Objective. To clarify the glucose-6-phosphate isomerase (GPI)-specific CD4؉ T cell lineage involved in GPI-induced arthritis and to investigate their pathologic and regulatory roles in the induction of the disease.Methods. DBA/1 mice were immunized with GPI to induce arthritis. CD4؉ T cells and antigen-presenting cells were cocultured with GPI, and cytokines in the supernatant were analyzed by enzyme-linked immunosorbent assay. Anti-interferon-␥ (anti-IFN␥) monoclonal antibody (mAb), anti-interleukin-17 (anti-IL-17) mAb, or the murine IL-6 receptor (IL-6R) mAb MR16-1 was injected at different time points, and arthritis development was monitored visually. After MR16-1 was injected, percentages of Th1, Th2, Th17, and Treg cells were analyzed by flow cytometry, and CD4؉ T cell proliferation was analyzed using carboxyfluorescein diacetate succinimidyl ester.Results. GPI-specific CD4؉ T cells were found to be differentiated to Th1 and Th17 cells, but not Th2 cells. Administration of anti-IL-17 mAb on day 7 significantly ameliorated arthritis (P < 0.01), whereas administration of anti-IFN␥ mAb exacerbated arthritis.Neither anti-IL-17 mAb nor anti-IFN␥ mAb administration on day 14 ameliorated arthritis. Administration of MR16-1 on day 0 or day 3 protected against arthritis induction, and MR16-1 administration on day 8 significantly ameliorated existing arthritis (P < 0.05). After administration of MR16-1, there was marked suppression of Th17 differentiation, without an increase in Th1, Th2, or Treg cells, and CD4؉ T cell proliferation was also suppressed.Conclusion. IL-6 and Th17 play an essential role in GPI-induced arthritis. Since it has previously been shown that treatment with a humanized anti-IL-6R mAb has excellent effects in patients with rheumatoid arthritis (RA), we propose that the IL-6/IL-17 axis might also be involved in the generation of RA, especially in the early effector phase.

IntroductionImmunization 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.MethodsArthritis was induced in DBA/1 mice with 300 μg 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)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 levels were assessed using cytometric bead array. Monoclonal antibodies to TNF-α, IFN-γ, IL-12, CD40L, inducible co-stimulator (ICOS), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig) were used to block TNF-α and IFN-γ production, examine clinical index in mice with GPI-induced arthritis, and determine anti-GPI antibody production.ResultsLarge amounts of TNF-α and IFN-γ and small amounts of IL-2 and IL-6 were produced by splenocytes from mice with GPI-induced arthritis. Anti-TNF-α mAbs and CTLA-4Ig suppressed TNF-α production, whereas anti-IFN-γ mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-γ production. A single injection of anti-TNF-α and anti-IL-6 mAbs and two injections of CTLA-4Ig reduced the severity of arthritis in mice, whereas injections of anti-IFN-γ and anti-IL-12 mAbs tended to exacerbate arthritis. Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.ConclusionTNF-α and IL-6 play an important role in GPI-induced arthritis, whereas IFN-γ 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.

Introduction Tumor necrosis factor-alpha (TNFα) plays a pivotal role in rheumatoid arthritis (RA); however, the mechanism of action of TNFα antagonists in RA is poorly defined. Immunization of DBA/1 mice with glucose-6-phosphate isomerase (GPI) induces severe acute arthritis. This arthritis can be controlled by TNFα antagonists, suggesting similar etiology to RA. In this study, we explored TNFα-related mechanisms of arthritis.

Human six-transmembrane epithelial antigen of prostate4 (STEAP4), an ortholog of mouse tumor necrosis factor-α-induced adipose-related protein (TIARP), plays a role in tumor necrosis factor (TNF)-dependent arthritis models. However, its role in rheumatoid arthritis (RA) is still obscure. This study explored such a role for STEAP4. The expressions of STEAP4, TNFα, and IL-6 were compared in synovia of RA and osteoarthritis patients. STEAP4 induction was examined in TNFα-stimulated fibroblast-like synoviocytes (FLS) in vitro. FLS (with/without TNFα stimulation) were also analyzed for IL-6 expression after STEAP4 knockdown, using siRNA or transfection with STEAP4-plasmid DNA. IL-8, cell proliferation, and apoptosis were also evaluated in STEAP4-overexpressing FLS. The expression of STEAP4 in joints correlated with TNFα expression, specifically in RA synovium. In the cultured FLS, STEAP4 protein expression was augmented by TNFα activation, and localized in endosomal/lysosomal compartments. STEAP4 downregulation by siRNA enhanced the expression of IL-6 mRNA, while STEAP4 overexpression suppressed IL-6 and IL-8 expression, inhibited cell proliferation, and induced apoptosis via caspase-3. The results indicated that human STEAP4 is regulated by TNFα in synovium, where it controls IL-6 secretion and proliferation of FLS, suggesting that STEAP4 might potentially suppress the pathogenesis of TNFα-induced arthritis such as RA.

IntroductionArthritis induced by immunisation with glucose-6-phosphate isomerase (GPI) in DBA/1 mice was proven to be T helper (Th) 17 dependent. We undertook this study to identify GPI-specific T cell epitopes in DBA/1 mice (H-2q) and investigate the mechanisms of arthritis generation.MethodsFor epitope mapping, the binding motif of the major histocompatibility complex (MHC) class II (I-Aq) from DBA/1 mice was identified from the amino acid sequence of T cell epitopes and candidate peptides of T cell epitopes in GPI-induced arthritis were synthesised. Human GPI-primed CD4+ T cells and antigen-presenting cells (APCs) were co-cultured with each synthetic peptide and the cytokine production was measured by ELISA to identify the major epitopes. Synthetic peptides were immunised in DBA/1 mice to investigate whether arthritis could be induced by peptides. After immunisation with the major epitope, anti-interleukin (IL) 17 monoclonal antibody (mAb) was injected to monitor arthritis score. To investigate the mechanisms of arthritis induced by a major epitope, cross-reactivity to mouse GPI peptide was analysed by flow cytometry and anti-GPI antibodies were measured by ELISA. Deposition of anti-GPI antibodies on the cartilage surface was detected by immunohistology.ResultsWe selected 32 types of peptides as core sequences from the human GPI 558 amino acid sequence, which binds the binding motif, and synthesised 25 kinds of 20-mer peptides for screening, each containing the core sequence at its centre. By epitope mapping, human GPI325–339 was found to induce interferon (IFN) γ and IL-17 production most prominently. Immunisation with human GPI325–339 could induce polyarthritis similar to arthritis induced by human GPI protein, and administration of anti-IL-17 mAb significantly ameliorated arthritis (p < 0.01). Th17 cells primed with human GPI325–339 cross-reacted with mouse GPI325–339, and led B cells to produce anti-mouse GPI antibodies, which were deposited on cartilage surface.ConclusionsHuman GPI325–339 was identified as a major epitope in GPI-induced arthritis, and proved to have the potential to induce polyarthritis. Understanding the pathological mechanism of arthritis induced by an immune reaction to a single short peptide could help elucidate the pathogenic mechanisms of autoimmune arthritis.