Lack of Tissue Inhibitor of Metalloproteinases-3 Results in an Enhanced Inflammatory Response in Antigen-Induced Arthritis
2005; Elsevier BV; Volume: 166; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)62483-2
ISSN1525-2191
AutoresMandana Mahmoodi, Solmaz Sahebjam, David Smookler, Rama Khokha, John S. Mort,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoTissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-α-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3−/− mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-α was measured by immunoassay. Compared to wild-type animals, Timp3−/− mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-α levels were greatly elevated in the Timp3−/− animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-α by reducing ADAM17 activity. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-α-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3−/− mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-α was measured by immunoassay. Compared to wild-type animals, Timp3−/− mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-α levels were greatly elevated in the Timp3−/− animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-α by reducing ADAM17 activity. The matrix metalloproteinases (MMPs) consist of a large family of proteolytic enzymes that mediate the degradation of many different components of the extracellular matrix.1Cawston T Matrix metalloproteinases and TIMPs: properties and implications for the rheumatic diseases.Mol Med Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar The expression and activity of these enzymes, which are thought to be important in the pathological destruction of joint tissue, are controlled at a number of key points. Various cytokines, growth factors, and other agents can stimulate the synthesis and secretion of these proteases2Mort JS Poole AR Proteases and their inhibitors.in: Klippel JH Crofford LJ Stone JH Weyland CM Primer of the Rheumatic Diseases. Arthritis Foundation, Atlanta2001: 72-81Google Scholar that are expressed as inactive proenzymes. The pro-MMPs require activation through proteolytic processing of the proregion,3Springman EB Angleton EL Birkedal-Hansen H Van Wart HE Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation.Proc Natl Acad Sci USA. 1990; 87: 364-368Crossref PubMed Scopus (620) Google Scholar and the active enzymes can be inhibited by tissue inhibitors of metalloproteinases (TIMPs), which bind to all known active MMPs with a 1:1 stoichiometry. All connective tissues produce members of the TIMP family4Murphy G Willenbrock F Tissue inhibitors of matrix metalloendopeptidases.Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (246) Google Scholar and these molecules play an important role in controlling connective tissue breakdown by blocking the action of active MMPs. Four members of the TIMP family have been described and these share similar secondary and tertiary structures, and are able to inhibit MMPs, although with different potencies. TIMP-1 is produced in response to a variety of external stimuli, such as growth factors and cytokines whereas expression of TIMP-2 is mostly constitutive.5Gomez DE Alonso DF Yoshiji H Thorgeirsson UP Tissue inhibitors of metalloproteinases: structure, regulation and biological functions.Eur J Cell Biol. 1997; 74: 111-122PubMed Google Scholar, 6Overall CM Regulation of tissue inhibitor of matrix metalloproteinase expression.Ann NY Acad Sci. 1994; 732: 51-64Crossref PubMed Scopus (84) Google Scholar However, TIMP-3 is unique in many ways. Unlike TIMP-1 and TIMP-2, which are present in soluble forms, TIMP-3 is bound within the extracellular matrix.7Leco KJ Khokha R Pavloff N Hawkes SP Edwards DR Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinct pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar, 8Yu WH Yu SC Meng Q Brew K Woessner JF TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix.J Biol Chem. 2000; 275: 31226-31232Crossref PubMed Scopus (272) Google Scholar It is an inhibitor of several ADAM's family members such as ADAM-12S,9Loechel F Fox JW Murphy G Albrechtsen R Wewer UM ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3.Biochem Biophys Res Commun. 2000; 278: 511-515Crossref PubMed Scopus (279) Google Scholar and tumor necrosis factor (TNF)-α convertase (TACE, ADAM-17)10Amour A Slocombe PM Webster A Butler M Knight CG Smith BJ Stephens PE Shelley C Hutton M Knäuper V Docherty AJP Murphy G TNF-α converting enzyme (TACE) is inhibited by TIMP-3.FEBS Lett. 1998; 435: 39-44Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 11Lee MH Knäuper V Becherer JD Murphy G Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-α convertase.Biochem Biophys Res Commun. 2001; 280: 945-950Crossref PubMed Scopus (52) Google Scholar that are not inhibited by other TIMPs. In addition, it is an inhibitor of aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5).12Kashiwagi M Tortorella M Nagase H Brew K TIMP-3 is a potent inhibitor of ADAM-TS4 (aggrecanase 1) and ADAM-TS5 (aggrecanase 2).J Biol Chem. 2001; 276: 12501-12504Crossref PubMed Scopus (445) Google Scholar, 13Hashimoto G Aoki T Nakamura H Tanzawa K Okada Y Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4).FEBS Lett. 2001; 494: 192-195Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar TIMP-3 has also been shown to inhibit cell shedding of l-selectin,14Borland G Murphy G Ager A Tissue inhibitor of metalloproteinases-3 inhibits shedding of L-selectin from leukocytes.J Biol Chem. 1999; 274: 2810-2815Crossref PubMed Scopus (109) Google Scholar syndecan-1, and syndecan-4,15Fitzgerald ML Wang Z Park PW Murphy G Bernfield M Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.J Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (350) Google Scholar and interleukin-616Hargreaves PG Wang F Antcliff J Murphy G Lawry J Russell RG Croucher PI Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor.Br J Haematol. 1998; 101: 694-702Crossref PubMed Scopus (72) Google Scholar and M-CSF receptors.17Rovida E Paccagnini A Del Rosso M Peschon J Dello Sbarba P TNF-α-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation.J Immunol. 2001; 166: 1583-1589PubMed Google Scholar It is also the only member of the TIMP family in which mutations are implicated in human pathology, namely Sorby's fundus dystrophy, a degenerative eye disease.18Weber BHF Vogt G Pruett RC Stöhr H Felbor U Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy.Nat Genet. 1994; 8: 352-356Crossref PubMed Scopus (552) Google Scholar TIMP-3 has also been shown to be proapoptotic in both normal and cancer cell lines.19Baker AH Zaltsman AB George SJ Newby AC Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis.J Clin Invest. 1998; 101: 1478-1487Crossref PubMed Scopus (420) Google Scholar, 20Ahonen M Baker AH Kahari VM Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells.Cancer Res. 1998; 58: 2310-2315PubMed Google Scholar Although TIMP-3-deficient mice appear phenotypically normal and are fertile, they were found to develop alveolar air space enlargement with a concomitant impaired capacity for gas exchange,21Leco KJ Waterhouse P Sanchez OH Gowing KLM Poole AR Wakeham TW Mak TW Khokha R Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3).J Clin Invest. 2001; 108: 817-829Crossref PubMed Scopus (244) Google Scholar accelerated mammary gland apoptosis,22Fata JE Leco KJ Voura EB Yu HY Waterhouse P Murphy G Moorehead RA Khokha R Accelerated apoptosis in the Timp-3-deficient mammary gland.J Clin Invest. 2001; 108: 831-841Crossref PubMed Scopus (147) Google Scholar and cardiac dysfunction.23Fedak PWM Smookler DS Kassiri Z Ohno N Leco KJ Verma S Mickle DAG Watson KL Hojilla CV Cruz W Weisel RD Li RK Khokha R TIMP-3 deficiency leads to dilated cardiomyopathy.Circulation. 2004; 110: 2401-2409Crossref PubMed Scopus (141) Google Scholar Very recently, in line with the observations reported in the present study, they also show increased susceptibility to hepatic inflammation.24Mohammed FF Smookler DS Taylor SE Fingleton B Kassiri Z Sanchez OH English JL Matrisian LM Au B Yeh WC Khokha R Abnormal TNF activity in Timp3−/− mice leads to chronic hepatic inflammation and failure of liver regeneration.Nat Genet. 2004; 36: 969-977Crossref PubMed Scopus (241) Google Scholar These data suggest that TIMP-3 plays important roles in physiological and pathological processes. The special characteristics of TIMP-3 invite a more thorough focus on its biological roles. Because TIMP-3 is the only member of the TIMP family that is retained by the extracellular matrix, possesses a unique ability to inhibit aggrecanases-1 and -2 as well as TACE, and is present in cartilage and synovium,25Su S Grover J Roughley PJ DiBattista JA Martel-Pelletier J Pelletier JP Zafarullah M Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints.Rheumatol Int. 1999; 18: 183-191Crossref PubMed Scopus (64) Google Scholar, 26Apte SS Hayashi K Seldin MF Mattei M-G Hayashi M Olsen BR Gene encoding a novel murine tissue inhibitor of metalloproteinases (TIMP), TIMP-3, is expressed in developing mouse epithelia, cartilage, and muscle, and is located on mouse chromosome 10.Dev Dyn. 1994; 200: 177-197Crossref PubMed Scopus (127) Google Scholar it seemed likely that it might play a role in arthritis development. Also, it has been shown recently that TIMP-3 inhibits the breakdown of aggrecan in stimulated cartilage cultures, through inhibition of aggrecanase, the synthesis of which is induced by catabolic factors.27Gendron C Kashiwagi M Hughes C Caterson B Nagase H TIMP-3 inhibits aggrecanase-mediated glycosaminoglycan release from cartilage explants stimulated by catabolic factors.FEBS Lett. 2003; 555: 431-436Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The loss of aggrecan is considered to be a crucial early event in the pathogenesis of arthritis.28Arner EC Aggrecanase-mediated cartilage degradation.Curr Opin Pharmacol. 2002; 2: 322-329Crossref PubMed Scopus (141) Google Scholar, 29Nagase H Kashiwagi M Aggrecanases and cartilage matrix degradation.Arthritis Res Ther. 2003; 5: 94-103Crossref PubMed Google Scholar As an approach to better understand the functions of TIMP-3 in joint disease, we used an antigen-induced arthritis model in Timp3 knockout animals. In the present study we investigated the histochemical and immunohistological differences between null and matching wild-type mice and show that the major effect of TIMP-3 deficiency is increased production of TNF-α and concomitant joint inflammation, presumably due to the enhanced TACE activity. The production of Timp3−/− mice in the C57BL/6 background has been described previously.21Leco KJ Waterhouse P Sanchez OH Gowing KLM Poole AR Wakeham TW Mak TW Khokha R Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3).J Clin Invest. 2001; 108: 817-829Crossref PubMed Scopus (244) Google Scholar Wild-type C57BL/6 mice, used as controls, were from Jackson Laboratories (Bar Harbor, ME). Animals were given a standard laboratory diet and tap water ad libitum. Protocols for use of animals were approved by the local and McGill University animal care committees. Mice were injected subcutaneously with 100 μl of an emulsion containing 100 μg of methylated bovine serum albumin (mBSA), prepared as described by Mandell and Hershey,30Mandell FD Hershey AD A fractionating column for analysis of nucleic acids.Anal Biochem. 1960; 1: 66-77Crossref PubMed Scopus (510) Google Scholar in normal saline and an equal volume of Freund's complete adjuvant (Life Technologies, Inc., Gaithersburg, MD) over two sites in the flank. Heat-killed Bordetella pertussis (2 × 109 cells i.p.; Lee Laboratories, Grayson, GA) was administered as an additional adjuvant. Seven days later, booster injections of 100 μg of mBSA in Freund's incomplete adjuvant were administered subcutaneously, divided over two sites in the neck region, with an additional 2 × 109 killed B. pertussis intraperitoneally. Arthritis was induced on day 21 by an intra-articular injection of 60 μg of mBSA in 6 μl of normal saline into the right knee. The left knee, which received 6 μl of normal saline, was used as a control. Mice were sacrificed at days 3, 7, and 14 after intra-articular injection. Knee joints were dissected and then placed in periodate-lysine-paraformaldehyde fixative31McLean IW Nakane PK Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy.J Histochem Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3204) Google Scholar and decalcified, either by treatment in 10% formic acid for 24 hours followed by paraffin embedding (Oxford Labware, St. Louis, MO), or in 10% ethylenediamine tetraacetic acid (w/v in 0.1 mol/L Tris-HCl, pH 7.4) for 3 weeks, followed by rinsing in phosphate-buffered saline (PBS), infiltration in a 2:1 v/v of sucrose (20% w/v in PBS) and OCT compound (Sakura, Torrance, CA), then embedding in OCT alone, followed by snap-freezing.32Davoli MA Lamplugh L Beauchemin A Chan K Mordier S Mort JS Murphy G Docherty AJ Leblond CP Lee ER Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats aged 0–21 days: II. Two proteinases, gelatinase A and collagenase-3, are implicated in the lysis of collagen fibrils.Dev Dyn. 2001; 222: 71-88Crossref PubMed Scopus (41) Google Scholar In the case of OCT-embedded tissues, sections were cut with a cryostat at −20°C and were stored at −20°C for further use. In this study, we focused on the tibio-femoral compartment. Paraffin sections were deparaffinized with xylene and solutions of decreasing ethanol percentage. Staining was with 0.5% hematoxylin for 10 minutes, 0.02% Fast Green for 4 minutes (Chroma-Gesellschaft Schmid GMBH and Co., Stuttgart-Unterturkheim, Germany), acetic acid for 1 minute, and Safranin O (BDH Laboratory Supplies, Poole, UK) for 6 minutes. Proteoglycan depletion was determined by the decreased uptake of Safranin O dye. The slides were scored in a blinded manner by two different observers. Each slide was graded for three different parameters: 1) hyperplasia of the lining layer of the synovium (mostly due to proliferation of the fibroblast-like synoviocytes) and infiltration of the synovial membrane with predominantly mononuclear cells; 2) pannus formation and synovial invasion of the cartilage and cartilage destruction; and 3) Safranin O loss, each on a scale of 0 to 3.33Petrow PK Thoss K Katenkamp D Brauer R Adoptive transfer of susceptibility to antigen-induced arthritis into severe combined immunodeficient (SCID) mice: role of CD4+ and CD8+ T cells.Immunol Invest. 1996; 25: 341-353Crossref PubMed Scopus (33) Google Scholar, 34Ospelt C Neidhart M Gay RE Gay S Synovial activation in rheumatoid arthritis.Front Biosci. 2004; 9: 2323-2334Crossref PubMed Scopus (88) Google Scholar Safranin O loss was assessed (where 0, 1, 2, and 3 represent no loss, 30% loss, 60% loss, and 100% loss of red staining, respectively) by the method described by Van Meurs and colleagues.35van Meurs JBJ van Lent PLEM Holthuysen AEM Singer II Bayne EK van den Berg WB Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (115) Google Scholar A final score was assigned for each animal by calculating the sum of the above values. Each animal was scored at days 3, 7, and 14, after intra-articular injection, for joint swelling and difficulty in moving the joint, on a scale of 0 to 3. The sum of these scores was used as an arthritis score for each animal where 0 represents the normal joint and 6 the most severe arthritis. Decalcified sections were fixed with 4% formaldehyde and then digested with proteinase-free chondroitinase ABC (0.25 U/ml Tris acetate, pH 7.3; ICN Biomedicals, Aurora, OH) for 1 hour at 37°C to remove glycosaminoglycan chains. Subsequently, sections were treated with 0.3% H2O2 for 30 minutes followed by incubation with 1.5% normal goat or normal donkey serum (depending on the antibody being used) for 20 minutes. The sections were then incubated with primary antibody raised against either VDIPEN or NITEGE36Sztrolovics R Alini M Roughley PJ Mort JS Aggrecan degradation in human intervertebral disc and articular cartilage.Biochem J. 1997; 326: 235-241Crossref PubMed Scopus (215) Google Scholar peptides for 24 hours. Sections were then incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) or donkey anti-goat (Jackson ImmunoResearch, West Grove, PA) and antigens were detected using avidin-peroxidase staining (Vectastain Elite ABC kit and DAB kits, Vector Laboratories) and counterstained with hematoxylin. Staining was scored in a blinded manner and graded on a scale of 0 to 3: 0, no staining; 1, minor staining; 2, marked staining; and 3, maximal staining defined as staining of ∼50% of the total cartilage layer as previously described by Van Meurs and colleagues.35van Meurs JBJ van Lent PLEM Holthuysen AEM Singer II Bayne EK van den Berg WB Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (115) Google Scholar Mice were bled 1 hour after bacterial injection and serum-assayed for mouse TNF-α using the OptEIASet ELISA kit (Pharmingen, San Diego, CA). Statistical significance between scores was determined using the Mann-Whitney test. TNF-α levels were analyzed using the Student's t-test to compare mean values. A general inflammatory arthritis model involving direct injection of antigen (mBSA) into the joint of the previously sensitized animal was used because the type II collagen-37Banerjee S David CS Immunogentics of type II collagen-induced arthritis in mice.in: Farid NR The Immunogenetics of Autoimmune Diseases. vol. II. CRC Press, Boca Raton1991: 119-134Google Scholar and aggrecan38Mikecz K Glant TT Poole AR Immunity to cartilage proteoglycans in Balb/c mice with progressive polyarthritis and ankylosing spondylitis induced by injection of human cartilage proteoglycan.Arthritis Rheum. 1987; 30: 306-318Crossref PubMed Scopus (141) Google Scholar-induced models, which do not require such direct intervention, are ineffective in the C57BL/6 mouse strain. After intra-articular injection of mBSA, the joints of both Timp3−/− and wild-type mice showed arthritic changes, while the contralateral joints showed no swelling or other pathology. In the wild-type animals clinical symptoms tended to be at their highest between days 2 to 3 after intra-articular injection with joint swelling, difficulty using the affected limb, and occasional redness and warmth over the joint as reported by others.39Lewthwaite J Blake S Hardingham T Foulkes R Stephens S Chaplin L Emtage S Catterall C Short S Nesbitt A Role of TNF alpha in the induction of antigen induced arthritis in the rabbit and the anti-arthritic effect of species specific TNF alpha neutralising monoclonal antibodies.Ann Rheum Dis. 1995; 54: 366-374Crossref PubMed Scopus (29) Google Scholar By day 7, these symptoms begin to resolve. In Timp3−/− mice, swelling was much more apparent than for age- and sex-matched wild-type mice at days 2 and 3 (Figure 1). Differences in the use of the affected knee were also noticed in the same period. When the clinical scores were combined, a clear difference was seen between the two groups in the first few days of arthritis induction, however with time these differences became much less prominent, with the inflammation resolving and the clinical scores for both wild-type and Timp3−/− declining to almost normal values (Figure 2).Figure 2Arthritis score during first 2 weeks after intra-articular injection of mBSA. Arthritis severity in Timp3−/− mice was significantly higher than in wild-type mice at day 3 (n = 32, range 2 to 6 for Timp3−/− and 2 to 5 for wild-type; *P = 0.001) but not at days 7 (n = 25, range 1 to 4 for Timp3−/− and 1 to 4 for wild-type) and 14 (n = 13, range 0 to 2 for Timp3−/− and 0 to 1 for wild-type).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of histological sections demonstrated higher scores in Timp3−/− mice than wild-type on days 3 and 7 after arthritis induction (Figure 3). There was a significant difference in synovial inflammation especially on days 3 and 7 (Figure 4). When the scores for synovial infiltration with mononuclear cells and invasion, and loss of Safranin O staining are compared individually, the difference is mainly in synovial infiltration (Table 1). The differences in loss of Safranin O staining and invasion were not statistically significant, so it seems that the higher sum of scores in Timp3−/− was mainly due to increases in synovial inflammation.Figure 4Histological analysis of arthritic joints of wild-type (A, C, and E) and Timp3−/− (B, D, and F) mice on day 3 (A and B), day 7 (C and D), and day 14 (E and F). Safranin O and hematoxylin staining. Original magnifications, ×48.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Histological Grading—Individual ComponentsDay 3Day 7Day 14Synovial inflammationTimp-3−/−3 (3) P = 0.0183 (3) P = 0.0141.86 (1–3)Wild type1.33 (1–2)1.83 (1–2)2.5 (2–3)Safranin O lossTimp-3−/−1 (0–2)2.16 (1–3) P = 0.122 (0–3)Wild type0 (0)0.83 (0–3)1.8 (0–3)Synovial invasionTimp-3−/−0.25 (0–1)1.5 (0–3) P = 0.251.42 (1–3)Wild type0 (0)0.33 (0–1)1.66 (0–2)Values represent averages and ranges of values obtained. Only results for synovial inflammation on days 3 and 7 showed significant differences between the Timp-3−/− and wild type animals. n = 7, 12, and 13 for days 3, 7, and 14, respectively. Open table in a new tab Values represent averages and ranges of values obtained. Only results for synovial inflammation on days 3 and 7 showed significant differences between the Timp-3−/− and wild type animals. n = 7, 12, and 13 for days 3, 7, and 14, respectively. The degradation of the major cartilage proteoglycan, aggrecan, can be evaluated by immunohistochemical analysis using anti-neoepitope antibodies specific for the cleavage products remaining in the cartilage matrix after protease activity. Antibodies recognizing two different, well-characterized C-terminal epitopes were used. The epitope VDIPEN is produced by MMPs40Fosang AJ Neame PJ Last K Hardingham TE Murphy G Hamilton JA The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.J Biol Chem. 1992; 267: 19470-19474Abstract Full Text PDF PubMed Google Scholar but can also be generated in a multicleavage mechanism by the lysosomal cysteine protease cathepsin B41Mort JS Magny M-C Lee ER Cathepsin B: an alternative protease for the generation of an aggrecan "metalloproteinase" cleavage neoepitope.Biochem J. 1998; 335: 491-494Crossref PubMed Scopus (88) Google Scholar and as a minor cleavage product by ADAMTS-4,42Westling J Fosang AJ Last K Thompson VP Tomkinson KN Hebert T McDonagh T Collins-Racie LA LaVallie ER Morris EA Sandy JD ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain.J Biol Chem. 2002; 277: 16059-16066Crossref PubMed Scopus (78) Google Scholar whereas the epitope NVTEGE is only generated by aggrecanase action.43Sandy JD Neame PJ Boynton RE Flannery CR Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain.J Biol Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar Because these antibodies recognize the new C-termini present on the G1 region of aggrecan that remains localized in the extracellular matrix, and the NVTEGE epitope is located downstream of the VDIPEN epitope, the latter is expected to accumulate at the expense of the former if both activities are present. Although cleavage products were observed in Timp3−/− and wild-type animals, no significant difference was seen between the two groups for the VDIPEN epitope (Figure 5) or for the NVTEGE epitope, where much less staining was observed (data not shown). These findings agree with the lack of significant differences in loss of Safranin O staining. As shown previously by van Meurs and colleagues,35van Meurs JBJ van Lent PLEM Holthuysen AEM Singer II Bayne EK van den Berg WB Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (115) Google Scholar production of the VDIPEN epitope was closely related to loss of Safranin O staining. Circulating TNF-α levels were measured in the serum using an enzyme-linked immunosorbent assay. TNF-α levels in the blood are known to rise immediately after subcutaneous injection of inflammatory stimuli, returning to near base line after 3 hours.44Chorinchath BB Kong LY Mao L McCallum RE Age-associated differences in TNF-α and nitric oxide production in endotoxic mice.J Immunol. 1996; 156: 1525-1530PubMed Google Scholar One hour after subcutaneous injection of mBSA and intraperitoneal injection with killed B. pertussis we found a dramatic difference in TNF-α levels in sera (Figure 6). Timp3−/− mice had significantly higher TNF-α levels than wild-type animals (7500 compared to 2600 pg/ml, respectively). Compared to the other members of the TIMP family, TIMP-3 has several unique properties. In particular, it is the only member of TIMP family that is bound to the extracellular matrix, and is able to inhibit members of the ADAM and ADAMTS families. A protective role of TIMP-3 against aggrecan degradation in cartilage cultured, in vitro, was recently shown by Gendron and colleagues27Gendron C Kashiwagi M Hughes C Caterson B Nagase H TIMP-3 inhibits aggrecanase-mediated glycosaminoglycan release from cartilage explants stimulated by catabolic factors.FEBS Lett. 2003; 555: 431-436Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar where the effect of TIMP-1, -2, and -3 on degradation of aggrecan, as monitored by GAG release in bovine nasal and porcine articular cartilage stimulated by interleukin-1α or retinoic acid, was investigated. Only treatment with TIMP-3 [using the N-terminal inhibitory domain (N-TIMP-3)], showed a decrease in aggrecan release in a dose-dependent manner, whereas both TIMP-1 and TIMP-2 failed to show such an effect. Similar results would be expected in cartilage cultured under TNF-α stimulation but substantially higher levels of TNF-α are required to obtain comparable anabolic effects to those of interleukin-145Saklatvala J Tumour necrosis factor α stimulates resorption and inhibits synthesis of proteoglycan in cartilage.Nature. 1986; 322: 547-549Crossref PubMed Scopus (655) Google Scholar so it is not clear whether the levels of TNF-α present in the mouse joints in the present study would have had a major influence on aggrecan degradation. Timp3−/− mice develop various patholog
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