Comparison of the inhibitory effect of tocilizumab and etanercept on the progression of joint erosion in rheumatoid arthritis treatment.

We compared the efficacy of tocilizumab and etanercept in inhibiting radiographic progression of joint destruction in rheumatoid arthritis. Overall, 187 patients treated with etanercept or tocilizumab were selected. To adjust for baseline patient characteristics between the tocilizumab and etanercept treatment groups, a propensity score matching was performed. Radiographic progression of joint destruction was compared between patients treated with tocilizumab or etanercept. Clinical disease activity index (CDAI) and modified health assessment questionnaire (mHAQ) scores at the administration of biologic treatment and after 12 months of tocilizumab and etanercept therapy were measured and compared to radiographical parameters between the groups. Levels of C-reactive protein (CRP), matrix metalloproteinase-3 (MMP-3), CDAI, and mHAQ scores improved after 12 months of treatment in the two groups. Proportion of patients with no Sharp erosion score progression was significantly higher with tocilizumab treatment than with etanercept treatment (p = 0.032). Multivariate analysis demonstrated that Sharp erosion score was significantly associated with baseline CDAI (odds ratio, 1.05; 95% confidence interval, 1.003-1.099, p = 0.037). Tocilizumab treatment suppressed joint erosion progression compared to etanercept, and the progression correlated with baseline CDAI.

DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus.

Pathogens including autoantigens all failed to induce systemic lupus erythematosus (SLE). We, instead, studied the integrity of host's immune response that recognized pathogen. By stimulating TCR with an antigen repeatedly to levels that surpass host's steady-state response, self-organized criticality, SLE was induced in mice normally not prone to autoimmunity, wherein T follicular helper (Tfh) cells expressing the guanine nucleotide exchange factor DOCK8 on the cell surface were newly generated. DOCK8+Tfh cells passed through TCR re-revision and induced varieties of autoantibody and lupus lesions. They existed in splenic red pulp and peripheral blood of active lupus patients, which subsequently declined after therapy. Autoantibodies and disease were healed by anti-DOCK8 antibody in the mice including SLE-model (NZBxNZW) F1 mice. Thus, DOCK8+Tfh cells generated after repeated TCR stimulation by immunogenic form of pathogen, either exogenous or endogenous, in combination with HLA to levels that surpass system's self-organized criticality, cause SLE.

Conditional Upregulation of IFN-α Alone Is Sufficient to Induce Systemic Lupus Erythematosus.

The cause of systemic lupus erythematosus (SLE) is unknown. IFN-α has been suggested as a causative agent of SLE; however, it was not proven, and to what extent and how IFN-α contributes to the disease is unknown. We studied the contribution of IFN-α to SLE by generating inducible IFN-α transgenic mice and directly show that conditional upregulation of IFN-α alone induces a typical manifestation of SLE in the mice not prone to autoimmunity, such as serum immune complex, autoantibody against dsDNA (anti-dsDNA Ab), and the organ manifestations classical to SLE, such as immune complex-deposited glomerulonephritis, classical splenic onion-skin lesion, alopecia, epidermal liquefaction, and positive lupus band test of the skin. In the spleen of mice, activated effector CD4 T cells, IFN-γ-producing CD8 T cells, B220+CD86+ cells, and CD11c+CD86+ cells were increased, and the T cells produced increased amounts of IL-4, IL-6, IL-17, and IFN-γ and decreased IL-2. In particular, activated CD3+CD4-CD8- double-negative T cells positive for TCRαß, B220, CD1d-teteramer, PD-1, and Helios (that produced increased amounts of IFN-γ, IL-4, IL-17, and TNF-α) were significantly expanded. They infiltrated into kidney and induced de novo glomerulonephritis and alopecia when transferred into naive recipients. Thus, sole upregulation of IFN-α is sufficient to induce SLE, and the double-negative T cells expanded by IFN-α are directly responsible for the organ manifestations, such as lupus skin disease or nephritis.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

MMP-3 as a predictor for structural remission in RA patients treated with MTX monotherapy.

BACKGROUND: The study was undertaken to assess the efficacy of methotrexate (MTX) monotherapy on the radiographic progression of individual rheumatoid arthritis (RA) patients, each of whom had received MTX monotherapy for 3 years with an option to change to biological disease-modifying anti-rheumatic drugs (bDMARDs). We also looked for predictors of radiographic non-progression in these patients. METHODS: Rheumatoid patients (n = 161) were prospectively followed for 3 years while receiving low-dose MTX monotherapy unless disease was otherwise active and/or adverse events appeared. Their disease activity and radiographic progression were evaluated with reference to disease activity score 28 (DAS28), modified health assessment of questionnaire (mHAQ) and other indices. The change in van der Heijde-modified total Sharp score per year (∆TSS) was assessed using probability plots, in which the patients were classified into the subgroups showing structural remission (REM; ∆TSS ≤0.5), radiographic progression (∆TSS >3) or rapid radiographic progression (RRP; ∆TSS >5). RESULTS: MTX monotherapy, continued until disease became active and/or adverse event appeared, was associated with a significant improvement (p <0.0001) in the DAS28-ESR (3) scores, % DAS28 remission, and mHAQ scores each year, from baseline to 3 years. The mHAQ remission rate (∆mHAQ <0.5) and Boolean remission were also improved from 16 to 60 % and 0.8 to 24.0 %, respectively. We found that the ratio of patients classified as REM increased yearly from 62/161 (38.5 %) to 69/137 (50.4 %), while those classified as ∆TSS >3 decreased from 55/161 (34.2 %) to 28/137 (20.4 %) and those in RRP decreased from 35/161 (21.7 %) to 15/137 (10.9 %). Receiver operating characteristic (ROC) curve analyses showed that serum matrix metalloproteinase-3 (MMP-3) <103.7 ng/ml at outset predicts a patient subgroup that exhibits no radiographic progression. CONCLUSIONS: Half of rheumatoid patients treated with MTX monotherapy for 3 years exhibited structural remission, and this outcome can be predicted at the outset by lower serum MMP-3.

Induction of de novo autoimmune disease in normal mice upon repeated immunization with antigen.

There are many issues with animal models that represent human autoimmune disease or protocols to induce systemic autoimmunity, especially protocols to induce disease in normal mice not having a genetic disposition to autoimmunity. We describe here a novel and completely reproducible experimental technique that can induce systemic autoimmunity or systemic lupus erythematosus (SLE) in mice otherwise not prone to spontaneous autoimmune disease. This protocol involves the repeated immunization of mice with the same antigen. This rather simple technique enables us to perform exact and quantitative in vivo animal experiments with great accuracy.

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

IFN-γ-producing effector CD8 T lymphocytes cause immune glomerular injury by recognizing antigen presented as immune complex on target tissue.

We investigated the role of effector CD8 T cells in the pathogenesis of immune glomerular injury. BALB/c mice are not prone to autoimmune disease, but after 12 immunizations with OVA they developed a variety of autoantibodies and glomerulonephritis accompanied by immune complex (IC) deposition. In these mice, IFN-γ-producing effector CD8 T cells were significantly increased concomitantly with glomerulonephritis. In contrast, after 12 immunizations with keyhole limpet hemocyanin, although autoantibodies appeared, IFN-γ-producing effector CD8 T cells did not develop, and glomerular injury was not induced. In ß2-microglobulin-deficient mice lacking CD8 T cells, glomerular injury was not induced after 12 immunizations with OVA, despite massive deposition of IC in the glomeruli. In mice containing a targeted disruption of the exon encoding the membrane-spanning region of the Ig µ-chain (µMT mice), 12 immunizations with OVA induced IFN-γ-producing effector CD8 T cells but not IC deposition or glomerular injury. When CD8 T cells from mice immunized 12 times with OVA were transferred into naive recipients, glomerular injury could be induced, but only when a single injection of OVA was also given simultaneously. Importantly, injection of OVA could be replaced by one injection of the sera from mice that had been fully immunized with OVA. This indicates that deposition of IC is required for effector CD8 T cells to cause immune tissue injury. Thus, in a mouse model of systemic lupus erythematosus, glomerular injury is caused by effector CD8 T cells that recognize Ag presented as IC on the target renal tissue.

Pathogenesis of joint destruction in rheumatoid arthritis.

Synovial mesenchymal cells, matrix metalloproteinases (MMPs), and osteoclasts are the three major players directly responsible for the pathogenesis of rheumatoid joint destruction. First, synovial mesenchymal cells, internally driven by a transcription factor c-Fos/AP-1, not only directly invade cartilage and bone as a granulation tissue called "pannus" but also release inflammatory cytokine interleukin (IL)-1ß. IL-1ß induces MMPs and activates osteoclasts. Synovial cells can also present antigen to T cells to drive antigen-specific immune responses. Second, cartilaginous joint matrix can only be degraded after the first attack of collagen fibrils by MMPs, and importantly, most of the MMPs are under the control of c-Fos/AP-1 and IL-1ß as well. Third, differentiation of osteoclast is driven internally by NFATc1, where NFATc1 is under the control of TRAF6, c-Fos/AP-1 and osteoclastogenic signaling complex. IL-1ß has been shown to induce osteoclastogenesis directly and also indirectly via signaling through RANKL. Therefore, IL-1ß and c-Fos/AP-1 influence each other's gene expression and activity, resulting in an orchestrated cross-talk that is crucial to arthritic joint destruction, and thus, blockade of IL-1ß and/or c-Fos/AP-1 can be most promising as a therapeutic target, and in fact, a selective inhibition of c-Fos/AP-1 does resolve arthritic joint destruction.

Mathematical basis of affected sib-pair analysis: an extension to X-linked loci.

Most clinical genetic studies are done without knowing their mathematical basis. However, because the results of such studies rely on the correctness of the mathematical calculations involved, we cannot ignore the mathematics of genetic studies. In this study, the mathematical basis of the sib-pair method, which has been widely used in recent genome-wide studies, was extended to studies focusing on the X chromosome, and then applied to study a clinical microsatellite marker on the X chromosome. Our calculation involves classifying marker types on the X chromosome for an affected sib-pair and an unaffected sib, together with their sexual information and the possible parental marker types applicable to a genome-wide genetic study. The method proposed in this study was then applied to 41 Japanese rheumatoid families, and the locus DXS984 was found to be most likely to be linked with rheumatoid arthritis (RA). This locus, which we found nicely, was also highlighted in a previous study that used the Mapmaker/sibs program, so we can conclude that our calculation provides a solid foundation for understanding and confirming the results obtained using the sib-pair method.

Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3(+) CD69(+) T cells and a higher production of TNF-alpha from spleen cells. When arthritis is induced, Cry1(-/-)Cry2(-/-) mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-alpha, IL-1beta and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1(-/-)Cry2(-/-) mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti-TNF-alpha Ab significantly reduced the severity and halted the progression of the arthritis of Cry1(-/-)Cry2(-/-) mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1(-/-)Cry2(-/-) mice significantly reduced the trans activation of TNF-alpha gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Prospective study of methotrexate treatment for rheumatoid arthritis treated legitimately according to the government recommended 8 mg/week dose.

Clinical squeal of the treatment of rheumatoid arthritis patients with methotrexate (MTX) according to the Japanese government recommended dose of 8 mg/week was evaluated prospectively. A total of 176 patients with active RA attending Konan Kakogawa Hospital and Kobe University Hospital were enrolled. Patients' profile at the start of study was Class 2.0 +/- 1.1 and X-ray stage 2.6 +/- 1.0. The effects of MTX treatment were evaluated by the American College of Rheumatology (ACR) core set, disease activity score of 28 joints (DAS28), and European League Against Rheumatism (EULAR) response criteria. A modified Sharp method was used to evaluate the radiographs. The improvement in the clinical signs and symptoms of the ACR core set was maintained for a 24-month period (p < 0.05). The ACR20/50/70 and DAS28 were also improved at the 12- and 24-month assessments. However, 82 of 130 patients (63.5%) were found to be nonresponders at 24 months of MTX therapy, as evaluated by EULAR response criteria. The X-ray study showed that joint destruction progressed despite the treatment. Thus, long-term MTX treatment performed in accordance with the Japanese 8 mg/week regimen appears to be favorable in terms of the signs and symptoms of RA; however, it is clearly insufficient for and cannot halt the progression of rheumatic joint destruction.

Sjögren's syndrome patients presenting with hypergammaglobulinemia are relatively unresponsive to cevimeline treatment.

Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by sicca symptoms, including dry eyes and dry mouth. Cevimeline is used for the treatment of dry mouth in patients with SS. Here we prospectively tested the clinical effectiveness of cevimeline at increasing saliva secretion in patients with SS, and the results were compared with the clinical parameters of the patients. Saliva secretion was increased >160% in 17 of 30 (56.7%) patients (P < 0.005). When the clinical parameters were compared between the patients who responded to cevimeline treatment and those who did not respond to the treatment, the frequency of patients presenting with hypergammaglobulinemia was significantly higher in the nonresponder group (P < 0.05). It thus appears that cevimeline is effective in SS patients with milder disease activity.

Pathogenesis of rheumatoid arthritis and c-Fos/AP-1.

c-Fos/AP-1 controls the expression of inflammatory cytokines and matrix-degrading matrix metalloproteinases (MMPs) important in arthritis via promoter AP-1 binding motif. Among inflammatory cytokines, IL-1beta is the most important inducer of a variety of MMPs, and mainly responsible for cartilage breakdown and osteoclastogenesis. IL-1beta and c-Fos/AP-1 influence each other's gene expression and activity, resulting in an orchestrated cross-talk that is crucial to arthritic joint destruction, where TNFalpha can act synergistically with them. While how to stop the degradation of bone and cartilage, i.e., to control MMP, has long been the central issue in the research of rheumatoid arthritis (RA), selective inhibition of c-Fos/AP-1 does resolve arthritic joint destruction. Thus, the blockade of IL-1beta and/or c-Fos/AP-1 can be promising as an effective therapy for rheumatoid joint destruction in addition to the currently available TNFalpha blocking agents that act mainly on arthritis.

Self-organized criticality theory of autoimmunity.

BACKGROUND: The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in immune 'system', to explain the cause of autoimmunity. METHODOLOGY/PRINCIPAL FINDINGS: Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4(+) T cells led to the development of autoantibody-inducing CD4(+) T (aiCD4(+) T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4(+) T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8(+) T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE). CONCLUSIONS/SIGNIFICANCE: Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host's immune 'system' by repeated immunization with antigen, to the levels that surpass system's self-organized criticality.

Easy and accurate diagnosis of rheumatoid arthritis using anti-cyclic citrullinated peptide 2 antibody, swollen joint count, and C-reactive protein/rheumatoid factor.

OBJECTIVE: To determine an easy-to-use diagnostic criterion for early rheumatoid arthritis (RA) that may be useful for general physicians, using anti-cyclic citrullinated peptide (CCP) antibody. METHODS: We prospectively studied 435 patients who first visited the hospital with arthritic symptoms within 24 months, including 264 visitors within 6 months. The diagnosis was made on their first visit by examination and laboratory tests including anti-CCP antibodies, rheumatoid factor (RF) and C-reactive protein (CRP), and radiograph. RESULTS: The diagnostic specificity and positive predictive value (PPV) of anti-CCP2 assay were 94.9% and 87.8%, respectively, and those of anti-CCP2 plus RF were 96.9% and 90.9% for the patients who first visited having morning stiffness, arthralgia, and/or joint swelling within 3 months from onset (n = 165). For the patients who first visited later, but within 24 months from onset (n = 260), the diagnostic specificity and PPV were extremely high, 98.7% and 95.5%, when anti-CCP2 assay was coevaluated with RF, CRP, and more than 3 swollen joints. Respective combinations of anti-CCP2 assay plus either 2 of 3 measures were also highly specific. CONCLUSION: A diagnostic criterion including anti-CCP2 assay in combination with RF, CRP, and/or swollen joints is less sensitive but highly specific, and accurately predicts future development of RA among those with arthritic symptoms who first consulted doctors within 2 years after onset. It should be highly useful for the general physician without special techniques or devices.

Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative, but independent, signaling via ERK/MAPK and phosphatidylinositol 3-kinase/Akt.

OBJECTIVE: To determine whether angiopoietin 1 (Ang-1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell-signaling mechanisms of Ang-1 in the rheumatoid joint. METHODS: Expression of Ang-1, TIE-2 (a receptor for Ang-1), and matrix metalloproteinase 3 (MMP-3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3-kinase/Akt pathways and of NF-kappaB was determined by Western blotting and an NF-kappaB p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin-Rho and p21-activated kinase 1. Matrix degradation was examined by induction of proMMP-3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay. RESULTS: Ang-1 stimulated the ERK/MAPK and PI 3-kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang-1 activated NF-kappaB via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang-1 directly potentiated the extension of synovial cells in an ERK- and Akt-dependent manner by up-regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang-1 induced proMMP-3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix. CONCLUSION: Ang-1 stimulates the ERK/MAPK and PI 3-kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang-1/TIE-2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint.