Adenoviral Gene Transfer of Interleukin-1 in Combination with Oncostatin M Induces Significant Joint Damage in a Murine Model
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64330-1
ISSN1525-2191
AutoresAndrew D. Rowan, Hui Wang, Tim E. Cawston, Carl D. Richards,
Tópico(s)Signaling Pathways in Disease
ResumoOncostatin M (OSM) is an interleukin (IL)-6 family cytokine that we have previously shown can synergize with a number of proinflammatory cytokines to promote the release of collagen from cartilage in explant culture. However, the effects of this potent cytokine combination in vivo are not known. Using adenoviral gene transfer, we have overexpressed murine IL-1 (AdmIL-1) and murine OSM (AdmOSM) intraarticularly in the knees of C57BL/6 mice. Histological analyses indicated marked synovial hyperplasia and inflammatory cell infiltration for both AdmIL-1 and AdmOSM but not in control joints. This inflammation was even more pronounced for the AdmIL-1+AdmOSM combination with evidence of cartilage and bone destruction. Significant loss of both proteoglycan and collagen was also seen for this combination, and immunohistochemistry revealed an increased expression of matrix metalloproteinases (MMPs) with decreased tissue inhibitor of metalloproteinases (TIMPs) in both articular cartilage and synovium. Similar expression profiles for MMPs/TIMPs were found in IL-1+OSM-stimulated human articular chondrocytes. Taken together, these data confirm that, in vivo, OSM can exacerbate the effects of IL-1 resulting in inflammation and tissue destruction characteristic of that seen in rheumatoid arthritis. We provide further evidence to implicate the up-regulation of MMPs as a key factor in joint pathology. Oncostatin M (OSM) is an interleukin (IL)-6 family cytokine that we have previously shown can synergize with a number of proinflammatory cytokines to promote the release of collagen from cartilage in explant culture. However, the effects of this potent cytokine combination in vivo are not known. Using adenoviral gene transfer, we have overexpressed murine IL-1 (AdmIL-1) and murine OSM (AdmOSM) intraarticularly in the knees of C57BL/6 mice. Histological analyses indicated marked synovial hyperplasia and inflammatory cell infiltration for both AdmIL-1 and AdmOSM but not in control joints. This inflammation was even more pronounced for the AdmIL-1+AdmOSM combination with evidence of cartilage and bone destruction. Significant loss of both proteoglycan and collagen was also seen for this combination, and immunohistochemistry revealed an increased expression of matrix metalloproteinases (MMPs) with decreased tissue inhibitor of metalloproteinases (TIMPs) in both articular cartilage and synovium. Similar expression profiles for MMPs/TIMPs were found in IL-1+OSM-stimulated human articular chondrocytes. Taken together, these data confirm that, in vivo, OSM can exacerbate the effects of IL-1 resulting in inflammation and tissue destruction characteristic of that seen in rheumatoid arthritis. We provide further evidence to implicate the up-regulation of MMPs as a key factor in joint pathology. Rheumatoid arthritis (RA) is an inflammatory disease in which extensive cellular infiltration into the synovium leads to hyperplasia of the tissue, finally resulting in both cartilage and bone destruction. The involvement of proinflammatory cytokines, in particular interleukin (IL)-1 and tumor necrosis factor-α (TNF-α), has been found to play a key role in promoting disease pathogenesis.1Dayer JM Bresnihan B Targeting interleukin-1 in the treatment of rheumatoid arthritis.Arthritis Rheum. 2002; 46: 574-578Crossref PubMed Scopus (84) Google Scholar, 2Manicourt DH Poilvache P van Egeren A Devogelaer JP Lenz ME Thonar EJ Synovial fluid levels of tumor necrosis factor-α and oncostatin M correlate with levels of markers of the degradation of crosslinked collagen and cartilage aggrecan in rheumatoid arthritis but not in osteoarthritis.Arthritis Rheum. 2000; 43: 281-288Crossref PubMed Scopus (104) Google Scholar IL-1 is a pleiotropic cytokine with multiple biological actions including the migration of inflammatory cells, synovial cell proliferation, and production of cytokines and matrix metalloproteinases (MMPs).1Dayer JM Bresnihan B Targeting interleukin-1 in the treatment of rheumatoid arthritis.Arthritis Rheum. 2002; 46: 574-578Crossref PubMed Scopus (84) Google Scholar It is overexpressed in RA cartilage and synovial membranes, and raised levels of IL-1 in synovial fluids and sera correlate with disease activity and cartilage/bone destruction in RA.3Chu CQ Field M Allard S Abney E Feldmann M Maini RN Detection of cytokines at the cartilage/pannus junction in patients with rheumatoid arthritis: implications for the role of cytokines in cartilage destruction and repair.Br J Rheumatol. 1992; 31: 653-661Crossref PubMed Scopus (209) Google Scholar, 4Rooney M Symons JA Duff GW Interleukin 1β in synovial fluid is related to local disease activity in rheumatoid arthritis.Rheumatol Int. 1990; 10: 217-219Crossref PubMed Scopus (82) Google Scholar, 5Eastgate JA Symons JA Wood NC Grinlinton FM di Giovine FS Duff GW Correlation of plasma interleukin 1 levels with disease activity in rheumatoid arthritis.Lancet. 1988; 2: 706-709Abstract PubMed Scopus (449) Google Scholar Anti-IL-1 therapy in animal models and in humans with RA has been shown to significantly reduce arthritis incidence, inflammation, and joint destruction,1Dayer JM Bresnihan B Targeting interleukin-1 in the treatment of rheumatoid arthritis.Arthritis Rheum. 2002; 46: 574-578Crossref PubMed Scopus (84) Google Scholar, 6van de Loo FA van den Berg WB Gene therapy for rheumatoid arthritis: lessons from animal models, including studies on interleukin-4, interleukin-10, and interleukin-1 receptor antagonist as potential disease modulators.Rheum Dis Clin North Am. 2002; 28: 127-149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar suggesting that the effector pathways of joint damage are IL-1-mediated. 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Destruction of cartilage and bone is proteinase-mediated, and the MMPs are thought to play a critical role. 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Anti-MMP-13, TIMP-1, and TIMP-2 polyclonal antibodies were raised in-house.7Cawston TE Curry VA Summers CA Clark IM Riley GP Life PF Spaull JR Goldring MB Koshy PJT Rowan AD Shingleton WD The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint.Arthritis Rheum. 1998; 41: 1760-1771Crossref PubMed Scopus (174) Google Scholar Anti-MMP-8 polyclonal antibodies and recombinant human OSM were from R&D Systems (Abingdon, UK), anti-MMP-3 polyclonal was a kind gift from H. Nagase (Kennedy Institute, London, UK), while anti-type II collagen was from Southern Biotechnology Associates, Inc. (Birmingham, AL). Human IL-1α and OSM were generous gifts from GlaxoSmithKline (Stevenage, UK) and J. Heath (Birmingham University, Birmingham, UK), respectively. Replication-defective recombinant adenovirus, engineered to overexpress murine IL-1β (AdmIL-1) and murine OSM (AdmOSM), as well as Add170 (used as control), were as described previously.33Kolb M Margetts PJ Anthony DC Pitossi F Gauldie J Transient expression of IL-1β induces acute lung injury and chronic repair leading to pulmonary fibrosis.J Clin Invest. 2001; 107: 1529-1536Crossref PubMed Scopus (614) Google Scholar, 34Kerr C Langdon C Graham F Gauldie J Hara T Richards CD Adenovirus vector expressing mouse oncostatin M induces acute-phase proteins and TIMP-1 expression in vivo in mice.J Interferon Cytokine Res. 1999; 19: 1195-1205Crossref PubMed Scopus (28) Google Scholar, 35Bett AJ Haddara W Prevec L Graham FL An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.Proc Natl Acad Sci USA. 1994; 91: 8802-8806Crossref PubMed Scopus (690) Google Scholar VECTASTAIN Elite ABC kits PK 6102 and 6106 were from Vector Laboratories (Burlingame, CA). All other reagents were commercially available analytical grade obtained from Merck (Poole, UK) or Sigma (Dorset, UK). C57BL/6 mice were housed until 12 to 14 weeks of age. Intraarticular injections of adenovirus vectors (1 × 106 or 5 × 106 pfu/joint for each vector) were as previously described.10Langdon C Kerr C Hassen M Hara T Arsenault AL Richards CD Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo.Am J Pathol. 2000; 157: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Briefly, anesthesia was maintained with isofluorane, knees were swabbed with 70% ethanol and a 5-μl volume (treatment) was injected into the synovial space. The contralateral knee was treated with Add170 (control). Each treatment included 4 joints and animals were sacrificed 7 days after administration. To compare the effects of the AdmIL-1+AdmOSM combination to either vector alone, Add170 was used such that the total dose of vector was equivalent for each treated knee (ie, a total of 2 × 106 or 1 × 107 pfu/joint). A further 4 mice were injected with PBS (vehicle) only into the right knee and the contralateral knee was left untreated. This group was used to confirm that Add170-treated joints were unchanged from either PBS or untreated joints as found previously.10Langdon C Kerr C Hassen M Hara T Arsenault AL Richards CD Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo.Am J Pathol. 2000; 157: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11de Hooge AS van de Loo FA Bennink MB de Jong DS Arntz OJ Lubberts E Richards CD van den Berg WB Adenoviral transfer of murine oncostatin M elicits periosteal bone apposition in knee joints of mice, despite synovial inflammation and up-regulated expression of interleukin-6 and receptor activator of nuclear factor-κ B ligand.Am J Pathol. 2002; 160: 1733-1743Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Previous studies have validated this adenoviral delivery system as an effective means of cytokine overexpression in synovial tissues.10Langdon C Kerr C Hassen M Hara T Arsenault AL Richards CD Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo.Am J Pathol. 2000; 157: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11de Hooge AS van de Loo FA Bennink MB de Jong DS Arntz OJ Lubberts E Richards CD van den Berg WB Adenoviral transfer of murine oncostatin M elicits periosteal bone apposition in knee joints of mice, despite synovial inflammation and up-regulated expression of interleukin-6 and receptor activator of nuclear factor-κ B ligand.Am J Pathol. 2002; 160: 1733-1743Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 34Kerr C Langdon C Graham F Gauldie J Hara T Richards CD Adenovirus vector expressing mouse oncostatin M induces acute-phase proteins and TIMP-1 expression in vivo in mice.J Interferon Cytokine Res. 1999; 19: 1195-1205Crossref PubMed Scopus (28) Google Scholar, 36Goossens PH Huizinga TW Adenoviral-mediated gene transfer to the synovial tissue.Clin Exp Rheumatol. 2002; 20: 415-419PubMed Google Scholar Knee joints were dissected separately from the limbs and fixed overnight with 7% formaldehyde in PBS (pH 7.4). Subsequently, joints were decalcified in 10% ethylenediaminetetraacetate (EDTA) in PBS for 10 days, and wax-embedded. Sections (5 μm) were stained with hematoxylin & eosin (H&E), or safranin O (for proteoglycans) with hematoxylin counterstaining, or analyzed using immunohistochemistry. H&E sections were graded based on a pathological scoring system essentially as described.37Staite ND Richard KA Aspar DG Franz KA Galinet LA Dunn CJ Induction of an acute erosive monarticular arthritis in mice by interleukin-1 and methylated bovine serum albumin.Arthritis Rheum. 1990; 33: 253-260Crossref PubMed Scopus (64) Google Scholar Formalin-fixed paraffin sections were deparaffinized, rehydrated, and treated with 0.1% trypsin at 37°C for 20 minutes or microwave boiling for 5 minutes, followed by 3% H2O2 for 15 minutes. Sections were blocked (1.5% normal sheep or rabbit serum) for 30 minutes, and then incubated with primary antibody for 90 minutes at room temperature. After sequential incubations with biotinylated secondary antibody and avidin-biotin complex (both for 30 minutes), the signal was developed using 3, 3′-diaminobenzidine tetrahydrochloride chromogenic solution (DAKO, Ely, UK) with hematoxylin counterstaining. The density of safranin O and type II collagen staining in articular cartilage was analyzed by Image-Pro Plus (MEDIA Cybernetics, Silver Spring, MD). Images of stained sections were captured using a JVC 3-CCD color video camera (Victor Company of Japan, Ltd., Tokyo, Japan) and displayed on a computer monitor. For each joint, four measurements of articular cartilage apposition (two on the femoral side and two on the tibial side) were performed in a standardized manner. Bonferroni's pairwise multiple comparison test or the Mann-Whitney U-test were used where appropriate. Values of P ≤ 0.05 were considered significant. Chondrocytes were isolated from bovine nasal cartilage and human articular cartilage as previously described.38Shingleton WD Ellis AJ Rowan AD Cawston TE Retinoic acid combines with interleukin-1 to promote the degradation of collagen from bovine nasal cartilage: matrix metalloproteinases-1 and -13 are involved in cartilage collagen breakdown.J Cell Biochem. 2000; 79: 519-531Crossref PubMed Scopus (46) Google Scholar Human tissue was obtained following joint replacement surgery with full ethical approval. Primary cells were incubated in supplemented Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum at 1 × 106 cells/T-25 cm2 tissue culture flask.38Shingleton WD Ellis AJ Rowan AD Cawston TE Retinoic acid combines with interleukin-1 to promote the degradation of collagen from bovine nasal cartilage: matrix metalloproteinases-1 and -13 are involved in cartilage collagen breakdown.J Cell Biochem. 2000; 79: 519-531Crossref PubMed Scopus (46) Google Scholar When the cells reached 80% to 90% confluence, the medium was removed and replaced with serum-free medium overnight, and then cultured in DMEM containing test cytokines for the indicated time periods. Total cellular RNA was isolated and purified using the RNeasy kit (Qiagen, Crawley, UK). Equal amounts (15 to 20 μg) of RNA were fractionated on 1% agarose gels as described.17Rowan AD Koshy PJT Shingleton WD Degnan BA Heath J Vernallis AB Spaull JR Life PF Hudson K Cawston TE Synergistic effects of gp130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.Arthritis Rheum. 2001; 44: 1620-1632Crossref PubMed Scopus (122) Google Scholar Following transfer of RNA to a GeneScreen Plus membrane (Perkin Elmer Life Sciences, Boston, MA) and UV cross-linking, blots were pre-hybridized for 2 to 3 hours and then probed for 18 hours at 42°C with full-length cDNA probes labeled by random priming with α[32P]dCTP. Radio-labeled cDNA-mRNA hybrids were visualized by phosphorimager (Molecular Dynamics, Chesham, UK). The signal for GAPDH was used to normalize for RNA loading. Sections from all treated joints were stained with H&E and the morphology assessed. The contralateral control sections (Add170 treated) showed no evidence of joint damage (Figure 1A) as previously described.10Langdon C Kerr C Hassen M Hara T Arsenault AL Richards CD Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo.Am J Pathol. 2000; 157: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar AdmIL-1 alone caused a mild to moderate inflammatory response in the joints, involving diffuse mononuclear cell infiltration with evidence of moderate synovial hyperplasia, pannus formation and cartilage and bone erosions (Figure 1B), similar to that seen for AdmOSM (Figure 1C) alone.10Langdon C Kerr C Hassen M Hara T Arsenault AL Richards CD Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo.Am J Pathol. 2000; 157: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar With the AdmIL-1+AdmOSM combination, more severe pathological changes were observed compared to each cytokine alone (see Figure 1D). These changes were even more pronounced in animals receiving a five-fold higher adenoviral titer of AdmIL-1+AdmOSM (Figure 2). Pronounced synovial hyperplasia and pannus formation were evident, as were indications of cartilage erosion (Figure 2, A and B). Severe peri- and intraarticular soft tissue inflammation with a marked (inflammatory) cell infiltration into the synovial space were also seen (Figure 2, C and D). Within the inflamed synovium there was evidence of angiogenesis (Figure 2E) and there appeared to be an accumulation of proliferative cells ju
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