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Objective: Obese patients with rheumatoid arthritis (RA) report more joint swelling and tenderness and often have poorer responses to therapy than nonobese patients. The aim of this posthoc analysis of the MUSICA trial was to compare imaging and clinical disease activity measures in obese and nonobese patients with RA., Methods: MUSICA evaluated methotrexate (MTX) 20 mg/week versus 7.5 mg/week in combination with adalimumab (ADA) in RA patients with an inadequate response to MTX. Patients were categorized by baseline body mass index as normal (< 25), overweight (≥ 25 to < 30), or obese (≥ 30). Synovial vascularity and hypertrophy, swollen and tender joint counts (SJC and TJC), American College of Rheumatology (ACR) responses, and low disease activity (LDA), defined as Clinical Disease Activity Index < 10 and 28-joint count Disease Activity Score using C-reactive protein (DAS28-CRP) < 3.2, were assessed at weeks 12 and 24., Results: Patient characteristics were similar among groups at baseline. Obese patients had numerically smaller changes from baseline to weeks 12/24 in SJC, TJC, DAS28-CRP, and synovial hypertrophy and vascularity versus nonobese patients. Significantly fewer obese patients reached ACR20/50 at weeks 12 and 24, and LDA at Week 12; this difference was especially apparent in patients receiving 7.5 mg/week MTX but was no longer significant at Week 24., Conclusion: Obese patients with RA had worse clinical and ultrasonographic responses than nonobese patients, which were partly overcome with time. Obese patients may experience better and faster clinical improvements if ADA is initiated with high-dose (20 mg/week) rather than low-dose MTX. [ClinicalTrials.gov: NCT01185288].

Objective: To determine the efficacy of 2 tight control treatment strategies aiming at Simplified Disease Activity Score (SDAI) remission (SDAI ≤ 3.3) compared to 28-joint count Disease Activity Score (DAS28) remission (DAS28 < 2.6) in the prevention of arterial stiffness in patients with early rheumatoid arthritis (RA)., Methods: This was an open-label study in which 120 patients with early RA were randomized to receive 1 year of tight control treatment. Group 1 (n = 60) aimed to achieve SDAI ≤ 3.3 and Group 2 (n = 60), DAS28 < 2.6. Pulse wave velocity (PWV) and augmentation index (AIx) were measured at baseline and 12 months. A posthoc analysis was also performed to ascertain whether achieving sustained remission could prevent progression in arterial stiffness., Results: The proportions of patients receiving methotrexate monotherapy were significantly lower in Group 1 throughout the study period. At 12 months, the proportions of patients achieving DAS28 and SDAI remission, and the change in PWV and AIx, were comparable between the 2 groups. In view of the lack of differences between the 2 groups, a posthoc analysis was performed at Month 12, including all 110 patients with PWV, to elucidate the independent predictors associated with the change in PWV. Multivariate analysis revealed that achieving sustained DAS28 remission at months 6, 9, and 12 and a shorter disease duration were independent explanatory variables associated with less progression of PWV., Conclusion: With limited access to biologic disease-modifying antirheumatic drugs, treatment efforts toward DAS28 and SDAI remission had similar effects in preventing the progression of arterial stiffness at 1 year. However, achieving sustained DAS28 remission was associated with a significantly greater improvement in PWV. [Clinical Trial registration: Clinicaltrial.gov NCT01768923.].

Objective: To retrospectively assess changes in disease activity and skeletal damage in children with chronic nonbacterial osteomyelitis (CNO) after infliximab and methotrexate, with or without zoledronic acid or nonsteroidal antiinflammatory drug (NSAID) monotherapy, using a standardized magnetic resonance imaging (MRI) approach., Methods: Treatment-related changes in clinical and MRI measures from aggressive therapy and NSAID monotherapy groups (n = 9 per group) were evaluated using nonparametric methods., Results: Pain, physical function, physician global assessment, inflammatory markers, nonvertebral inflammatory lesion number, and maximum bone edema score all improved significantly with aggressive therapy (p < 0.03), whereas only the maximum soft tissue inflammation severity decreased (p = 0.02) with NSAID monotherapy. Vertebral deformities and physeal damage did not worsen in the aggressive therapy group but 1 in the NSAID group had worsening of growth plate damage., Conclusion: An aggressive treatment regimen in CNO improved clinical and imaging measures of disease activity without progression of skeletal damage.

Objective: To assess the effects on bone mineral density (BMD) of prolonged anti-tumor necrosis factor (anti-TNF) therapy in patients with spondyloarthritis (SpA); to compare the BMD changes to those observed in SpA patients not treated with anti-TNF; and to identify the predictors of these changes., Methods: Fifty-nine patients with SpA according to the European Spondylarthropathy Study Group criteria who were treated with anti-TNF therapy for at least 4 years were included. Thirty-four patients with SpA from an international longitudinal observational study (OASIS cohort) were used as a control group. Lumbar spine and hip BMD were measured by dual-energy x-ray absorptiometry at baseline, after 1 year, and after at least 4 years., Results: Over an average 6.5 years' followup, the increase in BMD was 11.8% (± 12.8%) at the lumbar spine (p < 0.0001) and 3.6% (± 9.3%) at the great trochanter (p = 0.0001) in patients treated with anti-TNF. At the lumbar spine, the increase was similar in patients with and those without syndesmophytes. BMD changes were significantly higher in the anti-TNF group than in the control group at lumbar spine (p < 0.0001), at femoral neck (p = 0.002), and at trochanter (p = 0.011), but not at total hip (p = 0.062). Multivariate analysis showed that the predictors of lumbar spine BMD changes in the total population were the use of anti-TNF (p < 0.0001) and, in the anti-TNF therapy group, the 1-year lumbar spine BMD change (p = 0.007)., Conclusion: This study shows that prolonged anti-TNF therapy increases lumbar spine and trochanter BMD. This effect should be taken into account before introducing antiosteoporotic treatment in these patients.

Objective: To determine whether methotrexate (MTX) affects the expression of genes involved in the transport [SLC19A1 (RFC1), ABCB1 (MDR1), ABCC1 (multidrug resistance proteins 1), ABCG2 (BCRP)], metabolism [γ-glutamyl hydrolase (GGH), folylpolyglutamate synthetase (FPGS)], and mechanism of action of MTX [thymidylate synthase, MTR, MTRR] in rheumatoid synovium., Methods: Synovial tissue samples were obtained from 20 patients with rheumatoid arthritis (RA). Gene expression was undertaken using quantitative real-time PCR., Results: All the genes examined were expressed in all samples. Expression of SLC19A1, GGH, FPGS, ABCC1, and MTRR was significantly higher in patients receiving MTX compared to those not receiving MTX (p < 0.05). The ratio of FPGS:GGH gene expression was 2.7 ± 0.51 ng/ml GAPDH (range 0.67-9.58)., Conclusion: Genes involved in the transport, metabolism, and mechanism of action of MTX are expressed in rheumatoid joint synovium. These data provide evidence that MTX has the potential to be polyglutamated within the joint. The higher expression of FPGS compared to GGH in synovial tissue might favor production of long-chain MTX polyglutamates. Thus MTX has the potential to exert its therapeutic effects at the primary site of the inflammatory process in RA.

Objective: Tumor necrosis factor-α (TNF-α) antagonists bring about significant improvement in chronic inflammatory diseases such as rheumatoid arthritis (RA) and spondyloarthritis (SpA). There is some evidence that they can also have negative myocardial effects, but to date this issue has not been clarified. We evaluated changes in electrocardiographic measures [QT interval, corrected, dispersion, and dispersion corrected (QT, QTc, QTd, QTdc, respectively)] in patients with RA or SpA treated with anti-TNF agents (infliximab and etanercept), those treated with other biological agents (rituximab), and with methotrexate., Methods: We studied 38 consecutive patients with RA (21 patients) or SpA (19 patients) being treated with TNF-α antagonists, 8 patients with RA being treated with rituximab, and 13 patients (8 with RA and 5 with SpA) taking methotrexate. Electrocardiographs (ECG) were performed on all participants at baseline and 12 months after initiation of treatment, and the QT, QTc, and QTd were calculated with standard procedures., Results: After 12 months of treatment, significant increases over baseline values were observed in the mean QT (p < 0.009), QTd (p < 0.0001), and QTdc (p < 0.0001) of the anti-TNF group, but no significant changes were observed in those taking rituximab. QT changes in the anti-TNF group were unrelated to the disease (RA vs SpA) or drug (infliximab vs etanercept), and none were associated with clinical manifestations of cardiac disease., Conclusion: In patients with RA and SpA, TNF-α antagonists seem to increase the QT and QTd measures. Although these changes were completely asymptomatic, ECG may be indicated in patients being considered for anti-TNF therapy to identify those at risk for cardiac complications.

Objective: To assess the risk of major malformation in the case of paternal exposure to methotrexate (MTX) at the time of conception., Methods: Using prospective data of our Teratology Information Service, we analyzed outcomes of paternal MTX exposure at the time of conception or up to 3 months before conception., Results: We report on the outcomes of 42 pregnancies involving 40 men treated with MTX at the time of conception. Twenty-three men were treated for an inflammatory disease (54.8%), 9 for psoriasis (21.4%), and 8 for a malignant disease (19.0%). Weekly dosages varied between 7.5 mg and 30 mg. The pregnancies resulted in 36 live births, 3 spontaneous abortions, and 3 voluntary abortions. No congenital malformation was observed at birth., Conclusion: Based on our results and case reports in literature, paternal MTX exposure at the time of conception does not seem to raise any major concern for offspring.

Objective: Randomized trials for uncommon diseases suffer from methodological challenges: difficulty in recruiting sufficient numbers of patients and low power to detect important treatment effects. Using traditional (frequentist) analysis, p values > 0.05 mean investigators are unable to reject the null hypothesis (of no treatment effect). The medical community often labels trials with p values > 0.05 as "negative." Our study demonstrates how Bayesian analysis conveys more relevant information to clinicians - using the example of methotrexate (MTX) in systemic sclerosis (SSc)., Methods: Data from 71 patients with diffuse SSc (n = 35 MTX, n = 36 placebo) in the trial were reanalyzed using Bayesian models. We examined 3 primary outcomes: modified Rodnan skin score (MRSS), University of California Los Angeles (UCLA) skin score, and physician global assessment of overall disease activity. Using noninformative prior probability distributions, the probability of beneficial treatment effects for each outcome and the probability of simultaneous benefit in outcomes were computed., Results: The probability that treatment with MTX results in better mean outcomes than placebo was 94% for MRSS, 96% for UCLA skin score, and 88% for physician global assessment. There was 96% probability that at least 2 of 3 primary outcomes were better on treatment., Conclusion: Bayesian analysis of uncommon disease trials allows for more flexible and clinically relevant interpretations of the data. From the trial data, clinicians can infer that MTX has a high probability of beneficial effects on skin score and global assessment.

Objective: To determine that concomitant use of methotrexate (MTX) is required to achieve adequate suppression of bone destruction in treating rheumatoid arthritis (RA) with tumor necrosis factor-alpha (TNF-alpha)-inhibiting biologic therapy. We quantitatively compared the suppressive effects of treatment with a combination of infliximab and MTX and treatment with each of these 2 agents alone on bone destruction in SCID-HuRAg-pit mice., Methods: Tissue derived from human RA pannus was implanted with a slice of dentin subcutaneously in the backs of SCID mice (SCID-HuRAg-pit model). Infliximab was administered daily to SCID-HuRAg-pit mice using an osmotic pump for 2 weeks with or without oral administration of MTX. Histological changes in tissue and the pits formed on the dentin slice were examined 8 weeks after transplant. Serum concentrations of TNF-alpha and interleukin 6 (IL-6) were also measured., Results: Treatment with a combination of infliximab and MTX suppressed pit formation significantly, while treatment with neither infliximab alone nor MTX alone had a significant effect on pit formation. Synovial inflammation and serum TNF-alpha and IL-6 levels were suppressed by infliximab with or without MTX., Conclusion: This is the first evidence in an animal model of arthritis that concomitant use of MTX is required to achieve adequate suppression of bone destruction when treating RA with a TNF-alpha-inhibiting biologic. Our findings suggest that infliximab suppresses bone destruction through a mechanism of action different from that mediating its antiinflammatory effects in the treatment of RA.

Objective: To evaluate peripheral blood expression of genes regulated by nuclear factor-kappaB (NF-kappaB), a key mediator of tumor necrosis factor-alpha (TNF-alpha) signaling, in patients with rheumatoid arthritis (RA) before and during treatment with anti-TNF-alpha or methotrexate (MTX). We analyzed association of gene expression with disease activity, rheumatoid factor (RF), age, sex, disease duration, treatment modality, and clinical response., Methods: Sixty patients consented for RNA analysis at baseline and after 2 and 6 weeks of treatment. Disease activity was quantified using Disease Activity Score (DAS28) and C-reactive protein (CRP). Expression of 67 TNF-alpha-responsive, NF-kappaB-regulated genes was measured using Affymetrix arrays and RT-PCR., Results: Expression of 34 genes was associated with DAS28-CRP, notably S100A12/calgranulin C, IL7R, and aquaporin 3. No association was observed with age, sex, RF, or disease duration. Expression of 16 genes changed in a manner that differed significantly between treatment groups. Eleven were reduced in anti-TNF-alpha-treated patients relative to MTX, while 5 were increased. The majority of these observations were confirmed using RT-PCR. Gene expression was not associated significantly with change in disease activity., Conclusion: NF-kappaB-dependent gene expression in peripheral leukocytes is highly correlated with RA activity as measured by DAS28-CRP. Expression of many genes responds differentially to anti-TNF-alpha versus MTX, suggesting fundamentally different effects on the NF-kappaB pathway. This peripheral blood expression signature provides candidate markers that could lead to development of a simple, minimally invasive pharmacodynamic assay for RA treatments directed at the NF-kappaB pathway. Combination of gene expression data with clinical scores and serum markers may provide more sensitive and predictive measures of RA disease activity.

Objective: To investigate the clinical efficacy, safety, tolerability, and toxicity profile of a metabolically stable antifolate, CH-1504, compared to methotrexate (MTX) in the treatment of rheumatoid arthritis (RA)., Methods: A 24-week open-label trial of MTX and CH-1504 was performed in 20 patients with RA., Results: Improvements in clinical and laboratory indicators were observed in both study groups. Improvement in the CH-1504 group was greater than in the MTX group. Both treatments were generally well tolerated; however, the liver function test abnormalities and gastrointestinal related adverse events expected with this class of medication were not seen with CH-1504., Conclusion: CH-1504 appears to be clinically efficacious and may possess a superior safety and tolerance profile compared to MTX.

Objective: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). In bone metabolism, it is known that IL-6 is produced and secreted by osteoblasts, and that IL-6 induces osteoclast formation and stimulates bone resorption. Various bone inflammatory agonists such as tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, prostaglandin D2 (PGD2), PGE2, and PGF2alpha, which play important roles in the pathogenesis of RA, induce IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Low dose methotrexate (MTX) is currently used for treatment of patients with RA. We investigated the effect of MTX on IL-6 synthesis induced by these agents in MC3T3-E1 cells., Methods: Cultured cells were pretreated with various doses of MTX, and then stimulated by these inflammatory agonists. The IL-6 in the conditioned medium was measured by IL-6 enzyme immunoassay., Results: MTX significantly suppressed IL-6 synthesis stimulated by these agonists in a dose-dependent manner, although MTX alone had no effect on the levels of IL-6. In addition, MTX significantly inhibited the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. MTX reduced the levels of IL-6 induced by 12-O-tetradecanoylphorbol 13-acetate, a direct activator of protein kinase C (PKC), suggesting that MTX inhibits PKC signals for IL-6 synthesis., Conclusion: MTX suppresses IL-6 synthesis stimulated by various inflammatory agonists in osteoblasts.

Objective: To test whether methotrexate (MTX) therapy of rat adjuvant arthritis (AA) prevents loss of bone mineral density (BMD) and loss of adipose and lean body mass compared to pair-fed controls with untreated rat AA (positive controls) and rats without AA (negative controls)., Methods: AA was induced by a Mycobacterium butyricum injection at the base of the tail of 5-week-old female Lewis rats. The MTX-treated group was injected with adjuvant and then treated twice weekly with MTX (1.0 mg/kg/wk intraperitoneally). To control for the effects of AA on appetite and weight, food given to control animals and MTX-treated rats with AA was limited to that consumed by rats with untreated AA. At 42 days post-adjuvant injection, the animals were sacrificed and tibial BMD was measured. Body composition was analyzed for percentage fat, protein, ash, and water., Results: There was no difference in ankle edema score or ankle width between the negative controls and MTX-treated group at necropsy. BMD was significantly higher in the negative controls versus positive controls and MTX-treated and in MTX-treated versus positive controls. There was significantly less body fat and protein and greater body water in the positive controls and MTX group compared to the negative controls., Conclusion: MTX prevents loss of BMD in the tibia in the rat AA model compared to positive controls. While MTX is effective in lowering inflammation in rat AA, there are still significant losses in BMD and body composition, which may have implications for rheumatoid arthritis.

Objective: To analyze the influence of low dose methotrexate (MTX) on bone using data from a large multicenter, cross-sectional study on bone mineral density (BMD) in women with rheumatoid arthritis (RA)., Methods: We selected 731 female patients with RA divided into 2 groups on the basis of MTX use: never MTX users (n = 485) and MTX users for at least 6 months (n = 246). Demographic, disease, and treatment related variables were collected for each patient. BMD was measured at lumbar spine and proximal femur by dual energy x-ray absorptiometry. Osteoporosis was defined as BMD < -2.5 T-score., Results: The frequency of osteoporosis among never MTX users and MTX users was 29.1% and 28.3% (p = NS) for lumbar spine, and 34.8% and 37.8% (p = NS) for femoral neck, respectively. Mean T-score values at lumbar spine and femoral neck were comparable in the 2 groups, even after adjusting for age, menopausal status, body mass index (BMI), Health Assessment Questionnaire (HAQ) score, and steroid use. The generalized linear model showed that age, menopause, BMI, HAQ score, and steroid use were significant independent predictors of BMD at lumbar or at femoral level, whereas MTX use was not. Logistic procedure showed that only age, HAQ score, and BMI were significantly associated with the risk of osteoporosis., Conclusion: We found no negative effect of low dose MTX on BMD in women with RA.

Objective: To study the effects of ribomunyl, an oral ribosomal immunostimulant frequently used to prevent recurrent oropharyngeal and bronchopulmonary infections in children and adults, on adjuvant arthritis as well as its effect on methotrexate and cyclosporine treatment., Methods: Rats with adjuvant induced arthritis were preventively treated orally with the following drugs: cyclosporin A (CSA, 2.5 mg/kg/day); ribomunyl (RIB, 25 mg/kg/day, 4 days a week); methotrexate (MTX, 0.6 mg/kg/week), and the combinations CSA + RIB, MTX + RIB, CSA + MTX, CSA + MTX + RIB for the period of 50 days from adjuvant application. Levels of serum albumin, serum nitrite/nitrate concentrations, hind paw swelling, arthrograms, and bone destruction were measured in rats as variables of the inflammation and destructive arthritis associated changes., Results: Preventive treatment with low doses of CSA and MTX significantly inhibited both markers of inflammation and arthritis. The combination CSA + MTX was more effective on all evaluated measures than any of its components alone. RIB alone improved arthrogram scores and decreased serum nitrite/nitrate concentrations in arthritic rats. The combination of RIB with the low dose of CSA or MTX enhanced the beneficial effects of these drugs. The best preventive effect was observed with the combination of 3 agents, CSA + MTX + RIB., Conclusion: Our results show ribomunyl to have the potential to improve some inflammation and arthritis associated changes in rat adjuvant arthritis and to enhance the preventive effect of MTX and CSA and their combination.

Objective: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg)., Methods: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day)., Results: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures., Conclusion: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.

Objective: To investigate the dose response relationships of methotrexate (MTX) therapy in rat adjuvant arthritis (AA), an animal model of rheumatoid arthritis (RA)., Methods: Female Lewis rats were fed a defined diet and were treated with 0, 0.3, 1, 2, 3, 5, and 10 mg MTX per week beginning 3 days after adjuvant injection and lasting 6 weeks. The presence or absence of arthritis, and its degree were measured by hindpaw edema scores, ankle widths, and radiographic and histopathologic scores., Results: The 2, 3, 5, and 10 mg MTX per week doses resulted in deaths before the end of the protocol and suppressed normal body weight gain. Tissue destruction, measured by radiographic and histopathologic scores, was reduced in a dose dependent manner with increasing MTX dose. Suppression of inflammation, measured by ankle widths and radiographic and histopathologic scores, reached a maximum at the 1 mg MTX dose and declined at higher doses., Conclusions: Suppression of tissue destruction and inflammation in rat AA does not occur in a concerted fashion as the dose of MTX increases. The implications of these findings to human disease remain to be determined.

Objective: To determine if methotrexate (MTX) induces human lung fibroblast (HFL-1) and epithelial (BEAS 2B) cell lines to release eosinophil chemotactic activity (ECA)., Methods: HFL-1 and BEAS 2B cell supernatant fluids were evaluated for ECA by a blind well chamber technique., Results: HFL-1 and BEAS-2B cells released ECA in a dose and time dependent manner in response to MTX. Partial characterization revealed that ECA was partly heat labile, trypsin sensitive, and ethylacetate extractable. Thus the culture supernatant fluids were evaluated for known eosinophil chemotactic factors. Although several were released constitutively, granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly increased in response to MTX from both cell types. Consistent with these observations, ECA from both cell types was inhibited by GM-CSF antibodies., Conclusion: These data suggest that lung fibroblasts and epithelial cells may modulate eosinophil recruitment into the lung by releasing ECA in response to MTX.

Objective: We investigated the antiproliferative and antiinflammatory effects of methotrexate (MTX) on differentiating/differentiated cells, namely cultured human monocytic myeloid cells (THP-1), and primary cultures of synovial macrophages from patients with rheumatoid arthritis (RA)., Methods: We evaluated early and late apoptosis as well as natural cytokine inhibitor production, such as the interleukin 1 (IL-1) receptor antagonist (IL-1ra) and the soluble tumor necrosis factor receptor (sTNFr)., Results: Within THP-1 cells we observed a significant (p < 0.001) dose-dependent inhibition of proliferation (at 24-48 and 72-96 h) and a significant presence of apoptosis (at 24-48 h) with MTX concentrations of 500, 100, and 75 microg/ml compared with untreated controls. No significant changes were observed with 5 microg/ml or 500 to 50, and 5 ng/ml. A significant increase of IL-1ra (p < 0.001) was observed with MTX concentrations of 5 microg (51.43 +/- 2.53 vs 16.22 +/- 5.19 pg/ml control) and 500 ng (36.43 +/- 3.3 vs 16.22 +/- 5.19 pg/ml control) at all the tested times. No significant changes were observed for the sTNFr p75. Evaluating the RA synovial macrophages, we obtained no significant effects on cell proliferation and apoptosis with MTX treatment at 24 h and at the concentration of 50 microg/ml (achievable in the serum with low dose MTX treatment in RA). No significant changes were observed for the IL-1ra and no detectable levels for the sTNFr p75 were detected after treatment with MTX., Conclusion: This study shows that the antiproliferative and antiinflammatory effects of MTX on human cultured monocytes are dose-dependent. The antiproliferative activity seems to be mediated by cell apoptosis and the antiinflammatory activity seems to be related to cytokine inhibitor release.

Objective: To study the modulation of CD69 expression on peripheral blood (PB) and synovial fluid (SF) lymphocytes by interleukin 15 (IL-15) and several other cytokines and chemokines widely detected in the rheumatoid microenvironment. The effect of cyclosporin A (CSA) or methotrexate (MTX) in the cytokine mediated regulation of CD69 was analyzed., Methods: CD69 expression on lymphocytes was assessed by flow cytometry after incubation with different cytokines, chemokines, phorbol myristate acetate, or calcium ionophore in the presence or absence of CSA, MTX, or both. The effect of IL-15 and SF supernatants in maintaining CD69 expression on SF lymphocytes was also assessed. IL-15 levels in SF supernatants were measured by ELISA., Results: IL-15 induced the greatest upregulation of CD69 expression on PB lymphocytes in a time and dose dependent manner. IL-15 was able to maintain a high CD69 expression on SF lymphocytes. SF supernatants from rheumatoid arthritis (RA), which contain significant amounts of IL-15, also reversed the CD69 downregulation of SF lymphocytes in culture. CSA, but not MTX, inhibited the CD69 upregulation mediated by IL-15 both in PB and SF lymphocytes., Conclusion: IL-15 appears to be responsible, at least in part, for the high CD69 expression on lymphocytes from the rheumatoid microenvironment. Consistent with the virtual absence of lymphocyte derived cytokines in RA synovium, the prevention of IL-15 mediated CD69 upregulation on lymphocytes may explain the effect of CSA in the treatment of RA.

Objective: The mechanism of action of methotrexate (MTX) in inflammatory joint disease is still unclear. We examined the possible interactions of MTX with the oxidative metabolism of rabbit articular chondrocytes., Methods: Cell cultures of articular chondrocytes enzymatically isolated from juvenile New Zealand white rabbits were incubated 24 h with either MTX (0.22 or 1.1 microM), bacterial lipopolysaccharide (LPS, 50 microg/ml), or both. Cytofluorometry was then performed using 2',7'-dichlorofluorescein diacetate (DCFH-DA), rhodamine 123 (Rh123), or propidium iodide (PI). These fluorochromes allow evaluation of cellular production of H2O2, mitochondrial membrane potential, and cell viability, respectively. In a separate experiment, we used the Griess colorimetric technique to evaluate cellular nitric oxide (NO) production., Results: Addition of MTX alone (0.22 or 1.1 microM) inhibited spontaneous production by chondrocytes of H2O2 (p < 0.01 and p < 0.001, respectively) and NO (p < 0.01 both concentrations). The LPS induced increase in H2O2, production was inhibited by MTX at 0.22 and 1.1 microM (p < 0.01 both concentrations), whereas the LPS induced increase in NO synthesis was not influenced by MTX, even at 1.1 microM. MTX did not significantly modify mitochondrial activity or cell viability., Conclusion: MTX at therapeutic concentrations in vitro inhibits the production of H2O2 and NO by unstimulated chondrocytes, and only the H2O2 overproduction by LPS stimulated chondrocytes. These properties may contribute to the therapeutic effect of MTX in RA.

Objective: To study the temporal relation between vascular inflammatory activity and synovial hyperplasia during the development of methylated bovine serum albumin (mBSA) antigen induced arthritis (AIA) in the rat, and to correlate these variables to changes in knee diameter. The influence of a single dose of indomethacin and methotrexate (MTX) on these measures was also determined., Methods: Vascular inflammatory activity was assessed as extravasation of radiolabelled albumin. Synovial hyperplasia was followed by measurements of the increases in wet and dry weight of the anterior part of the periarticular soft tissue and by routine histology., Results: The vascular inflammation peaked on Day 3 after antigen challenge. The pannus weight increased at a slower pace, peaking on Day 7. No major difference between the sexes was found in these responses. Both variables were attenuated by MTX or indomethacin, suggesting a dependence between them. The water content of the pannus increased in tandem with the tissue growth but did not correlate to vascular leakiness, and is thus explained by the structural properties of the pannus rather than by the formation of inflammatory edema. In histological sections, ingrowth of pannus and destruction of cartilage was visible from Day 3 until the end of the experiment., Conclusion: Proliferative response follows the inflammatory vascular inflammation over time. The knee diameter, which is the most commonly used clinical measurement, seems mainly to be a reflection of the former variable. The effects of MTX and indomethacin suggest that the pannus formation is induced by the inflammatory activity in this model.

Objective: Based on the hypothesis that blood flow in the inflamed joint is inadequate to maintain aerobic glycolysis, we sought to estimate the correlation between blood flow, glucose metabolism, and cellular proliferation rate in the arthritic joint., Methods: Experiments were performed on rats with antigen induced arthritis (AIA). Regional blood flows (RBF) were measured with the microsphere technique, glucose metabolism by determination of [14C]2-deoxy-D-glucose (2-DG) uptake, and the proliferative response as the incorporation of [3H]-thymidine., Results: In periarticular soft tissue of the arthritic knee the only significant change in the weight related RBF was an approximate 70% rise on Day 14 after arthritis onset. The RBF was lowest on Day 3 and the time course for the changes was inversely related to intensity of vascular inflammation. Weight related 2-DG uptake was more elevated than the RBF and peaked on Day 3. [3H]-thymidine incorporation in the soft tissue was only markedly enhanced on Day 3. Neither 2-DG nor [3H]-thymidine uptake was affected by treatment with methotrexate or indomethacin. In epiphyseal bone RBF was reduced on the first day of arthritis, but steadily increased thereafter., Conclusion: In AIA an intense vascular leakiness negatively affects the synovial blood. There is a marked enhancement of glucose metabolism, but only a minor part of this increase seems to be induced by increased cellular proliferation.

Objective: Our objective was to characterize the effect of methotrexate (MTX) on prostaglandin E2 (PGE2) synthesis in cultured human rheumatoid synovial cells. Prostaglandins (PG) are important mediators of inflammation and joint destruction in rheumatoid arthritis (RA). Two isoforms of cyclooxygenase (COX), the key enzyme in PG synthesis, have been characterized: a constitutively expressed form, COX-1, and an inducible form, COX-2. The mechanisms of action of low dose MTX in RA treatment are still poorly understood. As the clinical effects are often first noticed within a month of starting MTX therapy, an antiinflammatory action has been proposed., Methods: Adherent synovial cells were obtained by collagenase digestion of rheumatoid synovium, isolated from patients with RA undergoing synovectomy. Between passages 3 and 6, cultured synovial cells were incubated with or without MTX for 54 h, at various concentrations. Interleukin (IL)-1beta (1 ng/ml) was added or not for the last 6 h of incubation. Supernatants were harvested and assayed for PGE2 by enzyme immunoassay (EIA). Exogenous [1-14C]arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). COX-1 and COX-2 mRNA expression was determined by total RNA extraction and reverse transcription polymerase chain reaction., Results: Cellular viability was not affected by MTX. EIA showed that MTX decreased IL-1beta induced PGE2 production by synoviocytes in a dose dependent manner. RP-HPLC analysis confirmed the inhibition of PGE2 and (12S)-12-hydroxy-5,8,10-heptadecatrienoic acid production. COX-1 and IL-1beta induced COX-2 mRNA expression were not inhibited by MTX., Conclusion: MTX has an inhibitory effect on IL-1beta stimulated production of PGE2 by cultured human rheumatoid synoviocytes, without affecting either COX mRNA expression. Among various biochemical and immunologic events, MTX could have an antiinflammatory action by decreasing PGE2 release.

Objective: We reported beneficial effects of methotrexate (MTX) treatment on clinical variables in the murine model of experimental systemic lupus erythematosus (SLE). We now follow the kinetics of several cytokines that are involved in experimental SLE, to find out whether MTX treatment has an effect on cytokine production., Methods: SLE was induced in naive BALB/c female mice by injection of the human monoclonal anti-DNA antibody bearing the common idiotype, 16/6 Id. Six weeks after booster injection, when high levels of autoantibodies were observed, MTX treatment was started (2 mg/kg once a week) for 7 months. Cytokine production by macrophages or splenocytes from these mice was determined monthly by either ELISA or bioassays., Results: A significant increase in the production of tumor necrosis factor-alpha was determined as early as 2 weeks after injection. Interleukin 1 (IL-1) production increased gradually, starting one month after disease induction. These proinflammatory cytokines reached very high levels that were maintained through disease course. MTX treatment reduced production of these cytokines to normal levels throughout the experimental period. Increased levels of IL-2 and interferon-gamma (IFN-gamma), the Th1 type cytokines, were detected 2-4 months after disease induction. The secretion of the latter then dropped to levels lower than those observed in control mice. Treatment with MTX reversed levels of the cytokines to levels observed in healthy mice, IL-4 and IL-10, the Th2 type cytokines, predominated later in disease course, 5 months after immunization. Two to 3 months later production of IL-4 dropped to levels lower than those of control mice. IL-10 secretion remained higher than in controls throughout the experiment. The production of both IL-4 and IL-10 in MTX treated mice was also similar to that of control mice., Conclusion: The restoration of the cytokine profile to normal levels observed in the MTX treated mice from the initial steps of disease induction suggests its beneficial effects in SLE might be mediated through modulation of cytokine production.

Objective: Clinicians do not often employ washout periods before prescribing a change in therapy for rheumatoid arthritis (RA). As a result, the observed effectiveness or lack of effectiveness of a new drug actually represents the effectiveness of that drug had the patient been taking placebo minus the residual effectiveness of the old drug., Methods: We studied new starts of selected disease modifying antirheumatic drugs (DMARD) and prednisone in 2,898 patients with RA from 8 ARAMIS data bank centers, broken into subgroups on the basis of immediately prior therapy. Therefore, we examined the hypothesis that the chances of a treatment being observed effective depend upon the immediately preceding treatment. Using intent-to-treat analysis, we analyzed the effects upon Health Assessment Questionnaire (HAQ) disability and pain scores an average of 9 months after the new drug start., Results: Methotrexate reduced disability significantly except after intramuscular gold or hydroxychloroquine and it reduced pain significantly after all prior therapies. Hydroxychloroquine reduced disability significantly after nonsteroidal antiinflammatory drugs (NSAID) only, but disability increased after intramuscular gold; pain was decreased only after NSAID only. Prednisone had no consistent effect upon disability but was consistently associated with decreased pain. Greatest effectiveness was always seen with a new drug start after NSAID only treatment versus after DMARD treatment., Conclusion: The effectiveness of a newly started RA treatment after 9 months may be substantially influenced by immediately prior treatment. This finding provides an additional reason for concern about direct extrapolation of clinical trial data into clinical practice.

Objective: To investigate the effects of salazosulfapyridine (SASP) and methotrexate (MTX) on interleukin (IL)-1beta treated rabbit chondrocytes., Methods: Normal rabbit chondrocytes were cultured to confluency. IL-1beta was added to serum-free culture medium in the presence or absence of SASP or MTX. After 2 days' incubation, the effects were evaluated from the responses of metalloproteinases, glycosaminoglycan (GAG), and prostaglandin E2 (PGE2)., Results: SASP and MTX suppressed GAG and collagenase release into the culture medium from IL-1beta stimulated rabbit chondrocytes in a dose dependent manner. Only SASP suppressed stromelysin and PGE2 release., Conclusion: SASP may have a protective effect on cartilage degradation of patients with rheumatoid arthritis.

Objective: To learn whether rheumatoid factor (RF), HLA-DR4, or current therapy influences clonal expansion of B lymphocytes (B cells) in persons with rheumatoid arthritis (RA)., Methods: We measured clonal expansion by analysis of cell surface staining for immunoglobulin light chains. Double staining methods detected a B cell marker (CD19) plus either kappa or lambda on peripheral blood lymphocytes from subjects with RA (n = 26) and controls (n = 26). The difference between frequency histograms of surface kappa and lambda staining was determined by the Kolmogorov-Smirnov statistic D that represents the fraction of clonally expanded B cells., Results: The mean D value in RA was over 50% higher than in the controls [0.225 +/- SD 0.155 versus 0.144 +/- 0.025 (p < 0.0001)]. Ten subjects with RA had values exceeding +2 SD for controls (p = 0.0007). Mean D correlated with RF titer (Spearman's rank correlation coefficient rSp + 0.53, p = 0.006). All 10 high D values were found in the RA subgroups with positive serum tests for RF and with the HLA-DR4 positive genotype. The channel of maximal difference between kappa and lambda staining was higher in the RA group than in controls, showing that clonal expansion was most marked among brightly staining cells. Patients with RA currently receiving low dose methotrexate (MTX) tended to have higher D values than those not receiving MTX (mean 0.29 versus 0.18, respectively, p < 0.025). The RA group currently receiving MTX had a higher frequency of abnormal D values (7 of 11 versus 3 of 15 not currently receiving MTX, p = 0.03). This probably reflects preferential use of MTX for severely affected individuals. Confirmatory studies to detect clonal immunoglobulin gene rearrangements were attempted in selected individuals with high D values, but none was demonstrated in total leukocytic or B cell enriched fractions., Conclusion: Findings consistent with B cell clonal expansion occur in about 40% of persons with RA, particularly in the subgroups with positive serum tests for RF and with the HLA-DR4 genotype. However, the clonal expansion level must be below the sensitivity of confirmatory methods.

Objective: The mechanism of methotrexate (MTX) action in rheumatoid arthritis (RA) is unclear. We assessed the influence of MTX on neutrophil superoxide production evaluated by ferricytochrome c reduction., Methods: Neutrophils were collected from MTX treated patients with RA (MTX-RA), patients with RA without medication (RA) and healthy donors, cocultured with MTX or MTX-RA serum., Results: Polymorphonuclear leukocytes (PMN) from MTX-RA showed decreased superoxide production when compared with cells collected from patients with RA and controls. Control PMN superoxide production was inhibited (36%) by MTX-RA serum incubation. This reduction was accompanied by clinical improvement. MTX had no activity in the in vitro assays., Conclusion: MTX treatment may interfere with neutrophil superoxide production.

Objective: To explore the potential role of fibroblasts in the pathogenesis of psoriasis and its related arthritis. Specifically, we analyzed the cell cycle of psoriatic fibroblasts obtained from skin and synovium by flow cytometry, and we also studied their response to several growth factors., Methods: Fibroblast cultures were established from normal and psoriatic skin, uninvolved and involved, and synovium. NIH-3T3 cells were also used as indicator cells in some of the experiments. Fibroblasts DNA cell cycle analysis was performed by flow cytometry, and the data was analyzed by using the "Cytologic DNA applications software version 2." In addition, fibroblasts were stimulated with growth factors including epidermal growth factor, transforming growth factor-beta, and platelet derived growth factor., Results: A significant increase of S and G2-M phase values in confluent cultures of psoriatic fibroblasts in both skin and synovium compared to normal fibroblasts was found. Psoriatic fibroblasts also exhibited a greater proliferative response to growth factors compared to normal fibroblasts., Conclusion: Data obtained clearly showed a significant intrinsic in vitro alteration in skin and synovium fibroblasts from patients with psoriasis.

Objective: To evaluate the influence of methotrexate (MTX) on the frequency of postoperative complications in patients with rheumatoid arthritis (RA)., Methods: We conducted a randomized unblinded prospective study in 64 patients with RA treated with MTX and who underwent orthopedic surgery. Two groups of patients were constituted: in Group A (32 patients), MTX was interrupted 7 days before the surgery; in Group B (32 patients), MTX was not discontinued., Results: Fifty surgical procedures were performed in Group A and 39 procedures in Group B. No postoperative infection was observed in any group. A prolonged wound healing was noticed in 6 cases in Group A and in 4 cases in Group B (not significant)., Conclusion: We suggest that the interruption of MTX is not required in patients with RA when an orthopedic surgical treatment is planned. However a large prospective study is needed to make a definitive conclusion.

The effect of methotrexate (MTX) on proliferation and on interleukin 1 stimulated secretory activities of human synovial fibroblasts in culture was investigated. MTX caused a dose dependent inhibition of growth over the concentration range 0.07-2.2 microM with a half-maximal effect at 0.37 microM. INhibition was competitively relieved by coaddition of leucovorin. Cell growth was fully restored after MTX pretreatment of 24 h but not after 48 h, even on subsequent leucovorin addition. Cell viability was unaffected by MTX treatment. MTX had no effect on interleukin 1 stimulated production of prostaglandin E, hyaluronic acid and collagenase. Our results raise the possibility that one of the mechanisms contributing to the therapeutic effects of MTX in patients with rheumatoid arthritis may involve modulation of synovial fibroblast growth.

In the last decade, methotrexate (MTX) has emerged as a useful second line agent for a variety of arthritides. However, there still exists some reluctance for its wider use mainly because of concerns about its liver side effects. We describe our clinical experience with this drug in psoriatic arthritis (PsA). The study group included 24 men and 16 women, with a mean age of 47 years (16-75), with oligoarticular (13) or polyarticular (27) involvement, with a mean disease duration of 12 years (1-36). Patients received a mean dose of 11.2 mg of MTX orally/week during a mean period of 34 months (6-132). Seven had been previously treated with other second line agents. Thirty-eight patients had an excellent or good response. In them, the erythrocyte sedimentation rate dropped in a mean of 38 mm/h. Only 2 patients had a rather poor response. Two patients discontinued the medication because of side effects: leukopenia in one and stomatitis in the other. Eleven patients presented with liver test abnormalities: 3 mild, 6 moderate and 2 severe. Seven patients had 11 liver biopsies. Except for one, none had evidence of cirrhosis or inflammation. Indeed, no changes were observed in the histopathology in those with repeated biopsies. The case reported as cirrhosis occurred very early in the course of MTX therapy. He continued taking MTX treatment without further deterioration of liver chemistry and/or histology. It is concluded that MTX is an effective and safe agent in PsA. Results also indicate that it is not necessary to perform liver biopsies on a routine basis.

The inhibition of 5-lipoxygenase could be involved in the mechanism of action of methotrexate (MTX). We studied 8 patients with active rheumatoid arthritis (RA) immediately before and the day after the first dose of MTX (10 mg intramuscularly). Leukotriene B4 (LTB4) formation by polymorphonuclear leukocytes was significantly decreased (32%, p less than 0.01). This essentially involved released LTB4. A slight decrease was also obtained in omega-oxidation products. Similar results were obtained for plasma LTB4 (30%, p less than 0.02). A non-significant decrease in 5-HETE was noted. Conversely, 12-HETE was not modified. Our results suggest MTX has an effect on the 5-lipoxygenase pathway, particularly at the LTA4 epoxide hydrolase step, since 5-HETE and 6-trans-LTB4 isomers are not involved.

Interleukin 1 (IL-1) is an important mediator of immune regulation, inflammation and joint destruction in arthritis. The objective of our study was to investigate the effect of a number of representative antirheumatic agents on the activity of IL-1 and the production of both IL-1 and IL-1 inhibitor by human monocytes. IL-1 activity and production were measured by its effect on the proliferation of murine thymocytes; IL-1 inhibitor production was assayed by inhibition of IL-1 mediated thymocyte proliferation. Dexamethasone (10-100 nM) inhibited IL-1 production and IL-1 mediated thymocyte proliferation. High concentrations of indomethacin, aspirin, and 12 other nonsteroidal antiinflammatory drugs (NSAID) inhibited IL-1 activity, or production or both. Methotrexate inhibited IL-1 activity without affecting IL-1 production. D-penicillamine inhibited IL-1 activity and tended to inhibit IL-1 production. Gold compounds inhibited IL-1 activity, but only auranofin inhibited IL-1 production. Acetaminophen in high concentrations (1 mg/ml) inhibited IL-1 activity and production. Colchicine inhibited IL-1 activity but not IL-1 production. No drug tested induced, by itself, the production of IL-1 inhibitor or enhanced the production of IL-1 inhibitor.

We examined the effects of low dose parenteral methotrexate (MTX) on immunologic and inflammatory responses of potential importance in the pathogenesis of rheumatoid arthritis. Only 2 significant changes were noted: the number of esterase positive cells in the peripheral blood decreased, and the uptake of tritiated thymidine by incubated peripheral blood mononuclear cells fell markedly. These changes were present 48 h after an injection of MTX, before decreased disease activity became clinically apparent. We therefore conclude that they were direct effects of the drug itself.

Serum concentrations of hyaluronate may provide a clinically relevant, quantitative marker of disease in patients with active rheumatoid arthritis (RA). We studied the utility of serum hyaluronate in an animal model, with features reminiscent of human RA, in which LEW/N female rats were made arthritic by intraperitoneal injection of sonicated Lactobacillus casei. When serum hyaluronate was measured by an inhibition ELISA, a dose dependent correlation was found between the amount of L. casei injected and both joint score and serum hyaluronate in the chronic phase of the disease. A linear correlation between the chronic phase joint score and serum hyaluronate was observed (r = 0.69, p less than 0.001). Two orally administered compounds, flurbiprofen (20 mg/kg) and methotrexate (0.125 mg/kg), were effective in decreasing both variables. Thus, serum hyaluronate may have utility in evaluating the therapeutic efficacy of antirheumatic/antiinflammatory agents in vivo in the chronic phase of RA-like diseases.

We evaluated the effect of methotrexate (MTX) and methylprednisolone (MP) on reticuloendothelial system (RES) clearance of soluble model immune complexes (IC) in BALB/C mice. MTX was administered by intraperitoneal route either as a single dose (0.5 mg/kg) or as 10 alternate day doses (0.1 or 0.5 mg/kg), MP as a single intravenous (IV) bolus (50 mg/kg) with normal saline used as a control. Mice were then injected IV with radiolabelled IgG anti-DNP covalently crosslinked IC. Blood radioactivity was measured over a 3 h period at which time organ uptake, corrected for blood contamination, was determined. Clearance curves for each mouse were derived using the Marquardt-Levenberg curve fitting method. No significant differences in IC clearance or organ uptake were found between drug and control groups at any drug dose or time period. Our findings argue against an influence of MP and MTX on immunospecific clearance of soluble IC.

Time course studies in rat adjuvant arthritis have shown that resident peritoneal macrophages synthesize large amounts of interleukin-1 (IL-1) on Day 10-11 postadjuvant immunization. Expression of Ia antigen on these macrophages increased rapidly between Day 15 to Day 20 postadjuvant treatment and remained at these high levels on Day 25. If these adjuvant immunized rats were treated with low doses of methotrexate, resident peritoneal macrophages appeared to synthesize lower amounts of IL-1 and their Ia expression was decreased. Methotrexate, in vitro, has no effect on lipopolysaccharide induced IL-1 synthesis by resident peritoneal macrophages.

When mediators of inflammation such as complement component C5a or leukotriene B4 are introduced into an air pouch created in mice, these mediators induce the migration of neutrophils into the air pouch. Pretreatment of mice with low doses of methotrexate inhibits leukotriene B4 or C5a induced neutrophil migration into the air pouch. Inhibition of neutrophil chemotaxis by methotrexate may, at least in part, account for the rapid onset of antiinflammatory activity that was observed in clinical trials with methotrexate in rheumatoid arthritis.

Diminished splenic T-cell function, as measured by their response to phytohemagglutinin and Concanavalin-A, was noted only during the active phase of adjuvant induced disease. This diminished function is due to at least two types of suppressor cell: (a) cells which adhere to glass but not plastic, and which are sensitive to methotrexate in vitro; (b) cells which adhere to plastic and glass, and which are relatively insensitive to methotrexate in vitro. Methotrexate treatment in vivo inhibits adjuvant induced disease, and is associated with the absence of both types of suppressor cells in the spleen.

Adjuvant arthritis is inhibited by the interferon-inducing anti-viral agents statolon, pyran copolymer and tilorone, and by the passive transfer of interferon. The present experiments demonstrate that Virazole, a non-interferon-inducing, anti-viral agent also inhibits adjuvant arthritis. The responses of splenic lymphocytes to phytohemagglutinin and concanavalin A are diminished in active adjuvant disease, due to suppressor cells. Suppressor cells are absent during spontaneous, methodrexate or corticosteroid-induced remissions. Suppressor cells are also absent during Virazole-induced inhibition of adjuvant disease. Methotrexate and corticosteroids inhibit suppressor cells in preference to mitogen-responsive cells in vitro. The preferential inhibition with Virazole is superior to that noted with methotrexate or corticosteroids.

We demonstrated previously that variables of macrophage activation are associated with the development and progression of the arthritic lesion in the model of adjuvant induced arthritis. This association was investigated further by assessing the ability of antiarthritic agents to modulate variables of macrophage activation in direct comparison to effects on the arthritic lesion. Whereas indomethacin effectively reduced hindpaw edema, it had no significant effect on Ia expression or on any measurement of activation. Prednisolone inhibited hindpaw edema and the production of interleukin-1 (IL-1) by splenic macrophages. Only methotrexate inhibited hindpaw edema and all variables of macrophage activation (PGE2 and IL-1 production, cyanine dye accumulation) as well as the influx of Ia positive macrophages into synovial tissue.