Serosal Mast Cells Research Articles

Helicobacter pylori potentiates histamine release from serosal rat mast cells in vitro.

Helicobacter pylori seems to be involved in the etiology of peptic ulcer and chronic gastritis. Histamine is fundamental in gastric secretion modulation, and some features of H. pylori-associated gastritis (edema, vasodilatation, inflammatory cell infiltration) are typical of the histamine-mediated response. This in vitro study has been undertaken as a preliminary step, in order to find a possible link between H. pylori and histamine release. H. pylori isolated from gastric biopsies has been tested as whole washed bacterium, whole formalin-killed bacterium, and crude cell wall preparation with serosal mast cells obtained by density gradient centrifugation or elutriation from peritoneal and pleural washings of male Wistar albino rats. Histamine release was assayed fluorimetrically. No significant histamine release was obtained by testing the various bacterial preparations alone with mast cells. On the contrary, whole washed cells, whole formalin-killed cells and crude cell walls from H. pylori have been found to potentiate compound 48/80 or calcium ionophore A23187-induced histamine release. Crude cell walls showed the highest activity, whereas filtered supernatants from broth cultures constantly appeared inactive. The enhancement in histamine release differed between the different strains. The present in vitro study, which shows the potentiating effect of H. pylori on histamine release, may prove interesting; however, at present, clinical implications cannot be inferred and further studies as well as in vivo demonstrations are needed.

Three-dimensional models of four mouse mast cell chymases. Identification of proteoglycan binding regions and protease-specific antigenic epitopes.

Mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5 are serine proteases which are predicted to have chymotryptic specificity (chymases). They are bound to negatively charged heparin or chondroitin sulfate proteoglycans and are stored in secretory granules. Three-dimensional (3D) models of these four proteases were constructed with a comparative molecular modeling technique based on satisfaction of spatial constraints. The models were used to predict immunogenic epitopes and surface regions that are likely to interact with proteoglycans. Nine potential antigenic segments in the four chymases were identified on the basis of solvent accessibility, protrusion, flexibility, and sequence variability. These segments are suitable epitopes for preparation of protease-specific antipeptide immunoglobulin. Two regions with net charges ranging from +6 to +10 at neutral pH were found on the surfaces of mMCP-4 and mMCP-5. The two regions are located far from the substrate binding cleft at diametrically opposite ends of the folded proteases. A strong positive electrostatic potential surrounds the two regions. Thus, they are good candidates for binding sites that interact with heparin proteoglycan in the granules of serosal mast cells. In contrast, mMCP-1 and mMCP-2, which are present in granules of mucosal mast cells that contain chondroitin sulfate, lack one of these regions and have a lower charge density in the other. The differences between the 3D models provide a structural basis for the selective localization of specific chymases within mouse mast cells that contain different proteoglycans.

Inhibition of macrophage-mediated low density lipoprotein oxidation by stimulated rat serosal mast cells.

Mast cells and macrophages coexist in the human arterial intima where oxidation of low density lipoproteins (LDL) also takes place during atherosclerosis. To investigate whether mast cells play a role in macrophage-mediated oxidation of LDL, a model system was designed in which mast cells and macrophages were cocultured in incubation medium containing LDL. Stimulation of rat serosal mast cells to induce exocytosis of their cytoplasmic granules was found to inhibit macrophage-mediated oxidation of LDL. The inhibitory effect depended on the ability of mast cell-derived histamine, released from the exocytosed granules into the medium, to bind the copper ions necessary for propagation of the macrophage-initiated oxidation of LDL. In addition to binding free copper ions, the mast cell-derived histamine was also capable of inhibiting oxidation of LDL propagated by copper ions bound to the apolipoprotein B component of the LDL particle. The results indicate that mast cells may prevent cell-mediated oxidation of LDL and imply a potentially preventive role for the mast cell in atherosclerosis.

Helicobacter pylori potentiates histamine release from rat serosal mast cells

We have studied the effect ofHelicobacter pylori (H. pylori) and its bacterial products on mast cell histamine release evoked by compound 48/80, calcium ionophore A 23187 and cholic acid. No significant histamine release is obtained when the various bacterial preparations ofH. pylori are incubated alone with mast cells, but the release of histamine is dose-dependently and significantly enhanced when whole washed and formalin killed cells or crude cell walls are incubated with compound 48/80, calcium ionophore A 23187 or cholic acid. Crude cell walls show the highest activity whereas the filtered supernatants from broth cultures are consistently inactive.

New pharmacological profiles of loratadine: Effects on platelet aggregation and histamine release from repository cells

Loratadine is endowed with antiallergic activity. It reduces both the immunological release of histamine in active anaphylaxis of isolated guinea-pig mast cells and in anti-IgE challenged human basophils as well as the non-immunological release of histamine induced by compound 48/80 and calcium ionophore A 23187 in isolated purified rat serosal mast cells. Loratadine was also found capable of directly reducing platelet aggregation induced by different agonists in a concentration-dependent fashion. It is capable of blocking the potentiation afforded by histamine on human platelet aggregation induced by thrombin, collagen and arachidonic acid. The present results suggest a broader profile for loratadine beyond its original role as a non-sedative anti-H1-antihistaminic drug.

Metabolism of LDL in mast cells recovering from degranulation. Description of a novel intracellular pathway leading to proteolytic modification of the lipoprotein.

Rat serosal mast cells contain cytoplasmic secretory granules composed of a proteoglycan matrix in which histamine and neutral proteases are embedded. On stimulation, these granules are exocytosed, but some of them remain in the degranulation channels where on exposure to the extracellular fluid, they lose their histamine and a fraction of their proteoglycans. In vitro, such granule remnants efficiently bind low density lipoprotein (LDL) present in the incubation medium. After a lag period of about 10 minutes, the granule remnants, still within the channels and coated with LDL particles, are internalized by the parent mast cells. During subsequent recovery from degranulation, the apolipoprotein B of the intracellularly located remnant-bound LDL becomes efficiently (up to 70%) degraded by the proteolytic enzymes of the granule remnants. Since the granule remnants lack cholesteryl esterase activity, no LDL cholesterol is made available for cellular nutrition. Instead, selective proteolytic degradation of the bound LDL leads to formation of LDL particles enlarged by fusion on the granule remnant surface. In response to restimulation of the mast cells, about 50% of the fused LDL particles are exocytosed with the granule remnants. Of these, about one in five are expelled into the incubation medium. The granule remnants that again remain in the degranulation channels bind and internalize more LDL. This "round trip" of LDL in mast cells exposed to repeated stimulation constitutes a hitherto-unknown intracellular pathway for modification of LDL.

Inhibition of copper-mediated oxidation of LDL by rat serosal mast cells. A novel cellular protective mechanism involving proteolysis of the substrate under oxidative stress.

Rat serosal mast cells, when stimulated to exocytose their cytoplasmic granules, effectively blocked the copper-mediated oxidation of low density lipoproteins (LDLs) in vitro. This effect depended on the proteolytic activity of the formed extracellular granule remnants, since specific inhibition of chymase, the neutral protease that they contain, blocked the protective effect of the mast cells. The mechanism of this chymase-mediated inhibition of LDL oxidation was found to be binding of the copper ions present in the incubation medium by peptides released from LDL on proteolytic degradation of their apolipoprotein B (apoB) component. This was verified by demonstrating that addition of such peptides to LDL--copper ion mixtures completely prevented oxidation of LDL and that this protective effect could be overcome by adding copper ions in excess. Furthermore, proteolytic degradation of the apoB of LDL, with concomitant release of copper-containing peptides, left the partially degraded apoB without the copper ions necessary for propagation of LDL oxidation. These observations provide the first evidence for cell-mediated inhibition of LDL oxidation.

Intracellular degradation of Fc gamma RIII in mouse bone marrow culture-derived progenitor mast cells prevents its surface expression and associated function.

Although mouse interleukin-3-dependent, bone marrow culture-derived progenitor mast cells (BMMC) and a Kirsten sarcoma virus (KiSV)-immortalized mouse mast cell line (MC4w) both express on their surfaces receptors for the Fc portion of IgG (Fc gamma R), only MC4w degranulate upon Fc gamma R perturbation. As shown by surface iodination and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated proteins immunoprecipitated with the Fc gamma R-specific monoclonal antibody 2.4G2, a 26-kDa protein, identified as Fc gamma RIII by immunoblotting with antibody to Fc gamma RIII, was predominantly expressed on the surface of MC4w but minimally on BMMC. However, both BMMC and MC4w expressed mRNA for Fc gamma RIII as determined by RNA blot analysis, and both translated Fc gamma RIII as assessed by intrinsic radiolabeling and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated monoclonal antibody 2.4G2 immunoprecipitates. Pulse-chase analysis showed that intrinsically radiolabeled Fc gamma RIII was stable in MC4w cells but was degraded rapidly in BMMC and that newly synthesized Fc gamma RIII remained sensitive to digestion by endoglycosidase H in BMMC but rapidly became resistant in MC4w. These data suggest that the deficiency in surface Fc gamma RIII expression on BMMC is due to the degradation of Fc gamma RIII in the endoplasmic reticulum. Immunoprecipitation of surface Fc gamma R and Fc receptors for IgE (Fc epsilon RI) from digitonin-extracted cells followed by immunoblotting with antibody to Fc epsilon RI gamma-chain showed that gamma-chain is associated with surface Fc epsilon RI and Fc gamma R in MC4w, but only with Fc epsilon RI in BMMC, which lack surface Fc gamma RIII. Inasmuch as BMMC are progenitors of serosal mast cells, which, like MC4w, express surface Fc gamma RIII and undergo Fc gamma R-mediated activation, the data suggest that maturation of BMMC enables Fc gamma RIII to bypass degradation in the endoplasmic reticulum, resulting in the acquisition of functional Fc gamma RIII/gamma-chain complexes on the cell surface.

Isolation, characterization, and transcription of the gene encoding mouse mast cell protease 7.

A gene that encodes mouse mast cell protease (mMCP) 7 (also known as mouse mast cell tryptase 2) was isolated by genomic cloning with a cDNA that encodes mMCP-6, a tryptase in serosal mast cells. cDNAs encoding mMCP-7 were isolated from a bone-marrow-derived mast cell cDNA library. The mMCP-7 gene spans 2.3 kilobases and contains five exons rather than six, as found in the mMCP-6 and human mast cell tryptase I genes. Comparison of the 5' end of the transcript with the genomic sequence indicated that the region corresponding to the first intron in the mMCP-6 and human tryptase I genes is not spliced during transcription of mMCP-7 mRNA because of a point mutation at the intron 1 acceptor splice site; this results in a 5' untranslated region of 195 nucleotides, which is longer than that of any other known mast cell-specific transcript. mMCP-7 is 71-76% homologous with mMCP-6 and with dog and human mast cell tryptases, and it is the most acidic mast cell protease, with an overall net charge of -10. RNA blot analyses revealed that the mMCP-7 gene is transcribed in bone-marrow-derived mast cells but is not transcribed in mature serosal mast cells or in mucosal mast cell-enriched intestinal tissue of Trichinella spiralis-infected mice. Transcription of the mMCP-7 gene by differentiating bone-marrow-derived mast cells occurred within 1 week of bone-marrow culture but decreased dramatically after 3 weeks. Thus, the mMCP-7 gene displays a number of unusual structural characteristics and is distinctive in its transient and selective expression in immature mast cells maintained in interleukin 3-enriched medium.

Mast cell-mediated inhibition of reverse cholesterol transport.

Net cholesterol efflux from cholesterol-loaded macrophages, i.e., foam cells, was induced by incubating the foam cells with high density lipoprotein3 (HDL3). However, when the incubation system included rat serosal mast cells stimulated to trigger exocytosis of their cytoplasmic secretory granules, the ability of HDL3 to induce cholesterol efflux was largely lost. This loss was found to be due to the proteolytic action of chymase, the neutral protease of the granules, which degraded the apolipoproteins of HDL3, so rendering them unable to mediate cholesterol efflux from the foam cells. The observation defines a novel cell-dependent mechanism that blocks the initial steps of reverse cholesterol transport and suggests a role for mast cell chymase in cellular accumulation of cholesterol, an early stage in atherogenesis.

Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.

An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.

IL-10 induces transcription of the gene for mouse mast cell protease-1, a serine protease preferentially expressed in mucosal mast cells of Trichinella spiralis-infected mice.

Mouse bone marrow-derived mast cells (BMMC) obtained by culturing progenitor cells with rIL-3 express mouse mast cell protease (MMCP)-5 mRNA but not MMCP-1 mRNA or MMCP-4 mRNA. In terms of mast cell differentiation, these transcripts encode one early-expressed and two late-expressed chymases, respectively. cDNA and cRNA probes were used in RNase protection assays and RNA blot analyses to study the expression of these three homologous protease genes in cultured mast cells and in helminth-infected mice. Intestinal tissue from Trichinella spiralis-infected mice, containing high numbers of mucosal mast cells, had abundant amounts of MMCP-1 mRNA but only minimal amounts of the serosal mast cell transcript that encodes MMCP-4. Exposure of mouse BMMC to rIL-10-induced transcription of the MMCP-1 gene but not the MMCP-4 gene, and a cDNA encoding MMCP-1 was obtained from these rIL-10-treated cells. The expression of MMCP-1 mRNA in BMMC depended on the continuous exposure of these cells to rIL-10, and the level of MMCP-1 mRNA (but not MMCP-5 mRNA) was substantially higher in BMMC maintained in rIL-4 and rIL-10 than in rIL-3 and rIL-10 or in rIL-3, rIL-4, and rIL-10. Thus, whereas rIL-3 elicits transcription of early expressed genes in cultured mast cells, it suppresses the transcription of late-expressed genes. These in vitro and in vivo transcription studies also indicate that rIL-10 preferentially induces differentiation of mouse progenitor cells in a mucosal mast cell-specific lineage, and that expression of granule serine protease genes is regulated in a subclass-specific manner in mouse mucosal mast cells and serosal mast cells.

Influence of aging on mast cell production of a nitric oxide-like factor: Comparison between normal and hypertensive rats

Rat serosal mast cells (MCs) isolated from Wistar albino rats synthesize a nitric oxide (NO)-like factor which inhibits platelet aggregation, stimulates guanylate cyclase and modulates mast cell histamine release. Aging can affect the ability of MCs to generate and release chemical mediators. The aim of the present study was to evaluate the ability to generate a NO-like factor by MCs isolated from 2, 6 and 18 month old normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats (systolic blood pressure 180±2.5 mm Hg) by two bioassays: inhibition of platelet aggregation and stimulation of MC guanylate cyclase. MCs from SH rats produced less NO-like activity than MCs from normotensive WKY rats of the same age. In both strains aging decreased the capacity of MCs to generate the NO-like factor. These results suggest that NO synthesis and/or release from MCs is involved in some events associated with aging and hypertension.

Histamine secretion from mast cells stimulated with platelet activating factor (PAF)

Platelet activating factor (PAF) produced a dose-dependent release of histamine from rat peritoneal mast cells. The release was non-cytotoxic at 5 μM but cytotoxic at a concentration of 10 μM and above. Isolated tissue mast cells of the rat, guinea-pig and man showed varying responses to PAF but the release was again generally cytotoxic. The non-cytotoxic release from rat serosal mast cells stimulated with low concentrations of PAF was potently inhibited by phosphodiesterase inhibitors, cAMP-active drugs and the naturally occurring flavonoid quercetin. The release was also selectively inhibited by the specific PAF antagonist BN 50730 but not by BN 52021 or WEB 2086. These findings suggest that PAF may interact with the mast cell membrane to produce mediator release, rather than acting via a specific receptor.

Serosal mast cells maintain their viability and promote the metabolism of cartilage proteoglycans when cocultured with chondrocytes.

To determine the consequences of mast cell (MC)-chondrocyte interactions. Cocultured cells were analyzed histochemically, morphologically, biochemically, and functionally. Cocultured MC adhered to the chondrocytes and remained viable. Chondrocytes cocultured with nonactivated MC produced more proteoglycans than did chondrocytes cultured alone, and these proteoglycans possessed an intact hyaluronic acid-binding region. In contrast, most of the proteoglycans produced by chondrocytes cocultured with activated MC were degraded. These studies indicate that a complex interaction occurs in which the nonactivated MC stimulates biosynthesis and the activated MC degrades cartilage proteoglycans.

Correlation between Nitric Oxide Synthesis and Histamine Release in Mast Cells from Spontaneously Hypertensive Rats: Influence of Aging.

Isolated perfused hearts and serosal mast cells (MC) of 2- 6- and 18-month-old spontaneously hypertensive rats (SH) were compared to hearts and MC from Wistar-Kyoto (WKY) rats of the same age for their ability to release nitric oxide (NO). The relationship between histamine release and NO production was also investigated. In hearts and MC of 2-, 6- and 18-month-old SH rats, the production of NO is significantly lower than in hearts or MC of WKY rats of the same age. Concomitantly, the spontaneous release of histamine in cardiac perfusates and MC reactivity to various exocytotic stimuli (compound 48/80, calcium ionophore A 23187) is modified by aging and hypertension.

Inhibitory effects of organotin compounds on histamine release from rat serosal mast cells.

Tributyltin inhibited compound 48/80-induced histamine release from rat serosal mast cells in a dose-related manner. Triphenyltin and tripropyltin also showed a strong inhibitory effect on the histamine release and the degree of the inhibition was the same as that of tributyltin. The inhibitory effects of other organometals were much smaller than those of the organotins. Tributyltin and triphenyltin also showed a tendency to inhibit histamine release induced by concanavalin A + phosphatidylserine and calcium ionophore A 23187. The present results suggest that certain triorganotin compounds may have a specific inhibitory effect on histamine release by acting on the same process in each of the three stimulus responses.

Formation of histamine-releasing activity from albumin by medium conditioned by endotoxin-stimulated rat peritoneal macrophages.

Incubation of bovine serum albumin (BSA), rat serum albumin or rat plasma with medium conditioned by endotoxin stimulated rat peritoneal macrophages produced an activity that released histamine from isolated rat serosal mast cells. The amount of histamine-releasing activity (HRA) produced increased with the length of the incubation period, with the concentration of albumin, with the number of macrophages stimulated, and with the duration of exposure of the macrophages to endotoxin. Moreover, the formation of the HRA showed a dependency on the pH of the incubation medium with an optimum at pH 4.5. Boiling the medium conditioned by stimulated macrophages before its incubation with albumin or including the acid protease inhibitor, pepstatin with the conditioned medium prevented the formation of HRA. The generation of HRA was not inhibited by pretreatment of the macrophages with the inhibitor of protein synthesis, cycloheximide. Media from macrophages not stimulated with endotoxin failed to generate HRA. Histamine release from mast cells in response to the HRA was inhibited by pretreatment of the cells with antimycin A and deoxyglucose or by preincubation in Ca-free Locke's solution containing a calcium chelating agent. When injected intradermally into anesthetized Evan's Blue treated rats, the generated HRA produced a change in vascular permeability that was prevented by the H1 antagonist, diphenhydramine. Treatment of the HRA with carboxypeptidase A reduced its ability to stimulate histamine release from mast cells. Histamine-Releasing Peptide (HRP), a neurotensin-related octapeptide, shown previously by us to be formed by the action of cathepsin D or pepsin on albumin, was identified by radioimmunoassay in acid:acetone extracts of the histamine-releasing activity. It is concluded that the formation of HRA is due to the actions of enzymes released from macrophages acting on albumin. It is suggested that such histamine-releasing activity could be formed during the later stages of the inflammatory response and that HRP is one of the peptides present.

Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis.

Stimulation of rat serosal mast cells in vitro triggers exocytosis of secretory granules from their cytoplasm. Thereupon, the granules lose their perigranular membranes, and about 40% of the heparin proteoglycans and all of the chondroitin sulfate proteoglycans that they initially contained are released into the incubation medium. At physiologic ionic strength and calcium ion concentration, the solubilized heparin proteoglycans, but not the chondroitin sulfate proteoglycans, form insoluble complexes with the low density lipoproteins (LDL) present. We calculated that the heparin proteoglycans could bind approximately seven times their own mass (Mr about 1 x 10(6)) of LDL cholesterol. Using gold-labeled LDL, we observed massive phagocytosis of the heparin proteoglycan-LDL complexes by cultured mouse macrophages in vitro, which was inhibited by cytochalasin B. Uptake of LDL by mouse macrophages was 45-fold higher in the presence of solubilized heparin proteoglycans than in their absence, and continued unabated over a 72-h period, indicating that the uptake process was not under negative feedback control. Excess amounts of acetyl-LDL or polyinosinic acid inhibited the uptake of these insoluble heparin proteoglycan-LDL complexes, indicating that their phagocytosis was mediated by scavenger receptors of the acetyl-LDL receptor type. The experiments reveal the following pathophysiologic mechanism relevant to atherogenesis: stimulated mast cells secrete soluble heparin proteoglycans capable of forming insoluble complexes with LDL and thereby trigger uptake of LDL by macrophages through scavenger receptor-mediated phagocytosis.

Effect of mast cell chymase on the morphology of thyroid cells in vitro.

We studied the effect of mast cell chymase on the thyroid cells in culture. Rat serosal mast cells, similar functionally to connective tissue mast cells, were obtained after lavage of the peritoneal cavity and lyzed by freezing. The resulting lysate was used as crude enzyme preparation. Mast cell chymase was purified from the crude preparation by anion exchange chromatography. Crude and purified chymase incubated with thyroid cells induced cellular retraction, the appearance of long processes and gradual cell detachment from the substratum. The effect of the enzyme was not cytotoxic. The immunofluorescence studies of thyroid cells showed a decreased amount of polymerized actin and tubulin after incubation with chymase. Neutral protease inhibitor abolished the effect of crude and purified chymase on thyroid cell morphology. The above findings suggest that mast cell chymase may have a function in the control of cell morphology and cell-matrix interaction.