Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.     

Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

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.     

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. II. Inhibition of histamine, LTC4, and PGD2 release from primate bronchoalveolar mast cells and a comparison with rat peritoneal mast cells

As described in the preceding companion paper, bronchoalveolar lavage (BAL) of the primate Macaca arctoides infected with the nematode Ascaris suum yields a population of cells containing a high proportion of mast cells (21%). Nedocromil sodium, a new drug undergoing clinical evaluation for the treatment of reversible obstructive airways disease, inhibited the release of histamine, LTC4, and PGD2 from these cells challenged with antigen (with IC30 values of 2.1 X 10(-6) M, 2.3 X 10(-6) M, and 1.9 X 10(-6) M, respectively) and with anti-human IgE (IC30 values of 4.7 X 10(-6) M, 1.3 X 10(-6) M, and 1.3 X 10(-6) M, respectively). Cromolyn sodium was essentially inactive. Histamine release from rat peritoneal mast cells induced by anti-rat IgE was, however, inhibited by both nedocromil sodium and cromolyn sodium with IC30 values of 1.1 X 10(-6) M and 5.5 X 10(-7) M, respectively. Both compounds induce phosphorylation of a 78,000 m.w. protein in the rat peritoneal mast cell in the absence of any stimulus at the same concentrations as those required to inhibit histamine release stimulated by anti-IgE. This event may be part of a feedback mechanism to limit degranulation. Nedocromil sodium and cromolyn sodium were equipotent in their ability to inhibit anti-IgE-induced histamine release from rat peritoneal mast cells, but differed markedly in their ability to inhibit histamine release from macaque BAL cells.     

Secretory granule mediator release and generation of oxidative metabolites of arachidonic acid via Fc-IgG receptor bridging in mouse mast cells.

The releases of beta-hexosaminidase, LTC4, LTB4, and PGD2 after the bridging of Fc gamma R3 were assessed in mouse IL-3-dependent bone marrow-derived progenitor mast cells (BMMC), BMMC maintained in coculture with 3T3 fibroblasts separated by a filter to achieve maturation of the granules toward those of a serosal mast cell (SMC), and SMC that are the prototype of a mouse connective tissue mast cell. Bridging of Fc gamma R on BMMC with the 2.4G2 rat anti-Fc gamma RII/III mAb and anti-rat IgG elicited only 4% net release of beta-hexosaminidase and 4, 2, and 1 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively. Bridging of Fc-IgE receptors (Fc epsilon R) on BMMC yielded 35% net release of beta-hexosaminidase and 9, 4, and 3 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively. BMMC maintained in coculture responded to the bridging of Fc gamma R with statistically significant increases in the net percent release of beta-hexosaminidase to 16% and in the generation of immunoreactive LTC4 to 11 ng/10(6) cells, but without a significant change in the production of either LTB4 or PGD2. Bridging of Fc epsilon R on cocultured mast cells yielded a net percent release of beta-hexosaminidase and lipid mediator amounts and profile similar to those for BMMC. Bridging of Fc gamma R on purified mouse SMC resulted in a maximal net percent release of beta-hexosaminidase of 10% and the generation of 4, 1, and 17 ng/10(6) cells of immunoreactive LTC4, LTB4, and PGD2, respectively; the net percent release of beta-hexosaminidase and PGD2 generation were significantly greater than those obtained from BMMC. The Fc epsilon R-mediated net percent release of beta-hexosaminidase from purified SMC was 34%, with PGD2 being the predominant metabolite of arachidonic acid. That the predominant lipid mediator generated with activation by either Fc gamma R or Fc epsilon R is LTC4 for cocultured mast cells and PGD2 for SMC suggests that the mast cell phenotype rather than the receptor class being bridged determines the lipid mediator profile. The responsiveness to Fc gamma R bridging elicited by coculture of BMMC with fibroblasts in vitro and present in SMC derived in vivo relative to BMMC may relate to the previously measured increases in receptor number per cell, but may also involve the acquisition of an enhanced signal transduction capability, possibly through the increased expression of Fc gamma RIII.     

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.     

Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE

Anti-IgE-dependent activation of rat and human mast cells resulted in the preferential generation of the cyclooxygenase products prostaglandin D2 (PGD2) and prostaglandin I2 (PGI2) in the rat and PGD2 in the human. The average net generation of PGD2, determined by gas chromatography-mass spectrometry, was 13.1 ng/10(6) purified rat mast cells and 39.5 ng/10(6) dispersed, enriched human mast cells. After IgE-dependent activation, there was a linear relationship between the net quantities of PGD2 generated and of histamine secreted from dispersed human pulmonary cells when the number of mast cells was varied but the total number of cells was held constant, indicating that it is the number of mast cells participating in IgE-dependent activation, rather than total mast cell number, that determines PGD2 generation. A linear relationship was also shown between PGD2 generation, determined by radioimmunoassay, and the release of the granule marker beta-hexosaminidase from purified rat mast cells on the dose-response portion of the plot of their response to anti-IgE challenge. With higher concentrations of anti-IgE, PGD2 generation from rat mast cells plateaued, whereas net percent beta-hexosaminidase release increased further. In kinetic studies of rat mast cells activated with anti-IgE, the onset (1 to 2 min) and time of maximum generation (5 to 10 min) for PGD2 were delayed relative to the onset (15 to 30 sec) and completion (1 to 2 min) of beta-hexosaminidase release. Thus, the extracellular appearance of PGD2 during IgE-dependent mast cell activation represents a response additional to the secretion of granule-associated mediators.     

Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.     

Mast cell mediators stimulate synthesis of arachidonic acid metabolites in macrophages

Mast cells and macrophages were isolated from human lung tissues by using density gradient centrifugation, cell sorter, and adherence techniques. Passively sensitized mast cells in the absence of exogenous arachidonic acid (AA) released leukotriene (LT)C4, LTD4, PGD2, and thromboxane-B2 when challenged with Ag, and in the presence of AA, released 5-hydroxyeicosatetraenoic acid (HETE) and 15-HETE in addition to the above metabolites. Passively sensitized macrophages did not release significant amounts of AA metabolites when challenged with Ag. However, these cells released LTB4, LTC4, LTD4, LTE4, 5-HETE, PGE2 and 6-keto-PGF1 alpha when co-incubated with activated mast cells. During co-incubation, mast cells also generated greater amount of AA metabolites than when they were activated alone. The stimulatory action of mast cells on macrophages was shown to be due to the extracellular factor(s) present in the supernatant of the activated mast cells. Both heat and trypsin inhibited the biologic activity of mast cell-derived stimulatory factor. In addition, extraction of mast cells' materials with chloroform or ether showed no activity associated with the organic phase, suggesting it possibly possesses a protein nature, such as peptides, protease, or peptidase. These results suggest that mast cell-macrophage interaction might be important in the generation of multiple mediators in the airways during immediate hypersensitivity reactions.     

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Fc gamma RIIa, not Fc gamma RIIb, is constitutively and functionally expressed on skin-derived human mast cells

The expression of FcgammaR by human skin-derived mast cells of the MC(TC) type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of beta-hexosaminidase, PGD(2), leukotriene C(4) (LTC(4)), IL-5, IL-6, IL-13, GM-CSF, and TNF-alpha. FcgammaRIIa was consistently detected along with FcepsilonRI at the mRNA and protein levels; FcgammaRIIc was sometimes detected only by RT-PCR; but FcgammaRIIb, FcgammaRI, and FcgammaRIII mRNA and protein were not detected. FcgammaRIIa-specific mAb caused skin MC(TC) cells to degranulate and secrete PGD(2), LTC(4), GM-CSF, IL-5, IL-6, IL-13, and TNF-alpha in a dose-dependent fashion. FcepsilonRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC(4), which was not released by this agonist. Simultaneous but independent cross-linking of FcepsilonRI and FcgammaRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MC(TC) cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Immunologic release of heparin from purified rat peritoneal mast cells.

High m.w. [35S]heparin, labeled in vivo or in vitro, was released from purified rat mast cells by challenge with rabbit anti-rat F(ab')2, guinea pig anti-rat IgE, or calcium ionophore. The released and the residual heparin were isolated by Dowex 1 chromatography and were of comparable size by Sepharose 4B gel filtration. The majority of the released heparin was found by differential centrifugation to be granule-associated. Net percentage of mast cell heparin release, quantitated by metachromasia after isolation on Dowex 1 chromatography, correlated in a linear fashion with net percentage of histamine release, with heparin exhibiting a threshold requirement for onset of release. The correlation of histamine and high m.w. heparin release provides chemical support for the conclusion of others from ultrastructural studies that mast cell activation by immunologic means or by the calcium ionophore results in secretion of the whole granule.     

Biochemical and morphological modifications in dexamethasone-treated mouse bone marrow-derived mast cells

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.     

Antigenic stimulated release of arachidonic acid, lipoxygenase activity and histamine release in a cloned murine mast cell MC9

A cloned murine mast cell MC9 expresses phospholipase and lipoxygenase activity when stimulated with IgE and hapten. Addition of DNP-BSA to sensitized MC9 cells causes release of 58% of the cell histamine and 127 pmoles LTC4/10(6) cells. Prelabelling studies with [1-14C]-arachidonic acid showed that LTC4 production was proceeded by the release of arachidonic acid from membrane phospholipids. Approximately 8.7% of the cell arachidonic acid was released and half of this was converted to LTC4. The remaining radioactivity was converted to diHETES including LTB4 (15%), 5-HETE (10%), free arachidonic acid (10%), reesterified 5-HETE and arachidonic acid (8%) and prostaglandins (7%). This stimulation was dependent on hapten (DNP-BSA) and extracellular Ca++. Under identical conditions the calcium ionophore A23187 stimulated the release of 10.3% of the total cell arachidonic acid, and 51% of this was metabolized to LTC4. In addition the ionophore stimulated the release of 61% of the total cellular histamine.     

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.