A23187-induced Degranulation Research Articles

The peels of sacred Lotus (Nelumbo nucifera) rhizomes suppress allergic reactions by inhibiting the A23187-induced degranulation in rat basophilic leukemia (RBL[sbnd]2H3) cells

We isolated ten compounds from methanolic extract of the peels of sacred lotus (Nelumbo nucifera) rhizomes which were identified as β-sitosterol linoleate 1, β-sitosterol 2, lupeol 3, stigmasterol 3-O-β-D-glucoside 4, oleanolic acid 5, betulinic acid 6, pinoresinol 7, 4-hydroxybenzoic acid 8, catechin 9 and gallocatechin 10. All of the isolated compounds from the peels of sacred lotus rhizomes are reported for the first time, and were investigated for their anti-allergic activity. We found that three of them, stigmasterol 3-O-β-D-glucoside 4, oleanolic acid 5 and pinoresinol 7, were capable of inhibiting A23187-induced degranulation in RBL-2H3 cells with IC50 values 0.18 ± 0.01 mM, 0.28 ± 0.06 mM, and 0.27 ± 0.01 mM, respectively. With an exception to 4, compounds 5 and 7 achieved the anti-allergic effect without affecting the cells viability even at higher concentrations with their selectivity indices (SI) being >5. By reducing A23187-induced degranulation, it is suggestive of a mechanism attenuation of Ca2+ elevation. Our findings suggest that, the peels of sacred lotus rhizomes would be beneficial for providing an inexpensive source for the production of bioactive compounds with anti-allergic effect.

Anti-allergic potential of Typhonium blumei: Inhibition of degranulation via suppression of PI3K/PLCγ2 phosphorylation and calcium influx

BackgroundTyphonium blumei Nicolson & Sivadasan (Araceae) is a traditional Chinese medicinal herb possessing detumescent, detoxifying, and anti-inflammatory activities. It is used in Taiwan as a folk medicine to treat cancer and inflammatory diseases. Typhonium blumei is usually not distinguished from Typhonium roxburghii Schott and they are commonly used interchangeably. PurposeTo evaluate and compare the anti-allergic and anti-inflammatory properties of T. blumei and T. roxburghii, their composition profiles and molecular basis of the anti-allergic effect. MethodsThe methanolic plant extracts were partitioned with different solvents to obtain the nonpolar fractions. The anti-allergic activity of the nonpolar fractions was assessed by A23187- and antigen-induced degranulation assays using RBL-2H3 mast cells. Several molecular targets were investigated: FcεRI receptor expression by flow cytometry, calcium influx by live cells imaging fluorescent microscopy, cytokines mRNA expression by RT-PCR, and protein expression by Western blotting. The anti-inflammatory activity was evaluated using superoxide anion and elastase release assays in human neutrophils. TLC, NMR and GC–MS analyses were conducted to evaluate the chemical composition of the fractions. ResultsThe nonpolar fractions of both Typhonium species showed potent inhibitory activity in A23187-induced degranulation assay in RBL-2H3 cells. They also inhibited superoxide production and elastase release in human neutrophils. T. blumei nonpolar fractions inhibited antigen-induced β-hexosaminidase and histamine release. The nonpolar fractions of T. blumei significantly inhibited calcium influx upon activation with either A23187 or an antigen. The fractions did not affect FcεRI receptor expression, mRNA level of IL-4 and MCP-1 cytokine production or MAPK proteins expression, but did suppress the calcium signaling pathway via PI3K/PLCγ2. The active fractions were rich in fatty acids with palmitic, linoleic and α-linolenic acids identified as the major fatty acids in both plants. The content of omega-3 unsaturated fatty acids was higher in T. roxburghii nonpolar fractions compared to T. blumei. ConclusionBoth species possess potent anti-allergic and anti-inflammatory activities. The inhibition of degranulation in mast cells was attributed to calcium influx modulation. The obtained results support the traditional use of T. blumei in the treatment of inflammatory diseases as well as its substitution with T. roxburghii.

Inhibition of Mast Cell Degranulation by Phycoerythrin and Its Pigment Moiety Phycoerythrobilin, Prepared from <i>Porphyra yezoensis</i>

Phycoerythrin from the red alga Porphyra yezoensis has been demonstrated to possess antioxidative activity and is expected to have anti-inflammatory activity. In this study, we examined the effects of phycoerythrin and its pigment moiety, phycoerythrobilin, on the degranulation of mast cells. Phycoerythrin (0.01 mg/mL) and phycoerythrobilin (1 micromol/L) significantly inhibited calcium ionophore (A23187) induced release of beta-hexosaminidase from rat basophilic leukemia (RBL-2H3) cells (approximately 50% and 40%, respectively). Phycoerythrobilin significantly suppressed IgE-antigen-stimulated degranulation of RBL-2H3 cells, while phycoerythrin had no effect. A23187-induced degranulation of peritoneal exudate cells (PEC) prepared from rats treated orally with phycoerythrin (4 mg/kg) or phycoerythrobilin (1 micromol/kg) was significantly reduced compared to control PEC. These results suggest that phycoerythrin and phycoerythrobilin exhibit anti-inflammatory activities through the suppression of mast cells degranulation in vivo.

Protein kinase Cδ functions downstream of Ca 2+ mobilization in FcεRI signaling to degranulation in mast cells

Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.

Modulation of mast cell functions by in vitro ozone exposure.

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.

Influence of teicoplanin and vancomydn on degranulation by polymorphonuclear leucocytes stimulated by various agonists: an in-vitro study

The interaction between vancomycin and teicoplanin (50 and 100 mg/l) on human neutrophils was studied using fMLP- and A23187-induced degranulation of elastase, beta-glucuronidase and vitamin B12-binding-protein. Each of these proteins is a marker of a different population of granules. fMLP-induced degranulation of beta-glucuronidase was significantly impaired by pre-incubating the neutrophils with teicoplanin (without affecting cell viability) although the inhibition was at most 23% at 100 mg/l, a concentration not achieved in the serum of patients after normal doses. Calcium ionophore-induced degranulation of beta-glucuronidase and elastase were slightly impaired (Student two-tailed; P less than 0.1) by both glycopeptides (beta-glucuronidase) and teicoplanin only (elastase). Again, inhibition remained below 25%. Vancomycin and teicoplanin at high concentrations, seldom achieved in patients, can moderately impair neutrophil degranulation.

Dissociation of the 47-kilodalton protein phosphorylation from degranulation and superoxide production in neutrophils.

The aim for the present studies is to examine the relationship between the phosphorylation of the 47-kDa protein and some neutrophil responses such as degranulation, the synergistic effect of PMA on calcium ionophore-induced degranulation, superoxide generation, and the priming of the oxidative burst produced by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate 13-acetate (PMA). Incubation of neutrophils with the protein kinase inhibitor 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7) inhibits the phosphorylation of the 47-kDa protein produced by PMA and fMet-Leu-Phe but does not affect lysozyme release induced by the same stimuli or fMet-Leu-Phe-induced N-acetyl-beta-glucosaminidase release. Furthermore, the synergistic effect of PMA on the A23187-induced degranulation is not inhibited by H-7. Also, pretreatment of the cells with H-7 inhibits superoxide production produced by PMA but not by fMet-Leu-Phe. The inhibitory effect of H-7 is more pronounced on the rate than on the extent of PMA-induced superoxide release. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, HA-1004, a less potent protein kinase inhibitor, has no inhibitory effect on superoxide generation produced by fMet-Leu-Phe or PMA. In the case of superoxide production, the addition of low concentrations of PMA to rabbit neutrophils primes these cells to the subsequent stimulation by fMet-Leu-Phe, dramatically increasing the effect. Conversely, low concentrations of fMet-Leu-Phe prime the cells to PMA stimulation. The stimulation by PMA of cells primed with fMet-Leu-Phe is inhibited by H-7. Moreover, the priming effect by PMA is also inhibited by H-7. This inhibition is less pronounced at a low concentration of PMA. On the other hand, in the case of human neutrophils, the priming effect of PMA is not inhibited by H-7. These results suggest several points. First, phosphorylation of the protein identified on two-dimensional gel electrophoresis as having a molecular weight of 47 kDa and PI of 4.9 is not necessary for degranulation produced by either PMA or fMet-Leu-Phe. Second, the phosphorylation of the 47-kDa protein is not necessary for superoxide generation, at least in the case of fMet-Leu-Phe and possibly for other stimuli. Third, in rabbit neutrophils, both the priming and stimulation of superoxide production with PMA are inhibited by H-7. In human neutrophils, the priming by PMA is not affected by H-7.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of some compounds on human neutrophil degranulation and leukotriene B 4 and thromboxane B 2 synthesis

The effects of aspirin, indomethacin, phenylbutazone, eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA) and 3-amino-1-[3-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C) on human neutrophil degranulation induced by A23187 and F-Met-Leu-Phe (FMLP) have been studied. These effects have been compared with those on A23187 induced leukotriene B 4 (LTB 4) and thromboxane B 2 (TXB 2) synthesis by these cells to elucidate the relationship between LTB 4 formation and degranulation. All compounds inhibited TXB 2 synthesis by 50% at concentrations between 0.0016 and 50 μM. The synthesis of LTB 4 was inhibited by 50% by ETYA (1.9 μM) and by NDGA (0.52 μM). Degranulation induced by A23187 and FMLP was inhibited by 50% by ETYA (16 and 11 μM respectively) and by NDGA (1.5 and 6.5 μM respectively). In the case of ETYA the concentrations required to inhibit degranulation were significantly higher than those required to inhibit LTB 4 synthesis. In contrast, BW755C inhibited LTB 4 synthesis by 50% at 2.8 μM but did not affect A23187-induced degranulation and was only a weak inhibitor of FMLP-induced degranulation (50% inhibition at 89 μM). The effects of the above compounds on the ω-oxidation of LTB 4 by human neutrophils has also been studied to investigate the mechanism of action of these compounds. None of the above compounds affected the metabolism of LTB 4 by these cells suggesting that their actions are not as non-specific anti-oxidants. These data indicate that human neutrophil degranulation induced by FMLP and A23187 is independent of LTB 4 synthesis.