TSG-6

TSG-6 Hans-Georg Wisniewski Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA corresponding author tel: 212 263 0924, fax: 212 263 7933, e-mail: [email protected] DOI: 10.1006/rwcy.2001.0705. Chapter posted 5 November 2001

SUMMARY

BACKGROUND

TNF-stimulated gene 6 (TSG-6) is one of a number of genes activated by TNF and IL-1 during an inflammatory response. The TSG-6 gene encodes a 35 kDa secretory glycoprotein that is present in the synovial fluid, cartilage, and synovial lining of patients with various forms of arthritis. TSG-6 protein was also detected in sera of patients with bacterial sepsis, inflammatory arthritis, or systemic lupus erythematosus. Fibroblasts, chondrocytes, articular synovial cells, vascular smooth muscle cells, and cumulus-oocyte complexes have been reported to produce TSG-6 after stimulation with TNF, IL-1, or selected growth factors and hormones. TSG-6 protein consists of two structural domains, the N-terminal link module and the Cterminal CUB domain. The link module, the signature domain of the hyaladherin family of proteins, is responsible for the affinity of TSG-6 for hyaluronan. In addition, TSG-6 forms a stable complex with components of the plasma protein inter--inhibitor, a Kunitz-type serine protease inhibitor. Recombinant human TSG-6 protein was shown to exert a potent anti-inflammatory effect in a murine model of acute inflammation and treatment with TSG-6 resulted in amelioration of collageninduced arthritis in DBA/1J mice. Activation of the TSG-6 gene by proinflammatory cytokines, the presence of TSG-6 protein at inflammatory sites and its anti-inflammatory effect in experimental models of acute and chronic inflammation suggest a role for TSG-6 as an endogenous inhibitor of the inflammatory response.

Discovery

Cytokine Reference

TNF-stimulated gene 6 (TSG-6) was originally cloned from human fibroblasts by differential screening of a cDNA library established from TNF-stimulated FS-4 fibroblasts (Lee et al., 1990). The TSG-6 gene encodes a protein, also termed TSG-6, that was first detected in culture supernatants of TNF-stimulated FS-4 fibroblasts (Lee et al., 1992) and in synovial fluids of patients with various inflammatory joint disorders (Wisniewski et al., 1993). Besides the human TSG-6 gene, its rabbit, mouse, and rat homologs have been cloned from different cellular sources (Feng and Liau, 1993; FuÈloÈp et al., 1997; Yoshioka et al., 2000). The homology among the human, rabbit, and murine TSG-6 polypeptide sequences ranges from 94.2 to 97.5% (Lee et al., 1992; Feng and Liau, 1993; FuÈloÈp et al., 1997).

Alternative names The rabbit homolog of TSG-6 was originally described as PS-4.

Structure The TSG-6 cDNA encodes a polypeptide of 277 amino acids including a signal sequence of 17 residues. TNF-stimulated FS-4 fibroblasts, after

Copyright # 2001 Academic Press

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cleavage of the signal peptide and glycosylation, release TSG-6 as a glycoprotein with an apparent molecular mass of 35 kDa (Lee et al., 1992). TSG-6 protein consists of two distinct structural domains, termed link module and CUB domain. The two domains, together comprising 78% of the TSG-6 polypeptide, are flanked by short N- and C-terminal extensions. TSG-6 is the only known protein comprising these two domains. The N-terminal domain of TSG-6, formed by amino acid residues 36±127, represents a structural motif known as the link module (Kohda et al., 1996), which confers affinity for hyaluronan. It is shared by all members of the family of hyaluronan binding proteins termed hyaladherins (Toole, 1990, 1992) or hyalectans (Iozzo and Murdoch, 1996). The stretch of 113 amino acids comprising residues 135±247 is homologous to another structural module that has been termed CUB domain and is shared by a large and diverse group of proteins (Bork and Beckmann, 1993). This module has been defined exclusively on the basis of sequence homologies and the conservation of certain structural elements (Bork and Beckmann, 1993). Most of our knowledge about the potential functions of the CUB domain results from the analysis of the spermadhesins, a family of proteins consisting exclusively of CUB domains (TopferPetersen et al., 1998). Spermadhesins have been reported to bind heparin (Calvete et al., 1993; Sanz et al., 1993), glycoproteins (Sanz et al., 1992b; Dostalova et al., 1995a), phospholipids (Dostalova et al., 1995b; Ensslin et al., 1995), and soybean trypsin inhibitor (Sanz et al., 1992a). Based on their affinity for a variety of carbohydrates, spermadhesins may be considered a new group of animal lectins (TopferPetersen et al., 1998). So far no functions other than involvement in protein±carbohydrate and protein±protein interactions have been attributed to the CUB domain. The CUB domain of TSG-6 may also be involved in such interactions. However, the limited sequence homology between the CUB domain of TSG-6 and the CUB domains of other proteins precludes conclusions about any particular binding specificity.

Main activities and pathophysiological roles Transcriptional activation of the TSG-6 gene by TNF and IL-1 suggested an association with the inflammatory response (Lee et al., 1992). This association was confirmed by the demonstration

of the presence of TSG-6 protein in body fluids of patients with inflammatory and autoimmune disorders. TSG-6 was readily detectable in synovial fluids of patients with various joint diseases including rheumatoid arthritis, osteoarthritis, SjoÈgren's syndrome, polyarthritic gout, and osteomyelitis (Wisniewski et al., 1993). No TSG-6 protein was detectable in synovial fluids obtained at autopsy from accident victims without known joint disease. Articular synovial cells isolated from patients with rheumatoid arthritis expressed TSG-6 constitutively and responded to stimulation with either IL-1 or TNF with an additional upregulation of TSG-6 expression (Wisniewski et al., 1993). Chondrocytes of these patients also responded to stimulation with IL-1 or TNF with TSG-6 expression (Wisniewski et al., 1993; Maier et al., 1996). Expression of TSG-6 in IL-1stimulated chondrocytes obtained from patients with osteoarthritis was demonstrated in two additional studies (Margerie et al., 1997; Stove et al., 2000). Analysis of the distribution of TSG-6 in the synovium and cartilage of patients with rheumatoid arthritis and osteoarthritis using immunohistochemistry demonstrated the presence of this protein in the synovial lining, at the cartilage-synovial pannus junction, its association with blood vessels, expression by chondrocytes, and its presence in the surrounding cartilage matrix (Bayliss et al., 2001). These findings suggest that in inflammatory joint disease both synovial cells and chondrocytes, but probably also infiltrating mononuclear cells and vascular smooth muscle cells, contribute to the observed accumulation of TSG-6 protein in cartilage, the synovial lining, and in synovial fluid. TSG-6 expression is, however, not limited to inflammatory joint diseases. TSG-6 protein could be detected in the sera of patients with bacterial sepsis and in the sera of volunteers injected with bacterial lipopolysaccharide (LPS). In the latter sera, maximum levels of TSG-6 were observed 3±6 hours after the LPS injection (Lee et al., 1993b). The highest serum levels of TSG-6 protein were found in patients with systemic lupus erythematosus (SLE). Whereas only about 50% of SLE patients contained detectable amounts of TSG-6 in serum, some of those samples contained TSG-6 concentrations considerably higher than found in the sera of patients with sepsis or rheumatoid arthritis (Wisniewski and Vilcek, 1997). While its presence in patient materials suggests that TSG-6 plays some role in inflammatory conditions, its effects in experimental models of inflammation indicate an anti-inflammatory activity (see In vivo biological activities of ligands in animal models). The induction of TSG-6 by proinflammatory cytokines, its presence in synovial fluids and tissue samples from

TSG-6 3 joints affected by inflammation, and its antiinflammatory activity in experimental models of inflammatory diseases suggest that TSG-6 is an endogenous inhibitor of the inflammatory response. It is currently not clear if TSG-6 acts directly on cells and no cellular receptor for TSG-6 has been identified.

GENE AND GENE REGULATION

Accession numbers The GenBank accession numbers are M31165 for human TSG-6 (Lee et al., 1992), M86381 for rabbit TSG-6 (Feng and Liau, 1993), U83903 for murine TSG-6 (FuÈloÈp et al., 1997), and AF159103 for a partial coding sequence of rat TSG-6 (Yoshioka et al., 2000). These files contain both nucleotide and peptide sequences.

Chromosome location The human TSG-6 gene was mapped to chromosome 2 (Lee et al., 1993a) and its murine homolog was mapped to the 28.4±31.6 cM region of murine chromosome 2 (FuÈloÈp et al., 1997). The gene content of this region of murine chromosome 2 is homologous to its human counterpart (DeBry and Seldin, 1996). A single copy of the TSG-6 gene exists in humans (Lee et al., 1993a) and no close relatives of the gene are currently known. The murine TSG-6 gene consists of six exons separated by introns of 1.1±5.8 kbp (FuÈloÈp et al., 1997). The link module of murine TSG-6 is encoded by two exons and the CUB domain by three exons (FuÈloÈp et al., 1997).

Regulatory sites and corresponding transcription factors Results of nuclear run-on assays performed in normal human fibroblasts indicated that TSG-6 expression is controlled at the level of transcription (Lee et al., 1993a). Lee et al. (1993a) isolated a 1.3 kbp fragment of the 50 -flanking region of TSG-6 genomic DNA. Sequencing of this fragment led to the identification of TATA and CCAAT box sequences upstream of a single transcriptional start site. Deletion analysis revealed that the TSG-6 promoter region between the positions ÿ165 and ‡78 is sufficient for transcriptional activation by IL-1 and TNF in human fibroblasts. Transcriptional activation of TSG-6 by these cytokines was found to be cooperatively

regulated by NF-IL6 and AP-1 family transcription factors (Klampfer et al., 1994). Further analysis by site-directed mutagenesis showed that within this TSG-6 promoter fragment transcriptional activation is controlled by three cis-acting elements: two adjacent NF-IL6 sites and one AP-1 binding site located 5 bp upstream of the NF-IL6 sites (Klampfer et al., 1994). Mutations within either one of the two NF-IL6 sites completely abolished inducibility by either IL-1 or TNF, whereas inactivation of the AP-1 site reduced inducibility by IL-1 and virtually abolished the response to TNF (Klampfer et al., 1994, 1995).

PROTEIN

Description of protein The TSG-6 cDNA encodes a polypeptide of 277 amino acids. The 17 N-terminal amino acids form a cleavable signal peptide and the N-terminus of the mature polypeptide has been confirmed by microsequencing as Trp18 (Wisniewski et al., 1994). After cleavage of the signal peptide and N-glycosylation, TSG-6 is secreted as a 35 kDa glycoprotein (Lee et al., 1992). Based on sequence homologies, amino acids Gly36 to Cys127 form a distinct structural domain that has been named link module for its prominence in the cartilage link protein, a protein consisting of two tandem repeats of this domain (Kohda et al., 1996). Separated from the N-terminal domain by a short spacer of seven amino acids, residues Cys135 to Met247 form the second domain of TSG-6, a socalled CUB domain (Bork and Beckmann, 1993).

Discussion of crystal structure The solution structure of the link module of TSG-6 has been solved by NMR spectroscopy (Kohda et al., 1996). This study revealed a compact fold containing two  helices and two antiparallel sheets, each comprising three strands. The expected positions of two disulfide bonds within the link module of TSG-6 (Lee et al., 1992) were confirmed by Kohda et al. (1996). The overall structure of the link module is stabilized by a large hydrophobic core formed by amino acid residues contributed by different strands. Structural studies of the link module of TSG-6 have shed light on the interation of this domain with its ligand hyaluronan. Comparison of the NMR spectra

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of the link module of TSG-6 with the spectra of its complex with a hyaluronan octasaccharide have resulted in the identification of a binding surface on the link module of TSG-6 that is formed by amino acid residues that are not closely associated in the peptide sequence (Kahmann et al., 2000). Specifically, Lys46, Tyr94, Asn102, Phe105, Lys107, and Tyr113 have been implicated in binding to hyaluronan (Kohda et al., 1996; Kahmann et al., 2000). This prediction was tested by site-directed mutagenesis of the amino acid residues listed above and 15 additional residues in the link module of TSG-6 (Mahoney et al., 2001). Analysis of the affinity of 21 mutants of the link module of TSG-6 confirmed that Lys46, Tyr94, Phe105, and Tyr113 are essential for hyaluronan binding, while mutations of Asn102 or Lys107 did not impair hyaluronan binding. In addition, a mutation of Tyr47 also resulted in complete loss of hyaluronan binding by the link module of TSG-6 (Mahoney et al., 2001). Therefore, Lys46, Tyr47, Tyr94, Phe105, and Tyr113 (corresponding to Lys11, Tyr12, Tyr59, Phe70, and Tyr78 in the isolated link module, using the numbering system of Mahoney et al. (2001)) form the hyaluronan binding site of TSG-6. The structure of the CUB domain of TSG-6 has not been solved. However, structural data are available for the spermadhesins PSP-I/PSP-II and aSFP, proteins consisting exclusively of CUB domains (Romao et al., 1997; Varela et al., 1997). Analysis of the crystal structure of these proteins revealed that the CUB domain comprises 10 strands arranged in two sheets of five strands. Each sheet of the CUB domain sandwich consists of two parallel and four antiparallel strands. The sheets are arranged around a hydrophobic core of aromatic and hydrophobic amino acid residues. The 15 hydrophobic and four aromatic residues that form the core of the CUB domain and represent the CUB domain signature are well conserved in TSG-6, suggesting a similar fold. In addition, CUB domains share two exposed charged residues (Varela et al., 1997). These residues are also present in the CUB domain of TSG-6 (Asn191 and Arg208). Two disulfide bridges are well conserved in all spermadhesins and most other CUB domains, including the CUB domain of TSG-6 (Bork and Beckmann, 1993; Varela et al., 1997). These disulfide bridges between Cys135 and Cys161 and between Cys188 and Cys210 have previously been suggested for the CUB domain of TSG-6 (Lee et al., 1992).

Important homologies The link module of TSG-6 shows varying degrees of homology to members of the hyaladherin family of

proteins, with the level of identity ranging from 37 to 40% (Lee et al., 1992). The CUB domain of TSG-6 shows homology to other proteins utilizing this structural module (Lee et al., 1992; Bork and Beckmann, 1993), with a maximum of 40% identity between TSG-6 and bone morphogenetic protein 1. These homologies are in general limited to the particular structural domain that is shared. As an exception, the homology between TSG-6 and the cellular hyaluronan receptor CD44 extends slightly beyond the C-terminus of the link module (Lee et al., 1992).

Posttranslational modifications The sequence of the TSG-6 polypeptide contains two N-glycosylation consensus sequences, Asn118-ArgSer and Asn258-Thr-Ser. Using limited treatment with N-glycosidase F, Lee et al. (1992) demonstrated that TSG-6 protein expressed by FS-4 fibroblasts after stimulation with TNF is N-glycosylated at two sites.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce In addition to normal human fibroblasts, TSG-6 gene expression was found to be inducible in freshly isolated mononuclear cells, in chondrocytes, and in synovial cells isolated from joints of patients with rheumatoid arthritis (Wisniewski et al., 1993; Maier et al., 1996). With the exception of synovial cells from patients with rheumatoid arthritis, TSG-6 mRNA or TSG-6 protein are not detectable in unstimulated cells, but are expressed after stimulation with TNF or IL-1. In addition, TSG-6 expression can be induced in cultured mononuclear cells by bacterial LPS (Wisniewski et al., 1993). Various reports indicate differences in the pattern of TSG-6 expression by fibroblasts and chondrocytes. For example, dexamethasone was reported to interfere with the stimulation of TSG-6 expression by IL-1 in fibroblasts (Klampfer et al., 1994) but did not inhibit the IL-1-induced TSG-6 expression in chondrocytes (Maier et al., 1996). While TSG-6 mRNA was not induced by IL-6 in fibroblasts (Lee et al., 1993a), IL-6 was found to be moderately effective in inducing the TSG-6 message in human chondrocytes (Maier et al., 1996). Lee et al. (1990) did not observe any

TSG-6 5 detectable TSG-6 expression in FS-4 fibroblasts after stimulation with a variety of growth factors including EGF, PDGF, and TGF . In contrast, Maier et al. (1996) reported a modest induction of TSG-6 mRNA in human chondrocytes by PDGF, bFGF, or TGF . Inducibility of the TSG-6 gene by growth factors is also in line with data obtained in rabbit embryonic smooth muscle cells. Feng and Liau (1993) cloned the rabbit homolog of TSG-6 as a gene expressed in a serum-dependent fashion in embryonic smooth muscle cells. They observed that TSG-6 mRNA was induced after stimulation of quiescent embryonic smooth muscle cells by fetal bovine serum or by the growth factors aFGF, EGF, PDGF, and TGF . In contrast to the protein synthesis-independent induction of TSG-6 mRNA by TNF or IL-1 in human dermal fibroblasts (Lee et al., 1990), induction of the rabbit TSG-6 mRNA by growth factors was found to be suppressed by inhibitors of protein synthesis and therefore presumably indirect (Feng and Liau, 1993). Stimulation of rabbit vascular smooth muscle cells with TNF (Feng and Liau, 1993) or IL-1 (Ye et al., 1997) also resulted in transcriptional activation of the TSG-6 gene. Expression of the rabbit TSG-6 gene was detected in a range of fetal tissues and organs including skeletal muscle, esophagus, lung, and kidneys, whereas very little TSG-6 expression could be detected in the same organs in adult animals, suggesting that, at least in the rabbit, TSG-6 expression is developmentally regulated (Feng and Liau, 1993). In the rat, expression of the TSG-6 protein was induced in blood vessels injured by a balloon catheter. In this model, TSG-6 expression was mostly observed in the neointima of the injured artery, with increasing levels of TSG-6 toward the lumen of the vessel (Ye et al., 1997). An increase in the TSG-6 mRNA and production of TSG-6 protein has also been reported in human vascular smooth muscle cells subjected to mechanical strain in culture (Lee et al., 2001). Expression of TSG-6 has also been reported in epithelial cells. Janssen et al. (2001) studied the expression of TSG-6 in human renal proximal tubular epithelial cells induced by IL-1 or glucose. An increase of the TSG-6 mRNA after stimulation of these cells with IL-1 was apparent within 3 hours and independent of protein synthesis while the increase of TSG-6 mRNA after addition of 25 mM D-glucose became detectable only after 48 hours and was inhibited by cycloheximide (Janssen et al., 2001). The murine homolog of the TSG-6 gene was cloned from a cDNA library established from cumulus± oocyte complexes (COCs) of mice stimulated with

chorionic gonadotropin (FuÈloÈp et al., 1997). The TSG-6 mRNA became detectable in COCs within 3 hours after stimulation of mice with gonadotropin and persisted for at least 16 hours (FuÈloÈp et al., 1997). The rat homolog of TSG-6 was cloned from ovaries of rats stimulated with chorionic gonadotropin (Yoshioka et al., 2000). Expression became detectable 2 hours after the stimulus and the highest levels of expression were observed 4±8 hours after the injection of the hormone. In situ hybridization of rat ovaries indicated that the TSG-6 mRNA is associated with the cumulus mass and the antral granulosa cells (Yoshioka et al., 2000).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The proinflammatory cytokines IL-1 and TNF have been shown to induce production of TSG-6 mRNA and protein in a variety of cell types including fibroblasts, peripheral blood mononuclear cells, synovial cells, chondrocytes, smooth muscle cells, and epithelial cells. Experiments with cycloheximide demonstrated that expression of the TSG-6 mRNA after stimulation with TNF or IL-1 does not require protein synthesis, characterizing it as a primary response gene (Lee et al., 1990). In addition, TSG-6 expression can be induced in cultured mononuclear cells by bacterial LPS (Wisniewski et al., 1993). In a study of the effects of several agents known to induce cytokine genes, only the phorbol ester TPA, calcium ionophore A23187, and the double-stranded RNA poly(I)poly(C) induced a weak expression of TSG-6 mRNA in normal human fibroblasts (Lee et al., 1990). PDGF, bFGF, or TGF have been found to stimulate TSG-6 expression in articular chondrocytes (Wisniewski et al., 1993; Maier et al., 1996), and aFGF, EGF, PDGF, and TGF induced TSG-6 expression in vascular smooth muscle cells (Feng and Liau, 1993). In human vascular smooth muscle cells mechanical strain has been identified as a stimulator of TSG-6 expression (Lee et al., 2001). D-Glucose has been demonstrated to stimulate expression of TSG-6 in cultured proximal tubular epithelial cells of the human kidney (Janssen et al., 2001). Finally, chorionic gonadotropin has been demonstrated to induce the expression of TSG-6 in expanding murine cumulus cell±oocyte complexes (FuÈloÈp et al., 1997) and in the ovary of the rat (Yoshioka et al., 2000).

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IN VITRO ACTIVITIES

In vitro findings The presence of the link module, a domain associated with hyaluronan binding and the signature domain of the hyaladherin family of proteins, suggested that TSG-6 might have affinity for hyaluronan. This was confirmed experimentally (Lee et al., 1992; Kohda et al., 1996; Parkar and Day, 1997; Kahmann et al., 2000). In addition, it was reported that the isolated link module of TSG-6 binds selectively to chondroitin-4-sulfate, but not to chondroitin-6sulfate (Parkar and Day, 1997), and that TSG-6 does not bind to chondroitin-4,6-sulfate, heparin, dermatan sulfate, or keratan sulfate (Lee et al., 1992). Besides its affinity for selected glycosaminoglycans, TSG-6 also interacts with inter--inhibitor (II), a serine protease inhibitor present in plasma. II, a glycoprotein with an apparent molecular mass of 220 kDa, consists of three polypeptide chains linked by a chondroitin-4-sulfate chain (Enghild et al., 1989, 1991). The interaction between recombinant TSG-6 and purified II results in the formation of a stable complex of 120 kDa comprising TSG-6 and components of II (Wisniewski et al., 1994). The interaction between TSG-6 and II is apparently not speciesspecific and human TSG-6 has been found to form complexes with bovine, murine, and rabbit II (Wisniewski et al., 1994). The TSG-6/II complex is stable during SDS-PAGE, in 8 M urea, and it resists treatment with hyaluronidase (Wisniewski et al., 1994). However, the TSG-6/II complex has been found to be sensitive to treatment with condroitin sulfate lyase and II treated with this enzyme failed to form a complex with TSG-6 (Wisniewski et al., 1994). TSG-6 has been reported to synergize with II in the inhibition of plasmin, a protease that, besides its role in fibrinolysis, has been associated with cellular invasion of tissues in inflammation and cancer and with the activation of matrix metalloproteinases (Wisniewski et al., 1996).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS Effects of recombinant TSG-6 in two experimental models of inflammation have been described. In the first model, the murine air pouch model, an inflammatory agent is injected into an artificially induced air pouch on the back of mice, resulting in

a local acute inflammation (Edwards et al., 1981; Magilavy, 1990). Recombinant TSG-6 protein, injected into the air pouches of mice together with a proinflammatory stimulus like carrageenan or IL-1, exerted a potent and dose-dependent inhibitory effect on the infiltration of the air pouch lumen by neutrophils (Wisniewski et al., 1996). Heat-treated TSG-6 protein completely lost this activity. In a direct comparison of the anti-inflammatory activity of TSG6 with that of dexamethasone in this model, TSG-6 proved to be as effective as dexamethasone in the inhibition of the IL-1-induced neutrophil infiltration. Histological analysis of the air pouch lining and the underlying tissue showed that TSG-6 treatment resulted in a significant amelioration of the edema and of the tissue destruction induced by the injection of carrageenan (Wisniewski et al., 1996). In the second model, Mindrescu et al. (2000) examined the effect of TSG-6 on collagen-induced arthritis (CIA) in mice. CIA is an autoimmune polyarthritis inducible in susceptible strains of mice, rats, and in primates by immunization with heterologous or homologous collagen II (Trentham et al., 1977; Courtenay et al., 1980; Boissier et al., 1987; Myers et al., 1997; Anthony and Haqqi, 1999). CIA shares a variety of features with human rheumatoid arthritis and it has therefore been widely used as an experimental model for the human disease. Male DBA/1J mice developing collagen-induced arthritis were treated with 12 intraperitoneal doses of recombinant TSG-6 (9 mg/kg daily), beginning 3 days before the expected onset of disease symptoms. Treatment with recombinant TSG-6 protein had a potent and persistent ameliorative effect manifested by decreases in disease incidence, arthritis index, and footpad swelling. Histological examination of affected joints in TSG-6-treated animals revealed little pannus formation and cartilage erosion, signs of which were conspicuous in control mice. Animals treated with recombinant TSG-6 developed significantly reduced levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine collagen II (Mindrescu et al., 2000). These data support the conclusion that TSG-6 is an endogenous modulator of the inflammatory response.

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