A23187-induced Histamine Release Research Articles

Effect of an Anti-SRS-A Agent, NZ-107, on Airway Responses Induced by Ovalbumin and A23187 in the Guinea-pig

The effects of the anti-SRS-A agent NZ-107 on antigen-(ovalbumin) and calcium ionophore A23187-induced airway responses in the guinea-pig have been investigated. In the presence of 5 microM indomethacin, NZ-107 (3 microM) did not affect the peak response in ovalbumin-induced contraction but did inhibit the prolonged response following the peak response in the tracheal strip. A higher concentration of NZ-107 (10 microM) completely blocked both peak and prolonged responses. Inhibitory effects of NZ-107 on ovalbumin responses were less in the lung parenchymal strip. The potency of NZ-107 in inhibiting ovalbumin-induced tracheal contraction was not changed in the absence of indomethacin but was reduced in the presence of 45 mM serine-borate, an inhibitor of the conversion of LTC4 to LTD4. NZ-107 inhibited A23187-induced contractions in both tracheal and parenchymal strips but in both cases the inhibitory potency was less than that on ovalbumin response. NZ-107 was a more potent inhibitor of ovalbumin-induced SRS-A release than histamine release in lung fragments but was ineffective in inhibiting A23187-induced SRS-A and histamine release. NZ-107 at a concentration of 10 microM more effectively inhibited LTC4- and histamine-induced tracheal contractions than it did LTC4 in the presence of 45 mM serine-borate. These results suggest that NZ-107 selectively inhibits antigen-induced SRS-A responses in airway tissues of the guinea-pig.

950 THE EFFECT OF HONEY ON MAST-CELL DEGRANULATION: A POSSIBLE ROLE IN PAINFUL BLADDER SYNDROME/INTERSTITIAL CYSTITIS (PBS/IC)

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2011950 THE EFFECT OF HONEY ON MAST-CELL DEGRANULATION: A POSSIBLE ROLE IN PAINFUL BLADDER SYNDROME/INTERSTITIAL CYSTITIS (PBS/IC) Brian Birch, Tamsin Murray, Alan Cooper, and Bashir Lwaleed Brian BirchBrian Birch Southampton, United Kingdom More articles by this author , Tamsin MurrayTamsin Murray Southampton, United Kingdom More articles by this author , Alan CooperAlan Cooper Portsmouth, United Kingdom More articles by this author , and Bashir LwaleedBashir Lwaleed Southampton, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.895AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Painful Bladder Syndrome/Interstitial cystitis (PBS/IC) is a debilitating condition of uncertain pathogenesis and a low treatment success. Honey has long been used for the treatment of wounds and has more recently been demonstrated to have beneficial effects on wound healing. The mechanisms include antibacterial properties, cytokine interaction and anti-oxidant effects. Mast cell activity appears to be an important mediator of inflammation in PBS/IC. It is postulated that honey has an effect on mast cell degranulation that would render it a potentially useful agent for intravesical use in this condition. METHODS The LAD2 mast cell line was used to study both spontaneous and provoked degranulation, measured as histamine release. The cells were pre-treated with a range of medicinal grade honeys and challenged with the mast cell activator Calcium ionophore A23187 and released histamine measured (by an ELISA assay). Control preparations included a clover nectar, a honey-like mixed sugar syrup and agents commonly used topically against PBS/IC (Uracyst™, Cystistat™, Gepan™, RIMSO-50™ and heparin). RESULTS Both spontaneous and A23187 induced histamine release were inhibited by honey in a dose dependent fashion from LAD2 cells. There were marked variations in this effect between honey types (between 60% and 100%). Syrup and clover nectar inhibited release by 40% and 36% respectively. Histamine release was not inhibited by Uracyst™, Cystistat™ or Gepan™ and only marginally by RIMSO-50™ and heparin; the maximum inhibition obtained by any drug at any dilution was 24%. CONCLUSIONS A constituent of most honeys inhibits spontaneous and stimulated mast-cell degranulation in a cell line model. This effect was not reproduced using other agents currently employed in clinical practice. Honey, therefore, has distinct potential as an intravesical agent both used alone and in combination. It warrants further investigation as a possible intravesical agent in the treatment of PBS/IC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e381-e382 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Brian Birch Southampton, United Kingdom More articles by this author Tamsin Murray Southampton, United Kingdom More articles by this author Alan Cooper Portsmouth, United Kingdom More articles by this author Bashir Lwaleed Southampton, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of Hop Extracts on Nasal Rubbing and Sneezing in BALB/c Mice

The effects of hop extracts (Humulus lupulus L.) on histamine release from rat peritoneal mast cells and human basophilic KU812 cells were studied. Hop water extract (HWE) and XAD-4 50% methanol fraction of HWE (MFH) inhibited histamine release from rat mast cells induced by compound 48/80 at concentrations of 100 and 10 mug/ml, respectively. Almost the same findings were observed with A23187-induced histamine release from KU812 cells. Next, we studied the effects of hop extracts on antigen-induced nasal rubbing and sneezing in sensitized BALB/c mice. HWE caused a significant inhibition of nasal rubbing and sneezing at a dose of 500 mg/kg. MFH also inhibited nasal rubbing and sneezing dose-dependently. A significant difference was observed from 100 mg/kg in nasal rubbing and 200 mg/kg in sneezing. The effects of both extracts became clear after repeated administration. HWE and MFH significantly inhibited both nasal rubbing and sneezing, respectively, after consecutive treatment for 15 d at smaller doses compared with single administration. This finding indicates that the active component of hop is included in MFH, which was absorbed to Amberlite XAD-4 and eluted with 50% methanol. These results clearly demonstrated that hop extracts may be effective in the relief of symptoms of allergic rhinitis.

Lysophosphatidic Acid Induces Histamine Release from Mast Cells and Skin Fragments

The present study examined whether lysophosphatidic acid (LPA) induces histamine release and the role of Rho-associated protein kinase (ROCK) in histamine release utilizing Y-27632, an inhibitor of ROCK. Rat peritoneal mast cells and mouse skin fragments were challenged with LPA; subsequently, histamine contents were measured by spectrofluorometric assay. LPA-induced histamine release from mast cells and skin fragments occurred in a time- and dose-dependent manner Pretreatment with Y-27632 inhibited LPA-induced histamine release in a dose-dependent fashion. LPA-induced histamine release was not influenced by calcium-free conditions; however, A23187-induced histamine release decreased significantly. TMB-8, an intracellular calcium antagonist, dose-dependently inhibited the histamine release under these conditions. Additionally, Y-27632 scarcely affected A23187-induced histamine release. These findings suggest that LPA-induced histamine release may be attributable to calcium release from intracellular stores. Moreover, ROCK participates in the extracellular calcium-independent process of LPA-induced histamine release.

The inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin G

Inhibitors of mast cell tryptase and chymase can be effective as mast cell stabilising compounds. Lactoferrin has been reported to inhibit tryptase activity, but its actions on other serine proteases of mast cells and its potential to alter mast cell function are not known. We have examined the ability of lactoferrin to inhibit mast cell tryptase, chymase and cathepsin G, and investigated its potential to modulate the activation of human mast cells. Enzymatically dispersed cells from human skin, lung and tonsil were challenged with anti-IgE or calcium ionophore A23187, following incubation with recombinant human lactoferrin, and histamine release determined. IgE-dependent histamine release from skin mast cells was inhibited by up to 50% following incubation with lactoferrin (50 or 500 nM). Tonsil mast cells were also stabilised by lactoferrin, but not those from lung. Calcium ionophore A23187-induced histamine release was not altered by lactoferrin. A double-labelling immunocytochemical procedure revealed the presence of lactoferrin in 4–6% of mast cells, and this proportion increased to 40% following incubation with lactoferrin. Lactoferrin did not inhibit cleavage of synthetic substrates by tryptase and chymase directly, though it was able to diminish the ability of heparin to stabilise tryptase. Cathepsin G activity was inhibited by lactoferrin. The ability of lactoferrin to inhibit IgE-dependent activation of human mast cells and modulate protease activity suggests that the release of this neutrophil product may have a role in the downregulation of allergic inflammation.

Release of non-mast cell histamine from rat aorta

We previously reported that A23187 induces release of histamine from bovine intrapulmonary vein and provided pharmacological evidence against an involvement of mast cells as the source of histamine. This study was conducted to test more definitively the hypothesis that histamine is released from non-mast cell sources in blood vessels. The effects of A23187 on release of histamine were determined using rat aorta which does not contain mast cells. Aortic rings were mounted for recording of isometric tension, and following exposure to A23187 or vehicle, histamine in the bathing media was measured using enzyme immunoassay. A23187 (100 nmol/l – 10 μmol/l) induced concentration-related release of histamine from rings with endothelium. The accumulation of histamine in the bathing media induced by 10 μM A23187 reached plateau at 60 min (6.2 ± 1.1 pmol/mg) and was markedly and significantly higher than vehicle control (0.4 ± 0.1 pmol/mg, p < 0.05). Destruction of endothelium significantly inhibited A23187-induced histamine release (5.5 ± 1.5 pmol/mg with endothelium, 1.1 ± 0.3 pmol/mg without endothelium, p < 0.05). The results demonstrate that A23187 induces release of histamine from rat aorta which does not contain mast cells and that the release of histamine is largely dependent on the presence of endothelium.

Effects of nitric oxide and hydrogen peroxide on histamine release from RBL-2H3 cells

We have studied the effect of nitric oxide (NO) and hydrogen peroxide (H2O2), two reactive oxygen species (ROS) on histamine release (HR) from RBL-2H3 cells, a rat mucosal-type mast cell line. Marked HR was elicited by antigen (DNP-HSA), calcium ionophore A23187, sodium fluoride or phospholipase C, but not with compound 48/80 or 1,2-dioctanoyl-sn-glycerol. The NO-synthase substrate l-arginine and its inactive enantiomer (d-arginine), each on its own, induced a small but significant increase in HR above the basal level. However, the NO-donors (sodium nitroprusside or NaNO3) or the NO-synthase inducer lipopolysaccharide did not induce HR. Moreover, methylene blue (MB), which inhibits guanylate cyclase and Nω-nitro-l-arginine (L-NA), an inhibitor of NO synthase, were also without effect on either the basal HR or the l-arginine-induced HR. HR induced by A23187, DNP-HSA, sodium fluoride or phospholipase C was markedly reduced by MB, but mildly by L-NA (both at 1–100 μM). H2O2 (0.01–1.0 mM) on its own did not induce HR, but it had a potent inhibitory effect on DNP-HSA- or A23187-induced HR, which was not reversed by L-NA (1–100 μM). Taken together, it seems that neither the stimulatory nor the inhibitory effects of the NO-related compounds on HR can be attributed to NO, but rather to other mechanisms. The inhibition of HR by H2O2 also does not involve NO and suggests a negative feedback regulatory role for the peroxide in the allergic inflammation.

Effects of cold stress and epinephrine on degranulation of peritoneal mast cells in rats.

We studied the effects of histamine liberators calcium ionophore A23187 and substance 48/80 on mast cells during cold stress and epinephrine load. Under the effect of both stress factors, ionophore A23187-induced histamine release from mast cells underwent more pronounced changes than that stimulated by substance 48/80. Cold stress and epinephrine load produce different changes in functional activity of Ca2+ channels in mast cell membranes.

Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells.

Flavonoids have a variety of activities including anti-allergic activities, and are known to inhibit histamine release from human basophils and murine mast cells. The effects of luteolin, a flavone, on the immunoglobulin (Ig) E-mediated allergic mediator release from human cultured mast cells (HCMCs) were investigated and compared with those of baicalein and quercetin. HCMCs were sensitized with IgE, and then treated with flavonoids before challenge with antihuman IgE. The amount of released mediators was determined as was mobilization of intracellular Ca2+ concentration, protein kinase C (PKC) translocation and phosphorylation of intracellular proteins were detected after anti-IgE stimulation. Luteolin, baicalein and quercetin inhibited the release of histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), and granulocyte macrophage-colony stimulating factor (GM-CSF) from HCMC in a concentration-dependent manner. Additionally, the three flavonoids inhibited A23187-induced histamine release. As concerns Ca2+ signalling, luteolin and quercetin inhibited Ca2+ influx strongly, although baicalein did slightly. With regard to PKC signalling, luteolin and quercetin inhibited PKC translocation and PKC activity strongly, although baicalein did slightly. The suppression of Ca2+ and PKC signallings might contribute to the inhibition of mediator release. The activation of extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinase (JNK), that were activated just before the release of LTs and PGD2 and GM-CSF mRNA expression in IgE-mediated signal transduction events, were clearly suppressed by luteolin and quercetin. In contrast, the flavonoids did not affect the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway. These results indicate that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation.

ATP modulates anti-IgE-induced release of histamine from human lung mast cells.

Adenosine 5'-triphosphate (ATP) is released from the cytoplasm under physiologic and pathophysiologic conditions and enters the extracellular space, where it acts on a group of recently cloned cell-surface receptors termed P2-purinoceptors (subtypes P2X and P2Y). We examined the effects of extracellular ATP, uridine triphosphate (UTP), the stable ATP analogues alpha,betamethylene-ATP (alpha,betamATP), beta,gammamethylene-ATP (beta,gammamATP), and 2-methylthio-ATP (2mSATP), and adenosine (10(-6)-10(-3) M) on histamine release from human lung mast cells (HLMC) induced by anti-IgE and the calcium ionophore A23187. None of the nucleotides or adenosine directly induced histamine release. Adenosine exhibited a bimodal effect, enhancing histamine release at 10(-6) to 10(-4) M (P > 0.05, NS) and inhibiting it at 10(-3) M (P < 0.05). ATP (10(-4) M) enhanced anti-IgE-induced histamine release (10.9 +/- 2.7% to 19. 2 +/- 2.9%, n = 20, P < 0.01), but not ionophore A23187-induced histamine release (n = 10). The adenine nucleotides consistently enhanced anti-IgE-induced histamine release; the rank order for this action was: ATP > 2mSATP > alpha,betamATP > beta,gammamATP, suggesting mediation by a P2Y-purinoceptor subtype. The selective P2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid failed to influence the effect of ATP, further supporting P2Y-purinoceptor mediation of anti-IgE-induced histamine release. UTP, an agonist at P2Y-purinoceptors, also significantly enhanced anti-IgE-induced histamine release. Application of the reverse transcription-polymerase chain reaction indicated that HLMC constitutively express the messenger RNAs encoding the P2Y1- and P2Y2-purinoceptor subtypes, and not that encoding the P2X7-purinoceptor (i.e., P2Z), a subtype implicated in ATP-induced histamine release in rodent peritoneal mast cells. The data produced in the study suggest that ATP plays an important modulatory role in histamine release from HLMC, and that it may therefore be mechanistically involved in human allergic/asthmatic reactions.

Inhibitory Effects of Sasa senanensis Rehder Extract (SE) on Calcium-Ionophore A23187-Induced Histamine Release From Rat Peritoneal Exudate Cells

The effects of Sasa senanensis Rehder extract (SE) by alkaline hydrolysis on histamine release from rat peritoneal exudate cells (PECs) were examined. Preincubation with SE for 5 min suppressed calcium-ionophore A23187-induced histamine release in a concentration-dependent manner. A23187 evoked a quick increase in cytoplasmic free calcium ([Ca2+]i) levels in the presence of extracellular calcium. Preincubation with SE also had an inhibitory effect on calcium influx increases induced by A23187. These results indicate that SE prevents degranulation from rat PECs by inhibiting [Ca2+]i level elevation.

Effects of toxin A from Clostridium difficile on mast cell activation and survival.

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-alpha). In contrast, 1 microg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 microg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

Exogenous Nitric Oxide Regulates the Degranulation of Human Basophils and Rat Peritoneal Mast Cells

This study was designed to investigate whether anti-IgE-induced or ionophore A23187-induced histamine release from human basophils is regulated by exogenous nitric oxide (NO), and to assess some similarities between the effect of NO on basophils and that on rat peritoneal mast cells (RPMC). The NO donor, sodium nitroprusside (SNP), inhibited A23187-induced histamine release from crude human basophils and crude RPMC in a dose-dependent fashion. This downregulation was still observed when SNP was washed out just before the cell stimulation, indicating that the effect of SNP was irreversible. The downregulation disappeared in both purified cell populations after the removal of contaminating cells. However, when purified cells were preincubated with SNP in the presence of 5 mM N-acetylcysteine (NAC), increasing the bioavailability of NO, the downregulation was recovered. The presence of NAC significantly augmented the downregulation of SNP on A23187-induced histamine release from both crude cell populations. In contrast, SNP had no effect on anti-IgE-induced histamine release from either crude or purified basophil preparation in the absence of NAC, and SNP plus NAC inhibited anti-IgE-induced histamine release from both cell preparations. The same results were obtained with crude and purified RPMC preparations under the same conditions. These results show that SNP similarly downregulated exocytosis of basophils and RPMC, and acquired the potent effect in the presence of NAC, indicating that exogenous NO plays a part in the regulation of basophil and mast cell activation.

A three-step procedure for the purification of human basophils from buffy coat blood.

We report a method for basophil purification from buffy coats, which avoids positive selection of the cells and gives rise to good purity, yield and functional integrity of the cells. Buffy coat blood (concentrated leukocyte fraction derived from 450 ml venipuncture donations) obtained from healthy blood donors (n = 51). Basophils were enriched by a three-step process starting with Ficoll density centrifugation (1.6 +/- 0.1% basophil purity) followed by counter current centrifugal elutriation (17.7 +/- 1.4% basophil purity). The final stage involved negative selection using Dynal immunomagnetic beads directed against CD2, CD14, CD16 and CD19 positive cell contaminants. Functional integrity of which was assessed by comparing the anti-IgE or calcium ionophore A23187 induced histamine release from basophils obtained from each enrichment step. Furthermore, basophil morphology was investigated using light and electron microscopy. The final mean basophil purity of 67.3 +/- 1.4% with a yield of 3.5 +/- 0.5 x 10(6) basophils and a recovery of 21.8 +/- 2.4% was achieved. Net histamine release from basophils stimulated with optimal concentrations of anti-human IgE was 39.1 +/- 6.5% after Ficoll centrifugation, 41.6 +/- 7.7% following elutriation and 35.7 +/- 6.8% from the final purified fraction. Additionally, basophils enriched with our method showed intact morphology by electron microscopy and were functionally active to non-immunological stimulation. These results compare favourably with previous studies, which have often required the use of positive selection via the Fc epsilon RI receptor, which may result in cell degranulation, or cell sorting, which cannot be applied to large cell numbers. Our method provides a reproducible technique for basophil enrichment when large numbers of functionally intact basophils are required.

Effect of H1-antihistamines on histamine release from dispersed canine cutaneous mast cells

Abstract Objective Because of the implication of histamine in canine atopic dermatitis, H1-antihistamines may provide a valid alternative to glucocorticoid therapy. In vitro study of these drugs prior to clinical testing can allow the most promising compounds to be selected for trials and render trials with drugs of doubtful efficacy unnecessary. Sample Population Isolated canine cutaneous mast cells. Procedure Cells were preincubated with antihistamines at increasing concentrations and incubated with concanavalin A (1,000 µg/ml), calcium ionophore A23187 (1 µM), and substance P (100 µM). Compound 48/80 was not used because it proved to be cytotoxic. Results Generally, significant prodegranulating effect was not observed for most of the studied agents. Only terfenadine increased spontaneous histamine release at concentrations &gt; 30 µM. Cetirizine did not block histamine release at any of the studied concentrations. Ketotifen had a low inhibitory effect only at the highest concentration (100 µM) after concanavalin A- (23.6 ± 2.8%) and calcium ionophore A23187- (29.8 ± 3.0%) induced release. Terfenadine caused a concentration-dependent inhibitory effect after ionophore A23187- (48.1 ± 2.2%) and concanavalin A- (28.9 ± 2.3%) activation, but was inactive against substance P-induced release. In contrast, loratadine had potent dose-dependent inhibition of concanavalin A- and ionophore A23187-induced histamine release, with maximal effect of 85.6 ± 3.1 % and 62.6 ± 4.7%, respectively, at 100 µM concentration. After substance P activation, histamine release was only slightly inhibited by loratadine (14.8 ± 1.1%). Conclusions This study documents the behavior of isolated canine cutaneous mast cells in the presence of nonimmunologic stimulation. Using this in vitro method, we were able to determine that loratadine is the only antihistamine that has potent inhibition of histamine release from dog cutaneous mast cells without a substantial prodegranulating effect. Loratadine is, therefore, a good candidate for clinical testing. (Am J Vet Res 1997;58:293–297)

Effects of Clostridium botulinum C2 toxin-induced depolymerisation of actin on degranulation of suspended and attached mast cells.

We studied the effects of C. botulinum C2 toxin, which ADP-ribosylates G-actin, on mast cell degranulation. C2 toxin inhibited degranulation of suspended rat peritoneal mast cells induced by compound 48/80 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) maximally by about 50 and 90%, respectively. Inhibition by C2 toxin occurred in a time- and concentration-dependent manner. Half-maximal inhibition of DNP-BSA-induced degranulation by C2 toxin occurred at about 0.015 ng/ml, whereas stimulation of mast cells induced by compound 48/80 was half-maximally inhibited at 0.15 ng/ml C2 toxin. C2 toxin also inhibited stimulated [3H]serotonin release from suspended mast cells. Phorbol 12-myristate 13-acetate (PMA)-induced histamine release of suspended mast cells was inhibited by C2 toxin by about 80-90%. C2 toxin had no effect on calcium ionophore A23187-induced histamine release. Toxin treatment of mast cells caused ADP-ribosylation of actin and depolymerisation of F-actin. Attachment of mast cells, which largely increased the diameter of the subcortical actin network, reduced degranulation stimulated by compound 48/80, antigen and calcium ionophore but not by PMA. Opposite to its effect on suspended cells, in adherent mast cells C2 toxin stimulated degranulation by compound 48/80, antigen, and calcium ionophore but not by PMA. The data indicate that mast cell degranulation and responsiveness towards the actin-depolymerising C2 toxin depend largely on mast cell attachment.

Heterogeneity of Signal Transduction Mechanisms in Human Basophils and Human Skin Mast Cells

Basophils and mast cells play a crucial role in immunological and allergic processes due to the release of inflammatory mediators such as histamine. It has been suggested for a long time that the histamine release (HR) from these cells is closely related to protein kinase C (PKC) activity. However, the distinct role of PKC with its large variety of isozymes in different cell types and the actions of these isozymes in HR still remain unclear. Therefore, in the present study, we compared the effects of the two PKC inhibitors 7-O-methyl-UCN-01 (UCN-01-Me) and NPC 15%7 as well as two PKC activators, bryostatin 1 and 2, on anti-IgE and Ca²⁺-ionophore-induced HR from human basophils and isolated human skin mast cells (HSMC). In both HSMC and basophils, anti-IgE-induced HR was inhibited by PKC inhibitor UCN-01-Me pre-incubation dose-dependently. In stark contrast, A23187-induced HR was unaffected by UCN-01-Me in both cell types. In our experiments, the inhibitory efficacy of the compound NPC 15437 on HR was much lower than that of UCN-01-Me and showed no statistical significance. Both bryostatins 1 and 2 produced good dose-dependent inhibition of HR from HSMC stimulated with anti-IgE, whereas HR from basophils was potentiated with these compounds. The same effects were observed with basophils stimulated with A23187, where potentiation of HR was up to fourfold of the control at the highest concentrations of bryostatins, while HSMC showed a slight decrease in HR compared to non-bryostatin-treated controls. Basophils and HSMC showed very clear differences in HR when directly stimulated with the bryostatins, since no HR was observed from HSMC while in basophils the HR increased up to 47% of total histamine at the highest concentrations of bryostatins (1 µmol/1). HR from basophils was observed to be strictly dose-dependent. The differences in the cell reactions of the two cell types incubated with these four compounds indicate distinct biochemical roles of PKC in the cascades leading to degranulation of the cells. Furthermore, the experiments with UCN-01-Me support the hypothesis of PKC-β to play a substantial positive modulatory role for the degranulation of immunologically stimulated basophils.

Inhibitory effect of 1α,25-dihydroxyvitamin D 3 on mast cell proliferation and A23187-induced histamine release, also accompanied by a decreased c-kit receptor

Inhibitory effect of 1α,25-dihydroxyvitamin D 3 on mast cell proliferation and A23187-induced histamine release, also accompanied by a decreased c-kit receptor

Inhibitory effect of 1α,25-dihydroxyvitamin D3 on mast cell proliferation and A23187-induced histamine release, also accompanied by a decreased c-kit receptor

Using mouse peritoneal mast cells, we investigated the effects of 1 alpha,25-dihydroxyvitamin D3 (calcitriol) on mast cell proliferation and histamine release. Calcitriol did not affect IL-3/IL-4-dependent mast cell proliferation, but it selectively inhibited stem cell factor-dependent mast cell proliferation and colony formation. Immunohistochemical and immunoblot analyses revealed that calcitriol treatment reduced expression of purified peritoneal mast cell c-kit protein. Using a mast cell line, MC/9, both c-kit protein and c-kit mRNA transcript were seen to be reduced following calcitriol treatment. Calcitriol also reduced histamine release induced by calcium ionophore A23187. In contrast, anti-IgE antibody-dependent histamine release was not affected by calcitriol. Our results indicate that calcitriol inhibits mast cell proliferation and A23187-induced histamine release that might be associated with a decreased expression of c-kit receptor.

Ionic regulation of human basophil releasability. III. Effects of Na + and Ca 2+ on histamine release induced by different stimuli

The effects of Na + and Ca 2+ ions on histamine release from human basophils stimulated by anti-IgE, N-formyl-methionyl-leucyl-phenylalanine (FMLP), 4β-phorbol 120myristate 13-acetate (PMA) and Ca 2+ ionophore A23187 were evaluated. Isosmotic replacement of Na + in the extracellular medium with the non-permeant Na + analogue choline + or with glucose led to a significant increase in anti-IgE- (1/5000: 43.7±7.3% in high Na + vs 68.9±7.3% in low Na + mean± sem, n=8, P<0.001), FMLP- (1 μ m: 37.9 ± 2.3 % vs 49.5 ± 4.3%, n=8 P<0.01) and PMA-(160 n M: 12.7±0.9% vs 27.3±4.3%, n=8, P<0.05) induced histamine release, whereas A23187-induced histamine release was reduced (1 um: 90.4±2.4% vs 45.4±3.4%, n=8, P<0.0001). The progressive increase in extracellular Na + concentration was accompanied by a decrease of basophil response to anti-IgE, FMLP and PMA; in contrast, A23187-induced histamine release was up-regulated by Na + The Na +/H + exchanger monensin, in the concentration range of 10 −8−10 −4 M, exerted a dose-dependent inhibitory effect on anti-IgE-, FMLP- and PMA-induced histamine release, but not on A23187-induced histamine release. Extracellular Ca 2+ up-regulated the histamine release induced by all the above stimuli. Removal of extracellular Na + lowered the requirement of extracellular Ca 2+ for anti-IgE, FMLP- and PMA-induced histamine release. In contrast with previous observations showing that Na + supports histamine release from rat peritoneal mast cells and rat basophilic leukaemia cells, these results indicate that Na + strongly inhibits histamine release from human basophils stimulated by antiIgE, FMLP and PMA, whereas it enhances Ca 2+ ionophore A23187-induced histamine release. The effects of Na + which are probably related to modulation of membrane potential and/or intracellular pH, vary depending on the cell type and the stimulus employed for cell activation.