The in situ up-regulation of chondrocyte interleukin-1-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide.
OBJECTIVE
To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin-1-converting enzyme (ICE). Interleukin-18 (IL-18) was also studied and served as reference for the effects of ICE.


METHODS
An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N-iminoethyl-L-lysine (L-NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL-18, and protease inhibitor 9 (PI-9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 microM; a p38 mitogen-activated protein kinase [MAPK] inhibitor), PD 98059 (100 microM; a MAPK kinase 1/2 [MEK-1/2] inhibitor), NS-398 (10 ng/ml; a specific cyclooxygenase 2 [COX-2] inhibitor), and L-NIL (50 microM).


RESULTS
Both ICE and IL-18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI-9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L-NIL significantly decreased ICE and IL-18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L-NIL also increased the PI-9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK-1/2, and COX-2 inhibitors, but not by the iNOS inhibitor.


CONCLUSIONS
This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up-regulation of ICE and IL-18 synthesis while decreasing the level of the ICE inhibitor PI-9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK-1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.
Boileau
Christelle
C
Osteoarthritis Research Unit, Hôpital Notre-Dame, Centre hospitalier de l'Université de Montréal, 1560 rue Sherbrooke East, Montreal, Quebec H2L 4M1, Canada.
Martel-Pelletier
Johanne
J
Moldovan
Florina
F
Jouzeau
Jean-Yves
JY
Netter
Patrick
P
Manning
Pamela T
PT
Pelletier
Jean-Pierre
JP
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism*; D019902:Chondrocytes / Q000201:enzymology*; D004285:Dogs; D004789:Enzyme Activation / Q000502:physiology; D020382:Interleukin-18 / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010003:Osteoarthritis / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D020382:Interleukin-18; C102316:SERPINB9 protein, human; D015843:Serpins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020170:Caspase 1