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BACKGROUND: Low-dose methotrexate (LD-MTX) is the most commonly used drug for systemic rheumatic diseases worldwide and the recommended first-line agent for rheumatoid arthritis. Despite extensive clinical use for over 40 years, few data on adverse event (AE) rates derive from randomized placebo-controlled trials, where both causality and magnitude of risk can be inferred. OBJECTIVE: To investigate AE rates, risk, and risk differences, comparing LD-MTX to placebo. DESIGN: Pre-specified secondary analyses of a double-blind placebo-controlled randomized trial. SETTING: North America. PARTICIPANTS: Adults with known cardiovascular disease and diabetes or metabolic syndrome. INTERVENTION: Random allocation to LD-MTX (maximum of 20mg/week) or placebo. All subjects received folic acid 1mg per day for six days/week. MEASUREMENTS: The risk of specific AEs of interest as well as all AEs were compared across treatment arms, after blinded adjudication. RESULTS: 6,158 patients were enrolled and 4,786 randomized after an active run-in period; median follow-up was 23 months and median weekly dosage 16mg. Of the randomized subjects, 81.2% were male with a median age of 65.0 years and a median body mass index of 31.5 kg/m2. Of 2,391 subjects randomized to LD-MTX, 2080 (87.0%) experienced an AE of interest compared to 1951 of 2,395 (81.5%) randomized to placebo (HR 1.17, 95% CI 1.10 – 1.25). The relative hazards of gastrointestinal (HR 1.91, 95% CI 1.75 – 2.10), pulmonary (HR 1.52, 95% CI 1.16 – 1.98), infectious (HR 1.15, 95% CI 1.01 – 1.30), and hematologic (HR 1.15, 95% CI 1.07 – 1.23) AEs were elevated, comparing LD-MTX to placebo. With the exception of an increased risk of skin cancers (HR 2.05 (1.28, 3.28)), there was no difference between treatment arms for the risk of other malignancies, mucocutaneous, neuro-psychiatric, or musculoskeletal AEs. Renal AEs were reduced in subjects randomized to LD-MTX (HR 0.86, 95% CI 0.78 – 0.94). LIMITATIONS: The trial was conducted in non-rheumatic disease patients who tolerated LD-MTX during an active run-in. CONCLUSION: Use of LD-MTX was associated with small to moderate elevations in risks for skin cancer, gastrointestinal, infectious, pulmonary, and hematologic AEs, while renal AEs were decreased.

Objective The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. We undertook this study to perform a proteomic analysis of knee SF from OA patients and control subjects and to compare the results to microarray expression data from cartilage and synovium. Methods Age-matched knee SF samples from 10 control subjects, 10 patients with early-stage OA, and 10 patients with late-stage OA were compared using 2-dimensional difference-in-gel electrophoresis and mass spectrometry (MS). MS with a multiplexed peptide selected reaction monitoring assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity Pathways Analysis and compared to published synovial tissue and cartilage messenger RNA profiles. Results Sixty-six proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway. Differential expression of 5 proteins was confirmed by selected reaction monitoring assay. A focused analysis of transcripts corresponding to the differentially present proteins indicated that both synovial and cartilage tissues may contribute to the OA SF proteome. Conclusion Proteins involved in the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway are differentially regulated in SF from OA patients, suggesting that they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of 'wear and tear'. Although low-grade inflammation is detected in osteoarthritis, its role is unclear. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in complement component 5 (C5), C6 or the complement regulatory protein CD59a, we show that complement, specifically, the membrane attack complex (MAC)-mediated arm of complement, is crucial to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints from C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Further, MAC colocalized with matrix metalloprotease 13 (MMP13) and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints has a key role in the pathogenesis of osteoarthritis.