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.     

The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor

A variety of mast cell degranulating agents have previously been shown to induce mast cell hyperplasia in adult rats. In neonates 2.5 S nerve growth factor (NGF) induces a hyperplasia of both mucosal and connective tissue mast cells (MMC and CTMC). We have examined the role of the potent mast cell degranulating properties of NGF on its ability to induce mast cell hyperplasia. Administration of NGF in combination with the mast cell stabilizing agent disodium cromoglycate was found to abrogate the CTMC hyperplasia induced by NGF alone. Treatment of neonatal rats with the alternate degranulating agent compound 48/80 was found to induce a limited CTMC but not a MMC hyperplasia. A supernatant obtained by degranulating purified adult rat peritoneal mast cells with anti-IgE was found to induce hyperplasia of the CTMC population similar to that observed with NGF administration. However, this degranulation product supernatant only induced a limited MMC hyperplasia as judged by RMCP II content of the tissues. These results suggest that NGF has dual action inducing mast cell hyperplasia; CTMC hyperplasia being dependent on the ability of NGF to degranulate mast cells. MMC hyperplasia induced by NGF is independent of CTMC degranulation. Degranulation products from peritoneal mast cells act to increase both MMC and CTMC populations in the neonate. These data suggest that the CTMC population may be regulated by an autocrine positive feedback mechanism in vivo.     

Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs

This study investigated the capacity of conjugated linoleic acids (CLA) to reduce ex vivo antigen-induced release of eicosanoids in a type I hypersensitivity model. Guinea pigs were fed a diet containing 0.25% safflower oil (control) or 0.25% CLA [43% trans (t)10, cis (c)12; 41% c9, t11/t9, c11 18:2] for 2 wk before and during sensitization to ovalbumin (OVA). Lungs, tracheas, and bladders were incubated in physiological saline solution (PSS) for 1 h (basal mediator release) and challenged with OVA (0.01 g/l PSS) for 1 h (mediator release in response to antigen). Eicosanoids were quantified by HPLC/tandem mass spectrometry or enzyme immunoassay. CLA feeding resulted in no change in basal release but decreased eicosanoid release from sensitized tissues in response to antigen challenge in the following manner: thromboxane B(2), 6-keto-prostaglandin (PG)F(1alpha), PGF(2alpha), PGD(2), PGE(2) by 57-75% in lung, 45-65% in trachea, and 38-60% in bladder; and leukotriene C(4)/D(4)/E(4) by 87, 90, and 50% in lung, trachea, and bladder, respectively. These data indicate that feeding CLA reduces lipid-derived inflammatory mediators produced by this type I hypersensitivity model.     

Development of mucosal mast cells after injection of a single connective tissue-type mast cell in the stomach mucosa of genetically mast cell-deficient W/Wv mice

Mast cells may be classified into at least two phenotypically distinct populations: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). Mast cells in the peritoneal cavity of mice are typical CTMC, whereas mast cells in the mucosa of the stomach show morphologic characteristics of MMC. We investigated whether CTMC may change to MMC. A single peritoneal mast cell of WBB6F1-+/+ mice was identified under the phase-contrast microscope, picked up with the micromanipulator, and injected into the stomach wall of genetically mast cell-deficient WBB6F1-W/Wv mice. The cells with histochemical and electron microscopical features of MMC developed in the mucosa, and those with histochemical features of CTMC in the muscularis propria. This directly demonstrates that a certain proportion of CTMC may function as a bipotent precursor for both MMC and CTMC.     

Role of gastric mast cells in the regulation of central TRH analog-induced hyperemia in rats

RX 77368 (RX) increases gastric mucosal blood flow by a vagal cholinergic mechanism. The relative roles of mucosal and connective tissue mast cells (MMC and CTMC) were investigated in RX-injected rats. Blood flow and mast cell degranulation were measured after intracisternal RX. RX significantly increased gastric mucosal blood flow, and sequentially degranulated CTMC and MMC. Ketotifen or doxantrazole inhibited the hyperemic response. Ondansetron, RS-039604-90, or famotidine, but not ketanserin or pyrilamine, reduced hyperemia. Mast cells mediate RX-induced gastric hyperemia via 5-HT3, 5-HT4, and H2 receptors; initial increase depends upon CTMC whereas MMC contributes to the later response.     

Establishment and characterization of hybrid rat mast cells

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.     

Histochemical heterogeneity of dermal mast cells in athymic and normal rats

Summary Mucosal mast cells (MMC) and connective tissue mast cells (CTMC) of the rat contain different proteoglycans, which can be distinguished using histochemical methods. The chondroitin sulphate proteoglycan of the MMC, unlike the heparin of the CTMC, does not show fluorescent berberine binding, is susceptible to aldehyde fixatives and stains preferentially with Alcian Blue in a staining sequence with Safranin. The majority of the dermal mast cells are typical CTMC and are located in the deep part of the dermis. Subepidermal mast cells are comparatively few in normal rats but numerous in athymic rats and mice. These cells differ from other dermal mast cells in that they stain preferentially with Alcian Blue and they appear to contain little histamine. We examined some of the histochemical properties of the skin mast cells of female PVG-rnu/rnu rats and their heterozygous littermates aged from 5 to 29 weeks. The thiazine dye-binding of the subepidermal mast cells was partially blocked by formaldehyde fixation and only about half of them showed a weakly fluorescent berberine binding. The critical electrolyte concentration of the Alcian Blue staining of the subepidermal mast cells was between that of CTMC and MMC. Deaminative cleavage with nitrous acid abolished the staining of all skin mast cells, while that of the MMC was unaffected. There were no statistically significant differences in the staining patterns of the dermal mast cells between different ages or groups of rat. These results indicate that the subepidermal mast cells contain a heparin proteoglycan which is, however, different from that of the typical CTMC of other sites. They thus appear to represent a second example of a mast cell within a defined anatomical location exhibiting a distinct proteoglycan expression.     

Mast cells are involved in the gastric hyperemic response to acid back diffusion via release of histamine

Acid back diffusion into the rat stomach mucosa leads to gastric vasodilation. We hypothesized that histamine, if released from the rat mucosa under such conditions, is mast cell derived and involved in the vasodilator response. Gastric blood flow (GBF) and luminal histamine were measured in an ex vivo chamber. Venous histamine was measured from totally isolated stomachs. Mucosal mast cells (MMC), submucosal connective tissue mast cells (CTMC), and chromogranin A-immunoreactive cells (CgA IR) were assessed morphometrically. After mucosal exposure to 1.5 M NaCl, the mucosa was subjected to saline at pH 5.5 (control) or pH 1.0 (H(+) back diffusion) for 60 min. H(+) back diffusion evoked a marked gastric hyperemia, increase of luminal and venous histamine, and decreased numbers of MMC and CTMC. CgA IR cells were not influenced. Depletion of mast cells with dexamethasone abolished (and stabilization of mast cells with ketotifen attenuated) both hyperemia and histamine release in response to H(+) back diffusion. GBF responses to H(+) back diffusion were attenuated by H(1) and abolished by H(3) but not H(2) receptor blockers. Our data conform to the idea that mast cells are involved in the gastric hyperemic response to acid back diffusion via release of histamine.     

新生大鼠雌激素注射后睾丸肥大细胞的变化

新生大鼠注射雌二醇后,睾丸肥大细胞于第30天可见到,细胞数量随年龄增长而增多,生后4－6个月,睾丸网附近仍可见大量肥大细胞。睾丸内的肥大细胞比皮肤内的结缔组织肥大细胞（CTMC）小而与小肠粘膜的粘膜肥大细胞（MMC）相近,AB－S染色后基本着蓝色,硫酸小檗碱荧光染色后呈现中等强度黄色荧光,结果提示,新生大鼠雌激素注射后睾丸内肥大细胞的增多可能与免疫过程有关,睾丸内肥大细胞与CTMC和MMC皆有所不同。     

Cloning of the cDNA and gene for mouse mast cell protease 4. Demonstration of its late transcription in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the mouse and rat

Based on the amino-terminal amino acid sequence of the mature form of mouse mast cell protease 4 (MMCP-4), previously identified in peritoneal connective tissue mast cells (CTMC) and Kirsten sarcoma virus-immortalized mast cells (KiSV-MC), a 26-mer oligonucleotide probe was constructed and used to clone cDNAs for MMCP-4 from a KiSV-MC1 cDNA library. MMCP-4 is the first secretory granule serine protease of CTMC to be molecularly cloned. Using a cDNA probe derived from the 3'-untranslated portion of the MMCP-4 cDNA, the gene for MMCP-4 and a second highly related gene (mouse mast cell protease-like, MMCP-L) were cloned from a BALB/c mouse genomic DNA library and sequenced entirely, including approximately 2 kilobases of the 5'-flanking region. MMCP-4 and MMCP-L have five exons of identical length, four introns of nearly identical length, and approximately 900 base pairs of 5'-flanking DNA with sequence similarity by dot matrix analysis. By RNA blot analysis with gene-specific probes for MMCP-4 (bases 497-633 of the cDNA) and MMCP-L (bases 502-638 of the cDNA), mRNA for MMCP-4 was present in KiSV-MC5, CTMC, and the intestine of a mouse infected with the parasite Nippostrongylus brasiliensis markedly enriched for mucosal mast cells (MMC); MMCP-L mRNA was detected only in the intestine of the N. brasiliensis-infected mouse. MMCP-4 mRNA was not expressed in normal mouse intestine or in interleukin 3-dependent bone marrow-derived mast cells, which can serve as precursors to both MMC and CTMC. This finding suggests that MMCP-4 is transcribed relatively late in the development of both the CTMC and MMC subclasses and underscores the fact that mouse bone-marrow-derived mast cells are immature mast cells, rather than tissue culture equivalents of the MMC subclass.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

Hepatic mast cells in rats infected with Taenia taeniaeformis

Mast cells appeared in the liver around metacestodes of Taenia taeniaeformis by 13 days after infection (DAI) of rats. The cells often occurred in clusters. The population increased until 28 DAI, then gradually declined. These hepatic mast cells (HMC) were compared to intestinal mucosal mast cells (MMC) and connective tissue mast cells (CTMC) histochemically, morphologically and in their response in vivo to Compound (built|48/80) and dexamethasone. Hepatic mast cells were similar to MMC in that they stained strongly blue with Astra blue at pH 1·0, could not be demonstrated with 0·005% toluidine blue, disappeared after treatment with dexamethasone, and were unaffected by 48/80. Immunoglobulin-containing cells in the liver were characterized by immunofluorescence. Immunoglobulin E-positive, and to a lesser extent IgG_2a-, and IgG_2c-positive cells surround the parasites in increasing numbers until 28 DAI, then declined. Many IgE-positive cells were HMC, and the IgE was frequently located intracytoplasmically. These cells were clearly distinguishable from eosinophils which stained characteristically with Giemsa and did not react with the anti-IgE probe. The results suggest that mast cell progenitors may be induced to localize and proliferate at the host-parasite interface in the Taenia-infected livers, giving rise to a cell population comparable to the MMC often seen at parasitized mucosal surfaces.     

Tumor necrosis factors in a murine model of allergic peritonitis: effects on eosinophil accumulation and inflammatory mediators' release

In allergic disorders, the role of tumor necrosis factors (TNF) is not well established. We investigated the role of TNF in allergic peritonitis induced by ovalbumin (OVA) challenge in double TNF (TNF-alpha(-/-)/lymphotoxin-alpha(-/-)) knock out (TNF-KO) mice. In the peritoneal lavage of TNF-KO mice, mast cell number and histamine level (radioenzymatic assay) were similar to that in wild type (WT) mice. TNF-alpha (ELISA) and histamine were increased 1 h after challenge in WT mice. However, three days later eosinophil number and eosinophil peroxidase (EPO) levels (colorimetric-enzymatic assay) were found to be lower in TNF-KO mice. A second challenge three days after the first, increased EPO, histamine and IL-6 (ELISA) but did not alter eosinophil and mast cell numbers in both types of mice. On the other hand histamine and IL-6 were higher, while EPO was lower in TNF-KO mice. In conclusion, our findings show that TNF is involved in eosinophil accumulation and inflammatory mediators' release in a murine model of allergy.     

Genetically mast cell-deficient W/Wv mice as a tool for studies of differentiation and function of mast cells

Genetically mast cell-deficient W/Wv mice are useful for the analysis of mast cell biology, especially as recipients of bone marrow cells and skin pieces. Inasmuch as suspension and clonal cultures of mast cells have been developed, we combined these in vivo and in vitro systems. Suspension-cultured mast cells had morphological and biochemical characteristics similar to those of mucosal mast cells (MMC). However, i.p. injection of such cultured mast cells gave rise to development of cells with characteristics similar to those of connective tissue mast cells (CTMC). When peritoneal cells of normal +/+ mice were cultured in methylcellulose, pure mast cell colonies appeared. Cells from individual mast cell colonies were divided and injected into the skin and stomach wall of W/Wv mice; CTMC developed in the skin and MMC in the stomach mucosa. This indicates the presence of a common precursor for CTMC and MMC. Morphology of such bipotent mast cell precursors was studied by using micromanipulation. About 4% of morphologically identifiable peritoneal mast cells may function as the bipotent precursors. Although W/Wv mice showed a defect in resistance against ixodid ticks, injection of suspension-cultured mast cells normalized the defect. The four examples mentioned above indicate that combinations of in vivo and in vitro systems increase the usefulness of W/Wv mice.     

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.     

Endotoxin-induced lung injury in rats: role of eicosanoids

We studied lung vascular injury and quantitated lung eicosanoids in rats after intraperitoneal injection of Salmonella enteritidis endotoxin. Within 40 min after endotoxin injection (20 mg/kg), lung tissue thromboxane B2 doubled, although 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) increased by 8- to 10-fold. Lung 5-hydroxyeicosatetraenoic acid and leukotriene C4 were variably increased by endotoxin. The levels of all eicosanoids returned to base line 6 h after endotoxin challenge. Lung vascular injury, as assessed by the extravascular accumulation of 125I-albumin and water in isolated perfused lungs, was observed 90 min after endotoxin injection (0.02-20 mg/kg) in vivo. Inhibition of the cyclooxygenase pathway with indomethacin and the lipoxygenase pathway with diethylcarbamazine and 2-(12-hydroxydodeca-5,10-dinyl)-3,5,6-trimethyl-1,4-benzoqui none failed to attenuate endotoxin-induced lung injury. In addition, essential fatty acid deficiency, which markedly reduced lung tissue levels of 6-keto-PGF1 alpha, thromboxane B2, and leukotriene C4, did not protect against endotoxin injury. We conclude that although lung eicosanoids are activated during endotoxemia, they do not play a crucial role in the development of acute lung vascular injury in rats.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.     

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.     

Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.