Spontaneous Histamine Release Research Articles

Defining Optimal Basophil Passive Sensitisation Parameters

ABSTRACTBackgroundDetecting drug‐specific IgE (sIgE) is crucial for diagnosing immediate drug‐induced hypersensitivity reactions. Basophil activation tests serve as a method to determine the presence of drug‐sIgE, highlighting the importance of optimising the assay. Optimisation involves considering multiple factors to ensure sensitisation helps detect an antigen sIgE. The study investigates the complex factors influencing basophil responsiveness thresholds and aims to provide rules‐of‐thumb guidance for expected results.MethodsOpen and occupied FcεRI receptors were analysed by flow cytometry pre‐ and postdissociation of surface‐bound sIgE. Basophils were then sensitised with serial concentrations of penicillin (BPO)‐sIgE in serum or buffer and incubated for 1, 4 and 18 h with or without D2O and/or IL‐3. Basophil sensitivity was evaluated based on FcεRI receptor densities, sIgE/total IgE (tIgE) ratios, responses to BPO(21)‐HSA, and D2O and/or IL‐3 effects, with maximal responses determined using anti‐IgE human antibodies. These optimised conditions were tested with peanut‐sIgE and cat‐sIgE sera.ResultsBasophils from five donors were used. The FcεRI receptor expression initially averaged 155,000/cell (47,000–344,000/cell), with 35% (5%–79%) unoccupied, which postdissociation increased to 98% (82%–100%) unoccupied. Upon sensitisation, the average reloading with BPO‐sIgE was 39% (33%–48%). The ED50 (a measure of cellular sensitivity) was approx. 6000 BPO‐sIgE/cell, and the average maximal anti‐IgE antibody response was 58% (25%–68%). A 4‐h sensitisation at 4°C with IL‐3 pretreatment and 70% D2O allowed the detection of BPO‐sIgE/tIgE ratios as low as 0.02%–0.05% without spontaneous histamine release. Under the same conditions, responses were detected with 0.33% peanut‐sIgE and 0.1% cat‐sIgE ratios.ConclusionThis study outlines a method to assess basophil sensitisation, emphasising the minimum sIgE/tIgE ratio needed for basophil responsiveness. It considers factors like FcεRI open/unoccupied FcεRI receptors, sIgE/tIgE ratios and the effect of D2O and IL‐3. This sets a strong foundation for refining and advancing basophil activation functional assays.

Mathematical-based morphological classification of skin eruptions corresponding to the pathophysiological state of chronic spontaneous urticaria

BackgroundChronic spontaneous urticaria (CSU) is one of the most intractable human-specific skin diseases. However, as no experimental animal model exists, the mechanism underlying disease pathogenesis in vivo remains unclear, making the establishment of a curative treatment challenging.MethodsA novel approach combining mathematical modelling, in vitro experiments and clinical data analysis was used to infer the pathological state of CSU patients from geometric features of the skin eruptions.ResultsBased on our hierarchical mathematical modelling, the eruptions of CSU were classified into five categories, each with distinct histamine, basophils, mast cells and coagulation factors network signatures. The analysis of 105 real CSU patients with this classification by six individual dermatologists achieved 87.6% agreement. Furthermore, our network analysis revealed that the coagulation status likely determines boundary/area pattern of wheals, while the state of spontaneous histamine release from mast cells may contribute to the divergence of size and outline of the eruptions.ConclusionsOur multi-faceted approach was accurate in defining pathophysiological states of disease based on geometric features offering the potential to improve the accuracy of CSU diagnosis and better management of the disease in the clinic.

Basophils activation of patients with chronic spontaneous urticaria in response to C5a despite failure to respond to IgE-mediated stimuli.

Urticaria is characterized by the occurrence of wheals and flares in response to vasoactive mediators, such as histamine. Various studies have suggested the involvement of basophils in the pathogenesis of chronic spontaneous urticaria (CSU). However, histamine release from peripheral basophils in response to stimuli acting on the high affinity IgE receptor (FcϵRI) is impaired in many patients with CSU (non/low responders). We previously demonstrated that tissue factor (TF)s expressed on vascular endothelial cells in response to a combination of various stimuli, such as that of histamine and lipopolysaccharide (LPS), activates the extrinsic coagulation pathway and produces anaphylatoxin, complement 5a (C5a), which then activates basophils and mast cells via the C5a receptor (C5aR). We have revealed that histamine release was induced in response to C5a and formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), regardless of the response to anti-IgE antibody, the reduced numbers of basophils and severity of urticaria. Moreover, we found that spontaneous release of histamine ex vivo from basophils of patients with CSU is higher than that from healthy individuals. These results suggest that basophils and the complement system, which could be activated by coagulation factors, may play a critical role in the pathogenesis of CSU, especially in cases refractory to treatment involving the IgE/FcϵRI pathway.

FceRI density and spontaneous secretion from human basophils.

Mast cells and basophils depend on aggregation of the high-affinity IgE receptor, FceRI, to initiate secretion. A variety of studies have shown that FceRI densities vary 100 fold among subjects’ basophils and it has been speculated that high densities might be responsible for unusual behaviors of the cells, notably sensitivity to certain monomeric IgE antibodies or spontaneous release. These studies experimentally examined the density dependence of spontaneous release and signaling element expression in subjects’ basophils with FceRI densities ranging from approximately 6000 to 600,000 per cell. Through the use of numerical simulation, this study examined the expectations for spontaneous receptor aggregation and aggregate persistence at densities of FceRI ranging from 5000 to 500,000. Experimentally, FceRI density was not associated with greater spontaneous histamine release even when secretion was enhanced by the inclusion of deuterium oxide in the buffers. There was also no association of 15 activating or de-activating signaling elements with FceRI density. The numerical simulations demonstrated that at densities of ≈500,000 receptors, 13% of receptors were involved in transient aggregates at any given moment but that these aggregates rarely persisted for greater than 10 milliseconds. In contrast, a weak linear antigen aggregator, with ligand affinities known to induce secretion, would generate aggregates persisting for an average of ≈60 milliseconds. These results suggest that although a high density of FceRI likely produces a large number of transient aggregates, these aggregates do not persist long enough to induce signaling that results in secretion and do not induce the cells to alter their expression of several signaling elements known to be important in regulating secretion from human basophils. The results set some boundaries on the aggregation requirements for inducing histamine release from human basophils.

AN EVALUATION OF SPONTANEOUS HISTAMINE RELEASE AND THE LOW RESPONDERS IN A BASOPHIL HISTAMINE RELEASE TEST

We evaluated the clinical significance of the spontaneous histamine release ratio (SHR/T) and low responders in the automated basophil histamine release test (AllerportⓇ HRT). This study analyzed the outcomes of 101 oral food challenges (OFC) with egg, milk or wheat (challenge-positive: n=79) in relation to the SHR/T. The traditional HRT low responders (n=27) were separated into two groups:"LOW"responders (n=10), who showed a ≥10% concentration-dependent maximum histamine release in response to the anti-human IgE stimulation, and"NON"responders who did not fulfill the criteria (n=17). Among the 34 patients with ≥20% SHR/T, 32 patients (94%) had a positive OFC with a low threshold dose which provoked severe symptoms. Among the"LOW"responders, four cases showed ≥10% allergen-specific maximum histamine release. On the other hand, concentration-dependent histamine release was not seen in the"NON"responders, suggesting the basophil function was not detected in this subgroup. The present study suggested that SHR/T could be an indicator of basophil activation and hypersensitivity in vivo. We also suggested that significant basophil functions might be detected among the "LOW"responders, but not among the"NON"responders.

In vitro screening method for the evaluation of histamine release from rat peritoneal cells

Rat peritoneal mast cells offer a convenient approach for the evaluation of histamine release by chemicals. Peritoneal mast cells obtained by centrifugation on density gradients are routinely used for the assessment of histamine release. Manipulation of the cells during this purification step may lead to cell membrane damage and spontaneous release of histamine. The purpose of the present study was to evaluate two procedures of peritoneal cell purification. Rat peritoneal cells were purified (i) by three centrifugations in PIPES only (PIPES purification) or (ii) by three centrifugations in PIPES followed by density gradient centrifugation in Percoll (PIPES/Percoll purification). The effect of a reference compound 48/80 was evaluated at two or three concentrations (0.1, 1 and 10 μg/ml) under several experimental conditions

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Spontaneous basophil responses in food-allergic children are transferable by plasma and are IgE-dependent

Early studies showed that up to 80% of children with food allergy demonstrate high spontaneous basophil histamine release (SBHR).1 An association with IgE was suggested because basophils from food-allergic (FA) children additionally released histamine to the IgE-dependent histamine-releasing factor.2 However, the exact mechanism(s) responsible for SBHR, including the dependency for IgE, remains poorly understood. We recently reported that SBHR significantly decreased in children undergoing sublingual immunotherapy/oral immunotherapy (SLIT/OIT) for cow's milk allergy,3 prompting further investigations of this response and other markers of basophil hyperresponsiveness.

Mast cell expression of the serotonin1Areceptor in guinea pig and human intestine

Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

Cellular and Serologic Profiling of Adults with Peanut Allergen Sensitization

RationaleWe compared the expression of basophil and serologic measures in peanut-allergen (PA) sensitized adults with varying clinical manifestations.MethodsAdult subjects were evaluated by history, total IgE, peanut-specific IgE, IgE anti-Ara-h 1,2,3, and 8 (ISAC and ImmunoCAP), basophil spontaneous histamine release (SHR), PA-mediated basophil histamine release (BHR), and baseline basophil CD203c expression.ResultsAdults with confirmed peanut allergy (Group B, n=14) were compared to peanut-sensitized, but tolerant, adults(Group C, n=4). Peanut-specific IgE (median 15.2 vs. 1.03 kUa/L, p = 0.009) and specific to total IgE ratios (7.11% vs. 0.39%, p = 0.022) were increased in Group B compared to Group C, but not total IgE levels. Group B subjects demonstrated broader IgE reactivity to ∑Ara-h 1,2,3, and 8 by ISAC (28.11 ISU vs 0 ISU, p = 0.05) and ImmunoCAP (34.11 vs. 0.34 kUa/L, p = 0.019). Group B had markedly increased Arah-2 (15.4 vs. 0.055 kUa/L, p = 0.007) and similar Arah-h 8 ImmunoCAP reactivity (p=0.95). Basophil SHR was higher in Group B (6.3% v. 2.65%, p = 0.015), but not baseline surface CD203c expression. PA concentrations eliciting threshold and maximum BHR were several logs lower in Group B (p = <0.01).ConclusionsAdults with peanut allergy were found to have broader PA-specific IgE expression and heightened measures of basophil reactivity compared to subjects with asymptomatic sensitization. RationaleWe compared the expression of basophil and serologic measures in peanut-allergen (PA) sensitized adults with varying clinical manifestations. We compared the expression of basophil and serologic measures in peanut-allergen (PA) sensitized adults with varying clinical manifestations. MethodsAdult subjects were evaluated by history, total IgE, peanut-specific IgE, IgE anti-Ara-h 1,2,3, and 8 (ISAC and ImmunoCAP), basophil spontaneous histamine release (SHR), PA-mediated basophil histamine release (BHR), and baseline basophil CD203c expression. Adult subjects were evaluated by history, total IgE, peanut-specific IgE, IgE anti-Ara-h 1,2,3, and 8 (ISAC and ImmunoCAP), basophil spontaneous histamine release (SHR), PA-mediated basophil histamine release (BHR), and baseline basophil CD203c expression. ResultsAdults with confirmed peanut allergy (Group B, n=14) were compared to peanut-sensitized, but tolerant, adults(Group C, n=4). Peanut-specific IgE (median 15.2 vs. 1.03 kUa/L, p = 0.009) and specific to total IgE ratios (7.11% vs. 0.39%, p = 0.022) were increased in Group B compared to Group C, but not total IgE levels. Group B subjects demonstrated broader IgE reactivity to ∑Ara-h 1,2,3, and 8 by ISAC (28.11 ISU vs 0 ISU, p = 0.05) and ImmunoCAP (34.11 vs. 0.34 kUa/L, p = 0.019). Group B had markedly increased Arah-2 (15.4 vs. 0.055 kUa/L, p = 0.007) and similar Arah-h 8 ImmunoCAP reactivity (p=0.95). Basophil SHR was higher in Group B (6.3% v. 2.65%, p = 0.015), but not baseline surface CD203c expression. PA concentrations eliciting threshold and maximum BHR were several logs lower in Group B (p = <0.01). Adults with confirmed peanut allergy (Group B, n=14) were compared to peanut-sensitized, but tolerant, adults(Group C, n=4). Peanut-specific IgE (median 15.2 vs. 1.03 kUa/L, p = 0.009) and specific to total IgE ratios (7.11% vs. 0.39%, p = 0.022) were increased in Group B compared to Group C, but not total IgE levels. Group B subjects demonstrated broader IgE reactivity to ∑Ara-h 1,2,3, and 8 by ISAC (28.11 ISU vs 0 ISU, p = 0.05) and ImmunoCAP (34.11 vs. 0.34 kUa/L, p = 0.019). Group B had markedly increased Arah-2 (15.4 vs. 0.055 kUa/L, p = 0.007) and similar Arah-h 8 ImmunoCAP reactivity (p=0.95). Basophil SHR was higher in Group B (6.3% v. 2.65%, p = 0.015), but not baseline surface CD203c expression. PA concentrations eliciting threshold and maximum BHR were several logs lower in Group B (p = <0.01). ConclusionsAdults with peanut allergy were found to have broader PA-specific IgE expression and heightened measures of basophil reactivity compared to subjects with asymptomatic sensitization. Adults with peanut allergy were found to have broader PA-specific IgE expression and heightened measures of basophil reactivity compared to subjects with asymptomatic sensitization.

Serotonin and its 5-HT1 receptor in human mastocytosis

Context: Human mastocytosis is a rare disease, in which the serotonergic system may be involved.Objective: The objective of the present study was to examine the possible presence of serotonin (5-HT) and its 5-HT1A receptor (R) in the skin of patients with mastocytosis. In addition, the effect of the 5-HT1AR was tested on human mastocytosis cells, cultured in vitro.Materials and methods: The expression of 5-HT and 5-HT1AR in patients with urticaria pigmentosa and mastocytoma was studied using immunohistochemistry. The effects of 8-OH-DPAT, an agonist of 5-HT1AR, on the proliferation (cell number), viability, apoptosis, spontaneous release of histamine, as well as a possible 5-HT metabolism, in the human HMC-1 mast cell line, were investigated.Results: Both 5-HT and 5-HT1AR were expressed in the mast cells in biopsies of mastocytoma and urticaria pigmentosa, as well as in HMC-1 cells. However, no metabolism of 5-HT by the cell line could be detected by the methodology used. The 5-HT1AR agonist had no significant effect on the viability and number of HMC-1 cells, and was without effect on the apoptosis. At concentrations of 10−6 mol/L and 10−8 –10−10 mol/L (i.e. also at physiological concentrations), the agonist inhibited histamine release by these cells by as much as 30%.Conclusion: These findings indicate that 5-HT and its 5-HT1AR are expressed in human mastocytosis and that an agonist of the 5-HT1AR might be of value in the treatment of these patients.

The safety and efficacy of sublingual and oral immunotherapy for milk allergy

Oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) are potential therapies for food allergy, but the optimal method of administration, mechanism of action, and duration of response remain unknown. We sought to explore the safety and efficacy of OIT and SLIT for the treatment of cow's milk (CM) allergy. We randomized children with CM allergy to SLIT alone or SLIT followed by OIT. After screening double-blind, placebo-controlled food challenges and initial SLIT escalation, subjects either continued SLIT escalation to 7 mg daily or began OIT to either 1000 mg (the OITB group) or 2000 mg (the OITA group) of milk protein. They were challenged with 8 g of milk protein after 12 and 60 weeks of maintenance. If they passed the 60-week challenge, therapy was withdrawn, with challenges repeated 1 and 6 weeks later. Mechanistic correlates included end point titration skin prick testing and measurement of CM-specific IgE and IgG(4) levels, basophil histamine release, constitutive CD63 expression, CD203c expression, and intracellular spleen tyrosine kinase levels. Thirty subjects with CM allergy aged 6 to 17 years were enrolled. After therapy, 1 of 10 subjects in the SLIT group, 6 of 10 subjects in the SLIT/OITB group, and 8 of 10 subjects in the OITA group passed the 8-g challenge (P= .002, SLIT vs OIT). After avoidance, 6 of 15 subjects (3 of 6 subjects in the OITB group and 3 of 8 subjects in the OITA group) regained reactivity, 2 after only 1 week. Although the overall reaction rate was similar, systemic reactions were more common during OIT than during SLIT. By the end of therapy, titrated CM skin prick test results and CD63 and CD203c expression decreased and CM-specific IgG(4) levels increased in all groups, whereas CM-specific IgE and spontaneous histamine release values decreased in only the OIT group. OIT was more efficacious for desensitization to CM than SLIT alone but was accompanied by more systemic side effects. Clinical desensitization was lost in some cases within 1 week off therapy.

Mast Cell Fibroblastoid Differentiation Mediated by Airway Smooth Muscle in Asthma

Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5β1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.

The mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome

BackgroundMast cell activation is thought to be involved in visceral hypersensitivity, one of the main characteristics of the irritable bowel syndrome (IBS). A study was therefore undertaken to investigate the...

Cultured peripheral blood mast cells from chronic idiopathic urticaria patients spontaneously degranulate upon IgE sensitization: Relationship to expression of Syk and SHIP-2

Recently, signaling changes in the FcvarepsilonRI pathway involving inositol lipid phosphatases have been identified in the basophils of chronic idiopathic urticaria (CIU) subjects. Based on the profile of basophil FcvarepsilonRI-mediated histamine degranulation, we have segregated CIU subjects into two groups, CIU Responder (CIU R) or CIU Nonresponder (CIU NR). In the present study, we compared expression of SHIP-1, SHIP-2, and Syk protein to histamine release (HR) from mast cells (MC) cultured from the peripheral blood of CIU R, CIU NR, and normal subjects. The MC of CIU R donors contained significantly increased Syk and decreased SHIP-2 as compared to CIU NR (Syk: p=0.038, SHIP-2: p=0.038) and normals (Syk: p=0.042, SHIP-2: p=0.027). Spontaneous HR from CIU donors was increased two-fold compared to normals (p=0.04). In summary, our results suggest a possible predilection for urticarial MC to spontaneously degranulate upon IgE sensitization contributing to the increased pruritus associated with CIU.

Seasonal increase of spontaneous histamine release in washed leucocytes from rhinitis patients sensitive to grass pollen

The spontaneous histamine release (SHR) in basophils from patients sensitive to grass pollen has been studied before and during the 1987 grass pollen season. Nineteen patients were recruited on seasonal rhinitis symptoms, positivity for cutaneous tests and for serum-specific IgE with grass pollen. At the time of the biological investigations the patients were following a clinical trial of hyposensitization, including placebo, calcium phosphate and aluminium hydroxide-adsorbed grass pollen extract treatments. During the pollen season, grass pollen counts and clinical scores were checked over a 40-day period. Mean SHR was significantly higher during the pollen period than before, for the whole population of 19 patients (10.9% and 4.6%; P less than 0.005) as well as when the high SHR responders were excluded (5.5% and 3.6%; P less than 0.01). No significant correlation existed between SHR and clinical scores or treatments. SHR could be inhibited at 4 degrees C, in absence of CA++ or of oxidative metabolism and thus originated from cells actively secreting histamine.

Influence of the tyrosine kinase inhibitors STI571 (Glivec®), lavendustin A and genistein on human mast cell line (HMC‐1560) activation

The human mast cell line (HMC-1(560)) was used to study the effects of tyrosine kinase (TyrK) inhibition on histamine release in consequence of intracellular Ca2+ or pH changes. This is important since the TyrK inhibitor STI571 (Glivec) inhibits proliferation and induces apoptosis in HMC-1(560). HMC-1(560) cells have a mutation in c-kit, which leads to a permanent phosphorylation of the KIT protein and their ligand-independent proliferation. The TyrK inhibitors STI571, lavendustin A and genistein decrease spontaneous histamine release in 24-h pre-incubated cells. Results are compared with those of the mast cell stabiliser cromoglycic acid, which also drops spontaneous histamine release. When exocytosis is stimulated by alkalinisation, STI571 pre-incubated cells release more histamine than non-pre-incubated cells. Alkalinisation-induced histamine release reaches still higher levels in STI571 cells with activated protein kinase C (PKC) by PMA. We do not observe modifications on histamine release in cells, treated with PKC inhibitors (rottlerin, Gf109203 or Gö6976). Lavendustin A- and genistein 24-h incubated cells behave similar to STI571 cells, whereas cromoglycic acid does not show effects after stimulation with alkalinisation. Stimulation of exocytosis with the Ca2+ ionophore ionomycin does not modify histamine response in TyrK inhibited cells. Ca2+ and pH changes are observed after long-time incubation with STI571. Results show that pH is still higher in STI571 pre-incubated cells after alkalinisation with NH4Cl, whereas intracellular Ca2+ concentration remains stable. This work further strength the importance of pHi as a cell signal and suggest that STI571 has transduction pathways in common with other TyrKs.

STI571 (Glivec®) affects histamine release and intracellular pH after alkalinisation in HMC‐1560, 816

The human mast cell line (HMC-1(560, 816)) was used to study the effect of the tyrosine kinase inhibitor STI571 (Glivec) on exocytosis, intracellular Ca(2+) and pH changes, because STI571 inhibits the proliferation of HMC-1(560) and induces its apoptosis. This drug does not have these effects on HMC-1(560, 816). Exocytosis in HMC-1(560, 816) cells can be stimulated by alkalinisation with NH(4)Cl as well as with ionomycin. Surprisingly 24-h pre-incubation with STI571 decreases spontaneous histamine release of HMC-1(560, 816) cells, but increases the histamine response after alkalinisation and not after ionomycin-stimulation. After addition of NH(4)Cl, pH(i) has a higher increase in STI571 pre-incubated cells, without changing intracellular Ca(2+) concentration. Activation of PKC in combination with tyrosine kinase inhibition increases also histamine release in HMC-1(560, 816) cells. Strangely, STI571 pre-incubated cells with PKC inhibited by rottlerin show the same effects. In these cells, cytosolic pH increases more than in control cells. This is the first report of STI571 effect in HMC-1(560, 816) cells. It seems that different pathways modulate signals for proliferation and exocytosis. STI571 does not only inhibit KIT TyrK, but may also influence cytosolic pH after alkalinisation in both cell lines, HMC-1(560) and HMC-1(560, 816), and this ends in induced histamine release. This work is important since HMC-1(560, 816) cells are reported in 80% of aggressive systemic mastocytosis cases and the understanding of some signalling pathways involved in mast cell response could facilitate drug targeting.

An innovative technique for isolating functional mast cells from the heart

Cardiac mast cells are key players in pathological remodeling of the myocardium. However, the previously described procedures for isolating mast cells from the heart, which require enzymatic digestion of the myocardial tissue, result in a relatively low yield of mast cells with limited functionality. We have developed a protocol, whereby Hanks balanced salt solution is injected into the pericardial sac and then aspirated, drawing with it cardiac mast cells. The resulting preparation contains a greater number of mast cells of higher purity than could previously be obtained using traditional isolation techniques. Mast cells isolated by this new technique have low basal rates of spontaneous histamine release and are highly responsive to the secretagogue, compound 48/80. These cardiac mast cells were of the connective tissue type, based on the presence of chymase and tryptase demonstrated within the cells by immunofluorescence and western blotting. This cellular phenotype is consistent with the previously reported characterization of cardiac mast cells isolated from the heart by enzymatic dispersion techniques. Accordingly, this novel technique for the isolation of cardiac mast cells provides a powerful tool by which to characterize and study functional cardiac mast cells in an in vitro environment, allowing for development of a greater understanding of their role in myocardial disease. This study was supported in part by NIH R01-HL62228 (JSJ), R01-HL073990 (JSJ)

Src homology 2 domain–containing inositol 5-phosphatase 1 deficiency leads to a spontaneous allergic inflammation in the murine lung

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) controls the intracellular level of the phosphoinositide 3-kinase product phosphotidylinositol-3,4,5-trisphosphate and functions as a negative regulator of cytokine and immune receptor signaling. Emerging evidence suggests that the phosphoinositide 3-kinase pathway might be involved in allergic inflammation in the lung. However, the functional relevance of SHIP-1 in the T(H)2 activation pathway has not been established. SHIP-1(-/-) mice have spontaneous myeloproliferative inflammation in the lung, the nature of which has not been elucidated. We hypothesized that SHIP-1 plays an important role as a regulator in pulmonary allergic inflammation and in maintaining lung homeostasis. To test our hypothesis, we characterized the pulmonary phenotype of SHIP-1(-/-) mice. Analyses of lung histopathology and bronchoalveolar lavage cellularity revealed that the majority of SHIP-1(-/-) mice had progressive and severe pulmonary inflammation of macrophages, lymphocytes, neutrophils, and eosinophils; mucous hyperplasia; airway epithelial hypertrophy; and subepithelial fibrosis. These pathologic changes were accompanied by exaggerated production of T(H)2 cytokines and chemokines, including IL-4, IL-13, eotaxin, and monocyte chemoattractant protein 1, in the lung. Furthermore, the number of mast cells significantly increased, and many of these cells were undergoing degranulation, which was correlated with increased content and spontaneous release of histamine in the lung tissue of SHIP-1(-/-) mice. These findings provide strong evidence that mice lacking SHIP-1 have an allergic inflammation in the lung, suggesting that SHIP-1 plays an important role in regulating the T(H)2 signaling pathway and in maintaining lung homeostasis. SHIP-1 as a regulator might be a potential therapeutic target for controlling allergic inflammation in diseases such as asthma.