Anti-arthritic effect of methotrexate: is it really mediated by adenosine?

The mechanism of action for the anti-arthritic effect of methotrexate (MTX) was investigated in rats with antigen-induced arthritis (AIA). Arthritis intensity was quantified as area under the curve (AUC) for the joint swelling. The response to MTX was in several respects similar to what is seen in the clinic. The drug reduced the AUC in a dose-dependent manner after oral weekly (2-4 mg/kg/week) or daily (0.3 mg/kg/day) dosing. This effect was not affected by supplementation with an equal dose of folate. The model thus seemed suitable for this type of study. Supplementation with folate in excess abolished the effect of MTX. A structurally similar antifolate, aminopterin, also reduced the arthritis. The effect thus seemed to be due to folate antagonism although a complete inhibition of dihydrofolate reductase (DHFR) might not be essential. Hence, it could be that the main target is a process downstream of DHFR. It has been proposed that inhibition of AICAR-transformylase induce the release of adenosine with anti-inflammatory properties. Here the adenosine antagonist R-PIA reduced the arthritis but when MTX was combined with adenosine antagonists no attenuation of the anti-arthritic effect was seen. On the contrary, three adenosine agonists (8-p-sulphophenyltheophyllamine 30 mg/kg i.p. twice daily; 3,7-dimethyl-1-propargylxanthine, p.o. 3 mg/kg/day and 8-cyclopentyl-1,3-dipropylxanthine, 1.5 mg/kg/day p.o.) potentiated MTX. The specific thymidylate synthase inhibitor 5-fluourouracil (0. 3-3.0 mg/kg/day) had no anti-arthritic effect. Neither did our data support the hypotheses that syntheses of polyamines or cytokines were primary targets. It is thus possible that the mechanism of action is inhibition of a process downstream of DHFR but the release of adenosine seems not to be important.

Periarticular tissue levels of cytokine- and endothelin-1-like immunoreactivity during the course of antigen-induced arthritis in the rat.

OBJECTIVE AND DESIGN: To correlate the changes in periarticular tissue levels of pro-inflammatory cytokines and endothelin-1 (ET-1) to local pathophysiology in rats with antigen-induced arthritis (AIA). MATERIALS: The periarticular soft tissue was excised from sixty-six rats at different stages of AIA. METHODS: The samples were homogenized and the levels of immune like (LI) reactivities were determined. The levels in the arthritic joints were compared to those in non-arthritic tissue. Statistical significance was determined by ANOVA followed by Fisher PLSD. RESULTS: Compatible with a role in an early alarm reaction some mediators (tumor necrosis factor alpha-LI, interleukin-2-LI and interferon-gamma-LI) were elevated prior to the vascular inflammatory activity. The curve for ET-1-LI closely followed the intensity of the inflammation whereas these parameters preceeded the elevation of interleukin-1beta-LI. CONCLUSIONS: Transient waves of different mediators appear during the course of the arthritis. This indicates the presence of active downregulatory mechanisms. Here the model could differ from human rheumatoid arthritis.

Tissue and intracellular pH in normal periarticular soft tissue and during different phases of antigen induced arthritis in the rat.

OBJECTIVE: To measure intracellular and tissue pH in periarticular soft tissue during different phases of antigen induced arthritis in the rat. METHODS: pH was calculated using the following values: (1) the distribution of [14C]-dimethyl-oxazolidinedione; (2) the total tissue water and the extracellular space water volume, which was measured as [14C]-sucrose distribution in nephrectomized rats. Experiments were performed during both maximal inflammation (Day 3) and the restorative phase (Day 14). RESULTS: In all animals both tissue (pHt) and intracellular (pHi) pH were lower in arthritic joints than in the contralateral control. Mean pHt in control joints was 7.37+/-0.03. In arthritic rats it was 7.30+/-0.05 on Day 3 after challenge and 7.27+/-0.03 on Day 14. The pHi ranges were 6.86-7.81 for controls, 6.65-7.28 for arthritis Day 3, and 5.66-6.91 for arthritis Day 14. CONCLUSION: In this model there is a reduction in pH in the periarticular tissue of arthritic joints. The magnitude is, however, relatively small and the pannus tissue is not uniquely acidic in comparison with other compartments. There does not seem to be a correlation between pH and changes in metabolic balance, pannus formation, or healing.

Physiological characterization of mBSA antigen induced arthritis in the rat. I. Vascular leakiness and pannus growth.

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.

Physiological characterization of mBSA antigen induced arthritis in the rat. II. Joint blood flow, glucose metabolism, and cell proliferation.

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.