Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death. Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations. Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay. Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05). Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.
The aim of this study was to test the hypothesis that the proliferation of hydroxyapatite (HA)-induced human osteoblast cell line (HOS cells) may be up-regulated by exogenous nitric oxide (NO).
The aim of the present study was to determine the effect of nitric oxide (NO) on the production of prostaglandin E2 (PGE2) by a human osteoblast cell line (HOS cells) stimulated with hydroxyapatite. Cells were cultured on the HA surfaces with or without the presence of NO donors (SNAP and NAP) for 3 days. The effect of NO scavenger, carboxy PTIO, or endothelial nitric oxide synthase (eNOS) inhibitor, L-NIO, was assessed by adding this scavenger in the cultures of HA-stimulated HOS cells with or without the presence of SNAP. Furthermore, HOS cells were pre-treated with anti-human integrin alphaV antibody, indomethacin, a non-specific inhibitor, aspirin, a COX-1 inhibitor, or nimesulide, a COX-2 inhibitor, prior to culturing on HA surfaces with or without the presence of SNAP. The levels of PGE2 were determined from the 3 day culture supernatants. The results showed that the production of PGE2 by HA-stimulated HOS cells was augmented by SNAP. Carboxy PTIO suppressed but L-NIO only partially inhibited the production of PGE2 by HA-stimulated HOS cells with or without the presence of exogenous NO. Pre-treatment of the cells with anti-human integrin alphaV antibody, indomethacin or nimesulide but not aspirin suppressed the production of PGE2 by HA-stimulated HOS cells with or without the presence of NO. Therefore, the results of the present study suggest that NO may up-regulate the production of PGE2 by augmenting the COX-2 pathway initiated by the binding between HOS cell-derived integrin alphaV and HA surface.
Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with L-N(6)-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A(2) (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. L-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA(2) but not PI-3K-dependent fashion.
The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-gamma and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-gamma suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.
Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
The aim of the present study was to test the hypothesis that colchicine may alter Aggregatibacter actinomycetemcomitans-induced immune response and abscess formation in mice. BALB/c mice were either sham-immunized or immunized with heat-killed A. actinomycetemcomitans. Spleen cells were stimulated with heat-killed A. actinomycetemcomitans in the presence or absence of colchicine. Specific IgG subclass antibodies, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and cell proliferation were determined. The animals were sham-immunized (group I) or immunized with heat-killed A. actinomycetemcomitans (groups II-VII). Colchicine was administered intraperitoneally before (group III), on the same day of (group IV), or after (group V) the primary immunization and on the same day of (group VI) or after (group VII) the secondary immunization. All groups were challenged with viable A. actinomycetemcomitans. The levels of serum-specific IgG subclasses and both IFN-gamma and IL-4 before and after bacterial challenge were assessed. The diameter of skin lesions was assessed. The results showed that colchicine augmented splenic-specific IgG1 and IL-4 as well as cell proliferation but suppressed specific IgG2a and IFN-gamma levels. Enhancement of serum-specific IgG1 and IL-4 levels, suppression of specific IgG2a and IFN-gamma levels as well as DTH response, and delayed healing of the lesions were observed in groups IV and VI, but not in the remaining groups of animals. Therefore, these results suggest that colchicine may induce a T helper 2 (Th2)-like immunity specific to A. actinomycetemcomitans in vitro and that colchicine administered on the same day as the immunization may stimulate a non-protective Th2-like immunity in A. actinomycetemcomitans-induced infections in mice.
The aim of the present study was to determine the effect of nitric oxide (NO) on the production of cyclic AMP (cAMP) by a human osteoblast cell line (HOS cells) stimulated with hydroxyapatite. Cells were cultured on the HA surfaces with or without the presence of NO donors (SNAP and NAP) for 3 days. The effect of adenylyl cyclase inhibitor (SQ22536), NO scavenger (carboxy PTIO) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NIO), was assessed by adding these to the cultures of HA-stimulated HOS cells with or without the presence of SNAP. Furthermore, HOS cells were pre-treated with anti-human integrin alphaV antibody prior to culturing on HA surfaces with or without the presence of SNAP. The levels of cAMP and cGMP were determined from the 3-day culture supernatants. The results showed that the production of cAMP but not cGMP by HA-stimulated HOS cells was augmented by SNAP. SQ22536 and carboxy PTIO suppressed but L-NIO only partially inhibited the production of cAMP by HA-stimulated HOS cells with or without the presence of exogenous NO. Pre-treatment of the cells with anti-human integrin alphaV antibody suppressed the production of cAMP by HA-stimulated HOS cells with or without the presence of NO. Therefore, the results of the present study suggest that NO may up-regulate the production of cAMP, perhaps, by augmenting adenylyl cyclase activity initiated by the binding between HOS cell-derived integrin alphaV and HA surface.
The aim of this study was to determine the role of CD4 and CD8 cells on specific antibody production by murine Peyer's patch (PP) cells after oral immunization with Actinomyces viscosus in mice. Female DBA/2 mice were orally immunized with three low doses of heat-killed A. viscosus. Sham-immunized mice served as a control group. Mice were depleted of CD4 or CD8 cells by intraperitoneal injection of anti-CD4 or anti-CD8 antibodies daily for 3 days before oral immunization. One week after the last oral immunization, PPs were removed and cell suspensions were cultured with A. viscosus. Specific antibody production in the culture supernatants was assessed by enzyme-linked immunosorbent assay. The results showed that oral immunization with A. viscosus induced a predominant specific immunoglobulin A (IgA) response by PP cells and, to a lesser extent, IgM antibodies. Depletion of CD4 but not CD8 cells suppressed the production of specific antibodies. These results suggest that oral immunization with low doses of A. viscosus may induce the production of specific antibodies by murine PP cells in a CD4-cell-dependent fashion.
The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus 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 whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells).
Mucosal presentation of Actinomyces viscosus results in the induction of antigen specific systemic suppressor cells in mice. The aim of the present study was to determine the phenotype of the suppressor cells responsible for the induction of oral tolerance to low doses of A. viscosus. When CD8 cell-depleted DBA/2 mice were intragastrically immunized and systemically immunized with A. viscosus, the delayed type hypersensitivity response was suppressed but not the levels of antigen specific serum antibodies. Adoptive transfer of orally tolerized CD4(+) cells to CD4(+)-depleted mice resulted in suppression of delayed type hypersensitivity response but not of the levels of antigen specific serum antibodies. In contrast, adoptive transfer of orally immunized CD8(+) cells to CD8(+)-depleted mice resulted in partially suppressed delayed type hypersensitivity response but significantly inhibited the levels of antigen specific serum antibodies. When orally tolerized CD8(+) cells were cocultured with systemically immunized CD8(+) cell-depleted spleen cells, splenic specific antibodies were inhibited. However, no suppression of splenic specific antibodies could be observed in the cultures containing orally tolerized CD4(+) cells and systemically immunized CD4(+) cell-depleted spleen cells. The results of the present study suggest that oral tolerance of humoral and cellular immunity induced by low doses of A. viscosus may be mediated by CD8(+) and CD4(+) cells, respectively.
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 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.
It has previously been suggested that CD4+ T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingivalis in CD4-depleted BALB/c mice immunized with P. gingivalis outer membrane proteins (OMP).
Mucosal presentation of Actinomyces viscosus results in antigen-specific systemic immune suppression, known as oral tolerance. The aim of the present study was to determine the mechanism by which this oral tolerance is induced. DBA/2 mice were gastrically immunized with A. viscosus. Serum, Peyer's patch (PP) and spleen cells were transferred to syngeneic recipients which were then systemically challenged with the sameiA. viscosus strain. To determine antigen-specificity of cells from gastrically immunized mice, recipients which received immune spleen cells were also challenged with Porphyromonas gingivalis. One week after the last systemic challenge, the delayed type hypersensitivity (DTH) response was determined by footpad swelling and the level of serum IgG, IgA and IgM antibodies to A. viscosus or P. gingivalis measured by an ELISA. No suppression of DTH response or of specific serum antibodies was found in recipients which received serum from gastrically immunized mice. Systemic immune suppression to A. viscosus was observed in recipients which had been transferred with PP cells obtained 2 days but not 4 and 6 days after gastric immunization with A. viscosus. Conversely, suppressed immune response could be seen in recipients transferred with spleen cells obtained 6 days after gastric immunization. The immune response to P. gingivalis remained unaltered in mice transferred with A. viscosus-gastrically immunized cells. The results of the present study suggest that oral tolerance induced by A. viscosus may be mediated by antigen-specific suppressor cells which originate in the PP and then migrate to the spleen.
The aim of the present study was to determine whether Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) may stimulate nitric oxide (NO) production by murine spleen cells. Spleen cells derived from Balb/c mice were cultured in the presence of Pg-LPS or LPS from Salmonella Typhosa. The cell were also cultured in the presence of Pg-LPS with or without L-arginine, L-arginine plus NG-monomethyl-L-arginine (NMMA), or IFN-gamma. Furthermore, the plastic non-adherent spleen cells were stimulated with Pg-LPS and L-arginine. The results showed that Pg-LPS failed to stimulate splenic NO production by themselves. Exogenous L-arginine or IFN-gamma up-regulated the NO production of Pg-LPS-stimulated spleen cells, but the stimulatory effects of L-arginine were completely blocked by NMMA. It was also demonstrated that in the presence of Pg-LPS and L-arginine, splenic macrophages were the cellular source of NO. These results suggest, therefore, that P. gingivalis-LPS may induce murine splenic macrophages to produce NO in a L-arginine and an IFN-gamma-dependent mechanism.
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.