Carbohydrate heterogeneity of fibronectins. Synovial fluid fibronectin resembles the form secreted by cultured synoviocytes but differs from the plasma form.

Large quantities of fibronectin (Fn) are present in inflammatory synovial fluid. Inflammatory synovial fluid Fn, while indistinguishable from plasma Fn on the basis of reactivity to polyclonal antibodies, displays alterations in molecular size and charge. Since biochemical differences between plasma and synovial fluid fibronectins might be in part due to differences in glycosylation we have compared the carbohydrate composition of plasma Fn, synovial fluid Fn, and Fn from synoviocyte conditioned medium by biochemical assay, glycopeptide analysis, and binding to a series of lectins. Synovial fluid Fn has a greater carbohydrate content but contains less sialic acid when compared with plasma Fn. Glycopeptides formed from synovial fluid Fn are smaller than plasma Fn glycopeptides. These data suggest the presence of an additional N-linked oligosaccharide chain on synovial fluid Fn. In addition, synovial fluid Fn contains N-acetyl galactosamine indicating the presence of O-linked oligosaccharides. Synovial fluid Fn and Fn isolated from rheumatoid synoviocyte-conditioned medium display strong reactivity with the lectins wheat germ agglutinin (WGA) and peanut agglutinin (PNA), whereas normal and rheumatoid plasma Fn react weakly. The PNA reactivity of synovial fluid Fn is mediated by terminal beta-galactose residues on the gelatin-binding domain, whereas the enhanced WGA reactivity of synovial Fn is mediated by a sialic acid containing oligosaccharide located on a 27-kD C-terminal fragment. These data demonstrate domain-specific biochemical differences between plasma and synovial fluid fibronectins. These differences suggest a local origin for synovial fluid Fn and may contribute to functional differences between these forms of the protein.

Plasma fibronectin in systemic lupus erythematosus: relationship to clinical activity, DNA binding and acute phase proteins.

Plasma fibronectin was measured by ELISA in 25 samples from 22 patients with systemic lupus erythematosus (SLE). The mean fibronectin level for the entire patient group (654 micrograms/ml) was greater than that of normal controls (450 micrograms/ml), with highest levels observed in the subgroup of patients with severe disease activity (838 micrograms/ml) followed by those with moderate disease activity (732 micrograms/ml) (p = .04). Fourteen patients with other rheumatic disease had a mean fibronectin level of 407 micrograms/ml. Decreases in fibronectin levels corresponded to clinical improvement and reductions in DNA binding. Although elevated fibronectin levels did not correspond to any specific pattern of organ system involvement, fibronectin levels seem to parallel disease activity in certain patients. Future longitudinal studies of plasma fibronectin in SLE may further define its role as an indicator of disease activity.

Synthesis, secretion, and deposition of fibronectin in cultured human synovium.

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.

The immunoreactivity, ligand, and cell binding characteristics of rheumatoid synovial fluid fibronectin.

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.

Factors influencing the incorporation of fibronectin into synovial fluid cryoprotein.

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.

Glutamate oxidation of 6C3HED lymphoma: effects of L-asparaginase on sensitive and resistant lines.

L-Asparaginase (EC 3.5.1.1) inhibited respiration in sensitive, but not resistant, lines of murine lymphoma 6C3HED. Glucose, in these tumor lines, was principally converted to lactate, and very little was oxidized in the citric acid cycle or hexose monophosphate shunt. The cells derived 70-80% of their respiratory CO(2) from glutamine or glutamate. Asparaginase had no effect on the pattern of glucose utilization. The differential effect on oxygen consumption may result from the absence of asparagine synthetase in sensitive cells. Respiration may be inhibited by accumulation of the aspartate, the product of glutamate oxidation. Resistant lymphoma cells remove aspartate by converting it to asparagine. Sensitive cells, which lack asparagine synthetase, cannot make asparagine.