Chondrocyte death in experimental osteoarthritis is mediated by MEK 1/2 and p38 pathways: role of cyclooxygenase-2 and inducible nitric oxide synthase.

OBJECTIVE: To explore the mechanisms responsible for in situ induction of chondrocyte death in experimental dog osteoarthritic (OA) cartilage. The roles of 2 mitogen activated protein kinases (MAPK), MEK 1/2 and p38, nuclear factor-kappaB (NF-kappaB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the caspase cascade were investigated. METHODS: OA knee cartilage was obtained from dogs that had received sectioning of the anterior cruciate ligament and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in different inhibitors: Z-DEVD-FMK (caspase 3 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), PD 98059 (MEK 1/2 inhibitor). SB 202190 (p38 inhibitor), SN-50 (NF-kappaB inhibitor), NS-398 (COX-2 inhibitor), N-iminoethyl-l-lysine (L-NIL) (iNOS inhibitor). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for caspase 3, COX-2, iNOS, and nitrotyrosine. Morphometric analyses were performed. RESULTS: The significant level of chondrocyte death in OA cartilage was markedly decreased by caspase 3 and caspase 9 inhibitors. The two MAPK inhibitors, but not the NF-kappaB inhibitor, decreased chondrocyte death concomitant with the levels of caspase 3 and iNOS. COX-2 level was reduced by all 3 inhibitors. Specific inhibition of either COX-2 or iNOS reduced the level of chondrocyte death and caspase 3. There was evidence of crosstalk between these 2 latter systems; specific inhibition of COX-2 reduced the iNOS level, and selective inhibition of iNOS reduced COX-2 expression. COX-2 and iNOS seem to function in a positive autoregulatory manner that triggers transcription of their own biosynthetic machinery, since the specific inhibition of each system downregulates its expression. CONCLUSION: This study shows that in the early lesions of experimental OA cartilage in situ, activation of the caspase cascade is responsible for induction of chondrocyte death. Marked inhibition of cell death by caspase inhibitors indicates a significant participation of apoptosis in the phenomenon. This phenomenon is linked to the activation of at least 2 major kinase pathways, MEK 1/2 and p38. These pathways are responsible for upregulating the expression of iNOS and COX-2, each of which seems essential for the induction of apoptosis. Data are provided about possible regulation and interregulation of the COX-2 and iNOS systems by prostaglandin E2 and NO.

In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: suppression of collagenase 1 and interleukin-1beta synthesis.

OBJECTIVE: To study the therapeutic effectiveness of ML-3000, a new antiinflammatory drug that has balanced dual inhibitory activity against 5-lipoxygenase and cyclooxygenase, on the development of lesions in the experimental osteoarthritis (OA) dog model, and to determine the action of ML-3000 on the synthesis of collagenase 1 in cartilage and interleukin-1beta (IL-1beta) in synovial membrane. METHODS: The anterior cruciate ligament of the right stifle joint of 21 mongrel dogs was sectioned with a stab wound. Dogs were divided into 3 groups: group 1 (n = 7) received placebo; groups 2 (n = 7) and 3 (n = 7) were treated with therapeutic dosages of oral ML-3000 at 2.5 mg/kg/day and 5 mg/kg/day, respectively. The dogs began receiving medication the day after surgery and were killed 8 weeks later. The size and grade of cartilage erosions on both the condyles and plateaus were evaluated, and the severity of the cartilage lesions and synovial inflammation was examined histologically. Levels of collagenase 1 in cartilage and IL-1beta in the synovial membrane were measured by immunohistochemistry. In addition, levels of prostaglandin E2 (PGE2) in the synovial fluid and leukotriene B4 (LTB4) in cultured synovial membrane explants were determined using specific enzyme immunoassays. RESULTS: Serum levels of ML-3000 in treated dogs were within the therapeutic range. ML-3000 significantly decreased the size and grade of the cartilage lesions in tibials and plateaus, compared with placebo. At the histologic level, the severity of cartilage lesions was also decreased in the ML-3000-treated dogs versus the placebo-treated dogs in both the condyles and the plateaus. All 3 OA groups exhibited a notable and similar level of synovial inflammation. ML-3000 significantly decreased the level of PGE2 in synovial fluid and LTB4 production by synovium. It also markedly reduced the levels of collagenase 1 in cartilage and IL-1beta in synovial membrane. CONCLUSION: ML-3000 significantly reduced the development of lesions in experimental dog OA. The drug acts by reducing the synthesis of the inflammation mediators PGE2 and LTB4 and catabolic factors such as collagenase 1 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions. The effect of the drug on catabolic factors could possibly be related to its inhibitory action on LTB4 synthesis.

Modulation of TIMP-1 synthesis by antiinflammatory cytokines and prostaglandin E2 in interleukin 17 stimulated human monocytes/macrophages.

OBJECTIVE: To examine the regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1) synthesis by interleukin 17 (IL-17) stimulated human monocytes/macrophages in primary culture in the presence of prostaglandin E2 (PGE2) and antiinflammatory cytokines, and to compare this with the regulation of matrix metalloproteinase (MMP-9) production. METHODS: IL-17 stimulated human monocytes isolated from the peripheral blood of healthy donors were cultured in the presence of PGE2, cyclic adenosine monophosphate (cAMP) mimetics (IBMX, cAMP, forskolin, cholera toxin), or antiinflammatory cytokines (IL-4, IL-10, IL-13), or with protein kinase inhibitors of diverse specificity. MMP-9 and TIMP-1 were measured using specific ELISA, while expression of specific messenger RNA was determined by Northern blotting. RESULTS: IL-17 stimulated an increased level of MMP-9 production relative to TIMP-1 production in monocytes/macrophages. Stimulation was accompanied by upregulation of specific MMP-9 mRNA expression relative to TIMP-1 mRNA. Exogenous PGE2, cAMP, and cAMP-mimetics completely inhibited both basal and IL-17 induced MMP-9 synthesis, while only IL-17 induced TIMP-1 synthesis was abrogated. The same effect was found for the antiinflammatory cytokines. Both basal and IL-17 induced production of TIMP-1 involved p42/44 and p38 kinases and nuclear factor kappaB signaling pathways. CONCLUSION: The excess of MMP-9 over TIMP-1 production, and decreased inhibition of MMP-9 activity in chronic rheumatoid diseases, may result in cartilage degradation and joint destruction.

Carprofen simultaneously reduces progression of morphological changes in cartilage and subchondral bone in experimental dog osteoarthritis.

OBJECTIVE: To examine the effect of a nonsteroidal antiinflammatory drug, carprofen, on the structure and metabolism of cartilage and subchondral bone in the experimental osteoarthritic (OA) canine model. METHODS: Experimental Groups 1 and 2 received a sectioning of the anterior cruciate ligament (ACL) of the right stifle joint, and were administered carprofen (2.2 and 4.4 mg/kg/twice daily/po, respectively) for 8 weeks beginning 4 weeks postsurgery. Group 3 received ACL sectioning and no treatment. Group 4 was composed of unoperated normal dogs. Cartilage macroscopic lesions were assessed, and their histological severity was graded. Specimens of subchondral bones were fixed, decalcified, and stained with hematoxylin/eosin. The level of metalloprotease (MMP) activity in cartilage was measured. Osteoblast cells were prepared from the subchondral bone. The level of synthesis of osteoblast biomarkers (osteocalcin, alkaline phosphatase), as well as urokinase plasminogen activator (uPA) activity and insulin-like growth factor (IGF-1) in the culture medium, was estimated. RESULTS: Carprofen treatment decreased the width of osteophytes (p < 0.01), the size of cartilage lesions, and the histologic severity of cartilage lesions (p < 0.008). There was no difference in the levels of MMP activity in cartilage between OA and carprofen treated groups. In OA dogs, the subchondral bone plate was thinner and was the site of an extensive remodeling process with numerous lacunae. Dogs treated with carprofen showed a marked decrease in the remodeling activity with normal plate thickness, and subchondral bone morphology resembling that of normal dogs. Osteoblasts from untreated OA dogs showed slightly higher alkaline phosphatase activities and osteocalcin release that reverted back to normal upon carprofen treatment. Moreover, uPA activity and IGF-1 levels were increased in OA dogs and were significantly reduced in carprofen treated dogs. CONCLUSION: Under therapeutic conditions, treatment with carprofen could reduce the progression of early structural changes in experimental OA. Carprofen treatment also delays and/or prevents the abnormal metabolism of subchondral osteoblasts in this model. The hypothesis of a possible link between the protective effect of carprofen and its effect on subchondral bone is of interest in the context of therapeutic intervention.

The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2.

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.

Selective inhibition of inducible nitric oxide synthase reduces progression of experimental osteoarthritis in vivo: possible link with the reduction in chondrocyte apoptosis and caspase 3 level.

OBJECTIVE: To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase, on the progression of structural lesions in the experimental canine model of osteoarthritis (OA), and to explore the effect of L-NIL on the level of chondrocyte apoptosis and of important proteins involved in the apoptotic phenomenon, i.e., caspase 3 (inducer) and Bcl-2 (inhibitor). METHODS: The OA model was created by sectioning the anterior cruciate ligament. Dogs were placed into 4 experimental groups: unoperated dogs that received no treatment (controls), operated (OA) dogs that received placebo treatment, OA dogs that received oral L-NIL at 10 mg/kg/day, and OA dogs that received oral L-NIL at 1.0 mg/kg/day. In both L-NIL groups, treatment started immediately after surgery. The OA dogs were killed at 12 weeks after surgery. RESULTS: OA dogs treated with L-NIL showed a reduction in the size of osteophytes and a significant decrease in the severity of macroscopic and histologic cartilage lesions on both condyles and plateaus, compared with untreated OA dogs. L-NIL treatment also significantly decreased metalloprotease activity in cartilage. Immunohistochemical analysis revealed that the levels of chondrocyte apoptosis, caspase 3, and Bcl-2 were markedly increased in OA cartilage (P < 0.0001). A positive correlation between the levels of chondrocyte apoptosis and levels of caspase 3 was found (r = 0.54, P < 0.0001). OA dogs treated with the higher dosage L-NIL showed significantly reduced levels of chondrocyte apoptosis (P < 0.003) and caspase 3 (P < 0.04), but no effect on the increased level of Bcl-2 was demonstrated. CONCLUSION: 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.

Stimulation of 92-kd gelatinase (matrix metalloproteinase 9) production by interleukin-17 in human monocyte/macrophages: a possible role in rheumatoid arthritis.

OBJECTIVE: To examine the cellular mechanisms by which the proinflammatory cytokine interleukin-17 (IL-17) induces the synthesis of 92-kd gelatinase (matrix metalloproteinase 9 [MMP-9]) by human monocyte/ macrophages in primary culture. METHODS: IL-17-stimulated human monocytes isolated from the peripheral blood of healthy donors were cultured in the presence of antiinflammatory cytokines, neutralizing antibodies against IL-1beta, tumor necrosis factor alpha (TNFalpha), or IL-1 receptor antagonist, and with protein kinase inhibitors of diverse specificity. MMP measurements were performed using specific enzyme-linked immunosorbent assays, while the expression of specific messenger RNA was determined by Northern blotting. Detection of phosphorylated proteins and specific transcriptional factors was performed by Western blotting and by gel retardation experiments, respectively. RESULTS: Biologically active IL-17 was detected in the synovial fluid of patients with rheumatoid arthritis. IL-17-induced MMP-9 production in human monocyte/ macrophages was dependent on endogenous prostaglandin E2 synthesis and related to autocrine stimulation by TNFalpha, but was IL-1beta independent. This activation involves both p42/44 and p38 kinases and nuclear factor kappaB. IL-17-inducible activator protein 1 and signal transducer and activator of transcription 1/3 may transactivate the MMP-9 promoter. CONCLUSION: IL-17 may contribute to an unbalanced production of proinflammatory cytokines and MMP-9 in diseased articular joint tissues by interacting with the macrophages in the rheumatoid synovium.

IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages.

IL-17 is a newly described, T cell-derived cytokine with ill-defined physiologic properties. As such, we examined the release of proinflammatory mediators by human macrophages in response to recombinant human (rh) IL-17. IL-1beta and TNF-alpha expression and synthesis were up-regulated by rhIL-17 in a dose (ED50 was 50 +/- 9 ng/ml)- and time-dependent fashion, with cytokine accumulation reaching a zenith after 9 h. Release of IL-6, PGE2, IL-10, IL-12, IL-1R antagonist, and stromelysin was also stimulated by rhIL-17. IL-1beta and TNF-alpha mRNA expression levels were controlled by rhIL-17 in a complex manner with an initial 30-min inhibitory phase, and then up-regulation beginning at 1 h and reaching a plateau at about 3 h. The latter expression pattern closely mirrored the nuclear accumulation of the transcription factor nuclear factor-kappaB. cAMP mimetics isobutyl-1-methylxanthine (IBMX), forskolin, PGE2, and cholera toxin reversed rhIL-17-induced release of TNF-alpha, but had no consistent effect on induced IL-1beta synthesis. Induced release of TNF-alpha was also inhibited by serine/threonine protein kinase inhibitors KT-5720 (protein kinase A) and Calphostin C (protein kinase C), mitogen-activated protein kinase kinase inhibitor PD098059, and a nonspecific tyrosine kinase inhibitor, genistein. Calphostin C alone abrogated the rhIL-17-induced release of IL-1beta. The antiinflammatory cytokines IL-4 (p < 0.01) and IL-10 (p < 0.02) completely reversed rhIL-17-stimulated IL-1beta release, while IL-13 and TGF-beta2 were partially effective (59 and 43% diminution, respectively). IL-10 exerted a significant suppressive effect on IL-17-induced TNF-alpha release (99%, p < 0.02), while the inhibitory effects of IL-4, IL-13, and TGF-beta2 on TNF-alpha secretion were partial (48, 10, and 23%, respectively). The data suggest a pivotal role for IL-17 in initiating and/or sustaining an inflammatory response.