L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide.

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.

Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

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.

Protective humoral immunity induced by surface-associated material from Actinobacillus actinomycetemcomitans in mice.

The aim of the present study was to determine the role of antibodies specific to anti-surface-associated material from Actinobacillus actinomycetemcomitans (anti-SAM-Aa) in an infection induced by this periodontopathogen in mice. When SAM-Aa obtained by saline extraction of A. actinomycetemcomitans Y4 was separated on one-dimensional gel electrophoresis, this constituent contained antigen fragments with molecular weights ranging from 14000 to 79000. Immunoblot analysis revealed that increased antigen dose/immunization resulted in increased numbers of antigen epitopes recognized by serum antibodies of the immunized mice. Rapid healing of the primary lesions and high levels of specific IgG antibodies after challenge with live A. actinomycetemcomitans were seen in the immunized mice, especially at the highest-dose level of 100 microg/immunization. Transfer of SAM-Aa-immunized, but not the SAM-Aa-immunized and adsorbed, serum prior to challenge with live bacteria led to rapid healing of the lesions in the recipient mice. Increased phagocytosis of A. actinomycetemcomitans by murine macrophages (RAW264.7 cells) was observed when this periodontopathogen was opsonized by the SAM-Aa-immunized, but not SAM-Aa-immunized and adsorbed, serum. These results suggest that in mice, SAM-Aa antigens may induce protective antibodies by acting, at least, as an opsonin against challenge with live A. actinomycetemcomitans.

The effects of exogenous interleukin-12 on the induction of immune response to Porphyromonas gingivalis in vivo in mice.

The effects of treatment with exogenous interleukin-12 (IL-12) on the induction of immune response to Porphyromonas gingivalis, a black pigmented periodontopathic oral bacterium in mice, were determined in the present study. An increased footpad swelling representing a delayed type hypersensitivity (DTH) response to P. gingivalis in IL-12-treated mice could be observed, although increasing doses of IL-12 did not produce cumulative effects on this cellular Immune response. Multiple injections with IL-12 also resulted in elevated serum IFN-gamma levels. Treatment with this cytokine the day before, on and after immunization with heat-killed P. gingivalis augmented the levels of serum antigen-specific IgG2a and IgG3 antibodies, but had obviously little or no effects on those of serum antigen-specific IgG1 and IgG2b antibodies. The results of this study suggest that treatment with exogenous IL-12 In P. gingivalis-immunized mice may enhance DTH response and Th1 cell-associated antibody production.

The effects of interleukin-10 depletion in vivo on the immune response to Porphyromonas gingivalis in a murine model.

BACKGROUND: The immune response to Porphyromonas gingivalis in the mouse abscess model is known to be dependent upon CD4 T-cell activation and the regulatory role of cytokines. The role of interleukin-10 (IL-10) in this mouse model was examined in vivo. METHODS: One-week-old, female BALB/c mice were divided into 4 groups. Groups 1 and 2 were given intraperitoneal (i.p.) injections of phosphate buffered saline (PBS) weekly for 5 weeks. Group 3 was given an i.p. injection of rat immunoglobulin. Group 4 was injected with rat anti-IL-10 antibodies. At week 6, group 1 was sham-immunized with PBS, and groups 2, 3, and 4 were injected with P. gingivalis lipopolysaccharide (Pg-LPS) weekly for 2 weeks. One week after the final immunization, delayed-type hypersensitivity (DTH) was assessed by footpad swelling to Pg-LPS. The level of serum antibodies to Pg-LPS and IFN-gamma (IFN-gamma) was determined by enzyme-linked immunosorbent assay. Dorsal abscess formation induced by the injection of viable P. gingivalis was examined daily for 30 days. RESULTS: The footpad swelling of the anti-IL-10-treated group (group 4) was significantly higher than that of groups 1 to 3. Similarly, the serum IFN-gamma level in group 4 was much higher than that of the other experimental groups. There was no significant difference in serum IgG antibodies to Pg-LPS in any of the experimental groups. However, the level of IgM antibodies in group 4 mice was significantly lower than that in groups 2 and 3. In addition, serum IgG1 was suppressed in group 4 mice, while IgG2a antibodies were raised. However, there was no difference observed between the levels of IgG2b and IgG3 antibodies in any group of mice. The lesions in sham-immunized mice (group 1) persisted for 30 days, and those in group 2 and 3 were undetected by day 18 and 20, respectively. In sharp contrast, lesions in group 4 had healed completely by day 13. CONCLUSIONS: This study has shown that IL-10 depletion in vivo in P. gingivalis LPS-induced immune response in mice led to an elevated DTH response, an increase in serum IFN-gamma levels, and raised levels of IgG and IgG2a antibodies. Treatment with anti-IL-10 antibodies resulted in suppressed IgG1 and IgM responses and a more rapid healing of abscesses than in non-IL-10-depleted mice. These results suggest that IL-10 depletion in Pg-LPS-induced immune response in mice may lead to a Th1-like immune response and provide strong protection against a subsequent challenge with live P. gingivalis in an abscess model.

Effects of cytokines on Porphyromonas gingivalis-induced opsonophagocytosis of a murine macrophage cell line.

A murine macrophage cell line was incubated with opsonized Porphyromonas gingivalis and various concentrations of rIFN-gamma, rIL-4 and rIL-10. The number of phagocyted cells were microscopically assessed. The results showed that IFN-gamma upregulated macrophage phagocytosis to this periodontopathogen, and the upregulatory effects was abolished by anti-IFN-gamma antibodies. Neither IL-4 nor IL-10 had any effects on opsonophagocytosis by this cell line. These results suggest that up-regulatory functions of IFN-gamma on phagocytic activities of macrophages may play a crucial role in the induction of immune response to P. gingivalis.

The role of T lymphocytes in Sjögren's syndrome.

The exact role of T cells in the immunopathogenesis of Sjögren's syndrome (SS) is not well understood and is discussed herein. It seems plausible that this autoimmune disorder is associated strongly with the functions of autoantigen-specific CD4 cells. T cell receptor Vbeta gene usage appears to be unrestricted. Furthermore, elevated gene expression of T cell-derived cytokines such as IFN-gamma, IL-1, IL-6, IL-10 and IL-13 seen in salivary glands of SS patients and the animal models of this disorder suggests that the course of SS may be mediated by Th1 and Th2 cells. Defining the precise role of these CD4 cells subsets in SS would certainly provide insights into the establishment of immunotherapeutic bimodal.

The immunoregulatory roles of transforming growth factor beta.

Transforming growth factor-beta is a 25 kD polypeptide produced by both healthy and neoplastic cells. This cytokine effects the introduction of immune response, but its exact mechanism remains to be elucidated. In this paper the immuno-regulatory role of this cytokine on both the immunocompetent cells and accessory molecules is discussed. The growth factor-induced up and down regulation of the immune response suggest that transforming growth factor-beta can done some extent modulate certain pathological conditions, and hence suppressed production of this growth factor has potential benefits for bimodal immunotherapy.

The immunopathology of chronic inflammatory periodontal disease.

Chronic inflammatory periodontal disease is known to be under the control of the immune response. However, the precise mechanism of the immunopathogenesis of this lesion has not yet been fully elucidated. In this review, the regulatory role of both lymphoid and non-lymphoid cells as well as cytokines and accessory molecules in the course of chronic inflammatory periodontal disease is discussed. Finally, based upon previous evidences, an attempt to establish a model of chronic inflammatory periodontal disease is made herein.

The interleukin network in the immunopathogenesis of oral diseases.

Interleukins produced by both lymphoid and non-lymphoid cells play a crucial role in the immune response. This paper discusses the possible interleukin network in the immunopathogenesis of some oral diseases. In chronic inflammatory periodontal diseases and periapical inflammation, interleukins such as IL-1 and IL-6 may be responsible in tissue destruction. High levels of IL-12 but not IL-4 and IL-10 may reduce the course of candidal infection. The progression of HIV infection has been associated with the regulation of distinct cytokines; thus, the pathogenesis of Kaposi's sarcoma may be regulated by IL-6. In autoimmune-associated oral diseases such as lichen planus, the role of Langerhans cells in presenting autoantigens may parallel with increased levels of IL-6. It seems, therefore, that the course of these diseases is regulated by these polypeptides which may in turn modulate the disease severity. However, whether altered levels of interleukins in certain oral disorders can be used as a diagnostic marker requires further investigation.