Faculty Bibliography

OBJECTIVE: To determine why methotrexate (MTX) exacerbates rheumatoid nodules in some patients, despite the effective suppression of synovial inflammation. METHODS: Phorbol myristate acetate (PMA)-induced differentiation of monocytes into multinucleated giant cells was used as an in vitro model to study the effects of adenosine on nodulosis. RESULTS: MTX at 200-2,000 nM or the adenosine A1 agonist N5-cyclopentyl adenosine (CPA) (10(-12) to 10(-9) M) or the A2 antagonist 3,7-dimethyl-1-propargylxanthine markedly enhanced giant cell formation, whereas the adenosine A1 antagonist 8-cyclopentyl-dipropylxanthine completely reversed these effects. PMA, CPA, and MTX induced adenosine release by cultured monocytes at concentrations consistent with those associated with predominantly A1 effects. Furthermore, surface expression of A1 receptors was found to remain unchanged on the differentiating cells throughout the culture period. CONCLUSION: Agents that inhibit adenosine A1 receptors might be useful in the treatment of MTX-induced rheumatoid nodulosis, while still potentiating the A2-mediated antiinflammatory effects of MTX on synovitis

Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation

Methotrexate is one of the most effective and widely used medications in the treatment of rheumatoid arthritis. One poorly understood side effect of methotrexate is increased rheumatoid nodule formation, a phenomenon which has been reported to occur in some patients despite suppression of synovial inflammation. Using an in vitro model of nodulosis, induction of monocyte differentiation into multinucleated giant cells, we previously found that methotrexate promotes this inflammatory response by a mechanism dependent on adenosine A 1 receptor stimulation. In the current study, we tested the effects of an A 1 signal inhibitor, the commonly available anti-inflammatory medication colchicine, and found that it markedly inhibited nodulosis in vitro as well as in seven of fourteen patients in a clinical series