The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.
The aim of this study was to determine the role of macrophages in the Actinobacillus actinomycetemcomitans-induced murine immune response. BALB/c mice were given carrageenan solution by intraperitoneal injection before immunisation with heat-killed A. actinomycetemcomitans. Mice immunised with antigens and phosphate-buffered saline served as positive and negative controls, respectively. One week after the last immunisation, the delayed-type hypersensitivity (DTH) response was assessed by measurement of footpad swelling. Serum IgG and IgM anti-A. actinomycetemcomitans antibody levels and culture supernate levels of interferon (IFN)-gamma were determined by ELISA. The diameter of abscess formation was determined every 5 days. Sham-immunised spleen cells were transferred to carrageenan-untreated recipients (groups A and B) and to carrageenan-treated recipients (group D). Antigen-immunised spleen cells were transferred to carrageenan-untreated (group C) and carrageenan-treated (group E) recipients. The carrageenan-treated recipients in groups F and G received macrophages from antigen- and sham-immunised mice respectively. All mice except those in group A were immunised with antigen 24 h after cell transfer. After 1 week, a partial suppression of DTH response, reduced levels of IFN-gamma, serum IgG and IgM anti-A. actinomycetemcomitans antibodies and delayed healing were seen in carrageenan-treated mice when compared with the positive control. The immune response to A. actinomycetemcomitans in groups A, B and D was lower than that in groups C and E. Healing of the lesion in the former groups was also delayed when compared with the latter groups. The immune response and the healing of the lesion could be partially restored in carrageenan-treated mice that received antigen-pulsed macrophages (group F) but not in those that received naive macrophages (group G). These results suggest that macrophages play a partial role in the induction of the murine immune response to A. actinomycetemcomitans.
The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.
The aim of the present study was to determine the effect of immune status, age and genetic background on the induction of oral tolerance to Actinomyces viscosus. Suppression of delayed type hypersensitivity (DTH) response and antigen-specific serum antibody levels could be induced in DBA/2 mice intragastrically and systemically immunized with A. viscocus, suggesting the induction of oral tolerance. In contrast, this immune suppression could be abrogated if the animals had been systemically immunized prior to the induction of oral tolerance with the same bacterium. Long-term systemic immunization prior to intragastric immunization with A. viscocus suppressed DTH response only. Cell transfer of this group of animals also suppressed DTH response in the donors, indicating the action of suppressor cells for inhibition of DTH response. Furthermore, oral tolerance to A. viscocus failed to occur in mice aged at 3 days and 1, 2, 4, 6 and 36 weeks old. Mice bearing H-2(d) haplotype were the most susceptible to oral tolerization, followed by H-2(b) and H-2(k). Therefore, the results of the presence study suggest that the induction of oral tolerance to A. viscosus in mice may be dependence on the immune status and genetic background but not age.
The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW 264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.