Turnover of histamine in mucosal and connective tissue mast cells of the rat

Mast cells constitute a heterogenous cell system. The specific type of mucosal mast cell (MMC) of the gut differs with respect to a number of properties from the classical connective tissue mast cell (CTMC) found in, e.g. skin, tongue and the peritoneal cavity. This report summarizes recent findings concerning turnover rates of amines and heparin in the two cell types. The elimination rate of radiactively prelabelled histamine, 5-hydroxytryptamine (5-HT) and heparin from peritoneal CTMC was compared with the elimination of radiolabelled histamine from tongue, where histamine is stored in CTMC and duodenum where it is stored in MMC. The elimination of histamine from peritoneal CTMC was slow (t _1/2=23 days) and did not differ significantly from that of 5-HT (t _1/2=25 days) and heparin (t _1/2=35 days) suggesting a low degree of secretory activity in the normal rat. The elimination rate of histamine from the tongue was also very slow. The specific radioactivity of histamine in duodenum was decreasing more rapidly. This was explained by a dilution of the radioactivity since the histamine content increased during the experimental period, and also by MMC death. The results are compatible with the assumption that CTMC and MMC are secretory cells, but with low activity until recruited by adequate, immunological or other stimuli.     

IgE receptors from rat intestinal mucosal and peritoneal mast cells show mast cell subtype-specific differences

High- (alpha chain) and low-affinity IgE receptors from purified populations of rat intestinal mucosal (IMMC) and peritoneal mast cells (PMC) were characterized by SDS-PAGE. Receptor expression and molecular weight were compared. IMMC yielded 59-kilodalton (kDa) alpha chains of the high-affinity receptors and two forms (58, 50 kDa) of low-affinity receptors, whereas PMC possessed only 51-kDa alpha chains and 56-kDa low-affinity receptors. These differences extend the evidence for functional diversity between mast cell subtypes.     

Isolation and characterization of IgE receptors from rat intestinal mucosal mast cells

High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.     

Mucosal mast cells : III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells

Quercetin, a naturally occurring flavonol structurally related to the antiallergic drug disodium cromoglycate inhibits anaphylactic histamine release from MMC isolated from the small bowel LP of the rat previously infected with the nematode Nippostrongylus brasiliensis. This contrasts with our previous observation that cromoglycate is inactive in this system. The present effect is immediate and does not decrease on preincubation with the drug. The flavonoids acacetin , apigenin , chrysin , and phloretin also demonstrate significant activity but are less potent than quercetin. Catechin, flavone, morin, and taxifolin are inactive. These results resemble those previously reported for the human basophil. In contrast, all compounds with the possible exception of taxifolin demonstrate significant activity against rat PMC. Acacetin and chrysin are the most effective inhibitors and are more active than quercetin. Rutin (the glycane of quercetin) and phlorezin (the glycane of phloretin) are inactive in both systems. These results are discussed in terms of the functional heterogeneity of mast cells from different sources and identify a group of compounds other than doxantrazole (reported previously), which inhibit histamine secretion by MMC.     

IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells

Murine mesenchymal stem cells (MSC) have the ability to inhibit allogeneic immune responses. Two different mechanisms, either cell contact-dependent or independent, have been proposed to account for this immunosuppression. The focus of this study was to elucidate the involvement of soluble suppressive factors secreted by murine MSC in an inflammatory setting, and their role in MSC immunomodulation. In a non-inflammatory environment, bone marrow derived murine MSC constitutively expressed low levels of COX-2, PGE-2, TGF-beta1 and HGF, but not IL-10, PD-1, PD-L1 or PD-L2. These MSC were able to significantly reduce alloantigen driven proliferation in mixed lymphocyte reactions as well as mitogen driven proliferation. The pro-inflammatory cytokines IFN-gamma and TNF-alpha did not ablate MSC mediated immunosuppression. MSC expression of PGE-2, IDO and PD-L1 was differentially regulated by these cytokines. COX-2 and PGE-2 expression by MSC were upregulated by both IFN-gamma and TNF-alpha, and using a biochemical inhibitor this was shown to have an essential, non-redundant role in modulating alloantigen-driven proliferation. However, the surface expression of PD-L1 was induced by IFN-gamma but not TNF-alpha and similarly functional IDO expression was only induced by IFN-gamma stimulation. Blocking studies using neutralising antibodies and biochemical antagonists revealed that while PD-L1 induction was not essential, IDO expression was a prerequisite for IFN-gamma mediated MSC immunomodulation. These data demonstrate that murine MSC expression of immunomodulatory factors dramatically changes in a pro-inflammatory environment and that IFN-gamma in particular has an important role in regulating MSC immunomodulatory factor expression.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells.

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the 'state of equilibrium' at the cell surface caused by the superfusion (Uvn?s et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 microM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 microM. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Anthracycline-induced histamine release from rat mast cells

Comparisons were made of the ability of doxorubicin, daunorubicin, rubidazone and aclacinomycin A to release histamine from rat peritoneal mast cells. Preliminaryin vitro experiments indicated that doxorubicin (10^–6 to 2.5×10^–4 M), in contrast to compound 48/80 and the calcium inophore A23187, did not produce significant release under any condition tested when purified or unpurified rat mast cells were used. Inin vivo experiments, released histamine was measured in the cell-free supernatant of peritoneal fluid of rats after intraperitoneal injection of the agents. The time course of doxorubicin-induced histamine release from the peritoneum was rapid, with maximal release occurring within 4 to 6 min. Dose-response curves of the 4 agents over the range 10^–5 to 3.3×10^–3 M revealed that all caused histamine release, with 10^–3 M concentrations of each causing maximal release of comparable magnitude to that produced by 9.5×10^–6 M A23187. Treated mast cells recovered from the peritoneal cavity showed degranulation and vacuolization when examined by electron microscopy. Increased vascular permeability by the Evans-blue test was also noted with all 4 agents, and zones were of comparable size after injection of the highest concentration of each agent.The results indicate thatin vivo, doxorubicin, daunorubicin, rubidazone and aclacinomycin A cause a rapid release of histamine from rat mast cells and an increase in vascular permeability in rat skin. There also appeared to be a reasonable correlation between the blueing reaction and histamine release in the peritoneal cavity in that the doses that did not cause skin blueing also failed to cause histamine release. The lack of histamine release by doxorubicin from mast cell preparationsin vitro suggests that alterations to the doxorubicin molecule or the presence of other critical substances may be necessary for this activity to commence.     

Nasal mucosal mast cells and histamine in hay fever. Effect of topical glucocorticoid treatment

Symptomatic seasonal allergic rhinitis has previously been found to be associated with a redistribution of mast cells from the subepithelial stroma to the epithelial lining and the surface of the nasal mucosa. The present study was designed in order to elucidate the interaction between topical glucocorticosteroids, effective in the treatment of allergic rhinitis, and the migration of mast cells described earlier. Six patients treated prophylactically in the nose with budesonide were studied. Imprints and biopsies from the nasal mucosa were taken 2-3 weeks before and 2-3 weeks into the birch pollen season. The biopsies were used for light microscopy and tissue histamine determination. The morphologic studies showed, also in the actively treated patients, an increased number of metachromatically stained cells on the nasal mucosal surface of the same order of magnitude as previously reported for untreated patients. We did, however, find a decrease in the histamine content of the nasal mucosa, which was not associated with a decrease in the number of mast cells. Together with similar previous findings in the unstimulated allergic nasal mucosa these results suggest that glucocorticosteroids induce a decrease in the mast cell histamine pool, possibly due to an inhibition of the intracellular synthesis of histamine. This effect might contribute to the clinically beneficial effect of topical glucocorticosteroids in the treatment of hay fever.     

Auranofin inhibits histamine release from rat peritoneal mast cells.

The effect of auranofin on histamine release from immunologic and non-immunologic activated rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) was investigated. When MC/3T3 were preincubated with 2 x 10(-5) M auranofin and thereafter challenged with anti-IgE antibodies, a maximal inhibition of histamine release (86.2%) was obtained. Non-immunological histamine release induced by compound 48/80, substance P and bradykinin was inhibited to a lesser degree, i.e. 36.0%, 37.6% and 24.0% respectively. Simultaneous incubation of auranofin and the stimulating agents resulted in a higher inhibition of histamine release: anti-IgE antibodies--92.0%; compound 48/80--73.5%; substance P--46.1%. We conclude that auranofin effectively reduces histamine release from immunologic and non-immunologic activated mast cells. This may be relevant to the control of allergic reactions.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the ‘state of equilibrium’ at the cell surface caused by the superfusion (Uvnäs et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 μm and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 μm. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Inhibition of cholinergic histamine release in rat mast cells

Adrenaline inhibits the acetylcholine-evoked histamine release from isolated purified rat mast cells, in a dose-dependent fashion. The inhibitory effect of adrenaline is reversed by preincubating the cells with a beta-blocker, alprenolol, but not by preincubating them with an alpha-blocker, phentolamine. These results were confirmed by observations using an electron microscope and they suggest that adrenaline inhibits the cholinergic histamine release from rat mast cells acting upon beta-receptors.     

Mast cell heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cells

Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two-dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS-PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2-8; pI 9-9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule-associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (less than 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1-9) and, in general, they were more acidic (pI about 8.5-9) than those of PMC. Four major basic proteins (IB6-9) were all 24 kDa but were slightly different in isoelectric points. These and another 46-kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP-binding proteins in IMMC. In spite of remarkable differences between basic granule-associated proteins in PMC and basic proteins in IMMC, spots in the neutral-acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule-associated proteins with serine protease activity.     

Endothelins regulate mediator production of rat tissue-cultured mucosal mast cells. Up-regulation of Th1 and inhibition of Th2 cytokines

Mast cells have been shown to produce endothelin-1 (ET-1) and to express ET receptors. Thus, we postulated that ETs modulate mast cell mediator production in an autocrine manner. Rat tissue-cultured mast cells (RCMC-1) were incubated with exogenous ET-1 or ET-3, and beta-hexosaminidase release and TNF, IL-4, IL-10, IL-12, IL-13, macrophage inflammatory protein-1alpha (MIP-1alpha), and nitric oxide (NO) production were investigated. ET-1 and -3 induced the release of beta-hexosaminidase and TNF and of mRNA expression. An antagonist of the ET(B) receptor subtype abrogated ET-stimulated TNF release, although ET(A) and ET(B) receptors have been identified by immunocytochemistry. It is interesting that ET-1 and ET-3 inhibited (25-30%) mRNA expression of Th2-type cytokines (IL-4, IL-10, and IL-13) and increased IL-12 release (39% and 41%, respectively) without affecting MIP-1alpha and NO production. Thus, our data suggest that ETs may play an important role in modulating the cytokine network by regulating Th1/Th2 cytokine production by mast cells.     

Calcium efflux and histamine secretion from rat peritoneal mast cells

Purified rat peritoneal mast cells rapidly accumulated⁴⁵calcium from the external medium. The uptake was essentially unaffected by lanthanide ions but was almost totally prevented by metabolic inhibitors. Cells preloaded with⁴⁵calcium showed a steady efflux of the cation on transfer to a medium lacking the isotope. The efflux was unaffected by metabolic inhibitors but was totally dependent on extracellular sodium ions. These results indicate the operation of a sodium-calcium exchange mechanism for the extrusion of the divalent cation. Antigenic or pharmacologic stimulation of the mast cell led to a temporary suppression of calcium efflux during the period in which histamine release occurred. This effect was potentiated by phosphatidylserine and high concentrations of the lipid inhibited basal efflux. These results suggest that activation of the mast cell leads to an inhibition of calcium extrusion, thereby potentiating the induced rise in the intracellular concentration of the cation and thus augmenting the secretory response.     

Calcium pools involved in histamine release from rat mast cells

Basic secretagogues, antigen, concanavalin A, the ionophore A23187 and, to a lesser extent, anti-rat IgE produce a significant release of histamine from rat peritoneal mast cells in the absence of extracellular calcium. This release is due to the mobilization of intracellular reservoirs of calcium. The release is abolished by prolonged exposure to chelating agents, but is potentiated by brief exposure to these substances. It is suggested that the latter treatment removes calcium from a superficial, regulatory site and thus facilitates the mobilization of more internal pools of the ion. By analogy with smooth muscle, these regulatory sites may also modulate calcium influx into the cell. On the basis of these and other results, the possible calcium pools important in histamine secretion are discussed.     

Fluoroaluminates stimulate histamine secretion in the digitonin-permeabilized rat mast cells

The combination of NaF and AlCl3 stimulated histamine secretion from digitonin-permeabilized rat mast cells only in the presence of trace amount of Ca2+. When NaF plus AlCl3 were added together with a maximal dose of guanosine-5'-[gamma-thio]triphosphate (GTP gamma S), the combined effect was less than additive. The results suggest that the secretory effect of fluoroaluminates is, at least partially, mediated by GTP-binding protein, which is previously found to be activated by GTP gamma S.     

Mechanism of histamine release from rat peritoneal mast cells

Summary The present communication endeavours to elucidate the mechanism of histamine release from rat peritoneal mast cells induced by selective histamine liberators.Of the different enzymatic processes involved in secretion the following are considered: ecto-ATPase activity in the mast cell, pro-esterase-esterase conversion during histamine secretion, cyclic AMP and microtubule association/dissociation, phospholipase A₂ and the effect of phospholipid metabolites on secretion, N-methyl transferase and the methylation of phospholipids and the phosphorylation and desphosphorylation of proteins.