Inhibitory effects of apple polyphenol on induced histamine release from RBL-2H3 cells and rat mast cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 micrograms/ml and 25 micrograms/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Heterogeneous effects of protamine on human mast cells and basophils

To investigate the mechanisms of anaphylactoid reactions to protamine, we have examined the in vitro effects of increasing concentrations of protamine (10(-6)-3 x 10(-4) mol litre-1) on the release of preformed (histamine and tryptase) and de novo synthesized (peptide leukotriene C4 (LTC4) or prostaglandin D2 (PGD2)) mediators from human basophils and mast cells isolated from lung parenchyma, heart, skin and synovial tissues. Protamine 10(-6)-3 x 10(-4) mol litre-1 induced release of histamine, but not de novo synthesis of LTC4 from basophils. At concentrations from 10(-5) to 3 x 10(-4) mol litre-1 it induced histamine release from human heart (mean 6.5 (SEM 1.5)%), skin (17.7 (4.1)%) and to a lesser extent from synovial mast cells, but not from lung mast cells. Protamine also caused the release of tryptase from heart mast cells (12.8 (3.2) micrograms/10(7) cells), but did not induce de novo synthesis of LTC4 and PGD2 from lung and skin mast cells. In these experiments cross-linking of IgE by anti-IgE caused release of LTC4 or PGD2 from human basophils or mast cells. These results demonstrate that protamine acted as an incomplete secretagogue, causing the release of preformed mediators from human basophils and mast cells.     

Studies on cytosolic 5SrRNA-L5 protein particles (5SRNP) of rat liver

Effects of antiallergic drugs on bradykinin-induced histamine release and intracellular Ca2+ release from peritoneal mast cells were studied in rats. Bradykinin caused a concentration-dependent histamine release as well as Ca2+ release from the intracellular Ca store of peritoneal mast cells. Antiallergic drugs used in this study showed an inhibition of not only histamine release but also Ca2+ release. The Ca2+ release from the intracellular Ca store induced by bradykinin was more sensitive to antiallergic drugs than histamine release from mast cells. Mequitazine and terfenadine caused potent inhibitory effects on both responses, whereas effects of ketotifen and cromolyn sodium were relatively weak. In conclusion, histamine release from mast cells and intracellular C2+ release induced by bradykinin were inhibited by antiallergic drugs similar to those induced by substance P and compound 48/80.     

Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium

Endogenous proteolytic enzymes have been shown to be potential sources of airway inflammation inducing proinflammatory cytokine release from respiratory epithelial cells; however, whether any of the exogenous proteases from important allergen sources such as the house dust mite present in our environment behave in a similar fashion is unclear. To this end, we have investigated whether the mite cysteine and serine proteolytic allergens, Der p 1 and Der p 9, respectively, induced cytokine production from primary human bronchial epithelial cells and from the epithelial cell line BEAS-2B. Cells were exposed to mite proteases, and cells or supernatants were assayed for cytokine release, cytokine mRNA expression, and modulation of intracellular calcium ion concentration. Both proteases induced concentration- and time-dependent increases in the release of granulocyte-macrophage (GM)-CSF, IL-6, and IL-8 as well as an increase in the expression of IL-6 mRNA. Cytokine release and mRNA expression were first observed at 8 h and 2 h after protease exposure, respectively. The minimum concentration of each protease that was required to stimulate GM-CSF, IL-6, and IL-8 release was approximately 10 ng/ml. Cytokine release was initiated by 1 to 2 h of protease exposure, although maximum concentrations were detected only after a 24-h incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by both proteases at concentrations of > 2 microg/ml. The proteases also stimulated changes in the intracellular calcium ion concentration. All mite protease-induced phenomena were inhibited using appropriate protease inhibitors. These results suggest that the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium.     

Detection of micronuclei in peripheral erythrocytes of Cyprinus carpio exposed to metallic mercury

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 micrograms/ml), calcium ionophore A23187 (1 microM), and substance P (100 microM). 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 > 30 microM. Cetirizine did not block histamine release at any of the studied concentrations. Ketotifen had a low inhibitory effect only at the highest concentration (100 microM) 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 microM 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.     

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.     

Methacholine induces wheal-and-flare reactions in human skin but does not release histamine in vivo as assessed by the skin microdialysis technique

A number of investigations have indicated that cholinergic agonists release histamine from isolated mast cells and suggested that cholinergic stimulation releases histamine in vivo. The purpose of this study was to investigate whether the cutaneous wheal-and-flare reaction induced by methacholine challenge in human skin involves histamine release as measured by the skin microdialysis technique. Five hollow dialysis fibers were inserted intradermally in forearm skin in eight healthy subjects. Each fiber was perfused with Kreb's-Ringer bicarbonate at a rate of 3 microliters/min. Dialysates were collected in 2-min fractions before skin challenge and for 20 min after intradermal injection of methacholine 10(-3)-10(-1) M, the vehicle, and a positive control, codeine phosphate 0.3 mg/ml. Histamine was assayed spectrofluorometrically. Methacholine caused a statistically significant dose-related wheal-and-flare reaction, the flare reaction to methacholine 10(-1) M being comparable with that seen with codeine 0.3 mg/ml. No significant histamine release was observed with methacholine, cumulative histamine release of 16 +/- 8 nM by methacholine 10(-1) M being similar to vehicle responses of 15 +/- 9 nM. Histamine release by codeine was 2524 +/- 435 nM. In conclusion, methacholine-induced wheal-and-flare reactions in human skin appeared not to involve histamine release from skin mast cells.     

Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue. CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment.     

Identification of peptide fragments chemically cross-linked in cytochrome c oxidase from thermophilic Bacillus PS3

It has been suggested that differential histamine-releasing activity of an IgE-dependent histamine-releasing factor (HRF), which has recently been cloned, is related to carbohydrate difference in the IgE molecule. Lectins are able to recognize specific glycoforms and might therefore be useful in characterizing the proposed heterogeneity of IgE molecules. As one test of this hypothesis, we examined the histamine release potency of several well-characterized lectins on basophils passively sensitized with serum containing IgE molecules that support HRF-induced histamine release (IgE+) or serum that does not support release by this stimulus (IgE-). Histamine release was induced by challenging basophils with different concentrations of concanavalin A, Lens culinaris (LcH), and Pisum sativum (PSA). Dose-response curves revealed that LcH caused 30% histamine release at 2 micrograms/ml with IgE+ sensitized cells, whereas the same release with IgE- cells required sixfold higher concentrations. Similar values for PSA showed a sevenfold difference. With concanavalin A, the selectivity was reversed in that it was fourfold more active on IgE- -sensitized cells. Another pair of sera (IgE+ vs IgE-) revealed the same result for concanavalin A, but no difference occurred in LcH and PSA induced release between the IgE+ - and IgE- -sensitized cells. These contrasting findings with different pairs of IgE+ -and IgE- -containing sera indicated that the lectins LcH and PSA are not able to discriminate between IgE+ -and IgE- -sensitized cells as does HRF and therefore cannot be used to further define the proposed heterogeneity of IgE. this conclusion was supported by experiments in which basophil preparations from donors possessing natural sensitivity or insensitivity to HRF (having IgE+ or IgE- on their surface) were examined fro their response to these lectins. No difference was found in the sensitivity of the cells to challenge with LcH or PSA, and the response to concanavalin A was the opposite of that found when passively sensitized cells were used (30% histamine release at 0.9 vs 3.5 micrograms/ml for IgE+ vs IgE- donors, respectively). We conclude that oligosaccharide-specific lectins do not differentiate between HRF-reactive and HRF-nonreactive IgE molecules on basophils.     

The effects of anti-asthma drugs on mediator release from cultured human mast cells

BACKGROUND: A method for generating human mast cells in vitro was recently established. Little is known about the pharmacological profiles of allergic mediator release from cultured mast cells. OBJECTIVE: The main objective was to investigate the nature of cultured mast cells from a pharmacological point of view. We examined the effect of anti-asthma drugs on the release of histamine, sulfidoleukotrienes (LTs) and prostaglandin D2 (PGD2) from the cultured mast cells. METHODS: Using the method established by Saito et al. we cultured cord blood mononuclear cells in the presence of 80 ng/mL stem cell factor (SCF), 50 ng/mL interleukin-6 (IL-6) and 300 nmol/L prostaglandin E2 (PGE2), and obtained almost pure (> 99%) mast cells. We sensitized cultured mast cells with immunoglobulin E (IgE)-rich serum, and then treated them with some anti-asthma drugs before challenge with anti-human IgE. Released histamine, LTs and PGD2 were measured by high-performance liquid chromatography, commercial enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA) systems, respectively. RESULTS: The cultured mast cells released histamine, LTs and PGD2 following immunological stimulation through IgE. The mast cell stabilizing agents disodium cromoglycate (DSCG, 1 mmol/L) and azelastine (100 micromol/L) significantly inhibited the release of these three mediators. The beta-adrenoceptor agonists isoproterenol, salbutamol, and clenbuterol also inhibited all three mediators' release in a concentration-dependent manner. The non-selective and selective phosphodiesterase (PDE) inhibitors theophylline, rolipram, and cilostazol had no significant effect on mediator release at clinically useful concentrations. BAY x 1005 (a 5-lipoxygenase-activating protein inhibitor) inhibited the LTs release, whereas indomethacin (a cyclo-oxygenase I and II inhibitor) and NS-398 (a cyclo-oxygenase II inhibitor) inhibited PGD2 release. CONCLUSIONS: The present results indicate that cultured mast cells release histamine, LTs and PGD2 following IgE crosslinking. Anti-asthma drugs showed a characteristic suppression of the release of each mediator. The suppressive actions of these drugs are similar to their pharmacological actions on human lung mast cells. These results suggest that cultured mast cells are useful for the analysis of function and pharmacological profiles of lung mast cells.     

The effect of okadaic acid on histamine release, cell morphology and phosphorylation in rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells

To study the involvement of serine/threonine phosphatase in the signal transduction of mast cells, we examined the effects of okadaic acid (OA), an inhibitor of type-1 and -2A phosphatase on histamine release, cell morphology, calcium influx and protein phosphorylation of rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells. OA inhibited IgE-mediated histamine release from RBL-2H3 cells and human basophils dose-dependently. There was a remarkable enhancement of IgE-mediated histamine release when rat peritoneal mast cells were suboptimally challenged. OA induced a marked change of cell features, detached RBL-2H3 cells from plastic well and kept the 18- and 68-kD proteins phosphorylated. These findings show that phosphatase may play a role in the modulation of secretion in mast cells.     

Inhibition of immediate type allergic reactions by the aqueous extract of Kum-Hwang-San

We studied the effect of aqueous extract of Kum-Hwang-San (KHS) on mast cell-mediated immediate type allergic reactions. KHS (1-100 microg/site) inhibited concentration-dependently mast cell-dependent ear swelling response induced by compound 48/80 (200 microg/site) in mice by both topical and intradermal application. KHS (0.1-100 microg/site) inhibited concentration-dependently passive cutaneous anaphylaxis induced by anti-dinitrophenyl (DNP) IgE in rats by both topical and intradermal application. KHS also inhibited concentration-dependently the histamine release from the rat peritoneal mast cells (RPMC) by compound 48/80 and anti-DNP IgE. Moreover, KHS had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha (TNF-alpha) secretion from RPMC. These results indicate that KHS inhibits immediate type allergic reactions by inhibition of histamine release and TNF-alpha secretion from mast cells in vivo and in vitro.     

IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells

We have examined 3-week-old alcian blue positive cells (putatively mast cells) derived from mouse bone marrow for their expression of Fc epsilon RI. Using an indirect method of sensitizing the cells with immunoglobulin E (IgE) antibody (anti-DNP IgE) and detecting the level of bound IgE antibody by flow cytometry, we found that prolonged culture (1-5 days) with IgE, but not IgG, increased the total receptor density 6 +/- 1.9 fold. During the same period, histamine release in response to antigen (DNP-HSA) increased approximately 6-fold while the cell's response to either thrombin or ionomycin remained constant. The greatest up-regulation occurred in the first 2 days of culture. Using 2.4G2 to detect Fc epsilon RII RIII, we could not detect any up-regulation of this receptor. Culturing the cells for 1 h after sensitization did not result in any loss of cell surface IgE, suggesting a reasonably high affinity binding similar to that expected for Fc epsilon RI. This up-regulation was completely inhibited by co-culture with 2 micrograms/ml cycloheximide. These data suggest that IgE is capable of inducing a significant, protein synthesis, dependent up-regulation of its own high affinity receptor on mast cells/basophils.     

Transient sensitization to house-dust mites: a study on the influence of mite exposure and sex

This study aimed to evaluate the prognosis of a previous positive skin test to house-dust mites (HDM) in relation to environmental exposure. A total of 115 children, 50 from Stockholm and 65 from northern Sweden, all with a previous (average 2.5 years) positive Phazet (Pharmacia AB, Uppsala, Sweden) skin prick test (SPT) to extracts of Dermatophagoides pteronyssinus (Dpt) and/or D. farinae (Df) were included. Dust samples were collected from the children's mattresses, and the total (Dpt, Df, and D. microceras [Dm]) amount of major mite allergen was measured by ELISA (50 children) and expressed as microgram allergen per gram of dust, or was measured by microscopy (65 children). The results of microscopic mite counts were transformed to approximate allergen levels as 2 micrograms equals 100 mites per gram of dust. Of 115 originally SPT-positive children, only 48 (48%) remained positive at retest, while the majority (58%) were SPT negative after 2 years. Among the 67 converted children, 11 were still exposed to mite allergen, but only to low concentrations (only one converted child being exposed above the suggested threshold level [TLV] of 2 micrograms/g), compared to 15/48 children still SPT positive who were exposed above the TLV. This shows that continued mite exposure is a major risk factor (OR = 30, CI 4.8-184) for continued positive SPT to HDM. A minor risk factor for continued sensitization was sex, boys having a higher risk than girls (OR = 2.2, CI 1.0-4.8). In conclusion, a surprisingly high rate of SPT conversion occurred, mainly as a result of a favorable indoor environment with low exposure to HDM and, to a lesser degree, as a result of sex. The present results support the view that the risk level of exposure is 2 micrograms mite allergen per gram of dust.     

Effect of contignasterol on histamine release induced by anti-immunoglobulin E from rat peritoneal mast cells

In rat peritoneal mass cells induced by anti-immunoglobulin E (anti-IgE), contignasterol (1) inhibited histamine release in a dose-dependent manner. On the other hand, a reduction product of contignasterol (2) did not inhibit histamine release from mast cells induced by anti-IgE.     

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

BACKGROUND: The level of histamine in nasal lavage fluid has been used as an index of mast cell/basophil activation in a number of studies. Obviously, such an index can only be valid if changes in the secretory activity of nasal glands do not affect the level of histamine in lavage fluid (i.e. hypersecretion, without a simultaneous activation of mast cells/basophils in the nasal mucosa, must not increase the level of histamine). OBJECTIVES: To asses the effect of nasal hypersecretion on histamine levels in lavage fluid. METHODS: Nasal challenges were performed with methacholine and allergen in grass pollen-allergic patients and non-allergic controls. Nasal lavage fluid was collected before and repeatedly for nine hours after nasal challenge, and the level of histamine was compared with that of a specific mast cell-derived enzyme, tryptase. In addition, the effect of methacholine on basophils was examined in vitro. RESULTS: Allergen challenge of allergic patients produced sneezing and a significant increase in histamine and tryptase levels, whereas challenge of non-allergic subjects produced no such response. Interestingly, challenge with methacholine also induced a significant increase in histamine levels. This increase was seen in both allergic and non-allergic subjects and it was not associated with any sneezing or increase in tryptase levels, indicating that mast cells were not activated. Furthermore, stimulation of basophils with methacholine did not induce any histamine release in vitro. CONCLUSIONS: Apparently, there exists a pool of histamine in the human nose that can be transferred to lavage fluid during glandular hypersecretion. The source of this histamine is yet to be identified. As the level of histamine seems to be affected by the secretory activity of nasal glands, we question the use of this single mediator as an index of mast cell/basophil activation in nasal lavage studies.     

Shock and coagulation disorders in systemic mastocytosis

A 41-year-old man was admitted in circulatory shock of unknown aetiology (systolic pressure 50 mm Hg) and marked reddening of the upper part of the body as well as maculopapular rash over the whole body. After 1500 ml of colloidal solution had been infused the blood pressure rose to a level at which the patient's condition was no longer at risk. He reported having had a similar attack of flushing and circulatory collapse during the last few years, each time after drinking 3-41 of beer. Laboratory tests showed thromboplastin time 56%, partial thromboplastin time 130 s and thrombin time > 180 s. Three hours after admission the coagulation times had further deteriorated, but had become normal within 20 hours. After rest and after a provocation (hot bath) the serum concentrations were: heparin 0.21 U/ml and 0.85 U/ml; histamine 1.9 micrograms/ml and 2.3 micrograms/ml; serotonin 23 micrograms/ml and 38 micrograms/ml. Histological examination of an iliac crest bone marrow biopsy revealed dense collections of mast cells, as seen in systemic mastocytosis. A skin biopsy was diagnostic of urticaria pigmentosa.     

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.     

Dual regulation of the Na+/H(+)-exchange in rat peritoneal mast cells: role of protein kinase C and calcium on pHi regulation and histamine release

1. The purpose of this study was to compare the actions of phorbol 12-myristate 13-acetate (PMA) and ionomycin on Na+/H+ exchange activation and histamine release to that of compound 48/80 in order to study the possible relationship between pHi and secretion of histamine in rat peritoneal mast cells. 2. Resting pHi in mast cells suspended in a bicarbonate-free physiological salt solution amounted to 6.73 +/- 0.05 (mean +/- s.d., n = 52). 3. PMA (20 nM) induced a substantial but rather slow increase in pHi. This response was very sensitive to inhibition by staurosporine, very sensitive to inhibition by 5-(N,N-hexamethylene)amiloride (HMA), insensitive to the absence of extracellular calcium (without EGTA), and sensitive to partial depletion of intracellular calcium with EGTA. 4. Ionomycin (1 microM) induced a biphasic change in pHi that was sensitive to inhibition by HMA, insensitive to staurosporine. In the absence of extracellular calcium using EGTA, the biphasic response disappeared, leaving only a slow, and diminished change in pHi. 5. The effects of ionomycin and PMA on pHi were additive. 6. Addition of the secretagogue compound 48/80 (1 microgram ml-1) increased pHi, substantially, delta pHi amounting to 0.29 +/- 0.05 pH-units (n = 4). The biphasic pHi-response was insensitive to the absence of extracellular calcium (without EGTA). The initial fast response in pHi was, however, inhibited by HMA, not staurosporine. 7. The finding that staurosporine and HMA each inhibited approximately half of the compound 48/80-induced pHi-response, whereas both inhibitors completely abolished the compound 48/80-induced pHi-response seems to indicate that two independent pathways for the activation of the Na+/H+ exchange were stimulated by compound 48/80. 8. The histamine release induced via both PKC activation (using PMA) and calcium (using ionomycin) were much larger than the sum of each activation pathway, whereas in the absence of extracellular calcium using EGTA, the histamine release in response to PMA and ionomycin was completely abolished. 9. The compound 48/80-induced histamine release was partially sensitive to inhibition by HMA (approximately 30% inhibition) and partially sensitive to inhibition by staurosporine (approximately 50% inhibition). Preincubation with staurosporine and HMA before stimulation with compound 48/80 showed the same degree of inhibition as observed after staurosporine alone, even though this combination of drugs completely inhibited the pHi-response. Furthermore, compound 48/80-induced histamine release was not dependent on the presence of extracellular calcium (with and without EGTA). 10. In spite of the similarities in second messenger pathways for pHi regulation and histamine release, it is, however, not very likely that these two processes are directly related. It is, however, possible, that an increase in pHi plays a permissive, rather than an essential role for histamine release in rat peritoneal mast cells. This hypothesis was supported by the finding that preincubation with the Na+/H+ exchange-inhibitor HMA inhibited 30% of the compound 48/80-induced histamine secretion.     

Factors influencing immediate responses to intradermal allergen administration in patients with respiratory allergy

BACKGROUND: Immediate skin reactions to allergens are influenced by several factors, such as the amount of administered allergen, the level of specific IgE, releasability of mast cells and hyperresponsiveness of the target organ. METHODS: For the evaluation of factors influencing immediate skin response to intradermal allergen administration, we measured the wheal size 15 min after intradermal injection of 0.01-0.02 ml of the following agents: whole-body extract of Dermatophagoides farinae, 1,000 allergy units/ml; histamine, 0.1 mg/ml, and codeine sulfate, 0.09% in saline, and determined total IgE level, specific IgE and IgG subclass antibodies to D. farinae in 53 patients with respiratory allergy. RESULTS: Multiple regression analysis for factors influencing wheal size after intradermal injection of D. farinae, specific IgE antibody level to D. farinae and wheal size after intradermal administration of histamine showed statistically significant results (R2 = 0.42739, p = 0.0000; R2 = 0.50243, p = 0.0185, respectively). Multiple regression analysis for factors influencing wheal size after intradermal administration in the group with high levels of specific IgE to D. farinae (RAST class 3 or more) showed that wheal size after intradermal administration of codeine was the only factor exerting a statistically significant influence (p = 0.0119). CONCLUSION: Based on the above results, we can state that immediate responses to intradermal allergen administration were influenced by the level of specific IgE and hyperresponsiveness of the target organ to histamine, but that the immediate skin allergic responses in the presence of high levels of specific IgE were partially but significantly influenced by the releasability of skin mast cells.