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Objective Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein–coupled receptors (LPA receptors 1–6 [LPA1–6]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA1 on the development of arthritis. Methods Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription–polymerase chain reaction. The effects of abrogation of LPA1 on collagen-induced arthritis (CIA) were evaluated using LPA1-deficient mice or LPA1 antagonist. Migrating fluorescence-labeled CD11b+ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4+ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined. Results LPA1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA1-deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b+ macrophages from LPA1-deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA1 deficiency or LPA1 inhibition in vitro. Conclusion Collectively, these results indicate that LPA/LPA1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA1 may be a promising target molecule for RA therapy.

Objective It has been reported that polymyositis (PM) is driven by CD8+ cytotoxic T lymphocytes. The C protein–induced myositis (CIM) model we have established is similar to PM in pathology except that it undergoes spontaneous remission. We undertook the present study to delineate the roles of innate and acquired immunity in myositis. Methods C57BL/6 mice were immunized with recombinant C protein fragments together with Freund's complete adjuvant (CFA) and Toll-like receptor (TLR) ligands at hind leg footpads and tail bases. CIM mediated by adoptive transfer of T cells to naive mice was treated with cytokine antagonists. Results Second immunization with C protein fragments revealed no induction of tolerance. Injection of CFA and TLR ligands at the hind leg footpads reinduced myositis in the same legs. Interestingly, initial myositis was observed only in the CFA-treated forelegs. Transfer of C protein fragment–specific T cells from mice with CIM induced myositis in CFA- and TLR ligand–treated legs of recipient mice. CFA treatment resulted in the recruitment of macrophages producing inflammatory cytokines. Induction of myositis was inhibited by blocking interleukin-1 receptor or tumor necrosis factor α. Conclusion Myositis development requires activation of autoaggressive T cells and conditioning of muscle tissue. CIM regression is due to attenuation of local CFA-induced immune activation. These results are in accordance with a “seed and soil” model of disease development and might offer clues to decipher clinical aspects of PM.

Objective Histologic studies of the muscles suggest that cytokines are involved in inflammatory myopathy. The therapeutic effects of cytokine blockade are controversial, with anecdotal reports of clinical efficacy. The aim of this study was to discern the significance of interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) as therapeutic targets in polymyositis (PM) by studying their involvement and the effects of their blockade in C protein–induced myositis (CIM), a murine model of PM. Methods C57BL/6 mice were immunized with recombinant skeletal C protein fragments to induce CIM. The expression of IL-1 and TNFα in the muscles of mice with CIM was detected using immunohistochemical and real-time polymerase chain reaction analyses. After the onset of myositis, the mice with CIM were treated with recombinant IL-1 receptor antagonist (IL-1Ra), anti–IL-1R monoclonal antibody, recombinant TNF receptor (p75)–fusion protein (TNFR-Fc), or anti-TNFα monoclonal antibody. The muscles were examined histologically for the severity of myositis. Results IL-1α– and TNFα-positive macrophages were observed in the muscle tissue of mice with CIM as early as 7 days after immunization. IL-1α, IL-1β, and TNFα expression in the muscles increased as the severity of myositis peaked, at both the messenger RNA and protein levels. Continuous subcutaneous delivery of IL-1Ra resulted in suppression of established CIM. Intermittent delivery (1-day intervals) of anti–IL-1R monoclonal antibody suppressed myositis, while intermittent delivery of IL-1Ra did not suppress myositis. Treatment with anti-TNFα monoclonal antibody and with TNFR-Fc also reduced the severity of CIM. Conclusion IL-1 and TNF blockade ameliorated CIM after disease onset and should potentially be a new strategy for the treatment of inflammatory myopathy. As IL-1 blockade, treatment with anti–IL-1R monoclonal antibody appeared more feasible than the other approaches.

Objective The interaction between CXCL12 and its receptor, CXCR4, in the synovium of patients with rheumatoid arthritis (RA) is important for local inflammatory cell recruitment, angiogenesis, and cytokine production. CXCR7 was recently identified as an alternative receptor for CXCL12. We undertook this study to analyze the expression of CXCR7 in RA synovium and the pathogenic role of the CXCL12/CXCR7 pathway in RA. Methods CXCR7 expression in RA synovial tissue was analyzed using immunohistochemistry, while expression of CXCR4 and CXCR7 on human umbilical vein endothelial cells (HUVECs) was examined using quantitative reverse transcription–polymerase chain reaction, and CXCR7 expression was also analyzed by flow cytometry. Tube formation and rat aortic ring angiogenesis assays were used to assess the effects of CCX733 (a CXCR7 antagonist) and AMD3100 (a CXCR4 antagonist) on CXCL12-induced angiogenesis. The effect of anti-CXCR4 monoclonal antibody (mAb) was also analyzed using a tube formation assay. The effects of CCX733 in a murine model of collagen-induced arthritis (CIA) were also evaluated. Results CXCR7 was expressed on endothelial cells in RA synovium and also on unstimulated HUVECs. The expression of CXCR7 on HUVECs was markedly up-regulated by interleukin-1β (IL-1β) stimulation, and this overexpression was further enhanced by CXCL12 treatment. Incubation with CXCL12 also promoted angiogenic activity, with addition of IL-1β again augmenting the effect. CXCL12-induced angiogenesis was inhibited by both CXCR4 and CXCR7 antagonists and by anti-CXCR4 mAb. Furthermore, treatment with CCX733 significantly reduced the clinical arthritis scores and the numbers of vessels in the inflamed synovial tissue in mice with CIA. Conclusion CXCR7 and CXCR4 are both important for angiogenesis in RA synovium, making CXCR7 another potential target molecule for novel RA angiogenesis-blocking therapies.

Objective To explore new molecular targets in the treatment of polymyositis (PM) by examining a recently established murine model of PM, C protein–induced myositis (CIM), for involvement of an interleukin-6 (IL-6)/IL-17A pathway. Methods CIM was induced by immunizing wild-type mice as well as IL-6–null and IL-17A–null C57BL/6 mice with recombinant mouse skeletal C protein fragments. Some mice were treated with anti–IL-6 receptor (anti–IL-6R) monoclonal antibodies or control antibodies. Muscle tissue samples were examined histologically and immunohistochemically. Results The syngeneic C protein fragments successfully induced inflammation in the skeletal muscles of wild-type mice. IL-6 was expressed by mononuclear cells, especially in macrophages, infiltrating in the muscles. IL-6–null mice developed myositis with significantly lower incidence and milder severity than wild-type mice. In contrast, IL-17A–null mice were as susceptible to CIM as wild-type mice. Intraperitoneal administration of anti–IL-6R monoclonal antibodies, but not of control monoclonal antibodies, ameliorated CIM both preventively and therapeutically. Conclusion Our findings indicate that IL-6 is critically involved in the development of CIM. Although many other autoimmune models require IL-6 for differentiation of pathogenic T cells producing IL-17A, IL-17A was dispensable in CIM. Nevertheless, treatment with anti–IL-6R antibodies was effective. IL-6 blockade is potentially a new approach to the treatment of autoimmune myositis, via processes distinct from interference in the IL-6/IL-17A pathway.

Objective Triggering receptor expressed on myeloid cells 1 (TREM-1) is inducible on monocyte/macrophages and neutrophils and accelerates tissue destruction by propagating inflammatory responses in disease related to bacterial infections. Its blockade rescues the hosts in murine models of sepsis, to clear the bacteria without impairing the host defense. The aim of this study was to investigate the involvement of TREM-1 in an autoimmune, noninfectious disease. Methods Synovial tissue specimens from the joints of patients with rheumatoid arthritis (RA) and the joints of mice with collagen-induced arthritis (CIA) were examined for TREM-1 expression, using flow cytometric analysis. Expression of TREM-1 on macrophages was induced by lipopolysaccharide, with or without a cyclooxygenase inhibitor. Rheumatoid synovial cells were stimulated with agonistic anti–TREM-1 antibodies. Recombinant adenovirus encoding the extracellular domain of TREM-1 fused with IgG-Fc (AxCATREM-1 Ig) or synthetic TREM-1 antagonistic peptides were injected to treat CIA, and the clinical manifestations of the antigen-specific T cell and B cell responses were evaluated. Results TREM-1 was expressed on CD14+ cells in rheumatoid synovial tissue and synovial macrophages from mice with CIA. Unlike murine macrophages, human monocyte/macrophages did not depend on prostaglandin E2 for up-regulation of TREM-1. Agonistic anti–TREM-1 antibodies promoted tumor necrosis factor α production from rheumatoid synovial cells. Blockade of TREM-1 using AxCATREM-1 Ig and antagonistic peptides ameliorated CIA without affecting the serum levels of anti–type II collagen antibodies or the proliferative responses of splenocytes to type II collagen. Conclusion TREM-1 ligation contributes to the pathology of autoimmune arthritis. The results of this study implied that blockade of TREM-1 could be a new approach to rheumatic diseases that is safer than the presently available immunosuppressive treatments.

Objective To establish a new murine model of polymyositis (PM) for the understanding of its pathologic mechanisms and the development of new treatment strategies. Methods C protein-induced myositis (CIM) was induced by a single immunization of recombinant human skeletal C protein in C57BL/6 mice, as well as in CD4-depleted, CD8-depleted, and mutant mice as controls. Some mice were treated with high-dose intravenous immunoglobulin (IVIG) after disease induction. Muscle tissues were examined histologically. Results In mice with CIM, inflammation was confined to the skeletal muscles. Histologic examination revealed a common pathologic feature of CIM and PM, involving abundant infiltration of CD8 and perforin-expressing cells in the endomysial site of the injured muscle. Suppression of myositis was achieved by depletion of both CD4 and CD8 T cells. Despite the development of serum anti-C protein antibodies in wild-type mice, severe myositis was induced in mice deficient in B cells. Induction of myositis was suppressed in interleukin-1alpha/beta (IL-1alpha/beta)-null mutant mice, but not in tumor necrosis factor alpha (TNFalpha)-null mutant mice. Use of IVIG, a treatment with proven efficacy in PM, suppressed CIM in the subgroup of treated mice. Conclusion CIM mimics PM pathologically and clinically. Infiltration of CD8 T cells is the most likely mechanism of muscle injury, and IL-1, but not B cells or TNFalpha, is crucial in the development of CIM. IVIG has therapeutic effects in CIM, suggesting that the effects of IVIG are not mediated by suppression of antibody-mediated tissue injury. This murine model provides a useful tool for understanding the pathologic mechanisms of PM and for developing new treatment strategies.

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive T cell infiltration into the synovium. The accumulated T cells express type 1 cytokines, such as interferon-γ (IFNγ) and tumor necrosis factor α, and activated markers of inflammation, such as CD154 and inducible costimulator (ICOS). It is thought that chemokines contribute to T cell accumulation in the synovium. In this study, we examined the role of CXCL16 and CXCR6 in T cell migration and stimulation in RA synovium. Methods Expression of CXCL16 and CXCR6 was analyzed by immunohistochemistry, reverse transcription–polymerase chain reaction, Western blotting, and/or flow cytometry. Migration activity was assessed using a chemotaxis chamber. IFNγ production was analyzed by enzyme-linked immunosorbent assay. The effect of anti-CXCL16 monoclonal antibody on murine collagen-induced arthritis (CIA) was evaluated. Results CXCL16 was expressed in RA synovium. CXCR6 was expressed more frequently on synovial T cells than in peripheral blood. Moreover, CXCR6-positive synovial T cells more frequently expressed CD154 and ICOS than did CXCR6-negative T cells. Stimulation with interleukin-15 (IL-15) up-regulated the expression of CXCR6 on peripheral blood T cells, and then stimulation with CXCL16 induced migration of IL-15–stimulated T cells and enhanced IFNγ production. Furthermore, anti-CXCL16 monoclonal antibody significantly reduced the clinical arthritis score and reduced infiltration of inflammatory cells and bone destruction in the synovium of mice with CIA. Conclusion Our results indicate that CXCL16 plays an important role in T cell accumulation and stimulation in RA synovium and suggest that CXCL16 could be a target molecule in new therapies for RA.

Objective Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, inflammation, and hematopoiesis. Overproduction of IL-6 plays pathologic roles in rheumatoid arthritis (RA), and the blockade of IL-6 may be therapeutically effective for the disease. This study was undertaken to evaluate the safety and efficacy of a humanized anti–IL-6 receptor antibody, MRA, in patients with RA. Methods In a multicenter, double-blind, placebo-controlled trial, 164 patients with refractory RA were randomized to receive either MRA (4 mg/kg body weight or 8 mg/kg body weight) or placebo. MRA was administered intravenously every 4 weeks for a total of 3 months. The clinical responses were measured using the American College of Rheumatology (ACR) criteria. Results Treatment with MRA reduced disease activity in a dose-dependent manner. At 3 months, 78% of patients in the 8-mg group, 57% in the 4-mg group, and 11% in the placebo group achieved at least a 20% improvement in disease activity according to the ACR criteria (an ACR20 response) (P < 0.001 for 8-mg group versus placebo). Forty percent of patients in the 8-mg group and 1.9% in the placebo group achieved an ACR50 response (P < 0.001). The overall incidences of adverse events were 56%, 59%, and 51% in the placebo, 4-mg, and 8-mg groups, respectively, and the adverse events were not dose dependent. A blood cholesterol increase was observed in 44.0% of the patients. Liver function disorders and decreases in white blood cell counts were also observed, but these were mild and transient. There was no increase in antinuclear antibodies or anti-DNA antibodies. Anti-MRA antibodies were detected in 2 patients. Conclusion Treatment with MRA was generally well tolerated and significantly reduced the disease activity of RA.

Objective Rheumatoid arthritis (RA) is characterized by chronic inflammation of multiple joints. Large numbers of T cells, which produce type 1 cytokines, infiltrate into RA synovium. Chemokines and chemokine receptors are considered to contribute to the T cell infiltration. In this study, we examined the role of CX3CL1/fractalkine and its receptor CX3C chemokine receptor 1 (CX3CR1) in the T cell migration into RA synovium. Methods Using flow cytometry, immunohistochemistry, and reverse transcription–polymerase chain reaction, we analyzed CX3CR1 expression by peripheral blood and synovial T cells, and CX3CL1 expression in synovium from patients with RA. Cytokine and cytotoxic molecule expression by CX3CR1-positive T cells was analyzed by flow cytometry. Results CX3CR1 expression by peripheral CD4+ and CD8+ T cells was up-regulated in RA patients. The peripheral CD4+ and CD8+ T cells expressing CX3CR1 predominantly produced interferon-γ and tumor necrosis factor α, and expressed cytotoxic molecules such as granzyme A and perforin. Furthermore, CX3CR1+,CD3+ T cells infiltrated into RA synovium. CX3CL1, the unique ligand of CX3CR1, was expressed by endothelial cells and synoviocytes in RA synovium, but not in osteoarthritis synovium. Conclusion Our findings suggest that the interactions of CX3CL1 and CX3CR1 might contribute to the accumulation of CX3CR1+ T cells expressing type 1 cytokines and possessing cytotoxic granules in RA synovium.

Objective. To determine the involvement of human thioredoxi/adult T cell leukemia–derived factor (TRX/ADF) in Sjogren's syndrome (SS) and the correlation with Epstein-Barr virus (EBV). Methods. Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and the presence of EBV, using 6 normal tissues and 23 surgical specimens. The kinetics of expression of TRX/ADF induced by EBV was examined in vitro with peripheral blood B cells from EBV-seronegative donors. Results. Marked expression of TRX/ADF was found in the infiltrating B cells and the epithelial cells of salivary gland tissues from patients with SS (11 of 12 cases), but not in those from patients with other salivary gland inflammatory conditions (0 of 11 cases) or those of normal individuals (0 of 6 cases). In immunohistologic analyses, a striking topographic correlation between TRX/ADF and EBV was found. The coexistence of TRX/ADF messenger RNA and EBV DNA was detected by polymerase chain reaction (r = 0.75, P < 0.01). Peripheral blood B cells from EBV-seronegative donors showed de novo synthesis of TRX/ADF following in vitro infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRX/ADF was not detected in non-EBV-infected cells. Tumors in SCID mice reconstituted with mononuclear cells of salivary glands from SS patients, which were composed of human B cells carrying EBV DNA, were positive for TRX/ADF. Conclusion. These findings suggest that TRX/ADF expression closely reflects the intracellular event of EBV reactivation in SS. This is also the first report to show the ectopic in vivo expression of TRX/ADF in human autoimmune disease.

Objective. To investigate the expressions of costimulatory molecules CD86 (B7-2, B70) and CD28 by cells obtained from the synovial tissues (ST) and synovial fluids (SF) of patients with rheumatoid arthritis (RA). Methods. Monoclonal antibodies (MAb) against CD86 and CD28 were used for immunochemical study of synovia from 18 RA patients, 4 osteoarthritis (OA) patients, and 4 normal subjects. These MAb were also used for flow cytometry of isolated ST cells from 8 RA and 5 OA patients and of SF mononuclear cells from 5 RA and 5 OA patients. Results. Immunohistochemical examination revealed that CD86+ cells occurred in 11 of the 18 RA synovia, but in none of the 4 OA or 4 normal synovia. Most of the positive cells had macrophage-like morphology, and surrounded lymphoid aggregates. Most cells within lymphoid aggregates were stained positively for CD28. Flow cytometry showed that CD86+ cells comprised 2.9–33.4% (average 14.3%) of the total ST cells and 2.1–14.9% (average 6.1%) of the total SF mononuclear cells from RA patients. Approximately 40% of the CD86+ cells expressed CD14. A majority (mean 72%, range 57–89%) of the T cells in ST and SF expressed CD28. RA synovia expressed more CD86 molecules than did OA synovia (mean frequency of positive cells 14.3% versus 2.8%; mean fluorescence intensity 104.6 versus 40.9). Conclusion. This study has shown that the expression of the CD86 molecule was up-regulated in RA synovia. The CD86+ cells appeared macrophage-like and surrounded CD28+ cells in lymphoid aggregates. The simultaneous presence and close localization of CD86+ and CD28+ cells in the RA synovial compartment suggests that their interaction potentially contributes to the sustained immune activation of RA.

To reassess the role of autoantibodies to type II collagen in the pathogenesis of diseases, we studied antibodies from patients with rheumatoid arthritis (RA) and from patients with relapsing polychondritis for species specificity and collagen type specificity, using an improved enzyme-linked immunosorbent assay. Antibodies were found in the sera of 15% of the RA patients and 50% of the relapsing polychondritis patients, as well as in the cartilage of 69% of the RA patients examined. Reaction with both homologous and heterologous type II collagens was common. Analysis of 19 selected RA sera revealed that autoantibodies were generally associated with specific antibodies to some species of heterologous type II collagen. In contrast, antibodies found in 4% of the non-RA controls were specific for either bovine or chick type II collagen. These findings indicate that autoantibody formation in RA and relapsing polychondritis may occur as a result of an immune response to heterologous type II collagen. However, since RA and relapsing polychondritis patient sera differed in their reactivity with the cyanogen bromide-digested peptides, it is possible that the clinical manifestation of collagen autoimmunity might be influenced by the epitope specificity of the antibodies.

Potent interleukin-1 (IL-1) activity was detected in culture supernatants from synovium, obtained by arthroscopy, from rheumatoid arthritis (RA) patients but not from non-RA patients. Production of IL-1 by RA synovium correlated well with findings of inflammation on arthroscopy and HLA-DR expression in immunohistochemical staining. Furthermore, there was a positive correlation between IL-1 production from RA synovium and joint changes detected on roentgenograms. These findings strongly suggest that IL-1 might play an important role in the joint destruction in RA. Rheumatoid arthritis (RA) is a systemic disease characterized by marked inflammation in the synovium. Histologic studies of RA synovium demonstrate proliferation of the synovial cells and infiltration of lymphocytes in association with the destruction of cartilage and bone. However, it is not clear how synovitis causes joint destruction in RA. Altered immunoregulation in RA has been reported by various investigators (1-3). Decreased interleukin-2 (IL-2) production has been demonstrated both in the peripheral blood and the synovial fluid (3,4).

We sought evidence of Epstein-Barr virus (EBV) involvement in the mechanism of polyclonal B cell activation in 29 patients with Sjögren's syndrome (SS). Serum levels of anti-viral capsid antigen antibodies of both the IgG and the IgM class were significantly elevated in these patients. Excretion of EBV from the oropharynx was frequently observed, as well. Spontaneously transformed B cell lines that produced a large amount of transforming EBV were established preferentially from SS patients. We also found that an EBV-specific regulatory mechanism was impaired in these patients. Our findings thus provide evidence of the involvement of EBV in patients with SS.

Surface antigens of T lymphocytes in peripheral blood mononuclear cells from 19 patients with systemic lupus erythematosus (SLE), 14 patients with Sjogren's syndrome (SS), and 14 healthy control subjects were studied by 2-color analysis. The proportion of CD4+2H4+ cells, which correspond to a suppressor/inducer subset, was decreased significantly in both SLE and SS. In contrast, the percentage of CD4+4B4+ cells, which define a helper/inducer subset, showed no significant difference among the patient groups. These data suggest that quantitative defect of suppressor/inducer cells may play an important role in the immunoregulatory disturbance in SLE and SS.