Leukemia inhibitory factor production by rat mast cells

Leukemia inhibitory factor (LIF) is a pluripotent cytokine of importance in the regulation of immune and inflammatory responses. This cytokine may play an important role in neuronal development and bone metabolism. We have examined the ability of freshly isolated rat mast cells and mast cell lines to produce LIF at both the mRNA and bioactivity levels. Initial experiments demonstrated that two mucosal mast cell-like cell lines RBL.2H3 and RCMC9 endogenously produced low levels of LIF bioactivity. The production of this cytokine was examined using a hepatocyte-stimulating factor activity assay and confirmed by the use of neutralizing antibodies specific for LIF. This production was enhanced by treatment with the calcium ionophore A23187. No interleukin-6 production was observed by these cells either endogenously or following ionophore activation. Freshly isolated highly purified rat peritoneal mast cells also expressed mRNA for LIF. These results could have important implications for the role of mast cells in neuronal development, hematopoiesis bone metabolism and the acute-phase response.     

Morphological alterations in rat peritoneal mast cells by stem cell factor.

Stem cell factor (SCF) stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix. We found that the morphology of rat peritoneal mast cells (RPMC) altered after the addition of recombinant murine SCF (rmSCF) in vitro. The ability of rmSCF to enhance morphological alteration was dose dependent and completely abolished by anti-c-kit ACK2 monoclonal antibody. Exposure of RPMC to transforming growth factor-beta 1, wortmannin, genistein, herbimycin A, staurosporine, indomethacin and cytochalasin D before the addition of rmSCF antagonized rmSCF-induced morphological alteration. However, nordihydroguiaretic acid had no effect. Many RPMC appeared to respond also to nerve growth factor (NGF) but the total number of cells with altered morphology was much greater when the culture was stimulated by rmSCF than by NGF. We suggest that morphological alterations of mast cells by rmSCF is an important step for the participation in adhesion to tissue under resident physiological conditions.     

Factor-independent tissue cultured mast cells: establishment from rat peritoneal mast cells

We report here that extended culture of purified rat peritoneal mast cells (RpMC), typical of the connective tissue-type (CTMC), gives rise to continuously proliferative cell lines without the requirement of exogenous growth factors such as IL-3 and IL-4 or accessory cells. Two of the cell lines established, RCMC1 and RCMC2, are described here. Both cell lines have been maintained in continuous culture in vitro for over a year. Although these cell lines were derived from CTMC, they exhibit phenotypic characteristics of mucosal-type mast cells, i.e., they contain rat mast cell protease II (RMCP II), low levels of histamine and stain alcian blue+/safranin-. Previous studies have identified both high and low affinity receptors for IgE, designated Fc epsilon RI and Rc epsilon RII, respectively, on RpMC and rat basophilic leukemia (RBL) cells. At the early stages of cell culture, RCMC1 expressed predominantly Fc epsilon RI and a gradual increase in the expression of Fc epsilon RII has been observed with time in culture. By comparison, RCMC2 expressed predominantly Fc epsilon RII throughout its entire period of cell culture.     

Signaling pathway in nerve growth factor induced histamine release from rat mast cells

Objective and Design: We investigated a signal transduction pathway involved in NGF induced histamine secretion from mast cells. We compared this mechanism with the exocytosis induced by basic secretagogue compound 48/80.Materials and Methods: Isolated rat peritoneal mast cells were obtained from male Wistar rats. Histamine release was assayed spectrofl uorometrically.Results: We found that tyrosine kinase inhibitor genistein, phospholipase C (PLC) inhibitor U-73122, phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, but not MAP kinase inhibitors, SB203580 and PD98059, reduce histamine secretion in NGF provoked mast cell degranulation. In compound 48/80 mediated degranulation, we confi rmed only the involvement of tyrosine kinase and PLC, but not PI-3 kinase, PKC and MAP kinases.Conclusions: Our results indicate that release of histamine from mast cells after stimulation with NGF is regulated by tyrosine kinase, PLC, PI-3 kinase and PKC, but not by MAP kinases. This biochemical pathway differs from that provoked by compound 48/80.Received 16 February 2005; accepted by A. Falus 12 May 2005     

Serotonin release from rat brain mast cells in vitro.

Mast cells are primarily localized in connective tissues, where they secrete numerous mediators. They have also been identified in the mammalian central nervous system on the basis of their histochemical and morphological properties, but their role there remains unknown. A perfusion system was used to investigate in vitro mediator release from rat brain mast cells. Compound 48/80, the classic mast cell secretagogue of connective tissue mast cells, induced dose-dependent and non-cytotoxic release of serotonin, histamine and beta-hexosaminidase from mast cells in the rat thalamus and hypothalamus, but not in the cerebellum which was used as a negative control. Detailed studies were performed on thalamic mast cells, which were identified on the basis of metachromasia with Toluidine Blue and Safranin-positive staining with the Alcian Blue/Safranin technique. Their secretion was characterized by: (a) parallel release of serotonin, histamine and beta-hexosaminidase; (b) lack of dependence on extracellular calcium; (c) susceptibility to inhibition by disodium cromoglycate; and (d) lack of lactate dehydrogenase release. These results indicate that the morphology and secretory characteristics of thalamic mast cells resemble those of connective tissue mast cells. The ability of brain mast cells to secrete their mediators is discussed in the context of their possible involvement in brain pathophysiology.     

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.     

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.     

Creatine phosphokinase in rat mast cells.

The soluble cytoplasmic fraction of an homogenate from peritoneal rat mast cells, demonstrated a considerable amount of catalytic activity which promotes the transfer of phosphate from creatine phosphate to ADP. The plasma membrane, mitochondrial and microsomal fractions show negligible amounts of the catalyst. Enzyme activity is maximal at 37 degrees showing little activity below 17 degrees or above 45 degrees. The enzyme is strongly Mg2+-dependent, whereas it is only slightly activated by Ca2+. pH values between 7 and 8 are optimal and the enzyme is irreversibly inactivated below pH 4. The overall behaviour of the catalyst indicates it to be a creatine phosphokinase (CPK), an enzyme considered important to muscle and nerve tissues. The CPK is probably not encapsulated within the mast cells' perigranular membranes and is retained in the soluble cytoplasm during exocytosis. The possible role of CPK, as to whether it is assisting in maintaining proper levels of intracellular ATP during exocytosis, and/or whether it is associated with components of the mast cells' contractile apparatus, is discussed.     

Histamine release from mast cells of the rat

The relative activities of 12 agents inducing anaphylactoid oedema in the paws of two types of genetically different rats have been compared with those inducing histamine release from isolated peritoneal cells. Four groups were identified - group 1 where activities were much higher in R rats than in NR rats, doses in vivo being in all cases much lower than those required in vitro; group 2 where activities were similar in the two types of rat, doses in vivo again being relatively much lower; group 3 where activities were similar in both types of rat though doses in vivo were relatively high; group 4 where activities were similar in both types of rat, but doses in vivo were relatively extremely high. Only the activities of releasers in groups 1, 2 and 3 (represented by dextran, concanavalin A and antigen) required calcium ions in vitro and were potentiated by phosphatidyl serine; they were potentiated in vivo by insulin and inhibited by glucose.     

Arthritis transferred by cells derived from pre-inflammatory rat synovium.

Different cell populations isolated from rats during the period of latency of adjuvant arthritis were injected into the bloodstream of naive rats to test their ability to transfer articular disorders. Synovium-derived cells (synoviocytes) were able to induce arthritis in 3 out of 4 recipient animals, whereas peripheral blood leukocytes, peritoneal exudate macrophages, lymph node cells, synoviocyte lysates and synoviocytes from control animals were not able to do so. This model of cellular transferred arthritis is associated with antibody titres to hsp65 in rat sera. Our findings suggest a crucial role for synovial cells in the pathogenesis of adjuvant disease, which might be linked to their function as accessory cells.     

Interferon-alpha/beta inhibits IgE-dependent histamine release from rat mast cells.

Although mast cells and interferons are both involved in numerous immune and inflammatory responses, little is known about how microenvironmental factors such as interferons (IFNs) influence mast cell function. To study this question, sensitized peritoneal mast cells (greater than 98% purity) obtained from rats infected 4 weeks earlier with the parasite Nippostrongylus brasiliensis were preincubated for 24 hr with rat IFN-alpha/beta in RPMI-1640, then stimulated to degranulate with worm antigens. In the absence of antigen, IFN-alpha/beta had no noticeable effect on histamine release. However, in the presence of antigen, IFN-alpha/beta (150-1500 U/ml) inhibited histamine release in a dose-dependent manner (22.2 +/- 7.5% to 56.3 +/- 6.9%, n = 10). This inhibitory effect was neither heat (56 degrees for 1 hr) nor acid (pH 2 for 18 hr) labile, but was completely blocked by anti-IFN antibodies. In the presence of compound 48/80 (1 microgram/ml) or substance P (5 X 10(-5) M), IFN-alpha/beta was ineffective at modulating histamine release. Histamine release induced by antigen in the presence of the membrane phospholipid phosphatidyl-serine (30 micrograms/ml) was inhibited by IFN in a dose-dependent manner, but maximal inhibition (25.3 +/- 2.7%, n = 10) was reached at a lower concentration of IFN (750 U/ml) than when antigen was used alone. Therefore, rat IFN-alpha/beta appears to inhibit histamine release from rat mast cells in a dose- and stimulus-dependent manner and may do so by reducing the fluidity of the cell membrane.     

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.     

Evidence for morphologic diversity of human mast cells. An ultrastructural study of mast cells from multiple body sites

The ultrastructural features of 502 mast cells, including 34,187 granules, were studied from seven human tissue sites (lung, skin, colon, stomach, small bowel, breast parenchyma, and axillary lymph nodes). Granule areas and substructure were detailed according to the microenvironment (lung alveoli, lung bronchi, bowel mucosa, bowel submucosa, breast skin, breast parenchyma, and axillary lymph nodes). Although completely closed, discrete scrolls were present in some mast cell granules at all tissue locations, they were present more frequently in granules from bowel mucosa and lung (scroll-rich morphology). In contrast, most of the mast cell granules from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa were rimmed by incomplete scrolls forming parallel lamellae; centrally, amorphous granular material and/or grating/lattice-like structures occurred (scroll-poor morphology). In breast, the latter granules had a mean granule area almost twice that of all other sites. Individual mast cells having granules with both scroll-rich and scroll-poor features were common in all tissue sites and microenvironments. In fact, 7.8% of mast cells had granules showing at least one completely closed, discrete scroll and grating/lattice-like structures, sometimes within the same granule. These observations indicate that mixed forms occur and that there is considerable morphologic diversity between the predominant mast cell found in mucosa and lung (scroll-rich morphology) and the predominant mast cell found in skin, breast parenchyma, axillary lymph nodes, and bowel submucosa (scroll-poor morphology).     

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.     

Rosette-forming mast cells in rat anaphylaxis. Immunological characteristics of mast cell rosettes.

Peritoneal mast cells from immunized rats can form rosettes with antigen-coated sheep red blood cells. The receptor responsible for this active rosette formation is shown to be IgE cytophilic antibody: rosettes are inhibited by previous contact of mast cells with antigen, or with anti-IgE antiserum; the kinetics of mast cell rosettes following a primary immunization with ovalbumin and Bordetella pertussis vaccine is similar to the kinetics of reaginic antibody response. Furthermore, a reaginic serum can induce passive rosette formation. There is no correlation between cell-bound and circulating IgE.     

Canatoxin triggers histamine secretion from rat peritoneal mast cells

Canatoxin, a toxic protein present in the seeds ofCanavalia ensiformis, induces the secretion of serotonin, dopamine and insulin through activation of the lipoxygenase pathway. The purpose of the present study was to verify if canatoxin causes histamine release from rat peritoneal mast cells and to perform a detailed study of this phenomenon. Our results indicate that canatoxin is capable of activating mast cells to release histamine. The process is time- and concentration-dependent, occurs without cell damage and requires metabolic energy as well as the presence of divalent cations (Ca²⁺ and Mg²⁺). Optimal release occurs at 37°C and at physiological pH. Extremes of temperature (0°C and 45°C) inhibit the process. We conclude that canatoxin induces histamine release from rat peritoneal mast cells by an active secretory process.     

Mitoxantrone induces nonimmunological histamine release from rat mast cells

The antineoplastic drug mitoxantrone (MTX) elicits a fast nocytotoxic and nonimmunological histamine release from peritoneal and pleural rat mast cells. The non specific phosphodiesterase inhibitor isobuthylmethylxantine (1 mM) decreases the potency of MTX. Theophylline (10 mM) decreases both the potency and the efficacy of MTX-induced histamine secretion. The protein kinase C (PKC) activator, tetradecanoyl-phorbol-13-acetate (50 ng/mL), enhances the effect of MTX, whereas the non specific PKC inhibitor trifluoperazine (10 M) exerts no effect.Histamine release was also unaffected by substances acting on G-proteins, namely pertussis toxin (200 ng/mL), cholera toxin (300 mg/mL) and benzalkonium chloride (10 g/mL). The inhibition of protein phosphatases 1 and 2A by okadaic acid (1 M) does not modify the response. The results indicate that mitoxantrone elicits the exocytosis in mast cells by a mechanism similar to the parent compound adriamycine, but different to the polyamine compound 48/80.accepted by W. Lorenz     

Isolation and properties of mast cells from rat prostate

Inflammation Research -     

Histamine release from saponin-permeabilized rat mast cells by calcium

The first effect of receptor activation on the mast cell surface, initiating histamine secretion, is an increase in the cytosol Ca²⁺ concentration. It should then be possible to induce histamine secretion by calcium alone, if the calcium permeability of the cell membrane could be increased without any significant interference with the physiological cell functions. This was achieved in the present study by adding low concentrations of saponin (0.005% and 0.001% w/v) to the medium. When calcium was added to the saponinpermeabilized cells, around 40% histamine release occurred with 0.25 mM extracellular calcium (free Ca²⁺ 0.15 mM). The release was inhibited by antimycin A (1 M). Transmission electron microscopy showed formation of vacuoles containing granules stripped of their membranes, which characterize a secretory response. The observations are consistent with a limited increase in the calcium permeability of the cell membrane for a brief period. There was apparently an increase in the cytoplasmic calcium concentration, which acted through calmodulin, since the histamine release induced by calcium from the permeabilized mast cells could be inhibited by a calmodulin-antagonist, mepacrine (10–30 M).