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Therapeutic effects of antibodies to tumor necrosis factor-alpha, interleukin-6 and cytotoxic T-lymphocyte antigen 4 immunoglobulin in mice with glucose-6-phosphate isomerase induced arthritis.

Matsumoto I, Zhang H, Yasukochi T, Iwanami K, Tanaka Y, Inoue A, Goto D, Ito S, Tsutsumi A, Sumida T - Arthritis Res. Ther. (2008)

Bottom Line: Anti-TNF-alpha mAbs and CTLA-4Ig suppressed TNF-alpha production, whereas anti-IFN-gamma mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-gamma production.Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Clinical Immunology, Major of Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8575, Japan. ismatsu@md.tsukuba.ac.jp

ABSTRACT

Introduction: Immunization with glucose-6-phosphate isomerase (GPI) induces severe arthritis in DBA/1 mice. The present study was designed to identify the cytokines and co-stimulatory molecules involved in the development of GPI-induced arthritis.

Methods: Arthritis was induced in DBA/1 mice with 300 microg human recombinant GPI. CD4+ T cells and antigen-presenting cells from splenocytes of arthritic mice were cultured in the presence of GPI. Tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 levels were assessed using cytometric bead array. Monoclonal antibodies to TNF-alpha, IFN-gamma, IL-12, CD40L, inducible co-stimulator (ICOS), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig) were used to block TNF-alpha and IFN-gamma production, examine clinical index in mice with GPI-induced arthritis, and determine anti-GPI antibody production.

Results: Large amounts of TNF-alpha and IFN-gamma and small amounts of IL-2 and IL-6 were produced by splenocytes from mice with GPI-induced arthritis. Anti-TNF-alpha mAbs and CTLA-4Ig suppressed TNF-alpha production, whereas anti-IFN-gamma mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-gamma production. A single injection of anti-TNF-alpha and anti-IL-6 mAbs and two injections of CTLA-4Ig reduced the severity of arthritis in mice, whereas injections of anti-IFN-gamma and anti-IL-12 mAbs tended to exacerbate arthritis. Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.

Conclusion: TNF-alpha and IL-6 play an important role in GPI-induced arthritis, whereas IFN-gamma appears to function as a regulator of arthritis. Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.

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GPI-induced TNF-α and IFN-γ production from arthritic splenocytes in vitro. Spleens were removed from glucose-6-phosphate isomerase (GPI)-immunized DBA/1 mice (on day 8 after immunization), and then single-cell suspensions were prepared. MACS separated CD4+ T cells (1 × 106 cells/ml) were stimulated with 5 μg/ml GPI (or glutathione S-transferase [GST]) and antigen-presenting cells (APCs; 2 × 105 cells/ml, mitomycin treated) for 12 hours. The culture supernatants were collected and concentrations of tumor necrosis factor (TNF)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12p70 were measured by cytometric bead array. Data were averages of three independent experiments. Error bars represent ± standard error. *P < 0.05, by Mann-Whitney U-test.
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Figure 1: GPI-induced TNF-α and IFN-γ production from arthritic splenocytes in vitro. Spleens were removed from glucose-6-phosphate isomerase (GPI)-immunized DBA/1 mice (on day 8 after immunization), and then single-cell suspensions were prepared. MACS separated CD4+ T cells (1 × 106 cells/ml) were stimulated with 5 μg/ml GPI (or glutathione S-transferase [GST]) and antigen-presenting cells (APCs; 2 × 105 cells/ml, mitomycin treated) for 12 hours. The culture supernatants were collected and concentrations of tumor necrosis factor (TNF)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12p70 were measured by cytometric bead array. Data were averages of three independent experiments. Error bars represent ± standard error. *P < 0.05, by Mann-Whitney U-test.

Mentions: To identify the dominant cytokines at the onset of antigen-induced arthritis (day 8), we established the CBA array system using spleen CD4+ T cells plus mitomycin-treated APCs cultured in GPI. In this system, treatment of APCs with mitomycin is designed to kill autoreactive APCs. The results demonstrated the production of large amounts of TNF-α and IFN-γ by the spleen of arthritic mice (Figure 1). In contrast, cells cultured with control antigen (GST) instead of GPI did not produce these cytokines (Figure 1). APC plus antigen alone produced such amounts of cytokines. Very low but detectable levels of IL-2 and IL-6 were produced, but almost no T-helper-2 type cytokines (such as IL-4, IL-5, and IL-10) were detected (Figure 1). These results indicate that exposure to the GPI antigen results in induction of TNF-α and IFN-γ by immunocytes, and suggest that these cytokines could play a crucial role in the induction of arthritis in GPI-induced mice.


Therapeutic effects of antibodies to tumor necrosis factor-alpha, interleukin-6 and cytotoxic T-lymphocyte antigen 4 immunoglobulin in mice with glucose-6-phosphate isomerase induced arthritis.

Matsumoto I, Zhang H, Yasukochi T, Iwanami K, Tanaka Y, Inoue A, Goto D, Ito S, Tsutsumi A, Sumida T - Arthritis Res. Ther. (2008)

GPI-induced TNF-α and IFN-γ production from arthritic splenocytes in vitro. Spleens were removed from glucose-6-phosphate isomerase (GPI)-immunized DBA/1 mice (on day 8 after immunization), and then single-cell suspensions were prepared. MACS separated CD4+ T cells (1 × 106 cells/ml) were stimulated with 5 μg/ml GPI (or glutathione S-transferase [GST]) and antigen-presenting cells (APCs; 2 × 105 cells/ml, mitomycin treated) for 12 hours. The culture supernatants were collected and concentrations of tumor necrosis factor (TNF)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12p70 were measured by cytometric bead array. Data were averages of three independent experiments. Error bars represent ± standard error. *P < 0.05, by Mann-Whitney U-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483457&req=5

Figure 1: GPI-induced TNF-α and IFN-γ production from arthritic splenocytes in vitro. Spleens were removed from glucose-6-phosphate isomerase (GPI)-immunized DBA/1 mice (on day 8 after immunization), and then single-cell suspensions were prepared. MACS separated CD4+ T cells (1 × 106 cells/ml) were stimulated with 5 μg/ml GPI (or glutathione S-transferase [GST]) and antigen-presenting cells (APCs; 2 × 105 cells/ml, mitomycin treated) for 12 hours. The culture supernatants were collected and concentrations of tumor necrosis factor (TNF)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12p70 were measured by cytometric bead array. Data were averages of three independent experiments. Error bars represent ± standard error. *P < 0.05, by Mann-Whitney U-test.
Mentions: To identify the dominant cytokines at the onset of antigen-induced arthritis (day 8), we established the CBA array system using spleen CD4+ T cells plus mitomycin-treated APCs cultured in GPI. In this system, treatment of APCs with mitomycin is designed to kill autoreactive APCs. The results demonstrated the production of large amounts of TNF-α and IFN-γ by the spleen of arthritic mice (Figure 1). In contrast, cells cultured with control antigen (GST) instead of GPI did not produce these cytokines (Figure 1). APC plus antigen alone produced such amounts of cytokines. Very low but detectable levels of IL-2 and IL-6 were produced, but almost no T-helper-2 type cytokines (such as IL-4, IL-5, and IL-10) were detected (Figure 1). These results indicate that exposure to the GPI antigen results in induction of TNF-α and IFN-γ by immunocytes, and suggest that these cytokines could play a crucial role in the induction of arthritis in GPI-induced mice.

Bottom Line: Anti-TNF-alpha mAbs and CTLA-4Ig suppressed TNF-alpha production, whereas anti-IFN-gamma mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-gamma production.Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Clinical Immunology, Major of Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8575, Japan. ismatsu@md.tsukuba.ac.jp

ABSTRACT

Introduction: Immunization with glucose-6-phosphate isomerase (GPI) induces severe arthritis in DBA/1 mice. The present study was designed to identify the cytokines and co-stimulatory molecules involved in the development of GPI-induced arthritis.

Methods: Arthritis was induced in DBA/1 mice with 300 microg human recombinant GPI. CD4+ T cells and antigen-presenting cells from splenocytes of arthritic mice were cultured in the presence of GPI. Tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 levels were assessed using cytometric bead array. Monoclonal antibodies to TNF-alpha, IFN-gamma, IL-12, CD40L, inducible co-stimulator (ICOS), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig) were used to block TNF-alpha and IFN-gamma production, examine clinical index in mice with GPI-induced arthritis, and determine anti-GPI antibody production.

Results: Large amounts of TNF-alpha and IFN-gamma and small amounts of IL-2 and IL-6 were produced by splenocytes from mice with GPI-induced arthritis. Anti-TNF-alpha mAbs and CTLA-4Ig suppressed TNF-alpha production, whereas anti-IFN-gamma mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-gamma production. A single injection of anti-TNF-alpha and anti-IL-6 mAbs and two injections of CTLA-4Ig reduced the severity of arthritis in mice, whereas injections of anti-IFN-gamma and anti-IL-12 mAbs tended to exacerbate arthritis. Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.

Conclusion: TNF-alpha and IL-6 play an important role in GPI-induced arthritis, whereas IFN-gamma appears to function as a regulator of arthritis. Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.

Show MeSH
Related in: MedlinePlus