Abstract. TSG-6 cDNA was isolated by differential screening of a X cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts . We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells .

No abstract

No abstract

TSG-6 is a secreted 35-kDa glycoprotein, inducible by TNF and IL-1. The N-terminal portion of TSG-6 shows sequence homology to members of the cartilage link protein family of hyaluronan binding proteins. The C-terminal half of TSG-6 contains a so-called CUB domain, characteristic for developmentally regulated proteins. High levels of TSG-6 protein are found in the synovial fluid of patients with rheumatoid arthritis and some other arthritic diseases. Here we show that TSG-6 readily formed a complex with a protein present in human, bovine, rabbit, and mouse serum. This complex was stable during SDS-PAGE under reducing conditions, and in the presence of 8 M urea. The protein that binds TSG-6 was purified from human serum and identified as inter-alpha-inhibitor (I alpha I) by N-terminal microsequencing. Microsequencing of the complex itself revealed the presence of TSG-6 and two of the three polypeptide chains of I alpha I (bikunin and HC2). Experiments with recombinant TSG-6 and I alpha I purified from human serum showed that the TSG-6/I alpha I complex is rapidly formed even in the apparent absence of other proteins at 37 degrees C, but not at 4 degrees C. The TSG-6/I alpha I complex was cleaved by chondroitin sulfate ABC lyase, suggesting that cross-linking by chondroitin sulfate is required for the stability of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

summary Objective We investigated the relationship between the molecular weight (MW) distribution of hyaluronan (HA) in synovial fluid (SF) and risk of knee osteoarthritis (OA) progression. Methods HA MW was analyzed for 65 baseline knee SFs. At 3-year follow-up, knees were scored for change in joint space narrowing (JSN), osteophyte (OST) progression, or occurrence of total knee arthroplasty (TKA). HA MW distribution was analyzed using agarose gel electrophoresis (AGE), and its relationship to OA progression was evaluated using logistic regression. The association between HA MW and self-reported baseline knee pain was analyzed using Pearson's correlation coefficients. Results Knee OA was categorized as non-progressing (OST−/JSN−, 26 knees, 40%), or progressing based on OST (OST+/JSN−, 24 knees, 37%), OST and JSN (OST+/JSN+, 7 knees, 11%) or total knee arthroplasty (TKA, 8 knees, 12%). The MW distribution of HA in baseline SFs was significantly associated with the odds of OA progression, particularly for index knees. After adjusting for age, gender, BMI, baseline X-ray grade and pain, each increase of one percentage point in %HA below 1 million significantly increased the odds of JSN (odds ratios (OR) = 1.45, 95% CI 1.02–2.07), TKA or JSN (OR = 1.24, 95%CI 1.01–1.53) and the odds of any progression (OR = 1.16, 95% CI 1.01–1.32). HA MW distribution significantly correlated with pain. Conclusion These data suggest that the odds of knee OA progression increases as HA MW distribution shifts lower and highlight the value of reporting MW distribution rather than just average MW values for HA.

Objective. Expression of TSG-6 (tumor necrosis factor-stimulated gene 6) is induced by proinflammatory cytokines. This study was undertaken to examine the effects of local expression of TSG-6 in arthritic joints of TSG-6 transgenic mice, in the collagen-induced arthritis (CIA) model.Methods. We generated transgenic mice that harbored the TSG-6 gene under the control of the T cell-specific lck promoter. Arthritis was induced by immunization with bovine type II collagen (CII), and its progression was monitored based on the incidence of arthritis, the arthritis index, and footpad swelling. Anti-CII antibodies and cytokine production were determined by enzyme-linked immunosorbent assay. Gene expression arrays were used to compare gene expression profiles of transgenic and control mice at various stages of CIA.Results. TSG-6 was expressed in limbs of transgenic mice after immunization with CII, while its expression in nontransgenic animals was insignificant. The incidence of CIA was reduced in TSG-6 transgenic animals, its onset delayed, and all parameters of clinical arthritis significantly reduced. However, the immune response against CII was not significantly inhibited in TSG-6 transgenic mice.Conclusion. TSG-6 expression has been demonstrated in patients with rheumatoid and other forms of arthritis. Our data show that local expression of TSG-6 at sites of inflammation results in potent inhibition of inflammation and joint destruction in a model of autoimmune arthritis in mice. Therefore, it is likely that TSG-6 plays a similar modulatory role in human rheumatoid arthritis and related diseases and may have potential for the treatment of autoimmune arthritis in humans.

IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2.