Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of primate bronchoalveolar mast cells. I. IgE-dependent release of histamine, leukotrienes, and prostaglandins

Large numbers of functional mast cells were obtained by bronchoalveolar lavage (BAL) of Macaca arctoides monkeys that had been infected with the nematode Ascaris suum. These lavage cells, of which 21% were mast cells, released histamine, LTC4, and PGD2 in a concentration-dependent fashion when challenged with ascaris antigen or antibody to human IgE. However, there was no release of histamine when these cells were challenged with compound 48/80. The amount of mediator released was highly dependent on the sensitivity of the cells to immunologic challenge, but was generally in the range of 2 to 5 micrograms histamine (30 to 70% of total), 20 to 80 ng LTC4, and 100 to 300 ng PGD2 per 10(6) mast cells when maximally challenged. Other eicosanoids measured were released only in much smaller quantities. Maximal values were 4 ng LTB4, 2 ng PGE2, and approximately 10 to 20 ng PGF2 alpha per 10(6) mast cells. The amount of LTC4 and PGD2 released correlated with the release of histamine, the calculated regression line indicating that 18 ng LTC4 and 50 ng PGD2 were released per microgram of histamine released. This correlation suggests that the majority of the LTC4 and PGD2 released was probably mast cell-derived. Further support for this conclusion was given by the observation that when lavage cells were fractioned on continuous Percoll gradients, the ability to release LTC4 and PGD2 on immunologic challenge coincided with the peak of mast cells.     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Human basophil/mast cell releasability. VII. Heterogeneity of the effect of adenosine on mediator secretion

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than N6-(R-phenyl-isopropyl)-adenosine (R-PIA) inhibited in vitro anti-IgE-induced histamine and peptide leukotriene C4 (LTC4) release from human basophils in a concentration-dependent fashion. Micromolar concentrations of adenosine, NECA and R-PIA potentiated the anti-IgE-stimulated release of histamine and LTC4 from human lung parenchymal mast cells. Submillimolar concentrations of adenosine, NECA and R-PIA inhibited in a concentration dependent manner the release of histamine and prostaglandin D2 (PGD2) from skin mast cells challenged with anti-IgE. These results demonstrate marked heterogeneity of the modulatory effect exerted by adenosine on mediator release from human basophils and mast cells.     

Comparative functional characterization of mouse bone marrow-derived mast cells and peritoneal mast cells in response to non-immunological stimuli

The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cells in terms of their T-cell dependence and histochemical staining characteristics. Mast cell heterogeneity has been confirmed by functional characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium Ionophore A23187 on the other hand, released histamine in dose-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, disodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced histamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca Ionophore induced histamine release was blocked by Quercetin. The results indicate that response of mast cells at one anatomic site to a given stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity within a single species. So, it cannot be assumed that antiallergic compounds stabilizing mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.     

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

Mast cell heterogeneity in higher animals: a comparison of the properties of autologous lung and intestinal mast cells from nonhuman primates

The issue of mast cell heterogeneity has been investigated in nonhuman primates by a comparative examination of lung and intestinal mast cells. These cells were obtained in parallel from the respective tissues of individual monkeys by an identical enzymatic dispersion technique. Mast cells derived from the lungs differed from those derived from the intestine in that the majority of the former cell type could be stained with toluidine blue at pH 4 to 5, whereas the intestinal mast cells in the dispersed preparations required a more acidic pH (less than 1) to display metachromasia. In addition, the lung cells exhibited an increased content of the mast cell mediator histamine. Nonhuman primate lung mast cells were also quantitatively more responsive to an immunologic challenge than their intestinal counterparts in that they released a higher percentage of cellular histamine and generated more leukotriene C4 on stimulation. Considerable inter-animal variation was observed between the magnitude of mediator release from both mast cell types after anaphylactic activation, but evidence for the presence in nonhuman primates of the phenomenon of releasability was not obtained. The responsiveness of both cell types to a range of potential nonimmunologic secretagogues and anti-allergic agents, including compound 48/80, substance P, theophylline, and isoprenaline, was essentially similar. We conclude that mast cell heterogeneity in higher animals may be reflected more by cytochemical rather than by functional differences between mast cell classes.     

Regulation of adhesion of mouse bone marrow-derived mast cells to laminin

We have reported that mast cells adhere to laminin after activation with PMA. In this study, we demonstrate that the cross-linking of cell surface high-affinity IgE-R on mast cells derived from mouse bone marrow cultured for 3 wk in the presence of WEHI-3-conditioned media acts as a highly sensitive physiologic stimulus for this attachment and that receptor activation is also induced by calcium ionophore A23187. Adherence occurred at threefold log concentrations less of A23187 and Ag than required for histamine release in a selective subpopulation comprising 20 to 30% of the total cells. At higher concentrations of agonist that permitted histamine release, the time course for degranulation was shown to be more rapid than that of adherence. Adherence was inhibited by antibodies to laminin and laminin receptor and was calcium ion and temperature dependent. Treatment of cells with dibutyryl cAMP, which activates protein kinase A, inhibited both adherence and histamine release induced by Ag or calcium ionophore. Treatment of cells with staurosporin, which inhibits protein kinase C, also inhibited adherence and histamine release induced by calcium ionophore, but was not significantly active against either adherence or histamine release induced by Ag. It thus appears that agents which modulate intracellular signaling mechanisms are equally effective toward histamine release and adherence, suggesting these two events are intimately linked in stimulus secretion coupling. Specific cytokines stimulating mast cell adhesion to laminin could not be found; however, culture of mast cells with TGF-beta 1 was determined to enhance IgE-mediated adherence to laminin. Hence, the high-affinity IgE-R on the mast cell functions not only in exocytosis but also facilitates the process of mast cell adherence to laminin.     

Inhibition of compound 48--80 induced histamine liberation from mast cells by triton X-100]

Triton X-100 at concentrations preceding those which liberated histamine, produced dose-dependent inhibition of compound 48/80-induced histamine release from rat mast cells. Triton X-100 (0.00002 1/1) depleted ATP content in the mast cells and blocked compound 48/80-induced histamine release. The inhibition of compound 48/80-induced histamine release and depletion of the ATP content in the mast cells was reversed by glucose (10 mmole). It is concluded that inhibition by Triton X-100 of histamine release induced by compound 48/80 is dependent on inhibition of energy production.     

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity

Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.     

Antiallergic effects of Vitis amurensis on mast cell-mediated allergy model

In this study, we investigated the effect of the methanol extract of fruits of Vitis amurensis Rupr. (Vitaceae; MEVA) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases, such as asthma and sinusitis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. MEVA inhibited compound 48/80-induced systemic reactions and serum histamine release in a dose-dependent manner in mice. MEVA decreased immunoglobulin E (IgE)-mediated local allergic reactions, passive cutaneous anaphylaxis. MEVA dose-dependently reduced histamine release from mast cells activated by compound 48/80 or IgE. The inhibitory effect of MEVA on histamine release was mediated by the modulation of intracellular calcium. In addition, MEVA attenuated the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated secretion of tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-8 in human mast cells. The inhibitory effect of MEVA on these proinflammatory cytokines was p38 mitogen-activated protein kinase and nuclear factor-kappaB (NF-kappaB) dependent. Our findings provide evidence that MEVA inhibits mast cell-derived, immediate-type allergic reactions and involvement of proinflammatory cytokines, p38 MAPK, and NF-kappaB in these effects.     

Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.

The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.