Objective. To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated ␣-enolase, in rheumatoid arthritis (RA).Methods. Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, ␣-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated ␣-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay.Results. Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/ l in RA samples, 4.3 ng/ l in SpA samples, and <0.9 ng/ l in OA samples. Two-dimensional electrophoresis provided evidence that the ␣-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample.Conclusion. These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Due to its ability to inhibit pro-metastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse. In mice, high systemic TIMP-1 levels increased the liver susceptibility towards metastasis by triggering the formation of a pre-metastatic niche. This promoted hepatic metastasis independent of origin or intrinsic metastatic potential of tumor cells. High systemic TIMP-1 led to increased hepatic SDF-1 levels, which in turn promoted recruitment of neutrophils to the liver. Both inhibition of SDF-1-mediated neutrophil recruitment and systemic depletion of neutrophils reduced TIMP-1-induced increased liver susceptibility towards metastasis. This indicates a crucial functional role of neutrophils in the TIMP-1-induced pre-metastatic niche. Conclusion Our results identify TIMP-1 as an essential promoter of hepatic pre-metastatic niche formation.

Aggrecanases have been characterized as proteinases that cleave the Glu 373 -Ala 374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0 -9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.Destruction of articular cartilage is a feature of various arthritides, including rheumatoid and osteoarthritis, that results in joint impairment and disability. It is caused primarily by an elevation in proteolytic enzymes that degrade macromolecules of the cartilage extracellular matrix. Aggrecan degradation is initially observed followed by essentially irreversible collagen degradation. The proteinases that are responsible for aggrecan degradation in cartilage are matrix metalloproteinases (MMPs) 4 and "aggrecanases," members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family (1, 2). Aggrecanase activity was first defined as the ability to cleave the Glu 373 -Ala 374 bond in the interglobular domain (IGD) of the aggrecan core protein (3, 4). The first two proteinases shown to be capable of cleaving aggrecan at this site were ADAMTS-4 (aggrecanase 1) (5), and ADAMTS-5 5 (aggrecanase 2) (6). More recently, ADAMTS-1, -8, -9, -15, -16, and -18 were shown to cleave the Glu 373 -Ala 374 bond in the IGD at a high enzyme-substrate ratio (2, 7). Although this bond is also cleaved by MMP-8 (8) and MMP-14 (9) a...

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an A common feature of osteoarthritis (OA) 2 and rheumatoid arthritis is destruction of articular cartilage, which is characterized by a homeostatic imbalance between synthesis and degradation of the extracellular matrix (ECM). The destructive process is believed to be due to elevated activities of proteolytic enzymes that degrade macromolecules of the cartilage ECM such as aggrecan and type II collagen fibrils. Aggrecan is the major proteoglycan in articular cartilage, and it forms large aggregates by interacting with hyaluronan and link protein.Aggrecan monomers consist of a core protein with chondroitin sulfate (CS) and keratan sulfate (KS) polysaccharide chains. The core protein consists of several segments, including an N-terminal globular domain (G1), an interglobular domain (IGD), a second globular domain (G2), a long glycosaminoglycan (GAG) attachment region, including KS-rich and CS-rich (CS-1 and CS-2) regions, and a C-terminal globular domain (G3) (1). Aggrecans are highly hydrated because of their long negatively charged polysaccharide chains. Thus, within the collagen framework, they enable the cartilage to resist mechanical compression as a load-bearing surface. Aggrecan loss is therefore considered to be a crucial initial event in the development of arthritis, which is followed by essentially irreversible collagen degradation (2, 3). This pathological aggrecan degradation in articular cartilage is driven mainly by proteolytic enzymes termed aggrecanases and matrix metalloproteinases (MMPs) (4, 5).The aggrecanases are members of the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family (6). The first two proteinases identified to cleave the aggrecan core protein at the aggrecanase-specific Glu 373 -Ala 374 bond in the IGD were ADAMTS-4 (aggrecanase-1) (7) and ADAMTS-5 (aggrecanase-2) (8). Finding aggrecan fragments cleaved at the Glu 373 -Ala 374 bond in synovial fluids and cartilage from patients with OA and rheumatoid arthritis suggested that aggrecanases play an important role in cartilage destruction (9, 10). They are also the primary enzymes that cleave aggrecan in response to inflammatory cytokines in articular cartilage explant systems (11). Gendron et al. (12) have shown that cleavage of the Glu 373 -Ala 374 bond in interleukin-1-stimulated cartilage explants is blocked by ...