Objective. This study explored the therapeutic effect of interleukin‐1 receptor antagonist (IL‐1Ra), administered by gene transfer, on the progression of osteoarthritic (OA) lesions in an experimental dog model. Methods. Seventeen mature mongrel dogs were divided into 3 groups. Group 1 (n = 7) had an anterior cruciate ligament (ACL) section of the right knee through a stab wound incision. Groups 2 and 3 (n = 5 per group), had an ACL section of the right knee and partial synovectomy of the left knee. Each dog's synovium was subjected to enzymatic digestion, and the synovial fibroblasts were propagated in monolayer culture. Synovial cells from each dog were transduced in vitro using the retrovirus MFG with either the Escherichia coli β‐galactosidase (lac Z) gene (group 2) or the human IL‐1Ra gene (group 3). Two days after surgery, the dogs received intraarticular injections as follows: group 1 phosphate buffered saline (PBS) (2 ml); group 2 autologous cells (60 × 106 cells/2 ml of PBS) transduced with the lac Z gene; group 3 autologous cells transduced with the IL‐1Ra gene. Synovial fluid was aspirated at 2 weeks and 4 weeks. All dogs were euthanized at 4 weeks postsurgery. The right knees were dissected, and lesions were scored for macroscopic and microscopic changes. Synovial explants were dissected and representative specimens were used for histology or were cultured for 48 hours. The levels of IL‐1Ra in synovial fluid and synovial explant conditioned medium were measured by specific enzyme‐linked immunosorbent assay. Results. The level of IL‐1Ra in synovial fluid of group 3 was 202.8 ± 131.5 ng/ml (mean ± SEM) at 2 weeks and 2.8 ± 2.2 ng/ml at 4 weeks after surgery. Membrane explants isolated from dogs that received synovial cells transduced with the IL‐1Ra gene (group 3) actively produced IL‐1Ra (4.0 ± 2.0 ng/gm of tissue wet weight). The severity of OA cartilage lesions was similar in groups 1 and 2. In contrast, group 3 dogs had a marked reduction in macroscopic lesion severity on the tibial plateaus (P < 0.01 for grade; P < 0.04 for size) and femoral condyles. Moreover, the histologic lesion severity was decreased on both plateaus (P < 0.06) and condyles. Conclusion. This study showed that a local increase in IL‐1Ra production in OA knee joints by intraarticular injection of transduced synovial cells can reduce the progression of experimentally induced lesions.
This study shows that L-NIL reduces the progression of experimental OA. This effect could be related to a reduced level of chondrocyte apoptosis and is likely mediated by a decrease in the level of caspase 3 activity. A sparing effect of L-NIL on the increased level of Bcl-2 may also be a contributing factor.
Objective. To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS), on the progression of lesions in an experimental osteoar-thritis (OA) dog model. The effect of L-NIL on metal-loprotease activity, levels of interleukin-1P (IL-lp), prostaglandin E, (PGE,), and nitritehitrate in synovial fluid was determined. Methods. The OA model was created by sectioning the anterior cruciate ligament of the right stifle joint of mongrel dogs by a stab wound. Dogs were separated into experimental groups: Group 1 was made up of uuoperated dogs that received no treatment, group 2 were operated dogs with no treatment, and group 3 were operated dogs that received oral L-NIL (10 mg/kg/twice daily) starting immediately after surgery. The OA dogs were killed at 10 weeks after surgery. Results. Experiments showed that dog OA cartilage explants in culture produced an increased amount of NO (nitrite). Immunohistochemical study demonstrated that this was due to an increased level of iNOS in chondrocytes. OA dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated OA dogs (58% versus 92%) as well as in their size (mean ?z SEM 1.92 * 0.58 mm versus 5.08 k Supported in part by grants from thc Arthritis Society and MoiisantoISearle USA. 0.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions by-50% both on condyles and plateaus. 'The histologic severity of both the cartilage lesions and synovial inflammation was significantly decreased in the L-NIL-treated dogs. Treatment with L-NIL also significantly decreased both collagenase and general metalloprotease activity in the cartilage and the levels of IL-lp, PGE,, and nitritehitrate in synovial fluid. Conclusion. This study demonstrated the effectiveness of a selective inhibitor of iNOS, L-NIL, in attenuating the progression of experimental OA. The data suggest that L-NIL may act by reducing the activity of metalloproteases in cartilage and the production of IL-1p by synovium, both of which are known to play a major role in the pathophysiology of OA structural changes.
There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.
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