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Background: Anaphylaxis is a severe and potentially lethal allergic reaction whose incidence is increasing. Murine models can elucidate the underlying mechanisms and pave the way for appropriate therapeutic options. However, differences in strains and protocols hamper comparisons of data between researchers. We performed a parallel study of clinical and immune responses with 2 strains of mice, BALB/c and C3H/HeOuJ, in an allergen-induced systemic anaphylaxis protocol. Both strains have been widely used in allergy models, although they have not been compared in an intraperitoneal systemic model. Methods: Groups of 5-week-old female BALB/c and C3H/HeOuJ mice were intraperitoneally sensitized with peanut in the presence of adjuvants. Specific immunoglobulin (sIg) G1, sIgG2a, sIgE, total IgE, histamine release, and specific stimulated splenocyte cytokines, interleukin (IL)-4, IL-5, IL-10, IL-12, IL-13, and interferon (IFN)-γ, were assessed. At week 6, mice were intraperitoneally challenged with peanut. Anaphylaxis was evaluated by recognition of clinical symptoms and changes in body temperature. Results: All peanut-sensitized mice induced sIg and developed anaphylactic symptoms upon challenge. Nonetheless, the C3H/HeOuJ strain demonstrated earlier and persistently higher sIgG1 and sIgG2a production, elevated sIgE, and more severe clinical symptoms and histamine release than the BALB/c strain. In contrast, BALB/c exhibited higher release of IL-4, IL-5, IL-10, IL-13, and IFN-γ. Conclusions: Both models are suitable for studying anaphylaxis. Consequently, they could be used in research on the pathogenesis and therapy of anaphylaxis. However, according to the type of study performed, differences in the specific clinical, humoral, and cellular responses to antigens have to be considered.

Background: Shrimp-derived allergen has a serious impact on people’s health. Chitosan oligosaccharide (COS) has anti-allergic action but its function on shrimp allergen-induced allergy and related molecular mechanisms remain unclear. Methods: COS and its degrees of polymerization (DP) were selected to interact with shrimp tropomyosin (TM) and IgE was measured. A mouse model of food allergy was established by receiving shrimp TM intraperitoneally. The models were treated with different concentrations of COS. Fecal and serum histamine, serum IgE, IgG1 and IgG2a, and inflammatory cytokines were measured. Results: The main products for COS were DP2–6 with the contents of 6, 40, 26, 16, and 4%, respectively, and reacted with shrimp TM increasingly when COS DP was increased. Severe symptoms of food allergy were observed in the TM group (diarrhea, anaphylactic response, and rectal temperature). In contrast, COS treatment improved these symptoms significantly (p < 0.05). The sensitized mice were desensitized after they were treated with 1 mg/kg COS. COS treatment significantly reduced serum IgE and IgG1 levels, and increased IgG2a levels (p < 0.05). COS consumption decreased fecal and serum histamine. COS treatment reduced Th2 cytokine (IL-4, IL-5, and IL-13) levels and increased the Th1 cytokine (IFN-γ) level (p < 0.05). Conclusions: COS showed anti-allergy properties by regulating the levels of Th1 and Th2 cytokines.

Background: Sweating can worsen atopic dermatitis (AD). The purpose of this work was to study the associations between reactivity to autologous sweat and the clinical severity of AD as well as investigate the possible wheal-inducing factors of sweat. Methods: Intracutaneous skin tests with autologous sweat were performed on 50 AD patients and 24 control subjects. In skin biopsies, tryptase and PAR-2 were enzyme and immunohistochemically stained. The associations between skin test reactivity and sweat histamine concentration, tryptase or chymase activity levels, tryptase or PAR-2 expression and AD clinical severity or IgE levels were investigated. Results: The wheal reactions in the intracutaneous tests with autologous sweat were positive, weakly positive and negative in 38, 34 and 28% of the AD patients, respectively, and in 4, 46 and 50% of the healthy controls, respectively (p = 0.008). In AD, the wheal reaction was associated significantly with clinical severity, serum total and specific IgE levels and sweat tryptase activity, but not with sweat histamine and chymase. In nonlesional AD skin, the percentage of PAR-2+ mast cells (MCs) or the number of tryptase+ MCs did not differ significantly between the intracutaneous test reactivity groups. Conclusion: Reactivity to autologous sweat correlates with the clinical severity of AD, and tryptase may be one of the factors involved in the sweat-induced wheal.

Background: Mast cells are key immune effector cells which release chemokines, proteases, and other inflammatory mediators upon activation by immunological stimuli. The aim of this study was to investigate the effects of co-releasing proteases on the kinetics of release of the chemokine monocyte chemoattractant protein-1 (MCP-1) in immunoglobulin E (IgE)-mediated activation of human mast cells. Methods: Homogenous populations of mature and functional primary human mast cells were generated from CD34+ progenitors originated from buffy coats of healthy adult donors. The releases of MCP-1 from human mast cells in basal conditions and in response to FcεRI cross-linking were assessed at different time points. The effects of different types of protease inhibitors on MCP-1 release from these mast cells under stimulated or unstimulated conditions were also investigated. Results: Cultured human mast cells released MCP-1 in basal conditions and its levels increased in a time-dependent manner. When stimulated by FcεRI cross-linking, the levels of MCP-1 detected in the medium gradually decreased over time after the initial peak induction. Such a decline in MCP-1 levels after IgE-dependent activation was completely prevented by pretreatment with a cocktail of protease inhibitors or the specific tryptase inhibitor APC366. Conclusions: Direct regulation of MCP-1 expression by co-release of tryptase in cultured human mast cells upon IgE-dependent activation demonstrates a role of the serglycin:serine protease axis in modulation of inflammatory reactions through proteolytic degradation of mediators such as chemokines.

Background: Immediate hypersensitivity reactions to clavulanic acid (CLV) seem to be on the increase. Diagnosis is mainly based on skin testing and the drug provocation test (DPT), procedures that are not risk free. The aim of this study was to evaluate whether the histamine release test (HRT) could help evaluate patients with selective hypersensitivity to CLV. Methods: Eighteen patients with immediate selective hypersensitivity reactions to CLV (positive skin tests to CLV but negative to the major and minor determinants of benzylpenicillin and amoxicillin; negative DPT to benzylpenicillin and amoxicillin) and 21 controls with tolerance to CLV were included. Direct and passive HRT, using patient whole blood or ‘IgE-stripped' donor blood sensitized by patient serum, respectively, were performed by stimulating the blood with CLV, and basophil histamine release was detected by fluorometric determination. Results: The clinical symptoms were anaphylaxis (n = 6), urticaria (n = 9) and urticaria-angioedema (n = 3). The median time interval between the reaction and the study was 225 days (interquartile range, IQR: 120-387.5) and between drug intake and the development of symptoms 30 min (IQR: 6.25-30). We obtained similar data for both the direct and passive HRT, with a sensitivity and specificity of 55 and 85%, respectively, a positive predictive value of 76% and a negative predictive value of 69%. Conclusions: The sensitivity of both the direct and passive HRT for diagnosing patients with immediate allergy to CLV is less than 60%. However, the passive HRT has the advantage that it is based on the testing of serum samples that can be handled more easily than fresh blood samples.

Background: Zinc (Zn) affects many aspects of immune function, including thymic development and the activities of immune cells. Zn is also involved in many steps of high-affinity IgE receptor (FcεRI)-induced mast cell (MC) activation, which is required for degranulation and cytokine production. Intracellular Zn levels increase in mouse MCs after FcεRI stimulation. We previously reported that Zn distribution in a human MC line, LAD2, changed dramatically following FcεRI aggregation with synchrotron radiation microbeams. However, the kinetics of Zn distribution and the underlying mechanisms following FcεRI cross-linking remain unknown. Methods: We used cord-blood-derived MCs and LAD2 cells. Degranulation was assessed by β-hexosaminidase (β-hex) release. Extracellular Zn levels were determined by inductively coupled plasma atomic emission spectrometry or based on the fluorescence intensity of a Zn indicator. We also used RNAi to knockdown ZnT1 expression. mRNA expression levels were determined by real-time RT-PCR. Results: Zn was rapidly released from human MCs after FcεRI aggregation. The kinetics and optimal conditions for FcεRI cross-linking for Zn release were different from those for degranulation. Treating LAD2 cells with an intracellular Ca2+ chelator significantly inhibited IgE-mediated β-hex release but not Zn release. We investigated IgE-mediated β-hex and Zn release with specific inhibitors of signaling pathways. Zn and β-hex release were partly correlated with but also partly independent of IgE. Knockdown of the Zn efflux transporter, ZnT1, significantly inhibited Zn release from human MCs. Conclusions: Our results indicate that IgE-dependent Zn release from human MCs involves signaling cascades that are distinct from those of degranulation. Thus, Zn may have a unique function as a mediator of allergic inflammation.

The etiology of chronic urticaria (CU) remains elusive. Histamine-releasing factor (HRF) is reported to have a proinflammatory role in asthma and immediate hypersensitivity of the skin. The aim of this study was to examine the role of HRF in the pathogenesis of CU. Forty patients with CU were enrolled and their serum HRF concentrations were determined by ELISA. The results demonstrated that the concentrations of HRF and HRF-reactive IgE in the CU group were significantly higher than those in the control group, and there was a significant linear correlation between HRF and HRF-reactive IgE concentrations (r = 0.859, p < 0.001) in CU patients. Additionally, the HRF-reactive IgE concentration was significantly correlated with the disease activity (r = 0.693, p < 0.0001). HRF and HRF-reactive IgE alone failed to activate LAD2 cells. After being primed by the patient sera with the highest IgE concentrations and stimulated by HRF, β-hexosaminidase can be released from LAD2 cells. Our findings suggest that the synergistic actions of HRF and HRF-reactive IgE may play important roles in the pathogenesis of CU.

Background: The prevalence of allergic diseases is globally increasing. We have previously described that glycomacropeptide (GMP), a bioactive milk peptide, has therapeutic value in experimental models of skin hypersensitivity, anaphylaxis, and asthma, as it prevents an excessive T helper type 2 cell immune response. The aim of this study was to analyze the effect of GMP on key elements directly involved in the development or control of allergy, in order to improve the precise knowledge about its mechanism of action. Methods: Rats were systemically sensitized with ovalbumin and orally treated with GMP. Levels of Lactobacillus, Bifidobacterium, and Bacteroides were analyzed in their feces. Splenocytes were isolated and the production of transforming growth factor (TGF)-β by allergens was measured. Intradermal skin reactions were developed to evaluate in vivo activation of mast cells. Peritoneal mast cells were isolated and activated by the allergen, and histamine secretion was determined. Results: GMP administration increased the amount of intestinal Lactobacillus and Bifidobacterium of allergen-sensitized animals after 3 days of treatment. The increase in Bacteroides was also significant, but only after 17 days of GMP administration. Ten days after treatment cessation, Lactobacillus and Bacteroides were still elevated. GMP intake also elevated the production of TGF-β in the splenocytes of sensitized animals. In addition, treatment with GMP attenuated mast cell activation by the allergen and inhibited histamine secretion, without affecting the number of mast cells. Conclusions: The prebiotic action of GMP on allergy-protective microbiota, an increase in TGF-β production, and a reduction in mast cell response to allergens are novel mechanisms that explain the antiallergic activity of GMP.

Many techniques are being used to examine the status of circulating human basophils including the enhanced expression of a variety of cell surface proteins. There is accumulating evidence that there are at least two compartments containing these activation marker proteins but there are only some indications for the signaling requirements for each of the compartments. This study began with published reports by other investigators who potentially dissociated CD63 expression from anaphylactic degranulation with the p38 inhibitor, SB203580, a possible falsification of a previously proposed hypothesis regarding CD63 expression. To explore the signaling requirements for CD63, a variety of pharmacological agents were used to inhibit or enhance 4 endpoints of basophil activation. First, it was found that inhibition of both histamine release and CD63 expression with SB203580 was concordant. But it was also found that this agent had no effect on increased expression of CD203c and CD11b. Actin polymerization inhibitors caused marked enhancement of CD63 expression (concordant with their effects on degranulation) with no effect on expression of CD203c and CD11b. The third generation syk inhibitor, NVP-QAB205, showed a 5-fold lower potency for inhibiting expression of CD203c and CD11b than for CD63. Finally, while desensitization of CD11b and CD203c expression occurs, it is slower than desensitization of the CD63 response. Taken together, these various observations demonstrate a marked difference in the early signaling requirements for the CD11b/CD203c compartment and CD63 degranulation and provide support for the hypothesis that CD11b and CD203c reside in a similar compartment.

Background: Atopic dermatitis (AD) is the most common allergic inflammatory skin disease. The activation of innate immunity by house dust mite (Dermatophagoides farinae extract, DFE) allergen plays an important role in the pathogenesis of AD. We previously showed the inhibitory effect of an extract of Amomum xanthioides on allergic diseases, and isolated 1,2,4,5-tetramethoxybenzene (TMB) as a major active component. In this study, we investigated whether TMB relieves DFE-induced allergic inflammation symptoms. Methods: We established a DFE-induced allergic inflammation model in BALB/c mice by repeated skin exposure to DFE. To define the underlying mechanisms of action, we used a tumor necrosis factor-α and interferon-γ-activated human keratinocytes (HaCaT cell line) and mouse keratinocytes (3PC cell line) cell line model. Results: Oral administration of TMB suppressed allergic inflammation symptoms, such as histopathological analysis and ear thickness, in addition to serum IgE, DFE-specific IgE and IgG2a levels. TMB decreased the serum histamine levels and tissue infiltration of inflammatory cells, including mast cells and eosinophils. TMB also inhibited CD4+IFN-γ+, CD4+IL-4+, and CD4+IL-17A+ lymphocyte expansion in the draining lymph nodes and expression of the Th2 cytokines in the ear tissue. TMB significantly inhibited the expression of cytokines and chemokines by the downregulation of the mitogen-activated protein kinases and nuclear factor of activated cytoplasmic T cells in HaCaT cells. Conclusions: TMB improved DFE-induced allergic inflammation by suppressing the production of proinflammatory cytokines and chemokines. Our results suggest that TMB might be a potential therapeutic agent for AD.

Background: The diagnosis of food allergy (FA) is made by oral food challenge tests (OFCs) that occasionally produce serious symptoms in patients; therefore, whether to perform OFCs should be carefully considered. The utility of the histamine release test (HRT) in the diagnosis of childhood FA has not been fully examined. Methods: Sixty-four subjects with suspected hen’s egg allergy, cow’s milk allergy (CMA), and wheat allergy (WA) were enrolled. The diagnosis of FA was made based on the outcomes of OFCs or a convincing history of symptoms after food ingestion within 6 months before or after sample collection. HRT was performed using an HRT Shionogi kit. The threshold of histamine release (HRT threshold), which was defined as the minimum concentration of food antigen to induce a 10% net histamine release, was analyzed in association with FA diagnosis. Results: Receiver operating characteristic analysis showed that the HRT threshold was useful in the diagnosis of heated egg allergy (HEA), raw egg allergy (REA), CMA, and WA. We were able to determine the cutoff value for the HRT threshold in relation to outcomes of OFCs. The cutoff value was 6 ng/ml of egg white antigen in HEA and REA (p < 0.01), 40 ng/ml of milk antigen in CMA (p < 0.01), and 500 ng/ml of wheat antigen in WA (p < 0.05). The efficiency was 70.3% for HEA, 78.0% for REA, 77.6% for CMA, and 70.7% for WA. Conclusions: We conclude that the HRT threshold measurement for egg white, milk, and wheat antigen is related to outcomes of OFCs and is useful in determining when OFCs should be performed.

Background: Seminal vesicle protein number 4 (SV-IV) is a small, basic, multifunctional, intrinsically disordered secretory protein synthesized in large amounts by rat seminal vesicle epithelium under androgen transcriptional control. SV-IV-immunorelated proteins occur in other rat tissues and in humans. Methods: The in vitro effect of SV-IV on human FcΕRI+ cells was investigated by standard immunologic, biochemical and molecular biology procedures. Results: SV-IV-induced histamine release from human basophils and lung mast cells without any influence on leukotriene C4 release and cell migration. The histamine release rate was slower compared with that induced by anti-IgE, the temperature dependence of the event being similar. SV-IV-induced histamine release was Ca2+-dependent, suggesting a physiological interaction of the protein with FcΕRI+ cells. SV-IV and anti-IgE acted synergistically on the histamine release. SV-IV did not induce de novo synthesis of cytokines and growth factors (transforming growth factor-β1, interleukin-10, interleukin-13, tumor necrosis factor-α, vascular endothelial growth factor A) in FcΕRI+ cells. Conclusions: SV-IV protein induces in human FcΕRI+ cells the release of histamine, a proinflammatory, antiapoptotic and immunosuppressive biogenic amine. These data: (1) are consistent with the antiapoptotic and immunosuppressive properties of SV-IV; (2) confirm a regulatory feature of SV-IV on mammal inflammatory reactivity by either inhibiting the arachidonate cascade pathway or stimulating proinflammatory cytokine release from lymphocyte/monocytes and histamine from FcΕRI+ cells; (3) raise the possibility of a protective role of SV-IV on implanting hemiallogenic blastocysts against maternal reactive oxygen species and immunological attacks at the uterine implantation site.

Background: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. Objective: Determination of the levels of LTP in a wide range of apple cultivars. Methods: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). Results: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson’s correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4–253.2 (sELISA), 2.7–120.2 (cELISA) and 0.4–47.3 µg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. Conclusions: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, β-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

Background: Omalizumab is an established add-on therapy efficacious in allergic diseases with additional anti-inflammatory activity in the treatment of asthma. The evaluation of responders to anti-IgE treatment is critical to maximize benefit/risk/cost ratio. The aim of the study was to monitor the efficacy of anti-IgE treatment by ex vivo basophil histamine release. Methods: Seventeen patients with allergic asthma were enrolled and received omalizumab at a dose of ≧0.016 mg/kg/IgE every 4 weeks. Histamine release from basophils was evaluated fluorometrically after dose-dependent allergen challenge at baseline and after 16 weeks of treatment. Maximal histamine release and cellular sensitivity to the allergen were calculated. Clinical measurements consisted of body plethysmography, skin prick test, β2-agonist usage, serum free IgE levels, peripheral eosinophils and investigator ratings of global evaluation of treatment effectiveness. Results: Maximal histamine release and cellular sensitivity to the allergen were significantly decreased in the omalizumab group compared to placebo. These changes were accompanied by significant changes in the clinical markers airway resistance, β-agonist usage, skin prick test wheal area and investigator ratings of global evaluation of treatment effectiveness. Conclusions: Omalizumab therapy decreases basophil histamine release and cellular sensitivity with high effectivity of 95.8% (median). The decline of ex vivo basophil responses does not always parallel individual clinical improvement. Basophil-based stimulation tests should be further evaluated before being regarded as useful parameters monitoring omalizumab therapy.

Background: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of Gαi3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569–1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of Gαi3 fused with importation sequences, affect mast cell function. Methods: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. Results: We demonstrate that out of three importation sequences tested only Gαi3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to Gαi2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. Conclusion: ALL1, a transducible Gαi3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Background: The major storage protein in soybean seed is β-conglycinin and this protein has been identified as being responsible for food-allergic reactions in several species. However, the mechanism through which β-conglycinin induces an allergic reaction has not yet been elucidated. In addition, assessing the antigenic activity of β-conglycinin by studying the activity of a subunit has rarely been conducted. Therefore, the objective of the present study was to characterize the antigenic specificity of the β-conglycinin α′-subunit. Methods: We established an Escherichia coli expression system to obtain β-conglycinin α′-subunit. The fusion proteins were then used in a rat model to induce a hypersensitive reaction. Immunoblotting, IgE and IgG1 level, histamine release, and passive cutaneous anaphylaxis reactions and intestinal histology were tested to assess the allergenic activity of the β-conglycinin α′-subunit. Results: Pure β-conglycinin α′-subunit was obtained by expression in E. coli. The recombinant proteins were shown to have the same biological activity as the natural β-conglycinin α′-subunit using immunoblotting analysis. Both the IgE and IgG1 level in serum and the histamine concentration in the intestine were increased while passive cutaneous anaphylactic reactions were induced in Brown Norway rats by intragastric gavage with the α′-subunit. Histamine release of mast cells was also elevated in vitro. Conclusions: Our results indicate that the β-conglycinin α′-subunit possesses an intrinsic immune-stimulating capacity and that it can induce an allergic reaction. Moreover, this study showed that β-conglycinin α′-subunit-induced anaphylaxis is IgE mediated, and mast cell degranulation and histamine release are associated with anaphylactic symptoms.

Background: Anaphylaxis is associated with systemic vasodilation that causes low blood pressure and induces hypoxic brain damage. The sound of a Buk (Korean traditional drum) is similar to the human heart beat and affects blood pressure, heart rate, and the nervous system by increasing physiological excitation and sympathetic nervous system activity. So, this study focused on the effect of Buk music as a means of treating anaphylaxis. Methods: Mice were given an intraperitoneal injection of compound 48/80 (6.5 mg/kg, a mast cell degranulator). After compound 48/80 injection, mice were exposed to Buk music and white noise for 5 min in a sound isolation booth. The mortality rate was checked over the next 40 min. Levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the serum and brain tissues were analyzed by Western blotting, quantitative real-time PCR, and ELISA methods. Results: Exposure to Buk music significantly reduced compound 48/80-induced mortality and histamine release, as well as HIF-1α and VEGF levels compared with the compound 48/80 group or compound 48/80 and white noise group. Buk music also reduced levels of tumor necrosis factor-α, and significantly increased estrogen receptor-β mRNA levels. Conclusion: These results indicate that Buk music has potential for the treatment of anaphylaxis.

Background: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR). Methods: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA. Results: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion. Conclusions: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo.

Background: Studies using rush immunotherapy (RIT) have shown that rapid protection can be achieved using protocols allowing a fast increment of allergen dose. We examined the early effects of RIT on basophil numbers and expression of CD203c, production of interleukin (IL)-4 and IL-13 and histamine release by basophils in the peripheral blood of patients treated with immunotherapy and controls. Methods: Twelve patients treated with RIT and 4 untreated controls were included in the study. Any adverse events were evaluated during the incremental phase of RIT. Mononuclear cells were isolated before the start of RIT and 3 days, 1 week, 4 weeks and 3 months after the beginning of the treatment. Histamine release upon allergen stimulation, expression of CD203c and allergen-induced production of IL-4 and IL-13 by basophils were examined. Results: Significant decreases in blood basophil count (p = 0.02) were observed early in the treatment, returning to baseline values 1 week after the start of RIT. Similarly, histamine release decreased at day 3 (p = 0.02), but returned to pretreatment levels after 1 week. Also, the percentage of IL-4+ and IL-13+ basophils and levels of CD203c expression were markedly reduced early in the treatment. IL-4 and IL-13 production correlated with histamine release and CD203c expression. Histamine release and production of IL-4 and IL-13 by basophils before the treatment correlated with the severity of adverse events during the incremental phase of RIT. Conclusion: We report the decrease in blood basophil numbers, their lower activation status and the reduced production of IL-4 and IL-13 early in the course of RIT. This early suppression of basophil activation could be one mechanism behind the protective effect of RIT.

Background: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. Methods: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. Results: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. Conclusions: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.

Background: Peroxynitrite (ONOO–), the product of the reaction between the superoxide anion (·O2–) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO– generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO– on inflammatory mediator release (histamine and leukotrienes) from activated mast cells. Methods: Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin (OVA). The intracellular ROS formation was determined by following the oxidative production of 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca2+ levels by confocal scanning microscopy, and PLA2 activity using prelabeling of [3H]arachidonic acid. Results: ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen (50 µM). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L-methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca2+ influx, and the PLA2 activity evoked by mast cell activation. Conclusion: The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO–, and that this modulates the release of inflammatory mediators via Ca2+-dependent PLA2 activity.

Background: Ginseng (the root of Panax ginseng C.A. Meyer, Araliaceae) has been reported to possess various biological activities, including anti-inflammatory and antitumor actions. In this study, we investigated the antiallergic activity of ginsenosides isolated from ginseng. Method: We isolated ginsenosides by silica gel column chromatograghy and examined their in vitro and in vivo antiallergic effect on rat peritoneal mast cells and on IgE-induced passive cutaneous anaphylaxis (PCA) in mice. The in vitroanti-inflammatory activity of ginsenoside Rh1 (Rh1) in RAW264.7 cells was investigated. Results: Rh1 potently inhibited histamine release from rat peritoneal mast cells and the IgE-mediated PCA reaction in mice. The inhibitory activity of Rh1 (87% inhibition at 25 mg/kg) on the PCA reaction was found to be more potent than that of disodium cromoglycate (31% inhibition at 25 mg/kg); Rh1 was also found to have a membrane-stabilizing action as revealed by differential scanning calorimetry. It also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 cells, and the activation of the transcription factor, NF-ĸB, in nuclear fractions. Conclusion: The antiallergic action of Rh1 may originate from its cell membrane-stabilizing and anti-inflammatory activities, and can improve the inflammation caused by allergies.

Background: We have reported that canine mastocytoma-derived CM-MC cells are activated via canine IgG and express a high-affinity IgG receptor (canine FcγRI). The predicted amino acid sequence of the canine FcγRI α subunit was found to be 72% similar to that of humans. These results suggest that canine FcγRI have binding activity with human IgG and led us to investigate CM-MC activation via canine FcγRI and human IgG. Methods: The binding of human IgG to canine FcγRI was examined by flow cytometry using FITC-conjugated human IgG. [Ca2+]i increase or histamine release via canine FcγRI and the four human IgG subclasses was measured following aggregation of IgG-bound FcγRIs by anti-human IgG. To determine the binding activity of canine FcγRI with human IgG1 or IgG3, the displacement of 125I-labeled canine IgG from canine FcγRI was examined by unlabeled human IgG1 or IgG3. Results: The fluorescence intensity of CM-MC cells was markedly (about 50 times) elevated by incubation with FITC-human IgG compared with the fluorescence of the control cells. A significant (p < 0.01) calcium response and histamine release were observed following aggregation of canine FcγRIs bound with human IgG1 or IgG4. 125I-labeled canine IgG was displaced from canine FcγRI by preincubation with unlabeled total human IgG or human IgG1 dose-dependently, whereas no displacement was detected by preincubation with human IgG3. Conclusions: Canine FcγRI possesses a significant binding activity with human IgG1 or IgG4, while IgG2 or IgG3 did not significantly react with canine FcγRI on CM-MC cells.

Backgrounds: Approximately half of the Japanese asthmatics experience exacerbation of asthma after alcohol consumption. We previously reported that this phenomenon is probably caused by histamine release from mast cells by acetaldehyde stimulation. However, no reports have described the effects of acetaldehyde on human airway mast cells. The purpose of the present study was to demonstrate acetaldehyde-induced histamine release from human airway mast cells with subsequent airway smooth muscle contraction and to investigate the ensuing mechanisms. Methods: Human tissue samples were prepared from the lungs resected from patients with lung cancer. The effect of acetaldehyde on airway muscle tone and the concentration of chemical mediators released in the organ bath were measured before and after acetaldehyde stimulation. Mast cells were prepared from lung parenchyma by the immunomagnetic method and then stimulated with acetaldehyde to determine the chemical mediators released. Results: Acetaldehyde (>3 × 10–4M) increased airway muscle tone, which was associated with a significant increase in the release of histamine, but not thromboxane B2 or cysteinyl-leukotrienes. A histamine (H1 receptor) antagonist completely inhibited acetaldehyde-induced bronchial smooth muscle contraction. Acetaldehyde also induced a significant histamine release from human lung mast cells and degranulation of mast cells. Conclusions: The present results strongly suggest that acetaldehyde stimulates human airway mast cells to release histamine, which may be involved in bronchial smooth muscle contraction following alcohol consumption.

Protein Fv, an endogenous protein produced in the liver, is released in biological fluids during viral hepatitis. Acute and chronic viral hepatitis can be associated with cardiovascular derangements. Protein Fv induced the release of histamine, tryptase and the de novo synthesis of prostaglandin D2 and cysteinyl leukotriene C4 from mast cells isolated from human heart tissue (HHMC). Protein Fv absorbed with protein A-Sepharose coated with polyclonal IgG did not induce histamine secretion. The maximal percent histamine secretion induced by protein Fv correlated (rs = 0.60; p < 0.05) with that induced by anti-IgE, whereas there was no correlation between the release caused by proteins Fv and C5a. Preincubation of HHMC with protein Fv or anti-IgE caused complete cross-desensitization to subsequent challenge with heterologous stimulus. HHMC from which IgE had been dissociated no longer released histamine in response to anti-IgE and protein Fv. A human monoclonal IgE blocked both anti-IgE- and protein Fv-induced release. Three human monoclonal IgM VH3+ inhibited protein-Fv-induced secretion of histamine from HHMC, whereas monoclonal IgM VH6+ did not inhibit the release induced by protein Fv. Protein Fv acts as an endogenous immunoglobulin superantigen by interacting with the VH3 domain of IgE to induce the release of mediators from HHMC.

Mast cells are pivotal secretory cells primarily implicated in allergen-evoked inflammatory responses and are downregulated following experimental chemical diabetes. Here we tested the hypothesis that a decrease in the number and reactivity of mast cells would account for the hyporesponsiveness of diabetic rats to allergen-induced inflammation. We found that the anaphylactic release of histamine from sensitized ileum, trachea and skin tissues was clearly reduced in rats turned diabetic via intravenous administration of alloxan. Likewise, actively and passively sensitized diabetic rats mounted a weaker allergen-evoked pleural mast cell degranulation and protein extravasation, as compared to sensitized nondiabetic animals, which paralleled a marked reduction in the mast cell population in the pleural cavity. The number of mast cells enumerated in the mesentery from diabetic rats was also clearly reduced. The allergen-evoked plasma leakage in diabetic rats was restored by the transfer of mast cells from sensitized nondiabetic rats. Moreover, the adoptive transfer of sensitized mast cells from diabetics to naive animals led to a lower allergen-induced exudation as compared to the response noted after the transfer of sensitized naive mast cells. Purified mast cells from diabetic rats were hyporesponsive to antigen and compound 48/80 stimulation in vitro as attested by histamine release. Thus, our results show that the phenomenon of refractoriness of diabetic animals to allergen challenge appears to be accounted for by a reduction in the number and reactivity of mast cells.

Background: High osmolal ionic radiocontrast media (RCM) cause vascular release of endothelin-1 (ET-1) and activate mast cells. Iomeprol, a nonionic RCM, has recently been reported not to activate cardiac mast cells. This coronary angiography study was performed to extend those findings using another nonionic RCM, iopromide, and to further determine whether iopromide causes release of ET-1. Methods: Pulmonary artery plasma ET-1, histamine and serum tryptase were determined before and 30 min following angiography with iopromide in 11 subjects. ET-1, histamine and tryptase were measured using immunoassays. Results: The concentrations of ET-1 (1.36 ± 0.66 pg/ml), histamine (2.63 ± 1.15 nM), and β (M), and β (Conclusions: This study demonstrates the lack of release of ET-1 by iopromide. The lack of cardiac mast cell activation by iopromide is consistent with the report that iomeprol also does not activate cardiac mast cells.

Background: CD45, receptor-type protein tyrosine phosphatases (PTPases) are essential components of signaling through both the T cell receptor and the B cell antigen receptor. However, the functional significance of CD45 in the signaling pathway through the high-affinity immunoglobulin (Ig) E receptor has not yet been established. In this study, we demonstrate that the potent CD45 inhibitor negatively regulates IgE-dependent anaphylaxis and contact hypersensitivity reactions. Method: We have previously found that TU-572, 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]-5-isopropoxybenzimidazole, had a potent and selective inhibitory effect against PTPase activity of CD45. Using a CD45 inhibitor, we examined in vitro and in vivo IgE-mediated responses. Results: TU-572 potently inhibited histamine release from rat peritoneal mast cells and mouse systemic anaphylaxis reaction using monoclonal anti-dinitrophenyl (DNP) IgE and DNP-BSA. TU-572 also suppressed the immediate-type hypersensitivity response induced by repeated epicutaneous application of trinitrochlorobenzene in BALB/c mice. Conclusion: These findings revealed that the PTPase activity of CD45 played a critical role in signal transduction of IgE-mediated anaphylaxis in vitro and in vivo. PTPase inhibitors such as TU-572 are useful in the treatment of allergic diseases.

Background: Recent data suggest that normal tissue mast cells can express functional receptors for IgG under certain conditions. However, little is known about IgG receptor expression and functional consequences in mast cell neoplasms. Methods: In this study, neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CM-MC) and from a dog with visceral mastocytoma (VI-MC). Both cell populations were characterized morphologically and functionally. Results: Most cells proliferated constantly in suspension without particular supplements. Doubling times of CM-MC and VI-MC were 52.2 and 27.5 h, respectively. Both cell types were sensitive to formalin fixation, did not contain heparin and were tryptase and chymase positive. Electron microscopy showed fine granules with electron-dense content in both cell populations. The total histamine content of CM-MC and VI-MC was 0.25 and 0.10 pg/cell, respectively. Calcium ionophore A23187 and substance P induced dose-dependent histamine release, whereas compound 48/80 had no effect. Most significantly, both cell types, when sensitized with monomeric dog IgG, released histamine upon stimulation by anti-dog IgG. Conclusions: Dog mastocytoma-derived cells may be useful to study the regulation of neoplastic mast cell growth and differentiation, as well as IgG receptor-mediated activation in neoplastic mast cells. Further research is required to clarify the pathophysiological significance of constitutive expression of IgG receptors in neoplastic (canine) mast cells.

Background: Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Methods: Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 °C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Results: Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8–13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 μM) did not inhibit histamine release in response to NT1–12, bardykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. Discussion: It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

The mechanisms of action of cyclosporin A require further elucidation since this drug includes anti–inflammatory properties unrelated to its previously documented effect of T cells. A study was performed using enzymatically dispersed cells from nasal polyps of 7 subjects to examine the effects of cyclosporin A on the release of histamine, leukotriene C4/D4 (LTC4/D4) and thromboxane (TxB2) following stimulation by anti–IgE. Cells were resuspended and preincubated with cyclosporin A (0.1, 1 and 10 μM) or 0.1% DMSO (the vehicle used to dissolve cyclosporin A) for 20 min prior to challenge with 10 μg/ml ε–chain–specific anti–IgE for 45 min at 37°C. Histamine, LTC4/D4 and TxB2 were measured using EIA. Cyclosporin A significantly inhibited the release of histamine, LTC4/D4, and TxB2 in a concentration–dependent manner. IC30 values, histamine (5.1 μM), LTC4/D4 (7.8 μM) and TxB2 (6.2 μM), were determined. These data demonstrate new antiallergic properties of cyclosporin A using a novel in vitro model which mimics more closely allergic inflammation.

New sources of human and mouse mast cells, which were isolated from individual organs (i.e., lung, colon, synovium, skin, uterus, heart), developed from progenitors in vitro in the presence of stem cell factor and/or interleukin (IL)-3, or enriched from fetal or adult blood, spleen or bone marrow by cell sorting, have made possible new studies of the cell biology of mast cells. Advances resulting from these new mast cell sources as well as from new methods for labeling specific products in subcellular sites and structures in resting and functional mast cells are the subject of this review. Specific advances discussed are as follows: identification of an Fc epsilonRI+ c-kit- mouse basophil population from bone marrow and spleen that is associated with IL-4 production and an Fc epsilonRI- c-kit- granulated mouse mast cell progenitor in fetal blood; identification of hyperplasia and functional activation of human skin mast cells in vivo when exposed to recombinant stem cell factor and spontaneous degranulation in X-linked immunodeficient mouse mast cells; use of an enzyme-affinity-gold method to detect histamine in mature and immature human mast cell granules, in secretion and recovery of histamine during anaphylactic degranulation of human lung mast cells ex vivo, and in secretion of histamine in vivo by piecemeal degranulation of IL-4 transgenic mouse mast cells in inflammatory eye disease and of human gut mast cells in inflammatory bowel disease; use of immunogold methods to localize cyclooxygenase and tumor necrosis factor-alpha to subcellular structures in human and rat mast cells and to localize the Charcot-Leyden crystal protein in human basophils to aid in the identification of mast cells arising in mixed cellular populations; use of a low-density lipoprotein (LDL)-gold affinity method to demonstrate a rat mast cell granule-mediated uptake of LDL by macrophages in peritoneal fluid.

Mast cells have been identified in the choroid of numerous species including man. However, functional studies involving these human mast cells have not been reported. In the current studies, the secretory response of human choroidal mast cells to various stimuli was examined using monodispersed choroidal cell preparations.Monodispersed cell suspensions of human choroid were prepared from eye bank globes and the number, histamine content, and secretory response of mast cells in these preparations were determined. Choroids from 27 donors were used for these experiments.Cell suspensions contained an average of 15% mast cells. Mast cells stained positively with toluidine blue and exhibited the classical granular appearance upon electron microscopy. The amount of histamine contained in each mast cell was calculated to be 2.74+/-0.17 pg. Significant histamine release was observed following treatment with anti-human IgE, calcium ionophore A23187, concanavalin A, compound 48/80 and morphine.A method has been developed for obtaining monodispersed human choroidal mast cell preparations. The cells were functional as evidenced by their ability to release histamine upon immunological and nonimmunological stimulation. The degranulation noted following compound 48/80 and morphine challenge suggests that these human choroidal mast cells are analogous to connective tissue or chymase/tryptase-positive mast cells.

In comparison to adenosine 3',5'-cyclic monophosphate (cAMP), surprisingly little is known regarding the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the functional modulation of mast cells and basophils. In the present study, the ability of cGMP, cAMP, and a range of related compounds, to inhibit immunologically induced histamine release from these cells was investigated and compared to the anti-asthmatic drug disodium cromoglycate (DSCG). Exogenously applied cGMP produced a potent inhibition of histamine release from rat peritoneal mast cells, but cAMP had a negligible effect. The attenuation noted with cGMP was markedly reduced if cells were pretreated with the nucleotide before stimulation. Similar results were obtained with DSCG. The inhibitory and tachyphylactic effects noted with cGMP were mimicked by direct derivatives of the compound but not by a range of other cyclic or guanosine nucleotides. The time courses of the induced tachyphylaxis seen with cGMP and DSCG were similar, and cross-tachyphylaxis between the two compounds was observed. In addition, both cGMP and DSCG showed a comparable spectrum of activity against mast cells isolated from the mouse, guinea pig, and human. The parallel effects of the two agents suggests that they may inhibit mediator release from mast cells through similar mechanisms.

Monocyte chemotactic protein-1 (MCP-1)/monocyte chemotactic activating factor has a potent histamine-releasing activity for basophils and is a major component of IgE-independent histamine-releasing factors (HRF). In this study, we examined the effect of a panel of kinase inhibitors on MCP-1-induced histamine release from human basophils to characterize the signaling pathway used by this chemokine. Genistein (3 micrograms/ml), an inhibitor of tyrosine kinase, inhibited MCP-1-induced histamine release by 44%. Wortmannin is a specific inhibitor of phosphatidylinositol 3 kinase (PI-3 kinase). It blocked MCP-1-induced histamine release with an IC50 of 3.3 x 10(-8) M indicating a role of PI-3 kinase in this reaction. KT5926, an inhibitor of myosin light chain kinase, also inhibited histamine release in response to MCP-1 with an IC50 of 10(-6) M. Staurosporine, a potent inhibitor of protein kinase C, although being not specific, augmented MCP-1-induced histamine release by 31.9% at 10(-6) M. These results indicate the possible involvement of a series of kinases, including PI-3 kinase, in the signal transduction pathway used by MCP-1.

A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.

Oxatomide (OXA), a histamine H1 receptor antagonist, is effective in the treatment of patients with allergic rhinitis, some allergic skin disorders, and bronchial asthma. We have characterized the effect of OXA on the immunologic release of preformed (histamine and tryptase) and de novo synthesized mediators (leukotriene C4:LTC4 and prostaglandin D2:PGD2) from human basophils and mast cells purified (from 10 to 82%) from human lung parenchyma (HLMC) and skin tissue (HSMC). Preincubation (15 min, 37 degrees C) of basophils with OXA (10(-7)-10(-5) M) before Der p I antigen or anti-IgE challenge concentration-dependently (10-40%) inhibited the immunologic release of histamine and LTC4. OXA (10(-7)-10(-5) M) also inhibited (10-40%) histamine, tryptase and LTC4 release from HLMC activated by anti-IgE. In addition, OXA caused a concentration-dependent inhibition of histamine, tryptase and PGD2 release from HSMC immunologically challenged with a monoclonal antibody against the alpha chain of the high affinity receptor for IgE (anti-Fc epsilon RI) or anti-IgE. These results demonstrate that OXA exerts anti-inflammatory activities by inhibiting the release of preformed and de novo synthesized mediators from human basophils and mast cells.

The hydroxymethylglutaryl coenzyme A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/80 was not altered. Lovastatin (20 microM, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

The suppressive effects of cyclosporine A (CsA) and dexamethasone (Dxt) on antigen-specific IgE responses to ovalbumin (OA) were studied in BALB/c mice. The effects upon other isotypes were also analyzed. The antiovalbumin IgE response did not change when low doses of CsA [8 mg/kg intraperitoneally (i.p.)] were administered; IgA also remained unchanged, while IgG and IgG1 decreased significantly. At higher CsA doses (16 mg/kg i.p. or orally), a decrease was noted for all the ispotypes assayed. Dxt administered orally at 0.3 mg/kg selectively inhibited IgE and IgA but did not influence IgG or IgG1 levels. Delayed hypersensitivity reactions to OA were not modified by CsA, but were depressed by Dxt. Although CsA had not effect on passive cutaneous anaphylaxis in the rat, Dxt significantly reduced this reaction when it was administered 6 h before challenge. These results suggest that Dxt has more specific antiallergic activity than CsA.

All sections of human heart tissue demonstrate tryptase- and chymase-containing mast cells (HHMCs) which have for the first time been isolated, partially purified and studied in vitro. HHMCs contain similar histamine levels as lung and skin mast cells, but tryptase levels are lower than in skin and higher than in lung mast cells. Complement C5a causes rapid dose-dependent release of histamine from HHMCs, but they are refractory to substance P and fMLP. Cross-linking IgE receptors on HHMCs leads to arachidonic acid metabolism through both the cyclooxygenase and 5-lipoxygenase pathways. HHMCs and their vasoactive mediators may be involved in anaphylactic/anaphylactoid reactions in humans and in the pathogenesis of some cardiovascular diseases.

Histamine exhibits various biological effects in inflammatory and immunological reactions. To further define its potential role in allergic enteropathy and inflammatory bowel disease, both gut mucosal histamine levels and histamine release from endoscopic biopsy samples were measured. Tissue histamine content resulted from addition of the released amount of histamine and the remaining part of tissue histamine. The results demonstrate highly elevated mucosal histamine levels of the large intestine in allergic enteropathy. In inflammatory bowel disease histamine content and secretion were found to be significantly increased particularly in affected mucosa of Crohn's disease and ulcerative colitis than in unaffected tissue or in healthy controls. These findings give strong evidence that mast cell mediators like histamine play a role in the pathogenesis of these diseases. Mucosal histamine is thus concluded to contribute to the immuno-inflammatory reactions of the intestine found in these disease states and to reflect the degree of colonic inflammation in Crohn's disease and ulcerative colitis.

Basophils are known to release histamine and to produce leukotrienes (LTs) following both IgE-dependent and -independent stimuli. Although there exist a few reports which examined the relationship between histamine release and LTs production, their conclusions were not always in agreement with each other. In the present study, we examined the relationship between histamine release and LTs production from basophils in the presence or absence of 1 ng/ml of interleukin-3 (IL-3). Normal basophils released a smaller amount of histamine and LTs than atopic determatitis (AD) basophils, when basophils were stimulated with an optimal concentration of anti-IgE antibody. When we examined the relationship of histamine release and LTs production from AD donors induced through Fc epsilon RI, we found a significant exponential correlation between these two mediators (R2 = 0.58 in the absence of IL-3, R2 = 0.83 in the presence of IL-3). Although IL-3 enhanced both histamine release and LTs production from AD donors, the relationship between these two mediators was not affected. In conclusion, there was an exponential correlation between histamine release and LTs production from AD basophils, which was not affected by the pretreatment with IL-3.

Histamine-releasing factors (HRF) that release mediators from human basophils by interacting with IgE have been identified from different cell sources, including lymphocytes, monocytes, thrombocytes and endothelial cells. These factors are studied in view of their potential importance as a stimulus in chronic inflammation. In this report we investigated the qualitative variability of the histamine-releasing activity in the supernatants of activated mononuclear cells. Purified human mononuclear cells of 8 donors were activated with streptokinase/streptodornase (SK/SD) and the supernatants (HRF-MN) were tested for histamine-releasing activity (HRA) in both allergic (RAST positive for inhalant allergens) and nonallergic individuals. Four of the eight HRF-MN supernatants were discriminating, i.e. showing no histamine-release response with nonallergic individuals, whereas four supernatants were not. Two of the HRF-MN supernatants that exhibited discriminating properties were studied in more detail. The response to HRF-MN was tested (1) in a direct bioassay on basophils of allergic (RAST positive for inhalant allergens) and nonallergic individuals and (2) in an indirect bioassay with 70% pure basophils of RAST-negative donors after passive sensitization with sera of allergic donors. An association was found between the response to HRF-MN and the RAST for inhalant allergens: none (0/12) of the RAST-negative but 15/22 of the RAST-positive individuals were HRF-MN responders. The IgE dependency of HRF-MN was shown e.g. by inhibition of passive sensitization by preincubating a responder serum with monoclonal antibody (moAb) anti-IgE MH25-1. Our results are in contrast with findings of other investigators who use pooled supernatants and demonstrated HRF-MN responsiveness with both allergic and nonallergic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Because of the importance of cytokines in the regulation of allergic inflammation, we investigated the effects of SCF, IL-3, IL-5 and GM-CSF on immunological histamine release from sensitized human lung fragments as well as human leukocytes. SCF (0.2–20 ng/ml) caused a concentration-related enhancement of anti-IgE (1/100) induced histamine release from lung fragments reaching maximally 64% at 20 ng/ml. In contrast, enhancement produced by IL-5, IL-3 and GM-CSF (0.2-20 ng/ml) was quite marginal and reached at best around 20% at the higher concentration, IL-5 being slightly more effective than IL-3 and GM-CSF. Further, SCF potentiated histamine release whatever the level of immunological control whereas potentiation by IL-5 primarily occurred when the amount of histamine release induced by the immunological control ranged between 5 and 10%. SCF acted synergistically with IL-5, producing a greater enhancement of histamine release than the sum of each cytokine used alone. Both SCF and, to a lesser extent, IL-5 potentiated anti-IgE-mediated histamine release regardless of passive sensitization of lung fragments. Unlike what was observed with lung fragments, IL-3, GM-CSF and to a lesser extent IL-5, were potent enhancing agents of anti-IgE (1/2,000)-induced histamine release from leukocytes. Maximal enhancement produced by IL-3 (20 ng/ml), GM-CSF (2 ng/ml) and IL-5 (20 ng/ml) reached 92%, 78% and 61%, respectively. By contrast, SCF (0.2–20 ng/ml) was ineffective on human leukocytes. The combination of IL-3, GM-CSF and IL-5 did not potentiate more anti-IgE-induced histamine release than IL-3 alone at optimal concentration. When compared on the same tissue sample (lung fragments or leukocytes), the magnitudes of the modulating effects of IL-3, GM-CSF and IL-5 were correlated together but not with that produced by SCF. We conclude that SCF selectively potentiates lung mast cells degranulation while IL-5, IL-3 and GM-CSF, which are poorly effective on lung mast cells, strongly enhance basophilic histamine release. The correlation between IL-3, GM-CSF and IL-5 regarding the degree of their modulating effect on histamine release from basophils and lung mast cells, may suggest that they affect the degranulation through a partly similar mechanism.

Glycosphingolipids (GSLs) are physiological membrane components known to modulate various cellular functions. In order to study their influence on basophil mediator release washed leukocytes obtained from allergic asthmatics and nonatopics were preincubated with varying concentrations of different GSLs. Cells were washed and stimulated with anti-IgE, allergen, and calcium ionophore A23187. None of the GSLs induced mediator release by itself. Preincubation with the acidic gangliosides GM2, GM3, and GD1a significantly enhanced the release of both mediators when stimulated with anti-IgE or allergen but not with calcium ionophore A23187. Highest enhancement was seen with ganglioside GM2. Mediator release was higher in asthmatics than in nonatopics. The acidic galactosylceramide-sulfate (sulfatide) showed a significantly enhanced mediator release as well, whereas the neutral GSLs lactosyl ceramide and asialo GM2 did not influence mediator release. It is concluded that IgE receptor-dependent mediator release could be modulated by acidic GSLs.

The antiallergic compound, lodoxamide, was evaluated for its abilities to attenuate a local allergic reaction in rat conjunctiva in vivo and to inhibit rat conjunctival mast cell mediator release in vitro. Topically applied lodoxamide (0.01, 0.10 and 1.0%, w/v) dose-dependently reduced the allergic response (23, 43 and 72%, respectively) in vivo. Lodoxamide was more effective than cromolyn sodium, N-acetyl aspartyl glutamic acid (Naaxia) and levocabastine, and 25 (7-200) times more potent than nedocromil sodium in direct comparisons. Addition of lodoxamide (10 micrograms/ml) to sensitized conjunctival tissue in vitro immediately prior to antigen challenge significantly reduced the amount of histamine released by the tissue. These data suggest that lodoxamide's in vivo anti-allergic activity in the conjunctiva is associated with its ability to prevent allergic mediator release from mast cells contained in this same tissue.

We studied the leukotriene (LT) antagonistic activity of DS-4574 in vivo and the inhibitory effect of this compound on antigen-induced bronchoconstriction in actively sensitized guinea pigs. Bronchoconstriction induced by LTD4 was inhibited by intravenous and oral treatment with DS-4574 in a dose-dependent manner. Orally administered DS-4574 was also able to inhibit the bronchoconstriction mediated by intravenous administration of LTC4 and E4 and that by endogenous LTs. The inhibitory effect of DS-4574 showed similar potency to those of FPL-55712 and LY171883. In contrast, histamine-, acetylcholine- or 5-hydroxytryptamine-induced bronchoconstriction was not significantly affected by DS-4574. Moreover, DS-4574 given orally or intravenously inhibited antigen-induced bronchoconstriction in actively sensitized guinea pigs and this compound prevented antigen-induced mediator release from actively sensitized guinea-pig lung fragments. The anti-asthmatic effect of this compound appears to be associated with LT antagonism and inhibition of the release of chemical mediators. This study therefore shows DS-4574 to have orally effective LT antagonistic and anti-asthmatic activities. This compound may prove useful in the treatment of bronchial asthma.

Guinea pigs were sensitized with 2,4-toluene diisocyanate (TDI) by a repetitive application onto the bilateral nasal vestibules once a day for 5 consecutive days. When these animals were further treated with TDI for additional 9 weeks, a number of these animals exhibited external symptoms similar to those in human asthmatics, such as labored breathing characterized by prolonged expiration phases. Pathological examination of the lung of these animals clearly revealed contraction of bronchioles where hypertrophy of the smooth muscle and hypersecretion of mucin were evident. Furthermore, infiltration of numerous inflammatory cells, especially eosinophils, was observed in the peribronchial area. In these animals, the histamine-releasing activity of lung tissues was also demonstrated after challenge with TDI-guinea pig serum albumin conjugate. The histamine-releasing activity was found to be elevated to a maximum level during the first 2 weeks after sensitization and to remain at a constant level throughout the rest of the treatment period. Similar time courses were observed with respect to serum levels of TDI-specific antibodies, IgE and IgG. A survey of a series of 28 separate experiments using totally 565 animals indicated that the incidence of animals exhibiting an asthmatic response was 35% on average, ranging from 0 to 58.3%. Furthermore, the serum level of TDI-specific IgG, rather than that of TDI-specific IgE, was rather closely related to the incidence of asthmatic responses in TDI-treated guinea pigs.

The retinoic acid-stimulated mouse-transformed epidermal cell line Pam 212 generated soluble mediators (RA-Pam sup) which showed mast cell-proliferating activity and induced mast cell-like colonies from bone marrow cells. Both mucosal-type and connective tissue-type mast cells were demonstrated on histochemical analysis. The 3T3 fibroblast cell line or human trichilemmomal cell line (TL1) also generated similar molecules on retinoic acid stimulation. Anti-stem cell factor antibody presented a rather more potent inhibitory activity on RA-Pam sup-dependent mast cell colony formation than did anti-IL3 and -IL4 antibodies. RA-Pam sup only induced mast cell colonies in semisolid culture and failed to promote mast cell growth in a suspension culture. In addition, mast cell colonies showed close contact with fibroblast-like cells on the methylcellulose plate, but this finding was not observed in culture without RA-Pam sup.