Dapsone suppresses human neutrophil superoxide production and elastase release in a calcium-dependent manner.

BACKGROUND: Dapsone (4,4'-diaminodiphenyl sulphone) is a powerful therapeutic tool in many skin diseases including neutrophilic dermatoses. The drug has an outstanding therapeutic efficacy against many skin diseases characterized by neutrophil-rich infiltrates; however, mechanisms of its action are poorly understood. OBJECTIVES: We investigated the effects of dapsone on respiratory and secretory functions of human neutrophils triggered by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the physiological agonist C5a, and phorbol myristate acetate (PMA). METHODS: Human neutrophils were isolated from venous blood obtained from healthy donors. We detected extracellular production of superoxide (O(2) (-)) by cytochrome C reduction assay, and intracellular production of O(2) (-) by flow cytometry. Neutrophil elastase release was measured by the cleavage of the specific elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. Measurement of cytosolic free calcium concentration was performed using the calcium-reactive fluorescence probe, Fluo-3. RESULTS: Dapsone suppressed intra- and extracellular production of O(2) (-) and elastase release triggered by fMLP and C5a, but not by PMA. Both fMLP and C5a signalled the above pathways by inducing calcium influx, but PMA functions bypassed calcium influx. Dapsone was capable of antagonizing the induction of calcium influx. CONCLUSIONS: These findings suggest that one mechanism of the anti-inflammatory action of dapsone is inhibition of calcium-dependent functions of neutrophils including release of tissue-damaging oxidants and proteases in the affected skin.

Blockade of superoxide generation prevents high-affinity immunoglobulin E receptor-mediated release of allergic mediators by rat mast cell line and human basophils.

BACKGROUND: Previous studies have shown that rat peritoneal mast cells and mast cell model rat basophilic leukaemia (RBL-2H3) cells generate intracellular reactive oxygen species (ROS) in response to antigen challenge. However, the physiological significance of the burst of ROS is poorly understood. OBJECTIVE: The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems. METHODS: RBL-2H3 cells were directly stimulated with anti-rat FcepsilonRI alpha-subunit monoclonal antibody (mAb). For the analysis of human cell system, leucocytes were isolated by dextran sedimentation from healthy volunteers or from patients, and challenged either with anti-human FcepsilonRI mAb or with the relevant antigens. Superoxide generation was determined by chemiluminescence-based methods. The releases of histamine and leukotrienes (LT)s were determined by enzyme-linked immunosorben assay (ELISA). RESULTS: Cross-linking of FcepsilonRI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-FcepsilonRI mAb resulted in a rapid generation of superoxide anion, as determined by chemiluminescence using superoxide-specific probes. Similarly, leucocytes from patients generated superoxide anion in response to the challenge with the relevant allergen but not with the irrelevant allergen. Furthermore, diphenyleneiodonium (DPI), a well-known inhibitor of flavoenzymes suppressed the superoxide generation and the release of histamine and LTC4 induced by the anti-FcepsilonRI mAb or by allergen in parallel. CONCLUSION: These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon FcepsilonRI cross-linking either by antibody or by allergen challenge and that blockade of the generation prevents the release of allergic mediators. The findings strongly support the role of superoxide generation in the activation of mast cells and basophils under both physiological and pathological conditions. The findings suggest that drugs regulating the superoxide generation have potential therapeutic use for allergic disorders.

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release.

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.

Diphenyleneiodonium prevents reactive oxygen species generation, tyrosine phosphorylation, and histamine release in RBL-2H3 mast cells.

Mast cells play a central role in immediate allergic reactions mediated by immunoglobulin E. It has recently been reported that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. However, the physiological role of ROS is poorly understood. Here we demonstrate that mast cell model rat basophilic leukemia (RBL-2H3) cells generate ROS in response to antigen and the calcium-ionophore A23187 via activation of diphenyleneiodonuim (DPI)-sensitive enzyme and that blockade of ROS generation by DPI suppresses histamine release induced by either stimulant. Increased tyrosine phosphorylation of pp125(FAK) and a 77-kDa protein coprecipitating specifically with the kinase occurred in parallel with the secretion, and blockade of ROS generation by DPI also suppressed the tyrosine phosphorylation of both proteins. These findings suggest that ROS generated by a flavoenzyme-dependent mechanism may be involved in histamine release through the pp125(FAK) pathway.

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway.

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).

Preparation and characteristics of rumen-bypass microcapsules for improvement of productivity in ruminants.

Rumen-bypass microcapsules were prepared by a spray-dry method for protection against microbial hydrogenation in the rumen (neutral pH). Porous starch was used as the core material, and the microcapsules were prepared by a triple coating of Eudragit E100, AS-HF, and shellac. Capsules were generated with yield of about 48% and a mean particle diameter of 20-30 microm. The microcapsules had high stability in a neutral solution that mimicked a ruminal pH (pH 6.5). Moreover, when microcapsules were incubated in the presence of ruminal microorganisms, about 65% of the microcapsules were resistant to digestion in ruminal fluids, and protection of the inclusion substance was observed. In addition, the efficiency of release of these microcapsules was about 85% within only 30 min in the abomasal environment (pH 3.0).