents; Calcimycin; Chromones; Guinea Pigs; Male; Neutrophils; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Pyrazoles; Pyridines; SRS-A

To explore the possible role of platelet-activating factor (PAF) in the pathogenesis of bronchial asthma, circulating PAF and in vitro production of PAF were studied.. Radioimmunoassay kits were used in 15 children with acute asthmatic attacks, in 25 newly diagnosed asthmatic children, in 25 good and 18 poor responders to immunotherapy, and in 18 healthy controls.. The results demonstrated the following: (1) PAF was present in the blood of healthy controls. (2) New patients had much higher circulating PAF than did healthy controls (p < 0.005), and the circulating PAF decreased after immunotherapy in good (p < 0.005) but not in poor responders. (3) The circulating PAF increased up to 20 times that of healthy controls during acute asthmatic attacks. (4) The spontaneous and allergen-stimulated secretion of PAF were markedly increased in new patients and decreased to normal after successful immunotherapy (p < 0.005). (5) No increased spontaneous and allergen-stimulated production of PAF was found during acute attacks, but granulocytes from those patients still produced the greatest amount of PAF when stimulated with calcium ionophore A23187. (6) Although a major portion of allergen-induced PAF was secreted, less than 10% of ionophore-induced PAF was secreted.. The findings that the circulating PAF increased markedly during acute asthmatic attacks and the enhanced in vivo and in vitro productions of PAF decreased to normal after successful immunotherapy strongly suggest that PAF may be involved in the pathogenesis of bronchial asthma.

Topics: Acute Disease; Animals; Asthma; Calcimycin; Child; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Granulocytes; Humans; Immunotherapy; Mites; Osmolar Concentration; Platelet Activating Factor; Reference Values

Airways epithelial cells may be involved in the pathogenesis of asthma, but their role remains to be determined. Epithelial cells can release large amounts of 15-hydroxy-eicosatetranoic acid (15-HETE) and smaller amounts of prostaglandin E2 (PGE2) as well as fibronectin, a mediator involved in epithelial repair after injury. Epithelial cells obtained after bronchial brushing of 16 asthmatic (age 38 +/- 5 yr) and 11 normal subjects (age 36 +/- 5 yr) were studied. The percentage of epithelial cells was assessed by immunocytochemistry using an anti-cytokeratin antibody. The viability of the cells was assessed by trypan blue exclusion. The release of 15-HETE PGE2 and fibronectin was studied in resting cells and after A23187 calcium ionophore stimulation. Epithelial cells always comprised more than 86% of cells recovered, and the viability of epithelial cells was significantly (p < 0.001, Mann-Whitney U test) greater in normal subjects (54 +/- 5%) compared with asthmatic subjects (13 +/- 1%). The release of 15-HETE and fibronectin by resting epithelial cells was significantly greater in asthmatics (p < 0.05, Mann-Whitney U test) than in normal subjects. A23187 significantly (p < 0.05, Wilcoxon W test) increased the release of 15-HETE and fibronectin. There was no significant difference in the release of PGE2 by resting cells from either asthmatics or normal subjects, but challenge with A23187 induced a significant (p < 0.03, Wilcoxon W test) increase in PGE2 from cells of asthmatics but not from cells of normal subjects. This study shows that epithelial cells are activated and less viable in asthma and suggests a role for these cells in asthma.

Topics: Adult; Albuterol; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Bronchoscopy; Calcimycin; Cell Survival; Dinoprostone; Epithelial Cells; Epithelium; Fiber Optic Technology; Fibronectins; Humans; Hydroxyeicosatetraenoic Acids

There is increasing evidence for the role of basophils in the pathogenesis of bronchial asthma. To examine the presence of basophils in the airways of patients with fatal asthma by immunohistochemistry, we stained lung tissues from four post-mortem cases who had died from severe asthmatic attacks and four controls with a monoclonal antibody raised against tryptase (AA-1) and anti-IgE. Mast cells and basophils were identified in the bronchioles as AA-1- and anti-IgE-positive cells, and anti-IgE-positive cells, respectively. Airway mast cells were found beneath the basement membrane, near blood vessels in the submucosa, and adjacent to the submucosal glands, and scattered throughout the muscle bundles. There was a significant increase of mast cells in the asthma group compared with the control group (203.5+/-84.6/mm2, mean+/-s.d. vs 37.7+/-8.7/mm2, P<0.05, n=4). In contrast, basophils were observed in the airway lumen, in the bronchial epithelium and in the submucosa. The number of basophils in the bronchioles was 81.8+/-55.5/mm2 (n = 4); however, basophils were not found at all in the airways of the control group. Although eosinophils, B lymphocytes and macrophages bear low affinity IgE receptors and could react with anti-IgE, the location of these cells in the close sections did not correspond closely with basophils. The presence of basophils in lung tissues obtained from fatal asthma patients supports the view that basophils play a role in the pathogenesis of bronchial asthma.

Topics: Adult; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Asthma; Basement Membrane; Basophils; Biomarkers; Bronchi; Calcimycin; Calcium Signaling; Cells, Cultured; Child; Chymases; Coloring Agents; Female; Humans; Immunoenzyme Techniques; Ionophores; Lung; Male; Mast Cells; Middle Aged; Mucous Membrane; Receptors, IgE; Serine Endopeptidases; Tolonium Chloride; Tryptases

Among the cells which participate in amplification of the local inflammatory reaction in asthma, neutrophils (PMN) are pro-inflammatory cells that can generate inflammatory mediators and arachidonic acid derivatives in particular. In asthmatic patients (AP) with attacks, the capacity of blood PMN to produce 5-lipoxygenase metabolites was investigated and compared to the response in healthy subjects (HS). PMN from 6 AP and from 6 HS were stimulated by calcium ionophore A23187 and arachidonate 5-lipoxygenase metabolites were analyzed by reverse-phase HPLC. LTB4, 6-trans LTB4, omega OH-LTB4 and 5-HETE were identified. In AP, total LTB4 synthesis was enhanced as compared to synthesis with PMN in HS. But the total 5-HETE synthesis by PMN from AP was decreased. Thus, the inflammatory potential of PMN from AP was enhanced in comparison to HS. The anti-inflammatory effect of nedocromil sodium (NS) was studied in the 5-lipoxygenase metabolism of arachidonic acid. NS (10(-4) mol/l) inhibited total LTB4 synthesized by PMN in AP but not in HS. We conclude that NS affects leukotriene synthesis only in cells with enhanced inflammatory potential.

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Male; Middle Aged; Nedocromil; Neutrophils; Quinolones

Aspirin and nonsteroidal antiinflammatory drugs induce bronchospastic reactions in patients with aspirin-sensitive respiratory disease. Although the mechanism of this reaction is unknown, all drugs that induce the respiratory reaction also inhibit the cyclooxygenase enzyme. The ensuing changes in arachidonate metabolism are presumed to play a role in the pathogenesis of the reaction. We measured generation of leukotrienes and thromboxane by calcium ionophore stimulated blood monocytes. Before aspirin challenge, monocytes released significantly more thromboxane B2 in patients with aspirin sensitivity than in patients without aspirin sensitivity or in healthy control subjects (p < 0.02). During aspirin-induced bronchospasm, release of leukotriene B4 increased significantly (45.5%, p = 0.018), whereas release of thromboxane B2 decreased (-46.9%, p = 0.028). Two hours after ingestion of 60 mg aspirin, normal monocyte release of thromboxane B2 did not drop, whereas leukotriene B4 release increased. Monocytes formed only minimal amounts of leukotriene C4. We conclude that the profile of released eicosanoids from aspirin-sensitive monocytes is distinct from non-aspirin-sensitive subjects, and that these differences could contribute to the development of bronchospasm after aspirin ingestion.

Topics: Administration, Oral; Adult; Arachidonic Acid; Aspirin; Asthma; Bronchial Spasm; Calcimycin; Eicosanoids; Female; Humans; Leukotriene B4; Male; Middle Aged; Monocytes; Thromboxane B2

Leukotrienes are thought to be involved in allergen-induced airway responses. To test this hypothesis we used a newly described 5-lipoxygenase inhibitor, zileuton, and examined its effect on antigen-induced early and late bronchial responses, airway inflammation and airway hyperresponsiveness in allergic sheep. Early and late responses were determined by measuring specific lung resistance (SRL) before and serially for 8 h after antigen challenge. Airway inflammation was assessed by bronchoalveolar lavage performed before, 8 h after and 24 h after antigen challenge. Airway responsiveness was measured before and 24 h after challenge by determining the dose of inhaled carbachol that caused a 400% increase in SRL (PD400%). The sheep (n = 8) were challenged with Ascaris suum antigen once after vehicle treatment (methylcellulose) and once after treatment with zileuton (10 mg/kg in methylcellulose, p.o.) given 2 h before antigen challenge. Trials were separated by at least 21 days. Zileuton had no effect on the early bronchoconstrictor response to antigen but the drug inhibited the late bronchial response by 55% (P less than 0.05). Unlike the control trial, there was no significant increase in bronchoalveolar lavage eosinophils at 8 h post challenge in the zileuton-treated sheep. Furthermore, zileuton treatment blocked (P less than 0.05) the airway hyperresponsiveness seen 24 h after challenge. Ex vivo formation of leukotriene B4 was inhibited over several hours after a single oral dose of zileuton, indicating that the compound was acting as a 5-lipoxygenase inhibitor in vivo. These results suggest that 5-lipoxygenase metabolites contribute to allergen-induced late responses, airway inflammation and airway hyperresponsiveness in this animal model of asthma.

Topics: Administration, Oral; Animals; Antigens; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Calcimycin; Dose-Response Relationship, Drug; Hydroxyurea; Leukotriene B4; Lipoxygenase Inhibitors; Sheep

Leukotriene B4 levels were measured after stimulation by calcium ionophore A23187: (i) in peripheral, neutrophils (PMN) from allergic asthmatics, rhinitis and healthy subjects; (ii) in macrophages collected by bronchoalveolar lavage. LTB4 levels in PMNs were significantly higher in non-treated allergic asthmatics and non-treated subjects with rhinitis compared to controls. Beta-2 agonist-treated asthmatics showed a significantly decreased LTB4 production which was not different from those of controls. In vitro, LTB4 production decreased significantly after PMN incubation with Salbutamol (10(-6) mol l-1). LTB4 produced by AM collected by BAL was measured in non-treated (n = 5) and treated (n = 11) asthmatics with inhaled beta-2 agonist. AM collected from all controls and non-treated asthmatics produced LTB4. By contrast, no production of LTB4 was observed in the treated group. LTB4 production decreased when normal AM were incubated in vitro with Salbutamol (10(-8) mol l-1). These results suggest that biochemical differences occur in PMN and macrophages from subjects treated with beta-2 agonist, presumably in changing the 5-lipoxygenase pathway.

Topics: Adult; Albuterol; Asthma; Calcimycin; Female; Humans; In Vitro Techniques; Leukotriene B4; Macrophages, Alveolar; Male; Neutrophils

Human alveolar macrophages (AM) from bronchoalveolar lavage of asthmatic patients (AP) and healthy volunteers (HS) were compared for their respective capacities to produce lipoxins and leukotrienes when stimulated by calcium ionophore A23187 with or without 15(S)-HETE. The metabolites were analyzed using an isocratic RP-HPLC system and their formation profiles evaluated on the basis of chromatographic behaviour, UV spectral characteristics and co-elution with synthetic standards. Without 15-HETE, AM from AP produced more LTB4 and 5-HETE than those from HS. In the presence of 15-HETE, human AM were able to produce 5,15-diHETE and lipoxins. Moreover, the total amount of lipoxins synthesized by AM from AP was 2 fold higher than that synthesized by AM from HS, thus showing an enhanced cell activation via the 5-lipoxygenase (5-LO) pathway. These results presented AM as in vitro 15-HETE metabolizing cells and suggested some hypothesis about human AM 5-LO regulation mechanism. The enhanced 5-LO activity in AM from AP suggested that in vivo they could participate in cell to cell interaction mechanisms involved in inflammatory lung diseases and might also take up and transform 15-HETE predominantly released by airway epithelial cells.

Topics: Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelial Cells; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lung; Macrophage Activation; Macrophages, Alveolar; Middle Aged

Mast cells represent a small but important proportion of bronchoalveolar lavage cells and are directly exposed to environmental triggers including allergens. Histamine and PGD2 are mediators released during the activation of mast cells. Fourteen patients allergic to Dermatophagoides pteronyssinus were studied. After bronchoalveolar lavage the unfractionated cell pellet containing metachromatic cells was submitted to allergen challenge using three concentrations of a standardized Dermatophagoides pteronyssinus extract and one concentration of A23187 (2.5 microM). The release of histamine was measured by radioimmunoassay using a monoclonal antibody against acylated histamine and PGD2 was measured by enzyme immunoassay using a polyclonal antibody against methoxamine-PGD2. Histamine was released in 13/14 patients following stimulation of the cells with A23187 and 12/14 patients after stimulation with Dermatophagoides pteronyssinus extract. The release of histamine was 3.5-fold greater when cells were stimulated by the Dermatophagoides pteronyssinus extract than with A23187. PGD2 was released in 10/12 patients when cells were stimulated with A23187 and 6/14 patients in the case of Dermatophagoides pteronyssinus extract stimulation. In the latter case, the mean release was not significantly greater than baseline. For histamine, the maximum release usually occurred with the more concentrated extract whereas in the experiments where PGD2 was released, maximal generation usually occurred with the lowest concentration used. There was no correlation between the severity of asthma and the release of mediators. This study confirms the activation of metachromatic cells by allergen and shows some heterogeneity in the release of granule and membrane-derived mediators.

Topics: Adult; Allergens; Animals; Antigens, Dermatophagoides; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Forced Expiratory Volume; Histamine Release; Humans; Middle Aged; Mites; Prostaglandin D2; Severity of Illness Index

Morphological changes of basophils from atopic asthmatics were compared among antigen, anti-IgE and Ca ionophore A23187 stimulation. 1. Antigen induced rapid and marked increase of histamine release from basophils compared with Ca ionophore A23187. 2. The decrease in number of basophils following stimulation with the agents was significantly higher in antigen stimulation than in Ca ionophore A23187 stimulation. 3. The increased ratio of short to long axis diameter (L/Sb ratio) of the cells, which shows an increased motility of basophils, was significantly higher in antigen stimulation. The ratio did not change by stimulation with Ca ionophore A23187. 4. Stimulation of basophils by Ca ionophore A23187 induced marked increase in mean diameter (MD) of the cells. Activation of basophils by anti-IgE was similar to that by antigen, but slower in start and shorter in duration compared with antigen. The results show that an increase in motility is essential for release mechanism of chemical mediators from basophils in antigen and anti-IgE stimulation, but not in Ca ionophore A23187 stimulation.

Topics: Adult; Antibodies, Anti-Idiotypic; Antigens; Antigens, Differentiation, B-Lymphocyte; Asthma; Basophils; Calcimycin; Cell Movement; Female; Histamine Release; Humans; Immunoglobulin E; Male; Receptors, Fc; Receptors, IgE

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory properties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min, total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 +/- 3% versus 47 +/- 2% of the total synthesis, p less than 0.005), whereas intracellular free 5-HETE was decreased (13 +/- 3% versus 37 +/- 4%, p less than 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.

Topics: Adult; Asthma; Calcimycin; Cell Migration Inhibition; Chromatography, High Pressure Liquid; Esterification; Humans; Hydroxyeicosatetraenoic Acids; Middle Aged; Neutrophils; Platelet Activating Factor; Reference Values

Topics: Asthma; Bronchi; Calcimycin; Cell Survival; Dinoprostone; Epithelium; Fibronectins; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Middle Aged

1. We have investigated the capacities of peripheral leukocytes from atopic asthmatic (AA) (n = 7), atopic non-asthmatic (AN) (n = 7), and normal (N) (n = 7) subjects to generate the bronchoconstrictor and proinflammatory mediators leukotrienes (LTs) B4 and C4. 2. Mixed leukocyte preparations containing 61-84% neutrophils, 2.4-15% eosinophils, and 13-29% mononuclear cells were incubated in vitro at 37 degrees C in the presence of calcium ionophore A23187. Synthesis of LTB4 and LTC4 was quantitated by radioimmunoassay. 3. Both in dose-response experiments (0-10 microM A23187 for 5 min), and in time-course investigations (2 microM A23187 for 0-30 min), the mixed leukocytes of the AA and AN subjects generated on average 4- to 5-fold more LTB4 and 3- to 5-fold more LTC4 than the normal leukocytes (P less than 0.01 in all cases; ANOVA). 4. This enhanced LT synthesis by the AN and AA leukocytes was not due to differences in the counts of leukocyte sub-types, or to altered rates of LT catabolism between the subject groups. 5. LTB4 synthesis correlated significantly with LTC4 synthesis in the leukocytes of the AN and AA subjects (r = 0.81, n = 14, P less than 0.01), but not in those of the normal subjects (r = 0.19, n = 7, P greater than 0.05). 6. Our results demonstrate an up-regulation of the leukotriene synthetic pathway in the circulating leukocytes of atopic non-asthmatic and atopic asthmatic subjects, which may have important implications in the pathophysiology of asthma and allergy.

Topics: Adolescent; Adult; Asthma; Calcimycin; Cells, Cultured; Dose-Response Relationship, Drug; Female; Humans; Hypersensitivity; Leukocytes; Leukotrienes; Male; Middle Aged

Alveolar macrophages (AM) are among the cells involved in the bronchial inflammation of asthma. It has been shown that AM are a heterogeneous cell population in normal subjects. The heterogeneity of AM from 36 asthmatic patients and 23 normal subjects was studied using Percoll density fractionation. AM recovered from asthmatic patients are mainly in the lower density fractions (1.03 and 1.04 g/ml), whereas AM from normal subjects are in the higher density fractions (1.05 and 1.07 g/ml). Electron microscopic studies showed that low density AM of both asthmatic and normal subjects appear to have morphologic characteristics of activated cells by comparison with high density AM that present characteristics of quiescent cells in both asthmatic and normal subjects. The functional activity of AM fractions of asthmatic and control subjects was assessed using the release of the oxygen free radicals induced by opsonized zymosan and TxB2 generation by A23187. There was no difference between the five fractions of asthmatic or control subjects with regard to oxygen species release. The TxB2 generation was increased in the low density AM from asthmatics when compared with the same fractions of normal subjects. The hypodense cells produced less TxB2 than did cells of higher density in both asthmatic and normal subjects. The density of AM was correlated with the recent instability of the asthma but not with the severity of it. This study shows that AM from asthmatic subjects, when compared with those from control subjects, are heterogeneous, hypodense cells and that they predominate. Hypodense AM did not appear to be hyperresponsive in vitro and may have been already committed into the airways.

Topics: Adolescent; Adult; Aged; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cell Count; Cell Separation; Humans; Macrophages; Microscopy, Electron; Middle Aged; Pulmonary Alveoli; Superoxides; Thromboxane B2; Zymosan

In our previous report, we demonstrated that the functions of phagocytes and lymphocytes were defective in patients with systemic lupus erythematosus (SLE). In an attempt to further clarify the defective mechanisms of these cells, 25 active SLE, 10 bronchial asthma patients (BA) on corticosteroids and 25 age and sex-matched normal individuals were investigated for the expression of membraneous C3b receptors, ionophore-induced 45Ca(2+)-uptake, mitochondrial potentials and phagocytic activity of neutrophils. We found decreased expression of C3b receptors on SLE PMN in both resting (37.2 +/- 3.7% of the normal controls) and FMLP-stimulated (68.3 +/- 7.1% of the normal controls) conditions, whereas the C3b receptor expression on BA-PMN receiving long-term steroid treatment was not different from normal controls. This suggests that the defective phagocytosis of SLE PMN is in the recognition, but not in the ingestion phase because of the normal function of Ca(2+)-influx and mitochondrial activity in SLE PMN. On the other hand, hyporesponsiveness to PHA stimulation (stimulation index: 127.4 +/- 46.3 in SLE vs. 311.2 +/- 30.4 in normals, p = 0.0077) was a distinct cell-mediated immune abnormality in our SLE patients. We measured the membrane potential of individual cells using 3,3'-dihexyloxacarbocyanin and found hyperpolarization in resting SLE lymphocytes. However, the membrane polarization of SLE lymphocytes became lower than that of normal cells after PHA stimulation for 3 days. A similar tendency was also found in Na(+)-K(+)-dependent ATPase activity in SLE lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Asthma; Calcimycin; Calcium; Disease Susceptibility; Female; Humans; Immunologic Deficiency Syndromes; Infections; Lupus Erythematosus, Systemic; Lymphocyte Activation; Lymphocytes; Membrane Potentials; Middle Aged; Neutrophils; Phagocytosis; Receptors, Complement; Receptors, Complement 3b; Sodium-Potassium-Exchanging ATPase

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.

Topics: Antibodies, Anti-Idiotypic; Asthma; Basophils; Calcimycin; Histamine Release; Humans; Immunoglobulin E; Opsonin Proteins; Prostaglandin D2; SRS-A; Zymosan

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Asthma; Bronchi; Calcimycin; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Lung

We evaluated basophil releasability in two groups of allergic patients with positive skin tests to Dermatophagoides pteronyssinus major allergen (Der p l) (29 adults with bronchial asthma and 17 with allergic rhinitis) and in 31 age-matched normal donors. Both basophil reactivity (maximal percent histamine release) and basophil sensitivity (the concentration that causes 50% of maximal percent histamine release: HC50) to Der p l in patients with asthma were similar to those in patients with allergic rhinitis. On the contrary, basophil reactivity to anti-IgE was significantly higher in patients with asthma (58.0 +/- 3.6%) than in patients with allergic rhinitis (46.3 +/- 5.2%; p less than 0.05). Both groups of patients showed an increased releasability compared to control subjects (27.3 +/- 4.6%; p less than 0.001), whereas there were no significant differences in basophil sensitivity to anti-IgE among the three groups of donors. Differences were also found with respect to basophil reactivity and sensitivity to f-met peptide, whereas no differences appeared when basophils from the three groups of donors were challenged with the Ca2+ ionophore A23187. There was a significant correlation between basophil reactivity and sensitivity to Der p l and to anti-IgE in both asthmatic and allergic rhinitis patients. A significant correlation was found between basophil reactivity and sensitivity to anti-IgE and serum IgE level only in patients with bronchial asthma, whereas no correlations were found in patients with allergic rhinitis. There was no correlation between in vivo mast cell releasability and in vitro basophil releasability in response to Der p l in either group of allergic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Animals; Antigens; Asthma; Basophils; Calcimycin; Child; Female; Histamine Release; Humans; Immunoglobulin E; Male; Mites; N-Formylmethionine Leucyl-Phenylalanine; Rhinitis, Allergic, Seasonal; Skin Tests

The generation and metabolism of leukotrienes (LTs) B4, C4, D4, and E4 were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB4 and LTC4 in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB4, which are significantly higher in the AA blood than in N blood. Incubations of 3H-LTB4 in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB4 and 20-COOH-LTB4. LTC4 is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p less than 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC4 elicits significantly greater amounts of LTD4, and consistently higher levels of LTE4, in the AA blood. Parallel incubations of 3H-LTC4 in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB4 and LTC4 in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB4 and LTC4 generation and was equipotent in N and AA blood.

Topics: Asthma; Benzeneacetamides; Calcimycin; Chromatography, High Pressure Liquid; Female; Humans; Hydroxamic Acids; In Vitro Techniques; Leukocyte Count; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Radioimmunoassay; Reference Values

To evaluate the pathogenetic role of leukotrienes in bronchial asthma, the levels of leukotrienes released from leukocytes stimulated by calcium ionophore A23187 were measured by the HPLC-RIA method in 32 asthmatic patients (12 infectious type, 12 non-hyposensitized atopic type, and 8 hyposensitized atopic type). The following results were obtained: 1) The level of leukotrienes released from the leukocytes of asthmatic patients were higher than those of healthy subjects, but there was no significant difference in the levels of leukotrienes between mild and moderate asthmatic patients. 2) The levels of leukotriene B4 released from the leukocytes of the infectious-type asthmatic patients were higher than those of the atopic asthmatic patients (non-hyposensitized group). There was a tendency for levels of leukotriene C4 released from the leukocytes of the infectious-type asthmatic patients to be higher than those of the atopic-type asthmatic patients. 3) The levels of peptide leukotriene released from the leukocytes of the atopic hyposensitized patients were lower than those of the patients in the atopic non-hyposensitized group, but there were no significant differences in leukotriene B4 between the groups. In addition, the levels of leukotriene C4 released spontaneously from the leukocytes of the hyposensitized group of patients were lower than those in the non-hyposensitized group. 4) Though the mechanism of the efficacy of hyposensitization has not been completely clarified, the reduction of leukocyte ability to release peptide leukotrienes through immunotherapy seems to be part of this mechanism. These results suggest that leukotrienes may play some role in the pathogenesis of both atopic- and infectious-type bronchial asthma.

Topics: Adult; Asthma; Calcimycin; Female; Humans; Leukocytes; Leukotrienes; Male; Middle Aged; Stimulation, Chemical

In stable state asthmatic patients (AP) without any airway obstruction, the capacity of peripheral blood polymorphonuclear neutrophils (PMN) to produce 5-lipoxygenase metabolites and to migrate, was investigated and compared with the response in healthy subjects (HS). After calcium-ionophore A23187 stimulation, PMN from AP and HS produced LTB4, its hydroxylated derivatives: omega-OH-and omega-CO2H-LTB4) (omega-LTB4, i.e 6-trans-LTB4 and 5,6-diHETE isomers, and 5-HETE. We found an increase in LTB4 (+59%), omega-LTB4 (+39%), 6-trans-LTB4 (+128%), and free 5-HETE (+63%) generation of AP as compared with HS. Unstimulated migration was enhanced in AP (122 +/- 27 PMN/10 high power fields (hpf) in AP versus 74 +/- 25 PMN/10 hpf in HS, p less than 0.025) and suggested a greater capacity of PMN from AP to migrate. This was confirmed by the PAF-induced chemotaxis studies which showed, in AP, a greater PAF-sensitivity of PMN (10(-6) M versus 10(-5) M in HS) and a greater chemotaxis response (600 +/- 50 PMN versus 200 +/- 35 PMN in HS). In AP, we compared the capacity of PMN to generate LTB4 and 5-HETE with their capacity to migrate. We found an inverse correlation (r = 0.86, p less than 0.007) of intracellular free 5-HETE with chemotaxis to PAF.

Topics: Adolescent; Adult; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Asthma; Calcimycin; Chemotaxis, Leukocyte; Humans; Leukotrienes; Middle Aged; Neutrophils; Platelet Activating Factor

We determined the relationship of allergic disease to the number and activity of eosinophils and their production of leukotriene B4 and leukotriene C4 (leukotriene D4 equivalents). Granulocytes from allergic rhinitis (AR) subjects and asthmatics release more LTC4 than normals. Furthermore, eosinophils of asthmatics generate more LTC4 than those of AR subjects.

Topics: Adult; Asthma; Calcimycin; Eosinophils; Female; Granulocytes; Humans; Leukocyte Count; Leukotriene B4; Male; Middle Aged; Reference Values; Rhinitis, Allergic, Seasonal; SRS-A; Statistics as Topic

Releasability of human basophils and mast cells is an important parameter in allergic disorders. We compared IgE- and non-IgE-mediated releasability of human peripheral blood basophils with that of mast cells obtained from lung parenchyma (isolated by mechanical or enzymatic dissociation) and from bronchoalveolar lavage of normal and asthmatic donors. In a first study, the response to anti-IgE, Staph A, Con A, f-met peptide, and Ca2+ ionophore A23187 of basophils obtained from 52 donors was compared with that of mast cells isolated enzymatically (PMCE) or mechanically (PMCM) from lung parenchyma obtained during surgery. The histamine content of basophils (1.1 +/- 0.1 pg/cell) was significantly lower than that of PMCE (4.1 +/- 0.3 pg/cell; p less than 0.001) and PMCM (3.7 +/- 0.3; p less than 0.001). The maximal percent anti-IgE-induced histamine secretion in basophils (41.3 +/- 3.6) was higher than in PMCE (17.5 +/- 1.8) and in PMCM (13.8 +/- 1.5). Similarly, the response to Staph A and Con A was higher in basophils (29 +/- 3.9 and 31.6 +/- 4.9, respectively) than in PMCE (3.5 +/- 0.6 and 3.3 +/- 0.8, respectively) and PMCM (5.1 +/- 1.3 and 8.8 +/- 2.2, respectively). A positive correlation between the maximal percent of histamine release induced by anti-IgE and Staph A was found in basophils (rs = 0.61; p less than 0.001), whereas there was a negative correlation between the reactivity of PMCE (rs = 0.67; p less than 0.001) and PMCM (rs = -0.40; p less than 0.001) to anti-IgE and their reactivity to Staph A.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Aged; Asthma; Basophils; Bronchoalveolar Lavage Fluid; Calcimycin; Cell Separation; Concanavalin A; Female; Histamine Release; Humans; Immunoglobulin E; Lung; Male; Mast Cells; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Staphylococcal Protein A

Broncho-Vaxom (BV) inhibited in dose-dependent manner the release of histamine from and degranulation of isolated rat peritoneal mast cells stimulated with compound 48/80 and the ionophore A23187. Inhibition persisted after removal of BV from the incubation medium before stimulation, but did not occur when bovine serum albumin (BSA) was used instead of BV. Binding of BV to mast cells was observed by electron microscopy on cells that had been incubated with colloidal-gold labelled BV. There was no significant difference between the binding of BV gold and BSA gold to the mast cells. Washing before fixation removed most of the BV gold from the cells. This study establishes BV as an in vitro histamine release inhibitor.

Topics: Adjuvants, Immunologic; Animals; Asthma; Bacteria; Calcimycin; Cell Extracts; Histamine Release; In Vitro Techniques; Mast Cells; p-Methoxy-N-methylphenethylamine; Rats

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.

Topics: Adolescent; Adult; Asthma; Basophils; Benzhydryl Compounds; Calcimycin; Eosinophils; Female; Histamine H1 Antagonists; Histamine Release; Humans; Immunoglobulin E; Male; SRS-A; Terfenadine

Eosinophilic granulocytes as well as neutrophilic granulocytes produced leukotriene C4 (LTC4) on stimulation with 1 microM A23187 (Ca2+ ionophore). In healthy volunteers, the LTC4 production in eosinophils was about 3 times the production in neutrophils. Within 15 min greater than 90% of the LTC4 was released into the supernatant. Stimulation of eosinophils and neutrophils together resulted in a synergistic increase in LTC4 production of 306 +/- 40%. LTC4 synthesis by hypodense eosinophils was also enhanced if stimulated in the presence of neutrophils. These findings suggest a communication between eosinophils and neutrophils, which may play a role in bronchial asthma.

Topics: Asthma; Calcimycin; Cell Communication; Churg-Strauss Syndrome; Eosinophils; Humans; Leukocyte Count; Neutrophils; SRS-A

Topics: Asthma; Calcimycin; Eosinophils; Granulocytes; Humans; In Vitro Techniques; Leukotrienes

Incubation of eosinophils (EOS) with alveolar macrophage (AM) supernatants isolated from asthmatic subjects followed by stimulation with the calcium ionophore A23187 resulted in enhancement of the capacity of EOS to elaborate leukotriene C4 (LTC4) (mean enhancement 169 +/- 37%, n = 31). Pretreatment of EOS with AM supernatants derived from normal individuals did not enhance LTC4 generation as compared with control medium. Enhancement was maximal when EOS were preincubated with a 1:6 dilution of AM supernatants for 5 min at 37 degrees C and were then stimulated with 5 microM A23187 for 15 min. Separation of AM supernatants by size-exclusion HPLC using a TSK G3000 SW column resulted in a peak of enhancing activity with an estimated molecular mass of approximately 30,000 D. Further purification by anion exchange HPLC using a TSK DEAE 5PW column (pH 7.4) resolved the activity into a minor peak at 0.17 M NaCl and a major peak at 0.2 M NaCl. The activities were distinct from interleukin-1 and tumor necrosis factor. Resolution of the major peak of activity by reverse-phase HPLC using a C18 spherisorb ODS column and a slope gradient of 0 to 100% acetonitrile/0.1% trifluoroacetic acid demonstrated a single peak of activity that eluted at 41% acetonitrile. The enhancing activity was sensitive to trypsin and heat and was neutralized by a specific antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF). Pretreatment of EOS with recombinant GM-CSF primed the cells for enhanced LTC4 generation following subsequent stimulation with A23187. GM-CSF may play a role in the amplification of the eosinophilic inflammation in asthmatic airways.

Topics: Adolescent; Adult; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Eosinophils; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Macrophages; Male; Middle Aged; Pulmonary Alveoli; SRS-A; Stimulation, Chemical

Granulocytes and mononuclear cells were isolated from the blood of asthmatic and healthy children. Stimulation with ionophore A 23187 induced a significantly higher leukotriene C4 (LTC4) generation from granulocytes of asthmatic children than from granulocytes of healthy controls. In contrast, mononuclear cells from patients and controls did not differ in their ability to produce LTC4. Additional analysis showed that the difference in LTC4 generation of granulocytes was due to increased formation but not to decreased oxidative degradation of LTC4. Analysis of LTC4 generation of purified neutrophils and eosinophils revealed that LTC4 was generated almost exclusively by eosinophils and, in particular, the hypodense population. Granulocytes from patients with a history of severe asthma displayed a higher LTC4 formation than granulocytes from patients with less severe disease.

Topics: Adolescent; Asthma; Calcimycin; Child; Child, Preschool; Eosinophils; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Neutrophils; SRS-A

We attempted to develop a nonimmunologically induced asthma model using the calcium ionophore A23187. Inhalation of A23187 (0.001-0.005%) for 5 min in male Hartley guinea pigs caused a marked bronchoconstriction in a dose-dependent manner with negligible effect on systemic blood pressure. The A23187-induced bronchoconstriction was strongly inhibited by chlorpheniramine and FPL-55712. These results indicate that an asthma-like bronchoconstriction was induced by inhalation of A23187 in guinea pigs, and the main chemical mediators involved in this response would be histamine and peptidoleukotrienes.

Topics: Administration, Inhalation; Animals; Asthma; Calcimycin; Chlorpheniramine; Chromones; Disease Models, Animal; Guinea Pigs; Injections, Intravenous; Male; SRS-A

Topics: Adolescent; Albuterol; Asthma; Calcimycin; Child; Child, Preschool; Cromolyn Sodium; Enzyme Inhibitors; Granulocytes; Humans; Imidazoles; In Vitro Techniques; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Pancreatic Elastase; Superoxides; Tetradecanoylphorbol Acetate

1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma by the release of bronchoconstrictor mediators including leukotrienes. Previous studies have revealed the almost exclusive synthesis of leukotriene C4 (LTC4) by human eosinophils and of leukotriene B4 (LTB4), 20-OH-LTB4 and the non-enzymatically formed LTB4-isomers by neutrophils when stimulated in vitro with the calcium ionophore A23187 or opsonized zymosan (OZ). In this study we have investigated whether nedocromil sodium, a new anti-asthma drug, was capable of inhibiting A23187- and OZ-induced leukotriene formation by these cells. 2. Nedocromil sodium inhibited A23187- and OZ-induced LTC4 formation by eosinophils in a concentration-dependent manner (mean IC30 for A23187: 5.6 X 10(-5) M; mean IC30 for OZ: 6.3 X 10(-5) M), whereas it did not inhibit A23187- and OZ-induced LTB4 formation by neutrophils. 3. Extension of the preincubation time of the cells with the drug did not alter the observed inhibitory capacity. The optimal preincubation time was 5 min. 4. The in vitro inhibition of LTC4 formation by eosinophils by nedocromil sodium may be a valuable property of this drug in the treatment of asthma.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Asthma; Calcimycin; Eosinophils; Humans; Hypersensitivity; In Vitro Techniques; Leukotriene B4; Nedocromil; Neutrophils; Quinolones; Zymosan

Alveolar macrophages (AM) are the principal resident phagocytes in the human lung, and play a major role in local defence against environmental agents. It is now known that during asthma these cells take part in the amplification of the inflammatory mechanism. It has been demonstrated in vitro that they can be activated to generate leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), mediators with potent pharmacological properties. These two arachidonic metabolites were identified and quantified by reversed phase high performance liquid chromatography (HPLC) performed in cell suspensions, and in cell free supernatants. AM from asthmatics, after stimulation by the calcium ionophore A23187 or opsonized zymosan, released significantly (p less than 0.05) more LTB4 than those from healthy subjects. The increase in LTB4 release could be evidence for in vivo activation. On the other hand, the levels of 5-HETE in the AM from asthmatics were significantly (p less than 0.03) higher than those in cells from healthy subjects. This intracellular increase could be correlated with a greater migratory ability of these inflammatory macrophages, as observed for eosinophils. The clinical efficacy of nedocromil sodium may be partly related to the decreases in LTB4 releasability and intracellular 5-HETE levels observed only in AM from asthmatic patients.

Topics: Adolescent; Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Male; Middle Aged; Nedocromil; Pulmonary Alveoli; Quinolones; Zymosan

Topics: Adolescent; Adult; Asthma; Calcimycin; Calcium; Humans; Inflammation; Influenza A virus; Influenza, Human; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Respiratory Syncytial Viruses; Respirovirus Infections; Superoxides

Peripheral blood mononuclear cells (PBMC) were isolated from seven normal subjects, eight asthmatic subjects clinically sensitive to corticosteroids (CS), and eight asthmatic subjects clinically resistant to corticosteroids (CR). PBMC were cultured at 37 degrees C for 24 h in the absence or presence of 10(-16) to 10(-4) M hydrocortisone. Calcium ionophore (A23187)-activated neutrophils (PMN) primed by supernatants of PBMC from asthmatic subjects cultured in the absence of hydrocortisone generated approximately threefold more leukotriene B4 than PMN primed by supernatants of PBMC from normal subjects (P less than 0.05). Incubation of PBMC derived from CS subjects with 10(-8) M hydrocortisone completely inhibited the production of the enhancing activity (P less than 0.01), whereas in CR subjects hydrocortisone at concentrations up to 10(-4) M did not suppress the release of enhancing activity. The enhancing activity was produced by monocytes. Enhancing activity eluted with an Mr of 3,000 D and a pI of 7.1. It eluted at 10% acetonitrile after reverse-phase HPLC. The activity was destroyed by heating to 60 degrees C for 60 min and was sensitive to pronase treatment. The purified factor also enhanced superoxide generation by PMN which had been stimulated submaximally by phorbol myristate acetate.

Topics: Adrenal Cortex Hormones; Arachidonic Acid; Arachidonic Acids; Asthma; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Drug Resistance; Forced Expiratory Volume; Humans; Hydrocortisone; Isoelectric Point; Kinetics; Leukotriene B4; Lipoxygenase; Molecular Weight; Monocytes; Neutrophils; Peptides

Activation of neutrophils (PMN) within the airways results in the secretion of a number of products such as reduced oxygen metabolites that could contribute to the inflammatory response associated with asthma. However, mediators of allergy, such as histamine, prostaglandin E2 (PGE2), isoproterenol, and adenosine, may serve to mitigate this inflammation through feedback inhibition of neutrophil function. To test the hypothesis that PMN activation and feedback inhibition mechanisms may be abnormal in asthmatics, we compared both superoxide production and adenosine-induced suppression of superoxide production in 12 matched pairs of asthmatics and control subjects. PMN obtained from asthmatic patients generated significantly more superoxide in response to f-met-leu-phe (fMLP) than controls (2.94 +/- 55 nmol/5 x 10(5) PMN/5 min versus 1.38 +/- 0.35 at 2 x 10(-8) M fMLP and 3.81 +/- 0.68 nmol versus 2.04 +/- 0.45 nmol at 10(-7) M; p less than 0.01 for both). In contrast, the respiratory burst generated by two receptor-independent stimuli, the calcium ionophore A23187 and phorbol myristate acetate, was equivalent between control and asthmatic subjects. At 10(-6) M, 2-chloroadenosine induced a 19.5 +/- 5.1% inhibition of fMLP-stimulated superoxide production in PMN from patients with asthma as compared to 55.6 +/- 24.6% inhibition in PMN from control subjects (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Chloroadenosine; Adenosine; Asthma; Calcimycin; Cytochalasin B; Dinoprostone; Dose-Response Relationship, Drug; Humans; Isoproterenol; Lipopolysaccharides; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides

The aim of the current study was twofold: (1) to consider the applicability of fluorescence-activated cell sorting (FACS) to basophil isolation from small blood volumes and (2) to compare basophils obtained from children with asthma to basophils from healthy children. With FACS, basophil suspensions were prepared with a purity of 84% (range, 75% to 95%) and a recovery of 20% (range, 15% to 30%). The purified basophils had a total histamine content of 1.6 +/- 0.12 pg per cell, not differing significantly from total histamine content observed in "total" leukocyte suspensions (1.4 +/- 0.07 pg per basophil). The same was true for IgE receptor-mediated histamine release (29 +/- 4% versus 27 +/- 4%) and for ionophore A23187-induced histamine release (41 +/- 6% versus 51 +/- 9%). Sorted basophils from subjects with asthma released more histamine after IgE receptor activation (0.67 +/- 0.09 pg per cell) than basophils from healthy children (0.40 +/- 0.04 pg per cell; p less than 0.02). Expressed as percent release, no significant difference was observed (37 +/- 3.2% versus 30 +/- 2.7%). Ionophore A23187-induced histamine release did not differ significantly between subjects with asthma and control subjects, neither expressed as picograms per cell (1.21 +/- 0.17 pg per cell versus 1.02 +/- 0.11 pg per cell) nor expressed as percent release (66 +/- 4.4% versus 74 +/- 3.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens, Differentiation, B-Lymphocyte; Asthma; Basophils; Calcimycin; Calcium; Cell Separation; Flow Cytometry; Histamine; Histamine Release; Humans; In Vitro Techniques; Membrane Potentials; Receptors, Fc; Receptors, IgE

The effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861), a selective 5-lipoxygenase inhibitor, on immunological or non-immunological release of slow reacting substance of anaphylaxis (SRS-A) and histamine and its effects on experimental asthma were investigated. AA-861 showed a dose-dependent inhibition of SRS-A release, with no effect on histamine release from passively sensitized guinea pig, monkey (M. irus) and human lung fragments. An analysis of the anaphylactic diffusate from the human lung fragments, using the combined technique of high performance liquid chromatography and radioimmunoassay, revealed that AA-861 markedly suppresses biosynthesis of the leukotrienes. However, this drug inhibits the release of histamine as well as SRS-A from lung fragments of anaphylactic monkey (M. mulatta) and in the Ca ionophore-stimulated rat peritoneal cavity. AA-861 suppressed the anaphylactically-induced airway resistance in mepyramine- and cimetidine-treated guinea pigs. These results suggest that AA-861 may be clinically effective for treating allergy-related asthma by modulating the 5-lipoxygenase pathway and that an inhibitory mechanism of histamine release by AA-861 may be present in some species.

Topics: Animals; Asthma; Benzoquinones; Calcimycin; Chromatography, High Pressure Liquid; Female; Guinea Pigs; Histamine; Histamine Release; Hypersensitivity; Lipoxygenase Inhibitors; Macaca; Macaca mulatta; Male; Mites; Quinones; Radioimmunoassay; Rats; Rats, Inbred Strains; Species Specificity; SRS-A

Mast cells have been implicated in the pathogenesis of allergic asthma but their role in non-allergic asthma remains to be elucidated. The spontaneous and non-specific release of histamine by suboptimal doses of calcium ionophore A23187 was studied in bronchoalveolar lavage cells obtained from nine asthmatic and seven healthy individuals. Bronchoalveolar lavage was performed with saline, and total cells were incubated without any secretagogue (spontaneous histamine release) or after addition of 1.25, 2.5 and 5 microM of A23187 for 30 min (net maximal release). Histamine was titrated by using a very sensitive radioimmunoassay using a monoclonal antibody against acylated histamine. The spontaneous release was similar in asthmatic (20.6 +/- 8.2%) and healthy individuals (17.4 +/- 8.4%). The net maximal release of histamine was significantly greater in asthmatic patients (28.1 +/- 17.4%) than in normal subjects (10.3 +/- 8.9%). The release of histamine was significantly correlated to the release of PGD2 measured by enzyme immunoassay using a polyclonal antibody against methoxamine-PGD2 (Spearman rank test: 0.78, P less than 0.01). In eight subjects, the release of histamine by A23187 was studied in the presence of nedocromil sodium and it was observed that this drug significantly (P less than 0.05) decreased the net maximal release of histamine. This study shows that mast cells from asthmatic individuals have a greater releasability than those of normal subjects.

Topics: Adult; Aged; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Histamine Release; Humans; Mast Cells; Middle Aged; Nedocromil; Prostaglandin D2; Quinolones

The generation of LTB4 by peripheral blood neutrophils (PMN) isolated before and for as long as 6 h after exercise-induced asthma (EIA) has been analyzed. Three and 6 h after the development of EIA, PMN isolated from 10 asthmatic subjects and stimulated in vitro by 2 x 10(8) and 4 x 10(8) zymosan particles per 2 x 10(6) PMN demonstrated a 12- and 4-fold enhancement, respectively, in the production of immunoreactive LTB4 as compared with PMN isolated before exercise. At 6 h after EIA, there was a redistribution of generated LTB4 such that 30 to 40% of LTB4 produced by zymosan-activated PMN was released extracellularly as compared with 10% before exercise. There was no significant enhancement in the generation of LTB4 by unstimulated PMN at any time point after exercise. Resolution by reverse-phase high performance liquid chromatography (HPLC) of products from [3H]arachidonic-acid-labeled and zymosan-activated PMN demonstrated that, in addition to LTB4, there was enhanced metabolism to 6-trans-LTB4, omega-oxidation metabolites of LTB4 and 5-HETE. Stimulation of PMN with 10 microM A23187 revealed a 2-, 6-, and 5-fold enhancement in the production of LTB4, 6-trans-LTB4, and 5-HETE, respectively, at 6 h after EIA, as measured by integrated ultraviolet absorbance after HPLC. There was no significant enhancement in LTB4 generation by PMN in 6 asthmatic subjects after methacholine-induced bronchospasm, and after exercise in 6 subjects who did not develop asthma. The augmentation of PMN LTB4 generation in EIA correlated with the extent of the early decrease in SGaw.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Airway Resistance; Arachidonate 5-Lipoxygenase; Asthma; Asthma, Exercise-Induced; Bronchial Provocation Tests; Calcimycin; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Methacholine Chloride; Methacholine Compounds; Middle Aged; Neutrophils; Stereoisomerism; Zymosan

We evaluated the formation of leukotriene C4 (LTC4) by peripheral blood eosinophils of different densities obtained from asthmatic and normal subjects. When stimulated with 1 microgram/ml of the calcium ionophore A23187 for 15 min at 37 degrees C, eosinophils with densities greater than 1.093 g/ml from asthmatic and normal subjects released 19.1 +/- 4.2 ng LTC4/10(6) eosinophils and 23.9 +/- 5.0 ng LTC4/10(6) eosinophils, respectively. In contrast, lower density eosinophils (densities 1.093 g/ml or less) isolated from the asthmatic subjects released significantly less LTC4 than did eosinophils of similar densities from normal subjects (41.6 +/- 3.0 versus 79.0 +/- 6.7 ng LTC4/10(6) eosinophils, p less than 0.05). Differences could not be demonstrated between the two subject groups in LTC4 metabolism, time course of extracellular release of LTC4, or dose response to A23187, nor were interactions between eosinophils and neutrophils with regard to LTC4 release evident. Thus, hypodense eosinophils elaborate greater quantities of LTC4 than do eosinophils of normal density whether obtained from normal or asthmatic subjects. However, the finding that peripheral blood eosinophils from asthmatic subjects have decreased capacity for the synthesis of LTC4 compared with cells of similar densities isolated from normal subjects demonstrates that the capacity of eosinophils to produce LTC4 is regulated by factors that are not necessarily reflected in the cell density.

Topics: Asthma; Calcimycin; Cell Count; Eosinophils; Humans; Neutrophils; Reference Values; SRS-A

The effect of the thromboxane (TX) A2 synthetase inhibitor, OKY-046, on human leukocyte histamine release and bronchial hypersensitivity in asthmatic subjects was evaluated. It was found that OKY-046 inhibited IgE- and Ca2+ ionophore A23187-mediated leukocyte histamine release in a dose-dependent fashion (IC50: 1.0 and 3.0 X 10(-3) M, respectively) and that OKY-046 could diminish bronchial hypersensitivity, determined by leukotriene D4 inhalation, following a 2-week oral medication. These data suggest that the TXA2 synthetase inhibitor can produce favorable effects upon the course of immediate-type hypersensitivity reactions.

Topics: Acrylates; Asthma; Bronchial Provocation Tests; Calcimycin; Drug Evaluation; Female; Histamine Release; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Leukocytes; Male; Methacrylates; SRS-A; Thromboxane-A Synthase

Topics: Asthma; Calcimycin; Histamine Release; Humans; Hypersensitivity, Immediate; Leukocytes; Leukotriene B4; SRS-A

Topics: Age Factors; Animals; Antibodies, Anti-Idiotypic; Asthma; Basophils; Calcimycin; Dermatitis, Atopic; Deuterium; Deuterium Oxide; Histamine Release; Humans; Hypersensitivity; Immunoglobulin E; N-Formylmethionine Leucyl-Phenylalanine; Phenotype; Water

Topics: Adolescent; Adult; Asthma; Calcimycin; Child; Female; Histamine Release; Humans; Leukocytes; Male; SRS-A; Zymosan

Amoxanox has potent antiallergic activity because it inhibits the release of chemical mediators such as histamine and leukotrienes. We studied the in vitro effect of amoxanox on arachidonic acid metabolism, including the lipoxygenase and cyclooxygenase pathways. Amoxanox inhibited calcium ionophore A23187-induced formation of 5-HETE, LTB4, SRS-A (LTC4, LTD4 and LTE4), and 12-HETE in rat peritoneal resident monocytes. These results indicate that amoxanox inhibits 5- and 12-lipoxygenases. The compound, however, did not affect the formation of TXB2 or 6-keto-PGF1 alpha in guinea pig lung fragments and PGE2 or PGF2 alpha in bovine seminal vesicles, suggesting that it did not inhibit cyclooxygenase. These results show that the antiallergic action of amoxanox is associated, at least in part, with the reduction of leukotrienes due to the inhibition of lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Aminopyridines; Animals; Asthma; Calcimycin; Cattle; Guinea Pigs; Histamine H1 Antagonists; Hydroxyeicosatetraenoic Acids; Hypersensitivity; In Vitro Techniques; Leukotriene B4; Lipoxygenase Inhibitors; Lung; Male; Monocytes; Prostaglandins; Rats; Rats, Inbred Strains; SRS-A; Thromboxane B2

Arachidonic acid metabolites may play a role in the pathophysiology of bronchial asthma, influencing bronchial tone, airways inflammation and bronchial hyperreactivity. This study was designed to investigate the effect of nedocromil sodium, at a range of concentrations, on the metabolism of arachidonic acid released from alveolar macrophages (AMs) obtained by bronchoalveolar lavage in healthy and asthmatic subjects. The only effect of nedocromil sodium observed in this study was a slight decrease in LTD4 synthesis by AMs from asthmatic patients. This possible effect of nedocromil sodium on LTD4 metabolism deserves further investigation.

Topics: Asthma; Calcimycin; Chromatography, Thin Layer; Humans; Leukotriene B4; Nedocromil; Pulmonary Alveoli; Quinolines; SRS-A; Thromboxane B2

Peripheral polymorphonuclear leukocytes and mononuclear leukocytes from normal subjects and subjects with asthma were studied for their production of slow-reacting substance (SRS) after nonimmunologic stimulation with the calcium ionophore A23187. The amount of SRS generated was determined in both polymorphonuclear leukocyte fractions and mononuclear leukocyte fractions from four groups of subjects: 10 with extrinsic asthma, five with mixed-type asthma, 10 with intrinsic asthma, and 10 normal control subjects. In either cell type, the SRS produced was much more in cells from subjects with extrinsic asthma and less in cells from subjects with the mixed-type asthma, intrinsic asthma, and the normal subjects in order of decreasing quantity. These results indicate the amount of SRS generated by nonimmunologic stimulation with calcium ionophore is related to the degree of atopy and suggest the existence of an intrinsic cellular defect in cells from subjects with atopic asthma in addition to the effects of higher serum levels of IgE antibodies.

Topics: Adult; Asthma; Calcimycin; Humans; Hypersensitivity, Immediate; Leukocytes; Middle Aged; Neutrophils; SRS-A

The role of the eosinophilic granulocyte in immediate hypersensitivity reactions is generally believed to be a beneficial one since this cell may phagocytose mast cell granules and inactivate certain mast cell mediators. However it has become clear that the eosinophilic granulocyte also has potent secretory capacities, and by this property may contribute in a detrimental way to the allergic process. In studying the late phase allergen induced bronchoconstriction by means of bronchoalveolar lavage (BAL) an evident infiltration of eosinophilic granulocytes in the bronchioli in the beginning of the late phase asthmatic reaction was noticed. Since also eosinophil cationic protein (ECP) has been reported to be elevated in the lavage fluid an active secretory role of the eosinophil in the late phase asthmatic reaction seemed likely. Although the release of ECP and other granular proteins may contribute to epithelial damage and inflammation and thereby to an increase in bronchial hyperreactivity they do not explain the late phase bronchoconstrictive reaction. Since leukotrienes were thought possible candidates to cause this reaction it was decided to isolate eosinophils from human peripheral blood and to study their leukotriene synthesis pattern. To our surprise purified human eosinophils almost exclusively synthesize the strongly bronchoconstrictive leukotriene LTC4 in considerable quantities upon in vitro stimulation with either the calcium-ionophore A23187 or opsonized zymosan. These findings suggest that the eosinophil may play an active role in causing the late phase asthmatic reaction.

Topics: Asthma; Calcimycin; Eosinophils; Granulocytes; Humans; SRS-A; Time Factors; Zymosan

IgE-dependent basophil histamine release does not necessarily correlate with the amount of cell-bound IgE, thus it has been suggested that basophil "releasability" is an important, but yet undefined, factor in this secretory process. Because mast cell, and possibly basophilic leukocyte, mediator release contributes to airway reactivity, any enhancement of this secretory process would favor asthma provocation. To evaluate IgE-dependent basophil histamine releasability in asthma, suspensions of leukocytes were isolated from patients with an allergic and nonallergic component to their airway disease and stimulated with concanavalin A (Con A) (0.03 to 10.0 micrograms/ml) and anti-IgE (10 to 1,000 ng/ml). Basophil histamine release to Con A and anti-IgE was significantly greater in both allergic and nonallergic asthmatic patients when compared with normal subjects. In contrast, basophil histamine release to the calcium ionophore A23187 was similar in leukocytes from normal subjects and asthmatic patients, suggesting the observed abnormality in secretion may be limited to an IgE-dependent process. To further determine if basophil histamine releasability in asthma correlated to measures of airway reactivity, bronchial provocation with histamine was performed. An inverse correlation was found between the provocative dose of inhaled histamine required to produce a 20% decrease, PD20, in the FEV1 and the leukocyte histamine release to Con A (p less than 0.05) and anti-IgE (p less than 0.05). Thus, we have new evidence that enhanced IgE-dependent release of leukocyte histamine correlates with airway reactivity in asthma. The mechanism of basophil releasability and its relationship to the pathogenesis of airway reactivity in asthma have yet to be established.

Topics: Adult; Antibodies, Anti-Idiotypic; Asthma; Basophils; Bronchi; Bronchial Provocation Tests; Calcimycin; Concanavalin A; Female; Histamine; Humans; Immunization, Passive; Immunoglobulin E; Leukocytes; Male; Reference Values

Human eosinophils with a density of over 1.095 were isolated from peripheral blood by dextran sedimentation, centrifugation with Lymphoprep and density gradients with Percoll. After the cells (1 X 10(5)/ml) were incubated with 1 microgram/ml calcium ionophore A23187 for 20 min, leukotriene C4 (LTC4) content in the supernatant was measured by radioimmunoassay. The generation of LTC4 was significantly higher in the cells from extrinsic asthmatics (23.5 +/- 14.8 ng/10(6) cells, mean +/- SD, n = 26, P less than 0.01) and intrinsic asthmatics (24.6 +/- 20.6 ng/10(6) cells, n = 27, P less than 0.01 as compared with normal healthy subjects (8.3 +/- 7.7 ng/10(6) cells, n = 10). There was no significant difference in the generation of LTC4 between intrinsic and extrinsic asthmatics. These observations indicate that eosinophils from asthmatic patients have increased ability to release LTC4.

Topics: Adult; Aspirin; Asthma; Calcimycin; Eosinophils; Humans; Middle Aged; SRS-A

The leukotriene generating capacities of ionophore stimulated human eosinophils and neutrophils were compared using specific radioimmunoassays for LTB4 and LTC4. Mixed granulocyte preparations (neutrophils and eosinophils) produced both LTB4 and LTC4 in a time-dependent fashion which was maximal at 10 and 15 min, respectively. Following the separation of eosinophils (greater than 75%) and neutrophils (greater than 90%) by metrizamide gradients, LTC4 production was predominantly from eosinophils, whereas neutrophils were the principal source of LTB4. The concentrations of leukotrienes produced by the eosinophil and neutrophil rich cell preparations were directly proportional to the concentration of ionophore. Following purification of eosinophil derived products by RP-HPLC the LTC4 immunoreactivity corresponded to the elution profile of a synthetic LTC4 marker. Furthermore, in 32 atopic subjects (21 bronchial asthmatics and 11 non-asthmatics) the amounts of LTC4 produced by unseparated leucocytes were directly proportional to the percentage of eosinophils in the total cell suspension. Preferential generation of LTB4 by neutrophils was also demonstrated by immunoreactivity of ionophore stimulated supernatants subjected to RP-HPLC, as well as by its characteristic u.v. absorbance and GC-MS profile and the ability to promote directional neutrophil locomotion (chemotaxis). These experiments support the concept that eosinophils accumulate in tissues partly as a result of the response to neutrophil derived LTB4, and that these cells contribute to the production of sulphidopeptide leukotrienes with subsequent amplification of the acute allergic response.

Topics: Adolescent; Adult; Asthma; Calcimycin; Eosinophils; Granulocytes; Humans; Leukotriene B4; Middle Aged; Neutrophils; Radioimmunoassay; Rhinitis; SRS-A; Urticaria

The hypothesis studied is that an increased responsiveness in asthma is not limited to the airways. 40 asthmatic children were analysed for their bronchial responsiveness (BR) to exercise. 20 patients revealed bronchial obstruction after exercise while the remainder did not. These parameters were compared to the responsiveness of leucocytes, which was determined by their histamine "releasability". 20 healthy children served as controls. Release of histamine induced by calciumionophore aided calcium influx was significantly higher in both groups of asthmatics than in the healthy children (P less than 0.005). Similar findings were obtained by induction of microtubule aggregation due to deuterium oxide (D2O). The S-shaped dose-response relationship with D2O was shifted to the left in the patients with BR to exercise compared to patients without (P less than 0.025). It is concluded that the mean "releasability" of histamine from leucocytes is higher if BR increases. The histamine release due to both stimulants correlated well (P less than 0.01). This suggests that the "releasability" is determined by the responsiveness of the microtubules. To a large degree this may also apply to allergen-induced histamine release, as was revealed from studies with anti-IgE The differences in histamine release found in relation to BR due to exercise were also present if the patients were divided according to BR due to histamine.

Topics: Asthma; Bronchial Diseases; Calcimycin; Calcium; Child; Child, Preschool; Deuterium; Deuterium Oxide; Dose-Response Relationship, Drug; Histamine; Humans; Immunoglobulin E; Leukocytes; Physical Exertion; Respiratory Hypersensitivity; Water; Zinc

Topics: Asthma; Basophils; Bone Marrow Cells; Calcimycin; Cromolyn Sodium; Flavonoids; Histamine Release; Humans; Mast Cells; Neutrophils; Oxidative Phosphorylation; Oxygen Consumption; Quercetin

The effect of auranofin, an oral chrysotherapeutic agent, on histamine release from human basophils was studied. Auranofin inhibited IgE-mediated, anti-IgE-induced histamine release in a dose-dependent fashion with a concentration of drug required to produce 50% inhibition of 1.3 micrograms/ml. This compound also inhibited calcium ionophore A23187 and 12-0-tetradecanoyl-phorbol-13-acetate-induced histamine release with a concentration of drug required to produce 50% inhibition of 1.7 micrograms/ml and 0.9 micrograms/ml, respectively. Auranofin was less active for formyl-L-methionyl-L-leucyl-L-phenylalanine-induced release of histamine with 32 +/- 10% (mean +/- SEM) inhibition at 2 micrograms/ml. Auranofin effects were reversible when leukocytes preincubated with auranofin was washed with buffer. In contrast, gold sodium thiomalate enhanced IgE-mediated histamine release, suggesting that these two compounds exert their actions at different levels. These results suggest that auranofin may be beneficial to patients with allergic diseases such as bronchial asthma.

Topics: Asthma; Auranofin; Aurothioglucose; Basophils; Calcimycin; Gold; Gold Sodium Thiomalate; Histamine Release; Humans; Immunoglobulin E; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate

The hypothesis that bronchial hyperresponsiveness in asthma is influenced not only by environmental but also by intrinsic cellular factors was studied. We compared leukocyte responsiveness in asthmatic children with high (Group A) or low (Group B) bronchial responsiveness and in healthy children (Group C). Leukocyte responsiveness was assessed by the generation of superoxide anion (O-2) and the release of histamine after challenge with calcium ionophore A 12387 in the presence of calcium, and with deuterium oxide (D2O). Bronchial responsiveness was graded on the basis of response to inhaled histamine and exercise. The mean generation of O-2 and mean release of histamine by the calcium ionophore A 12387 and calcium were significantly greater in the asthmatics than in the healthy subjects (p less than 0.05). The mean release of histamine, but not the mean O-2 generation, was greater in Group A than in Group B (p less than 0.025). After D2O challenge a significant difference was found in the mean O-2 generation (p less than 0.05) and the mean histamine release (p less than 0.025) between asthmatic and healthy children. Between Groups A and B, the mean histamine release but not the O-2 generation was significantly different (p less than 0.025). The generation of O-2 and release of histamine were significantly correlated (r = 0.45, p less than 0.01). The results suggest that there is a basic intracellular abnormality that causes increased bronchial hyperresponsiveness.

Topics: Asthma; Bronchi; Bronchial Provocation Tests; Calcimycin; Child; Deuterium; Deuterium Oxide; Female; Histamine; Histamine Release; Humans; Ion Channels; Leukocytes; Male; Microtubules; Physical Exertion; Superoxides; Water

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.

Topics: Asthma; Basophils; Calcimycin; Histamine; Histamine Release; In Vitro Techniques; Influenza A virus; Interferons; Leukocytes; Pollen

Topics: Allergens; Asthma; Bronchi; Calcimycin; Histamine; Humans; In Vitro Techniques; Leukotriene E4; Lung; Muscle Contraction; Pyrilamine; SRS-A; Structure-Activity Relationship

The hypothesis studied is that increased responsiveness in asthma is not limited to the airways. Forty asthmatic children were analysed for their bronchial responsiveness (BR) to exercise. Twenty patients revealed bronchial obstruction after exercise while the remainder did not. These observations were compared with the responsiveness of leucocytes, which was determined by their histamine 'releasability'. Twenty healthy children served as controls. Release of histamine induced by calcium ionophore-aided calcium influx was significantly higher in both groups of asthmatics than in the healthy children (P less than 0 X 005). Similar findings were obtained by induction of microtubule aggregation due to deuterium oxide (D2O). The S-shaped dose-response relationship with D2O was shifted to the left in the patients with BR to exercise compared to patients without (P less than 0 X 025). The slope was increased in both patient groups compared with the healthy children (P less than 0 X 01). It is concluded that the mean 'releasability' of histamine release due to both stimulants correlated well (P less than 0 X 01). This suggests that the 'releasability' is determined by the responsiveness of the microtubules. This may also apply to allergen-induced histamine release, as was revealed from studies with anti-IgE. The differences in histamine release found in relation to BR due to exercise were also present if the patients were divided according to BR due to histamine. A significant relationship existed between the degree of BR to histamine and the responsiveness of the microtubules (P less than 0 X 02).

Topics: Asthma; Asthma, Exercise-Induced; Bronchial Provocation Tests; Calcimycin; Child; Deuterium; Deuterium Oxide; Dose-Response Relationship, Immunologic; Histamine; Histamine Release; Humans; Leukocytes; Water

This study was based on the premise that mediator release plays a role in the pathogenesis of allergen-induced as well as nonallergen-induced asthma. We studied histamine release from human basophils obtained from patients with asthma and from control subjects. These cells were challenged with several different stimuli: goat anti-human IgE-Fc, C5-peptide, N-formyl-methionyl-leucyl-phenylalanine (f-met peptide), Ca++ ionophore A23187, hyperosmolar mannitol, and D2O. Release induced by any one stimulus was unrelated to the response to any other stimulus. The basophils of patients with asthma and control subjects responded similarly to most stimuli: they were significantly less responsive to C5-peptide and f-met peptide, and significantly more responsive to D2O. The results suggest that there is a parameter of releasibility that must be defined for each separate stimulus, and that patients with asthma can be differentiated from normal persons by the response of their basophils to selected stimuli.

Topics: Adult; Asthma; Basophils; Blood Proteins; Calcimycin; Complement C5; Deuterium; Histamine Release; Humans; Immunoglobulin E; In Vitro Techniques; Mannitol; Middle Aged; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Oligopeptides; Steroids

Topics: Antigens; Asthma; Calcimycin; Cromolyn Sodium; Histamine Release; Humans; Immunoglobulin E; Leukocytes; Neutrophils; Piperidines; SRS-A; Thiophenes

Topics: Antigens; Asthma; Autacoids; Calcimycin; Histamine Release; Humans; Lung