Characterization of alpha 5-integrin-dependent mast cell adhesion following Fc epsilon RI aggregation.

BACKGROUND: Integrin receptors are engaged in the upregulation of mast cell adhesion to extracellular matrix components upon stimulation with cytokines and antigen. Fibronectin receptor containing the alpha 5-integrin subunit is critical for mast cell interaction with the extracellular matrix protein fibronectin (FN). METHODS: The murine MCP5/L mast cell line was employed to investigate the process of Fc epsilon RI-mediated mast cell adhesion to FN. RT-PCR and cytofluorimetric analysis were used to assess the expression of alpha 5 integrin in MCP5/L mast cells. Radiolabelled mast cells were sensitized with monoclonal IgE and used in adhesion assays. Anti-alpha 5-integrin antibody (Ab), monovalent hapten and metabolic inhibitors were used to characterize antigen-mediated mast cell adhesion to FN. RESULTS: Addition of antigen to IgE-sensitized cells resulted in transient upregulation of mast cell adhesion to FN with a maximum adhesion following 30 min of incubation. Mast cell adhesion was inhibited with anti-alpha 5-integrin monoclonal antibodies blocking FN receptor or with excess monovalent hapten preventing antigen-mediated IgE cross-linking. The presence of the protein kinase C (PKC) inhibitor staurosporine also inhibited mast cell adhesion in a dose-dependent fashion. The process of Fc epsilon RI-mediated upregulation of mast cell adhesion to FN was not associated with an increase in surface expression of mast cell FN receptors. CONCLUSION: The major FN receptor on MCP5/L mast cell surface, an integrin containing the alpha 5 subunit mediates a transient change in mast cell adhesiveness following IgE cross-linking. Fc epsilon RI-derived signals engage PKC and upregulate mast cell adhesion in a process which might involve changes in integrin avidity rather than integrin expression.

Inhibitory effect of wheat germ agglutinin on mouse mast cell adhesion to fibronectin.

BACKGROUND: Mast cells play a critical role in allergic and inflammatory responses. The interactions between these cells and extracellular matrix components influence the distribution of mast cells in tissues and their biological responsiveness. It has been reported that the lectin wheat germ agglutinin (WGA) inhibits mast cell mediator release. We decided to investigate whether adhesion to fibronectin (FN), another mast cell function, which is upregulated following FcepsilonRI cross-linking, is also inhibited by WGA. METHODS: Mouse bone-marrow-derived mast cell line MCP5/L was used. For FcepsilonRI-dependent mast cell activation, MCP5/L cells were sensitized with mouse IgE antibodies. WGA was added to cell suspensions simultaneously with a challenging agent and, after an appropriate incubation period, beta-hexosaminidase release and adhesion to FN were determined. RESULTS: Both FcepsilonRI cross-linking-dependent mast cell adhesion to FN and mediator release were dose-dependently inhibited by WGA; however, the lectin concentrations required to induce maximum inhibition of adhesion were significantly lower. Furthermore, WGA inhibited phorbol-myristate-acetate- and A-23187-mediated mast cell adhesion to FN, i.e. processes that do not engage FcepsilonRI. The effect of WGA on FcepsilonRI-mediated secretion was reversed by GlcNAc. In contrast, combination of GlcNAc and NeuNAc or N, N'-diacetylchitobiose was required to reverse the inhibitory effect of WGA on mast cell adhesion. CONCLUSION: The characteristics of WGA-mediated inhibition of MCP5/L mast cell adhesion to FN suggest that mast cell integrins are targets of the inhibitory action of WGA and the sugar moieties on these receptors might be important for receptor function.

Effect of somatostatin and octreotide on proliferation and vascular endothelial growth factor secretion from murine endothelial cell line (HECa10) culture.

Angiogenesis, development of new blood vessels, is required for normal tissue repair and also for tumor cell proliferation, extracellular matrix invasion, and hematogenous metastases. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that has been shown to play a key role in neovascularization. Inhibition of angiogenesis in vitro and in vivo was documented by administration of native neuropeptide somatostatin and its analog octreotide. We have studied the effect of somatostatin-14 (SRIF) and ocreotide (sandostatin) on proliferation activity and VEGF release from cultured murine endothelial cells HECa10 in vitro. SRIF in concentrations from 10(-9) to 10(-5) M and ocreotide in concentrations from 10(-9) to 10(-5) M diminished the proliferative activity of cultured cells vs controls. SRIF and ocreotide in concentrations from 10(-14) to 10(-6) M did not change the release of VEGF into supernatants of 24 or 72 h endothelial cell cultures. Although we showed the antiproliferative effect of SRIF and ocreotide on mouse endothelial cells, we were unable to demonstrate the inhibitory effect of tested peptides on VEGF secretion in vitro.

Murine mast cells exposed to mercuric chloride release granule-associated N-acetyl-beta-D-hexosaminidase and secrete IL-4 and TNF-alpha.

BACKGROUND: Mast cells, by virtue of their location within the skin, respiratory tract, and gastrointestinal system, are considered as potential targets for environmental agents with immunotoxic effects. Mercuric chloride (HgCl2), is a xenobiotic, which induces autoimmune glomerulonephritis and stimulates polyclonal IgE production. OBJECTIVE: We sought to determine the ability of HgCl2 to degranulate murine mast cells and promote cytokine secretion and whether this was an active biologic process. METHODS: Bone marrow-derived murine mast cells were exposed to HgCl2, and the release of N-acetyl-beta-D-hexosaminidase and secretion of IL-4 and TNF-alpha were measured. RESULTS: HgCl2 was found to directly activate murine mast cells to release the granule-associated enzyme N-acetyl-beta-D-hexosaminidase and to secrete the proinflammatory cytokines IL-4 and TNF-alpha. Cytokine secretion occurred hours after exposure to HgCl2 and required transcription and protein synthesis. The secretion of cytokines mediated by HgCl2 was additive to that which followed FcepsilonRI-induced mast cell activation. The IL-4 secretion by mast cells occurred at concentrations of HgCl2 (10(-6) mol/L to 10(-5) mol/L) comparable with those required to induce upregulation of IgE production in experimental animals. CONCLUSION: These findings demonstrate that HgCl2 will directly activate mast cells, which is followed by degranulation and IL-4 and TNF-alpha synthesis and secretion. These findings are consistent with recognition of HgCl2 as a biologically important environmentally derived immunotoxic agent for mast cells.

Inhibitory effect of thiophosphoric acid alkaloid derivatives from Chelidonium majus L. (Ukrain) on ovalbumin antigenicity and antiovalbumin IgE antibody response in mice.

The ability of the Chelidonium majus L. alkaloid derivative Ukrain (UK) to inhibit ovalbumin-induced sensitization was tested in BALB/c and F1(BALB/c x C57BL/6J) mice. UK introduced into the mice in the mixture with antigen (ovalbumin) and adjuvant (alum) inhibited the sensitization of mice, reflected in lower anti-OA IgE antibody response and decreased antigen-induced histamine release from mast cells isolated from peritoneal cavities of sensitized mice. The effect of UK on the antigenicity of ovalbumin (OA) in anaphylaxis was tested in heterologous passive cutaneous anaphylactic (PCA) reaction on rats. The results show that the OA prepared in the mixture with UK had a decreased ability to react with anti-OA IgE antibodies raised against native OA in mice and fixed on the surface of rat mast cells in heterologous PCA reactions. The results suggest that UK pretreatment of OA may affect its antigenic property and the ability to react with anti-OA IgE antibodies raised against the native IgE molecules.

Relations between Fc epsilon RI crosslinking-induced mast cell activation and adhesion to fibronectin.

Demonstration of murine mast cell adhesion to fibronectin (FN) following PMA-mediated cell activation raised the question whether crosslinking of high affinity IgE receptors on mouse mast cells might induce changes in adhesiveness of these cells to FN. Murine mast cells of line MCP5/L were used to investigate the effect of antigenic stimulation on cell adhesion to fN and mediator secretion. effect of antigenic stimulation on cell adhesion to FN and mediator secretion. Adhesion assays were performed using sensitized radiolabeled cells and FN- or BSA-coated 96-well plates. The presence of antigen in the concentrations up to 10 ng/ml resulted in concentration-dependent adhesion potentiation, which was detectable after 5 min, reached maximum at 30 min and persisted or decreased over the next 30 min. Adhesion potentiation decreased at antigen excess and was abolished by heat inactivation of IgE in the antiserum prior to cell treatment. External calcium ion and temperature dependence of adhesion together with the observation that RGD (Arg, Gly, Asp)--containing peptide blocked cell binding to FN suggests that FC epsilon RI crosslinking-induced adhesion potentiation involves an integrin type receptor on cell surface. Sensitized mast cells allowed to adhere spontaneously to FN released more histamine and beta-hexosaminidase upon antigen challenge. Hence, the results show the relations between IgE-induced mast cell activation, adhesion to FN and mediator secretion.

Histamine releasing activity and its inhibition by supernatants from cultured lymphoid cells of Nippostrongylus brasiliensis-infected rats.

The influence of Nippostrongylus brasiliensis infection on the capability of HRF (Histamine Releasing Factor) generation by rat lymphoid cells in vitro has been studied. Spleen cells and thymocytes of normal and Nippostrongylus brasiliensis-infected rats were cultured in the presence of nonspecific mitogen (PHA) or specific N. brasiliensis antigen (NbAg), and cell-free supernatants fractionated by Sephadex G-75 chromatography were tested on homologous mast cells for histamine releasing activity. The results show that PHA-stimulated lymphoid cells from both normal and infected rats produced a factor releasing histamine from mast cells. Histamine releasing activity was not detected when lymphoid cells of N. brasiliensis-infected rats were cultured in the presence of NbAg. Moreover, supernatants of these cultures diminished HRF-induced histamine release from mast cells, suggesting the production of factor(s) inhibiting this release. This histamine release inhibiting activity was detected in fractions in Sephadex G-75 chromatography of supernatants from the cultures of NbAg-stimulated thymocytes of infected rats.

Investigations on allergogenic properties of Tolpa Peat Preparation.

The ability of Tolpa Peat Preparation (TPP) to induce or enhance an allergic sensitization was tested on mice and guinea pigs. The levels of IgE antibody in the mouse sera and IgG1 as well as IgE antibody levels in guinea pig sera were evaluated by PCA (Passive Cutaneous Anaphylaxis) tests. TPP adsorbed on aluminium hydroxide gel (alum) and introduced into BALB/c mice by several subcutaneous injections was unable to stimulate the noticeable anti-TPP IgE antibody response. TPP introduced together with ovalbumin (OA) into the mice in the course of immunization with OA did not enhance anti-OA IgE antibody response. TPP adsorbed on alum and injected subcutaneously into guinea pigs was unable to induce noticeable IgG1a, IgG1b and IgE antibody response, and mast cells obtained from lung and mesentery of these animals did not release histamine when challenged with TPP in vitro at 37 degrees C. In conclusion, our results show that under the experimental conditions used in the present experiments TPP was unable to induce or enhance an allergic sensitization of mice and guinea pigs.

Influence of Tolpa Peat Preparation on the IgE-induced anaphylactic reactions in mice.

The ability of Tolpa Peat Preparation (TPP) to affect anaphylactic sensitization and mast cell secretory function was tested in BALB/c mice treated with TPP orally for 12 days. TPP in the doses of 20 and 50 mg/kg/day reduced histamine release from mouse peritoneal mast cells challenged with anti-IgE or concanavalin A in vitro. The treatment of mice with TPP from day 1 to day 12 of immunization with Ovalbumin (OA) absorbed on aluminium hydroxide gel resulted in a decrease of antigen-induced histamine release from mast cells of these mice in vitro and in decreased IgE antibody level in their sera. TPP introduced into OA-immunized mice showing developed IgE antibody response was less effective in decreasing anaphylactic histamine release from mast cells of these mice. In all experiments low doses of TPP used for oral treatment were more effective than high doses in inhibiting anaphylactic events in the mice.

Rat lymphoid cell--derived histamine and 5-hydroxytryptamine releasing factor.

The in vitro production of histamine releasing factor (HRF) by lymphoid cells of rats, both normal and infected with Nippostrongylus brasiliensis, has been studied. Spleen cells and thymocytes were cultured either alone or in the presence of mitogen (PHA, 10 and 50 micrograms/ml) and the dialysed cell-free supernatants were tested for histamine releasing activity on rat peritoneal and pleural mast cell in vitro. We found that spleen cells and thymocytes of normal rats stimulated with PHA in 24 h cultures generated a factor which released histamine and 5-hydroxytryptamine from mast cells, and this ability was potentiated following N. brasiliensis infection of rats - lymphoid cells donors. Pleural mast cells were more sensitive to the action of HRF than peritoneal cells. Rat HRF had an apparent m.w. of 50,000 to 70,000 daltons as determined by gel chromatography and was a heat stable protein inducing histamine release from homologous mast cells in a very rapid (complete in 1-2 min at 37 degrees C), dose and temperature dependent secretory process.

The immunomodulating preparation Ukrain does not induce anaphylactic sensitization in mice and guinea pigs.

The ability of Chelidonium majus L. alkaloids derivative Ukrain to induce an anaphylactic sensitization was tested on mice and guinea pigs. The levels of IgE antibody in the mouse sera, and IgG1a, IgG1b as well as IgE antibody levels in guinea pig sera, were evaluated by passive cutaneous anaphylaxis (PCA) tests. Ukrain alone or adsorbed on aluminium hydroxide gel (alum) introduced into BALB/c mice in several subcutaneous injections was unable to stimulate measurable anti-Ukrain IgE antibody response. Moreover, Ukrain introduced together with ovalbumin (OA) into mice in the course of immunization with OA induced lower anti-OA antibody response as compared to the response induced by OA alone. Ukrain adsorbed on alum and injected subcutaneously into guinea pigs did not induce measurable IgG1a, IgG1b and IgE antibody response. The present results suggest that the immunomodulating preparation Ukrain could be therapeutically safe at least as far its inability to induce anaphylaxis is concerned.

Concanavalin A-induced activation of hamster mast cells: morphological changes and histamine secretion.

Peritoneal mast cells of Syrian hamsters release histamine to the action of concanavalin A (Con A) in dose-dependent fashion. The rate of release was very rapid in the first seconds of cell activation and completed in 60 s after the challenge. Morphological changes concomitant to the lectin treatment, followed by electron microscopy, show that early signs of exocytosis are seen after 10 s. The process starts in peripherally located granules which swell, have a decreased density and form pores by fusion of the cellular membrane and the perigranular membranes. Then it spreads toward the cell interior by fusion of granules and forming intracytoplasmic cavities. Some extruded granules are also observed. Preincubation of lectin with rat IgE or with rat serum induced an inhibition of its histamine releasing action. Immunization increased the Con A-induced histamine release in young but not in older hamsters. An IgE-mediated mechanism is suggested for the parallel ultrastructural changes and histamine release effects induced by Con A on the hamster mast cell.

5-Hydroxytryptamine releasing activity of the supernatants from cultured mouse spleen cells.

Mouse spleen cells have been shown to produce a histamine releasing factor (HRF) after stimulation with mitogen or specific antigen in vitro. The supernatants from the cultures of mouse spleen cells released not only histamine but also 5-hydroxytryptamine (5-HT) from homologous mast cells in a dose-dependent manner. The time-course of this release was similar to that observed in antigen or anti-IgE reaction. Heavy water (D2O) enhanced supernatant-induced mediator release.

Histamine metabolism in lungs of rats infected with Nippostrongylus brasiliensis.

Several parameters connected to histamine metabolism and mast cell number were examined in the lungs of rats infected with the nematode Nippostrongylus brasiliensis. Histamine levels as well as mast cell numbers were found to be increased on day 14 after infection and were elevated during the whole time of the experiment. Histidine decarboxylase activity also reached a peak on day 14. There was no measurable activity of diamine oxidase in the lungs of parasitized and normal rats. It is postulated that the increase in histidine decarboxylase activity and histamine concentration observed in the present study is related to the process of mastocytosis.

The effect of disodium 4-chloro-2-iminodibenzoate (CCA) on IgE levels and anaphylactic shock.

The effect of disodium 4-chloro-2,2-iminodibenzoate (CCA) on IgE antibody response was examined in C3H/A and (BALB/c x C57BL/6J) F1 hybrid mice immunized with low doses of ovalbumin (OA) adsorbed on aluminium hydroxide gel. CCA administered orally at the doses of 5 and 50 mg/kg/day reduced IgE antibody production in these mice as determined by PCA test. High doses of CCA (100 mg/kg/day) given from day 7 before immunization of C57BL mice and during 1 week after immunization of mice with OA and Bordetella Pertussis Vaccine reduced the mortality of these mice subjected to anaphylactic shock on day 7 of immunization. CCA treatment was ineffective in anaphylactic shock of C57BL mice immunized with very high dose of OA, known to elicit little or no IgE antibody production but high IgG antibody response. The treatment of OA-immunized Guinea pigs with one oral dose of CCA (100 mg/kg) did not reduce mortality in protracted anaphylactic shock. Our results demonstrate that CCA inhibits IgE production as well as IgE mediated hypersensitivity reactions in mice.