Cartilage‐specific constitutive expression of TSG‐6 protein (product of tumor necrosis factor α–stimulated gene 6) provides a chondroprotective, but not antiinflammatory, effect in antigen‐induced arthritis

Abstract

Objective. To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor ␣-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice.Methods. Transgenic mice constitutively expressing TSG-6 protein in cartilage were generated. Cartilage-specific constitutive expression of TSG-6 protein was confirmed by in situ hybridization, Western blot analysis, and immunohistochemistry. Control and transgenic mice were immunized with methylated bovine serum albumin (mBSA), and arthritis was induced by the intraarticular injection of mBSA. Mice were monitored up to day 35 after the challenge, and knee joint sections were examined for loss of cartilage proteoglycan (aggrecan) using Safranin O staining and antibodies to neoepitopes generated by various metalloproteinases (MPs). The loss of aggrecan in Safranin O-stained sections was quantified by morphometric methods.Results. Tsg6/tnfip6 transgenic mice constitutively expressed tsg6/tnfip6 messenger RNA and corresponding TSG-6 protein in cartilage from embryonic life through adulthood, without any phenotypic abnormalities. These mice were used for AIA studies. Intraarticular injection of mBSA uniformly induced severe inflammation both in control (wild-type and an irrelevant transgenic line) mice and in tsg6/tnfip6 transgenic mice. In contrast to the mBSA-injected knee joints of control animals that were heavily damaged from day 5, the cartilage of transgenic mice that constitutively expressed TSG-6 protein remained intact for at least 1 week, and this was followed by a relatively reduced loss of aggrecan. Concomitant with the loss of aggrecan, MP-generated neoepitopes accumulated in unprotected joints. By day 35, the proteoglycan content returned to nearly normal levels in tsg6/tnfip6 transgenic mice, whereas it remained low in MP-damaged knee cartilage of control mice.Conclusion. TSG-6 protein is known to form a complex with inter-␣-inhibitor (I␣I), a potent serine protease inhibitor, which may be immobilized via the hyaluronan (HA)-binding domain of TSG-6 protein in the HA-rich extracellular matrix of cartilage. Thus, the local accumulation of TSG-6 protein and TSG-6 protein-bound I␣I in tsg6/tnfip6 transgenic mice may inhibit serine proteases and subsequent activation of MPs. It is suggested that this mechanism might protect

Enhanced neutrophil extravasation and rapid progression of proteoglycan‐induced arthritis in TSG‐6–knockout mice

“…The results of these in vivo experiments suggest that Tnfip6 plays a role in the control of leukocyte influx into arthritic joints (most likely via interference with CD44/HA-mediated adhesion events), and is involved locally in the protection of cartilage matrix from proteolytic damage through complex formation with the serine protease inhibitor I␣I (7,8). The most important message of our in vitro and in vivo studies is that Tnfip6 can directly modulate cell adhesion (36,37), PMN cell extravasation (Figures 2 and 7), and perhaps also, the 3020 SZÁ NTÓ ET AL production of proinflammatory mediators such as IL-6 ( Figure 5C).…”

“…The antiinflammatory properties of Tnfip6 have been mainly attributed to its ability to potentiate the antiplasmin activity of I␣I upon association with this protease inhibitor, leading to the subsequent downregulation of the activity of a number of matrixdegrading proteases (7)(8)(9)(10). However, a recent study (11) found that the isolated Link module domain of Tnfip6, which lacks the ability to associate with I␣I, could still inhibit the influx of neutrophils to the site of inflammation.…”

“…A sandwich ELISA was developed using purified A38 mAb for capture, and biotinylated TSG-6-CR21 polyclonal rabbit antibody (7,8) was used for detection of Tnfip6 in serially diluted serum samples. Peroxidase-conjugated streptavidin (Zymed, San Francisco, CA) followed by o-phenylenediamine and hydrogen peroxide were used for the colorimetric detection of plate-bound Tnfip6, and rMuTnfip6 served as a reference standard.…”

“…A chondroprotective effect could also be achieved in antigen-induced arthritis by cartilagespecific constitutive expression of the Tnfip6 transgene (8).…”

TNF?-stimulated gene product (TSG-6) and its binding protein, I?I, in the human intervertebral disc: new molecules for the disc

“…It also stimulates the proliferation of vascular smooth muscle cells [34], and TSG-6 has been detected in the inflamed blood vessel walls of RA and OA synovium [2]. In addition, TSG-6 has anti-inflammatory effects in vivo, being a potent inhibitor of neutrophil migration [7,32], and it has been found to be chondro-protective in mice models of arthritis [8,21]. Since angiogenesis and inflammation are both believed to be significant in disc herniation patients with sciatica [22], we hypothesised that TSG-6 could be important in the aetiopathogenesis of this disorder.…”