Faculty Bibliography

We examined fibronectin synthesis, secretion, and deposition in vitro by primary explants of rheumatoid synovium. Primary cultures initiated from tissue with monocytic infiltrates had higher levels of fibronectin synthesis; addition of dexamethasone at concentrations known to stimulate other tissue fibroblasts increased fibronectin synthesis and secretion. Newly synthesized fibronectin recovered from primary rheumatoid culture medium had a higher apparent molecular weight (240-245 kd), on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared with fibronectin recovered from passaged normal and rheumatoid cultures (230 kd). Primary rheumatoid explant cultures had a characteristic morphology which correlated with fibronectin deposition. Dense deposits of fibronectin extracellular matrix covered overlapping synoviocytes adjacent to esterase-positive mononuclear cells. Dexamethasone-treated cultures showed little fibronectin deposited as extracellular matrix and did not develop overlapping cellular networks. Characteristic patterns of fibronectin synthesis and deposition in primary rheumatoid cultures appear to result from interaction between fibroblastic and monocytic cells. This culture system may provide a model by which to study interactions between cells and extracellular matrix components that regulate synovial cell function.

Fibronectin promotes macrophage adherence and expression of Fc receptors, is chemotactic for fibroblasts, and is an opsonin for fibrin and denatured collagen. These properties suggest a role for fibronectin in the modulation of joint inflammation. Since structural modification of the fibronectin molecule has been shown to result in loss or de novo acquisition of opsonic and chemotactic activity, we determined the functional and immunochemical properties of fibronectin isolated from the inflamed joint. Eighty-six percent of synovial fluids obtained from patients with active rheumatoid arthritis (RA) contained fibronectin fragments, and 39% of the fluids no longer displayed the dimeric form. Compared with native fibronectin, RA peptides were as active in promoting synoviocyte chemotaxis and in glycosaminoglycan binding, but displayed lower affinity for fibrin and gelatin. Although comparable with intact protein in augmenting monocyte attachment to gelatin, the RA synovial fluid peptides did not augment monocyte attachment to fibrin. Analysis of whole synovial fluid and isolated fibronectins by enzyme immunoassay showed that the increased fibronectin immunoreactivity, previously reported in RA synovial fluid, measures intact and nearly intact protein and does not measure extensively degraded fragments.

Commercial xanthine oxidase, widely used for generation of oxygen radicals in vitro, is usually contaminated by proteolytic activity, which limits its utility in studies of oxygen radical damage to protease sensitive substrates. An easily prepared fraction of fetal calf serum was found to inhibit virtually all of the proteolytic contaminant without affecting superoxide generation. The effects attainable with the "purified" enzyme were demonstrated with two protease sensitive targets: proteoglycan subunit from cartilage and fibronectin from human plasma.

Methods of synovial fluid collection and processing known to affect cryoprotein formation were examined to investigate the proposed role of fibronectin in synovial fluid cryoprecipitation. Fibronectin, a nonimmunoglobulin, noncomplement synovial fluid protein was present in all resolubilized synovial fluid cryoproteins studied. Radiolabeled fibronectin was precipitated from rheumatoid synovial fluid to a significantly greater extent (10%) than from noninflammatory (osteoarthritic) synovial fluid (2.8%), normal plasma (1.3%), or normal serum (0.5%) (p less than 0.01). Clotting of synovial fluid reduced fibronectin concentration 44% and resulted in a reduction in the amount and percent incorporation of fibronectin into cryoprotein, whereas heparinization and hyaluronidase treatment increased cryoprecipitable fibronectin. Affinity depletion of synovial fluid fibronectin resulted in loss of C1q and reduction in IgG in the cryoprotein; however, fibronectin, C1q, and IgG could not be co-eluted from affinity matrices of gelatin and protein A-Sepharose. Cryoprotein formation from pathologic synovial fluid depends in part on fibronectin and appears to involve interactions between fibronectin and fibrinogen as well as immunoglobulin complexes and complement components.