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Objective C protein-induced myositis (CIM) is a mouse model of polymyositis in which activated antigen-specific CD8+ T cells injure the muscles. Animal models of autoimmunity that have been examined in the past for the effects of the type 1 cytokine interferon-γ (IFNγ) and the type 2 cytokine interleukin-4 (IL-4) are all mediated by pathogenic CD4+ T cells. In those models, the disruption of IFNγ leads to up-regulation of IL-17A, exacerbating the disease with neutrophil infiltration into sites of inflammation. This study was undertaken to investigate the roles of IFNγ and IL-4, as well as IL-17A in the absence of IFNγ, in CD8+ T cell-mediated CIM. Methods IFNγ(-/-) mice, anti-IL-17A antibody-treated IFNγ(-/-) mice, IFNγ(-/-) IL-17A(-/-) mice, IL-4(-/-) mice, and wild-type C57BL/6 mice were immunized with skeletal muscle C protein fragments to induce CIM. Muscle tissue specimens were examined histologically. Results IFNγ(-/-) mice developed myositis at a higher incidence and with greater severity than wild-type mice. Unlike wild-type mice, IFNγ(-/-) mice had infiltration of neutrophils into the endomysial sites of the affected muscles. IFNγ(-/-) mice treated with anti-IL-17A antibodies and IFNγ(-/-) IL-17A(-/-) mice developed myositis with an incidence and severity comparable to those in IFNγ(-/-) mice and showed neutrophil infiltration similar to that in IFNγ(-/-) mice. IL-4(-/-) mice developed CIM comparable to that in wild-type mice. Conclusion Our findings indicate that IFNγ, but not IL-4, plays a suppressive role in the development of CIM. Unlike in CD4+ T cell-mediated autoimmune disease models, IFNγ prevents factors other than IL-17A from exacerbating myositis and neutrophil infiltration in CD8+ T cell-mediated CIM.

Objective To investigate whether leucine-rich α2-glycoprotein (LRG) could be a biomarker for disease activity during interleukin-6 (IL-6) blockade treatment of rheumatoid arthritis (RA). Methods In 59 RA patients who were treated with tocilizumab for 24 weeks, serum LRG levels were determined by enzyme-linked immunosorbent assay. RA disease activity was evaluated by the Clinical Disease Activity Index (CDAI). Receiver operating characteristic (ROC) curve analysis was used to examine the diagnostic performance of LRG and other biomarkers. In monkeys with experimental autoimmune arthritis, swollen joint counts, joint pathologic changes, and blood levels of C-reactive protein (CRP) and LRG were evaluated after treatment with anti–IL-6 receptor antibody. Results Among tocilizumab-treated RA patients, those with active disease (CDAI >2.8) had significantly higher serum LRG levels compared to those whose disease was in remission. ROC curve analysis suggested that the LRG level was more useful than the CRP or matrix metalloproteinase 3 level or the erythrocyte sedimentation rate in discriminating between remission and active disease during therapy with tocilizumab. In monkeys treated with IL-6 blockade, joint scores were more closely correlated with LRG levels than with CRP levels. Histologic analysis of joints revealed that LRG levels correlated significantly with granulomatous tissue formation, cartilage degeneration, and bone destruction in IL-6 blockade–treated monkeys with low levels of CRP. Conclusion Under conditions of IL-6 inhibition, LRG was more useful than other biomarkers in discriminating between active and inactive disease in human RA and in detecting joint inflammation in experimental arthritis. LRG may serve as a convenient biomarker for RA disease activity during IL-6 blockade treatment.

Objective To determine whether injury and regeneration of the skeletal muscles induce an inflammatory milieu that facilitates the development and relapse of autoimmune myositis. Methods The quadriceps of C57BL/6 mice were injured with bupivacaine hydrochloride (BPVC) and evaluated histologically. Macrophages and regenerating myofibers in the treated muscles and differentiating C2C12 myotubes were examined for cytokine expression. Mice were immunized with C protein fragments at the base of the tail and in the right hind footpads (day 0) to evoke systemic anti–C protein immunity and to induce local myositis in the right hind limbs. The contralateral quadriceps muscles were injured with BPVC or phosphate buffered saline (PBS) on day 7 or after spontaneous regression of myositis (day 42). The quadriceps muscle in nonimmunized mice was injured with BPVC on day 7. The muscles were examined histologically 14 days after treatment. Results The BPVC-injured muscles had macrophage infiltration most abundantly at 3 days after the injection, with emergence of regenerating fibers from day 5. The macrophages expressed inflammatory cytokines, including tumor necrosis factor α, interleukin-1β, and CCL2. Regenerating myofibers and C2C12 myotubes also expressed the cytokines. The BPVC-injected muscles from nonimmunized mice had regenerating myofibers with resolved cell infiltration 14 days after treatment. In mice preimmunized with C protein fragments, the muscles injected with BPVC on day 7 as well as on day 42, but not those injected with PBS, had myositis accompanied by CD8+ T cell infiltration. Conclusion Injury and regeneration could set up an inflammatory milieu in the muscles and facilitate the development and relapse of autoimmune myositis.

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.

Among the heterogeneous antiphospholipid antibodies, many studies suggest that those directed to beta2-glycoprotein I (beta2GPI) are the major pathogenic antibodies in antiphospholipid syndrome (APS). They have been shown to activate the coagulation pathway via several mechanisms, activate platelets via thrombin formation, and suppress fibrinolysis. Additionally, we propose another possible mechanism that involves certain chemokines and results in platelet activation. This hypothesis is based on the observations that anti-beta2GPI antibodies stimulated monocytes to secrete inflammatory cytokines such as IL-1beta and TNF-alpha, which in turn stimulated vascular endothelial cells to express chemokines such as CX3CL1 and CCL5. CX3CL1 increased the ability of normal platelets to adhere to collagen at a high shear rate, while CCL5 induced platelet aggregation. Expression of tissue factor, IL-1beta, and TNF-alpha by monocytes stimulated with anti-beta2GPI antibodies, as well as CX3CL1 and CCL5 by vascular endothelial cells stimulated with IL-1beta or TNF-alpha were all suppressed by the NF-kappaB-specific inhibitor DHMEQ. These results suggest that the NF-kappaB pathway may be a potential therapeutic target relating to both the coagulation pathway and platelet activity.

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.

Crk-associated substrate lymphocyte type (Cas-L) protein, also known as human enhancer of filamentation 1 (Hef1) or neural precursor cell-expressed, developmentally down-regulated gene 9 (Nedd9), belongs to the Cas family of adapter proteins, which are involved in integrin signaling. Previous reports showed that Cas-L is expressed preferentially in lymphocytes and epithelial cells. Cas-L mediates signals from integrins, T-cell receptors, B-cells receptors, and transforming growth factor beta, leading to cell movement and cell division. Here, we report the expression of Cas-L in neutrophils. Cas-L was tyrosine-phosphorylated when human neutrophils were stimulated by fMLP, tumor necrosis factor-alpha (TNF), or lipopolysaccharide. The tyrosine phosphorylation of Cas-L in fMLP- or TNF- stimulated neutrophils was further enhanced by adhesion of the cells to their substrates. Cas-L was found to be localized at focal adhesions in stimulated neutrophils based on immunofluorescence microscopy. These findings suggest that Cas-L is one of the targets of inflammatory cytokines and is also modulated by cell adhesion process in neutrophils.

p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of a Src homology 3 (SH3) domain, a substrate domain (SD) and a Src binding domain (SBD). The SH3 domain of Cas associates with focal adhesion kinase (FAK), but its role in cellular function has not fully been understood. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking exon 2, which encodes the SH3 domain (Cas Δexon 2). In comparison to wild-type cells, Cas exon 2Δ/Δ cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas Δexon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by IκBα phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts.

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.

The tumor suppressor p53 has been shown to be essential in apoptosis induced by irradiation, deregulated c-Myc expression and anti-cancer drugs. The protein level of p53 was moderately increased when the B cell line WEHI-231 undergoes apoptosis by B cell receptor (BCR) crosslinking. However, overexpression of a dominant negative form of p53, p53DD, abolished DNA binding activity of p53 almost completely but failed to block BCR-mediated death of WEHI-231, suggesting that p53-mediated transactivation is not required for BCR-mediated apoptosis of WEHI-231. Moreover, B cells of p53-deficient mice underwent cell death upon BCR crosslinking as efficiently as those of normal littermates, indicating that p53 is not essential for BCR-mediated apoptosis of normal B cells. Although a previous report suggested that p53 is required for BCR-mediated apoptosis through its transactivation, our data strongly argue that p53 is not required for BCR-mediated apoptosis.

Systemic lupus erythematosus (SLE) is an autoimmune-mediated collagen disease that results in multiorgan failure. It is the collagen disease most frequently associated with neuropsychiatric symptoms, which have been hypothesized to stem from certain types of cognitive dysfunction. Subjects were 21 patients with SLE (one man, 20 women; aged 16-55 years; mean age, 35.1 ± 10.7 years) who were undergoing treatment in the rheumatology unit of a general hospital, and 17 healthy control subjects matched to the patient group with respect to age and gender (two men, 15 women; mean age, 35.9 ± 6.3 years). They were administered various tests of cognitive function including verbal reasoning, non-verbal reasoning, verbal memory, non-verbal memory, attention and mental flexibility, psychomotor speed and frontal lobe function. In addition, the SLE patients were tested for antiphospholipid antibodies. The SLE patients performed worse than the control group on immediate, delayed and interference of the Rey verbal test and paired associate tests of Wechsler Memory Scale, and their reaction time was slower in Trail A and Trail B tests. Moreover, these findings were more pronounced in the group with major neuropsychiatric symp- toms. However, no relationship was apparent between these deficits in cognitive function and the presence or absence of antiphospholipid antibodies. The results suggest that verbal memory and psychomotor speed underlie the neuropsychiatric symptoms seen in SLE patients.

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.

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H2O2. Analysis with anti-TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.

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.

The right atrial posterior septum, including the coronary sinus (CS) ostium, is an important landmark in radiofrequency catheter ablation therapy for supraventricular tachycardia or atrial flutter. The anatomical findings around the CS ostium would be useful to determine a target site or line during catheter ablation. The aim of the study was to test the ability of the imaging catheter to identify structures in the posterior septal area of the right atrium and to evaluate the feasibility of guidance for catheter placement in the CS using a cardioscope that we recently developed. In 12 anesthetized dogs, the cardioscope, consisting of a deflectable 7 Fr fiberoptic endoscope with an inflatable and transparent balloon, was introduced into the right atrium via the femoral vein. The cardioscope was manipulated to observe the right atrial posterior septum. A deflectable electrode catheter was inserted via the jugular vein and positioned in the CS under cardioscopic guidance. In 10 of 12 dogs, the right atrial posterior septum, including the CS ostium, and the tendon of Todaro could be anatomically identified by cardioscopy. It was possible to position an electrode catheter in the CS in all 10 dogs under direct vision without fluoroscopy. But the CS ostium could not be detected in the remaining two dogs, although the cardioscope was placed at as many sites as possible. No complication occurred. The balloon-tipped cardioscope appears to be useful in observing the right atrialposterior septum and in guiding an electrode catheter into the CS.

The typical fourth criterion for transient entrainment is defined when both a sudden shortening in conduction interval to and a distinct change in electrogram morphology at a bipolar recording site are demonstrated while performing overdrive pacing of a reentrant tachycardia from a single pacing site at two different constant rates. The purpose of this article was to test the hypothesis that if an intracardiac recording site showing both orthodromic and antidromic capture with entrainment pacing is located suitably distant from the circuit, sudden shortening in conduction interval to that site may occur without any significant change in the bipolar electrogram morphology (i.e., atypical form of the fourth criterion). Atrial overdrive pacing of orthodromic tachycardia was performed in 20 patients with either left anterior (12 patients) or left posterior (8 patients) accessory pathways. We investigated the effects of overdrive pacing from the proximal or distal coronary sinus, specifically effects on the electrogram interval and the electrogram morphology at the right atrial appendage. Overdrive pacing of orthodromic tachycardia from the proximal coronary sinus was performed in 10 of the 12 patients with left anterior accessory pathways; those 10 patients demonstrated the first entrainment criterion at the right atrial appendage site. Overdrive pacing of orthodromic tachycardia at still shorter cycle lengths demonstrated a sudden shortening in conduction interval to the right atrial appendage site. Despite shortening in conduction interval the morphology of the right atrial appendage electrogram was completely or almost identical to that during orthodromic tachycardia, indicating an atypical form of the fourth criterion. This criterion was not demonstrated in patients with left posterior accessory pathways. Thus, atypical fourth entrainment criterion was demonstrated during overdrive pacing of orthodromic tachycardia from the proximal coronary sinus only in patients with left anterior accessory path ways. Demonstration of atypical fourth criterion seems largely dependent on the location of the accessory pathway, the pacing, and the recording sites.

Nucleobindin (Nuc) was originally found to be an enhancement factor of anti-DNA antibody production secreted by a lymphoid cell line derived from a lymphoproliferative MRL/lpr mouse. It has been shown that Nuc has a unique structure containing a DNA- and two calcium-binding domains, and a leucine zipper motif, but its biological roles have not yet been fully elucidated. Expression of Nuc was first studied in human lymphocytes. Expression of Nuc mRNA in normal peripheral blood mononuclear cells was significantly increased upon mitogen stimulation. Anti-human Nuc monoclonal antibody H-1D8 immunoprecipitated Nuc protein in the nuclear extract of Molt-4 cells. Furthermore, in the immunohistochemical staining of tumor specimens from 108 patients with non-Hodgkin's lymphoma (NHL) with H-1D8, H-1D8-positive cells were observed in nearly all cases in varying frequency. According to the Working Formulation, the percentage of cases in which more than 90% of the tumor cells were stained with H-1D8 was 65% in the high grade of the histological malignancy, 54% in the intermediate grade, and 22% in the low grade; however, normal cells surrounding the tumor cells were virtually negative for H-1D8. These results showed that the level of Nuc expression in human lymphocytes reflects the status of activation or proliferation of the cells, thus providing a clue for the further investigation into biological roles of Nuc. In addition, it might be applicable to the clinicopathological estimation of NHL as a novel indicator.

Enlarged lymph nodes of mice with lpr mutation consist predominantly of CD4- CD8- (double-negative: DN) T cells. Among them, TCRBV8S3 (V beta 8.3) T cells are overrepresented as compared to those in single-positive (SP) T cells. To address the question of whether the expansion of oligoclonal T cells is responsible for the increase in TCRBV8S3 cells, we examined the TCRBJ gene repertoires of BV8S3 DN and SP T cells from multiple MRL lpr/lpr mice. The BJ repertoires of BV3 (V beta 3), BV8S1 (V beta 8.1) and BV8S2 (V beta 8.2) were studied for comparison with those of BV8S3 T cells. The employed method, which was based on a PCR-ELISA technique, was newly developed and allowed us to make a precise quantitation of TCRBJ gene usage of the multiple lymphocyte samples. The results showed that there were no biases of the BJ gene usage by BV8S3 DN T cells as well as other BV T cells. Furthermore, the BJ gene usage of CD4 and CD8 BV8S3 T cells was conserved by the DN T cells. It is suggested that the BV8S3 DN T cells were not expanded by specific antigens. The expansion may result from aberrant regulation specific to the BV8S3-expressing T cells.

Although both ceramide and interleukin-1β converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both antiFas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.

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.

Objective To investigate associations between continuous treatments with tumor necrosis factor (TNF) antagonists and risk for developing serious infections (SIs) over 3 years in Japanese patients with rheumatoid arthritis (RA) enrolled in the Registry of Japanese RA Patients for Long-Term Safety (REAL) database. Methods We analyzed 727 RA patients who had started either infliximab or etanercept (the anti-TNF group; 1,480.1 patient-years [PY]) and 571 RA patients who had started conventional nonbiologic disease-modifying antirheumatic drugs (the unexposed group; 1,104.1 PY) at the time of enrollment in the REAL. We assessed the occurrence of SIs within a 3-year observation period, including the period after switching to other TNF antagonists, and all SIs, unlimited to the first one in each patient as reported in other studies, to evaluate the real safety of TNF antagonists in daily practice. Results The incidence rate of SIs per 100 PY was 5.54 (95% confidence interval [95% CI] 4.44–6.84) in the anti-TNF group and 2.72 (95% CI 1.87–3.83) in the unexposed group. Poisson regression analysis revealed that the relative risk (RR) of continuous use of TNF antagonists for SIs after adjusting for baseline and time-dependent covariates was significantly elevated both overall (1.97, 95% CI 1.25–3.19) and for the first year (2.40, 95% CI 1.20–5.03), but not for the second and third years combined (1.38, 95% CI 0.80–2.43). The adjusted RR for SIs of etanercept compared to infliximab was not significantly elevated. Conclusion Continuous anti-TNF therapy was significantly associated with increased risks for developing SIs during, but not after, the first year.

Local cytokine gene expression in vivo was analyzed by direct analysis of RNA obtained from salivary gland tissues of MRL/lpr mice with autoimmune sialadenitis. The expression of cytokine genes were assessed by the reverse-transcriptase polymerase chain reaction, and by immunohistochemical analysis. The expression of interleukin-1(IL-1)beta and tumor necrosis factor was detected before the onset of inflammatory lesions in the salivary glands of mice of 1 or 2 months of age, and IL-6 mRNA expression was clearly detected at the time of onset of typical autoimmune sialadenitis at 3 months of age in MRL/lpr mice, and was up-regulated with advancing age. These results suggest that the overexpression of these inflammatory cytokine genes is involved in the development and progression of organ-localized autoimmunity in the salivary glands of MRL/lpr mice.

Synovial T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA) synovitis. We have quantitatively analyzed the T cell receptor (TcR) variable (V) region gene repertoire of freshly isolated synovial fluid (SF) T cells, comparing it with that of peripheral blood (PB) T cells in RA. The TcR V gene repertoire of PB and SF T cells in RA and osteoarthritis was heterogeneous. In contrast, V alpha 11 in SF was expressed to a greater degree in three of five RA patients, and increased levels of V beta 6, 1-3 were found in the SF of four of six RA, compared with paired PB. Of note, V beta 6, 1-3 was universally used in four RA patients with a disease duration of less than 10 years, irrespective of their HLA-DR types. This was in contrast to two other RA patients, suffering for more than 20 years, who showed different V alpha and V beta usages. beta-chain sequence analysis in RA patients with a preference for V beta 6, 1-3 has shown that a few clones dominated in SF, whereas polyclonality was observed in PB. These findings suggest oligoclonal expansion of T cells in response to specific antigen(s) in the SF of these patients with RA of relatively short duration. Concomitant use of two hydrophobic amino acids, leucine and valine in the D beta region was noticeable among the predominant SF clones. These two amino acids might directly contact a peptide specific for the induction of synovitis in RA patients. TcR-directed therapy may, therefore, be useful for the treatment of early RA synovitis.

We attempted to determine whether cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (endothelial-leukocyte adhesion molecule-1; ELAM-1), are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in Sjogren's syndrome (SS) patients. Both immunohistochemical analysis and the reverse-transcripts polymerase chain reaction (RT-PCR) were used to analyze the expression of VCAM-1, ICAM-1, ELAM-1, very late antigen 4 (VLA-4 [alpha 4,beta 1]), lymphocyte function-associated antigen-1 (LFA-1), interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta). Immunohistochemical analysis of salivary gland biopsies from SS patients showed a marked expression of VCAM-1 and ICAM-1 in the venules surrounded by infiltrated CD4+ CD45RO+ T cells. E-selectin was expressed on vascular endothelium with weak intensity. Increased levels of VCAM-1, ICAM-1, IFN-gamma, and IL-1 beta mRNA were demonstrated by RT-PCR, whereas E-selectin mRNA were weakly expressed in SS lacrimal and salivary gland tissues. This is in contrast with strong expression of ELAM-1 in IL-1 beta-stimulated human umbilical vascular endothelial cells (HUVEC) in vitro. Cytokine-mediated up-regulation of VCAM-1 and ICAM-1 that facilitates the recruitment of VLA-4 and LFA-1 expressing T cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in SS.

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K-Cre knock-in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR-155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency-induced osteoclastic suppression was dominant over Dicer deficiency-induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen-Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo.

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.