Osteoarthritis (OA), the most common age-related cartilage and joint disorder, is a degenerative disease characterized by degradation of the matrix and cell death resulting in the gradual loss of articular cartilage integrity (1, 2). The chondrocyte, which is the only cell type present in mature cartilage, is responsible for repairing damaged tissue. Although the primary etiology of the disease is undetermined, OA is now believed to involve a disruption of cartilage homeostasis in which proinflammatory stimuli induce an increase in chondrocyte catabolic processes. Consequently, the role of inflammation in the progression of OA has acquired important new dimensions (3–5).
Accumulating evidence indicates that mitochondrial damage may play a significant role in the pathogenesis of OA (6–8). Recent ex vivo studies have demonstrated mitochondrial dysfunction in human OA chondrocytes, and analyses of mitochondrial electron transport chain activity in these cells show decreased activity of mitochondrial respiratory chain complexes I, II, and III compared to normal chondrocytes (9). This mitochondrial dysfunction may affect several pathways that have been implicated in cartilage degradation, including defective chondrocyte biosynthesis and growth response, cartilage matrix calcification, and increased chondrocyte apoptosis, as well as augmentation of the inflammatory and matrix catabolism responses. Most of these processes are potentially related to the production of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) intermediaries (10–15). In this regard, a correlation has been found between the OA disease stage and the presence of oxidative stress (2, 16–18).
As both the predominant site for ROS production and the prime target of these molecules, mitochondria play a key role in oxidative stress. High levels of oxidative stress may also underlie mitochondrial respiratory chain inhibition, ATP decrease, and mitochondrial DNA (mtDNA) mutation, all of which are related to the severity of the inflammatory process (14, 19, 20). In fact, an augmentation of oxidative mtDNA damage has been observed in OA cartilage (14, 21).
Proinflammatory mediators may also alter mitochondrial function. Accordingly, we previously described the modulation of mitochondrial activity by interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and nitric oxide in normal human chondrocytes (19, 20). In this scenario, mitochondrial damage produces ROS and RNS that in turn reduce mitochondrial bioenergetics, favoring cell damage and death (14, 18, 22). In this sense, increasing experimental findings support a connection between mitochondrial dysfunction and inflammation (22–24). However, the role of mitochondrial dysfunction in OA-related inflammation is not yet fully understood.
The key mediators of inflammation, IL-8 and prostaglandin E2 (PGE2), are up-regulated in inflamed joint tissue (25–27). Both are spontaneously released by cartilage specimens from patients with OA at significantly higher levels than those released by normal cartilage specimens (4, 27). Specifically, IL-8–mediated inflammation can promote cartilage degradation through matrix metalloproteinase 3 (MMP-3) synthesis and altered chondrocyte differentiation and calcification in OA (28, 29). PGE2 contributes to hyperalgesia and the erosion of cartilage and juxta-articular bone (30). Overproduction of both mediators is likely induced by proinflammatory cytokines such as IL-1β and TNFα. Because it controls the transcription of a number of proinflammatory genes, the redox-sensitive NF-κB pathway is considered a key regulator of tissue inflammation, including IL-8 and cyclooxygenase 2 (COX-2) expression, in several cell types, notably OA chondrocytes (31).
Although current evidence indicates that mitochondrial dysfunction and inflammation are important players in the pathogenesis of OA, data are lacking to support the hypothesis that the connection between these processes amplifies and accelerates the mechanisms contributing to the impairment of cartilage and joint function. Recently, we demonstrated that mitochondrial dysfunction produced a slight increase in COX-2 expression and PGE2 production in normal human chondrocytes in vitro (12). In this study, we investigated the effects of mitochondrial dysfunction on exacerbating the inflammatory response induced by cytokines in normal human chondrocytes, which can play a crucial role in governing the onset and progression of OA. Furthermore, the effect of antioxidant treatment on the above processes was examined.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In recent years, accumulating evidence has indicated that mitochondrial damage may play a significant role in the pathogenesis of OA (6–8). In addition, the role of inflammation in the progression of OA has acquired important new dimensions (3, 4). In this sense, increasing experimental data support a connection between inflammation and mitochondrial dysfunction (22–24). Importantly, we previously demonstrated that this mitochondrial dysfunction per se may generate low-grade inflammatory and matrix degradation processes in normal human chondrocytes in vitro (12, 13). However, whether the preexisting mitochondrial dysfunction described in OA chondrocytes (9) intensifies cytokine-induced chondrocyte inflammation remained unknown.
In this study, we focused on the potential of mitochondrial dysfunction to increase the cytokine-induced inflammatory response in normal human chondrocytes. This is the first study to show that mitochondrial dysfunction in normal human chondrocytes increases the production of inflammatory mediators such as IL-8, COX-2, and PGE2 in response to cytokines through ROS generation and activation of the transcription factor NF-κB. In addition, we showed that resveratrol significantly reduced this inflammatory response.
IL-8 and PGE2 are 2 key players that are up-regulated in inflamed joint tissue (25, 26, 34, 35) and in other age-related inflammatory diseases, including Alzheimer's disease, cancer, and atherosclerosis. In fact, cartilage specimens from patients with OA spontaneously released PGE2 and expressed IL-8 mRNA in ex vivo culture at levels at least 50-fold higher and 15-fold higher, respectively, than those observed in normal cartilage (4, 27). This up-regulation may occur secondary to the activation of inflammatory cytokines (27, 36) but may also be independent of cytokine activation (37). With regard to the latter possibility, we recently demonstrated that the inhibition of mitochondrial respiratory chain activity induces a slight increase in COX-2 expression and PGE2 production in normal human chondrocytes (12).
In the present study, when mitochondrial dysfunction was induced in normal chondrocytes with inhibitors of mitochondrial respiratory chain complexes III and V (antimycin A and oligomycin, respectively), a slight but significant increase in IL-8 mRNA and protein levels was also observed, which was dependent on both dose and incubation time. These findings are consistent with those obtained in human liver slices, where mitochondrial injury induced by pharmaceutical inhibitors of fatty acid oxidation led to a significant increase in IL-8 gene and protein expression (37). Mitochondrial dysfunction also induces PGE2 liberation through 4-hydroxynonenal, a lipid peroxidation end product, which is produced abundantly in OA articular tissue and was recently identified as a potent catabolic factor in OA cartilage (38). In fact, 4-hydroxynonenal induces COX-2 expression and PGE2 release in human OA chondrocytes (38).
To examine the potential role of mitochondrial dysfunction in increasing the vulnerability of cells to a cytokine-induced inflammatory response, we evaluated whether mitochondrial dysfunction in chondrocytes increases the level of IL-8 and PGE2 production induced by cytokines. Numerous in vitro and in vivo studies have shown that IL-1β and TNFα are the primary proinflammatory and catabolic cytokines involved in the initiation and progression of articular cartilage destruction (5, 14, 20). Furthermore, the increased levels of catabolic enzymes, prostaglandins, ROS, nitric oxide, and other markers in OA fluids and tissue appear to be related to elevated levels of IL-1β and TNFα. In the present study, antimycin A and oligomycin treatment of normal human chondrocytes resulted in synergistic amplification of the inflammatory response induced by the cytokines IL-1β or TNFα. Hence, we observed that mitochondrial dysfunction increases the IL-8 expression and chemotactic activity induced by these cytokines. We also found that mitochondrial dysfunction aggravates the COX-2 expression and production of its metabolic end product, PGE2, induced by IL-1β.
The findings of the present study are supported by those obtained in other studies demonstrating that mitochondrial dysfunction increases the inflammatory response to different catabolic stimuli. In lung epithelial cells, preexisting mitochondrial dysfunction induced by antisense oligonucleotides to ubiquinol cytochrome c reductase core protein II in mitochondrial respiratory chain complex III increased mitochondrial ROS generation, resulting in a marked potentiation of ragweed pollen extract–induced accumulation of inflammatory cells in the airways (39). Interestingly, recent data also revealed that PGE2 promotes IL-1 expression in articular chondrocytes, thus amplifying the local inflammatory process (30). In addition, the authors of that previous report showed that combined treatment of IL-1 with PGE2 synergistically accelerated the expression of pain-associated molecules, including nitric oxide synthase and IL-6 (30). These findings confer even more physiologic relevance to our findings regarding the effects of mitochondrial dysfunction.
Our results demonstrate a significant mitochondrial dysfunction–related increase in the chondrocyte inflammatory response. Consistent with our results, OA chondrocytes are more responsive to IL-1β than are normal chondrocytes. The spontaneous production of PGE2 by human OA cartilage is notably greater than that by normal cartilage, and the addition of cytokines augments this effect to ∼30-fold greater production compared to that observed in normal cartilage treated with cytokines (27). Similar effects have been described with proMMP-3 and proMMP-9 (40). Specifically, mitochondria from OA chondrocytes have been reported to be more sensitive to the DNA-damaging effects of the proinflammatory cytokines IL-1β and TNFα than are mitochondria from normal chondrocytes (14). Our results, and those of other investigators, suggest that mitochondrial dysfunction may contribute to the inflammatory phenotype observed in OA through the expression of inflammatory mediators.
In a previous study, we demonstrated that chondrocytes stimulated with antimycin A or oligomycin produced ROS, triggering an inflammatory response that was significantly reduced by pretreatment with the ROS scavenger NAC (12). In this sense, the mitochondrial respiratory chain is one of the most important sites for ROS generation. The cumulative oxidative stress caused by ROS has been implicated as a key factor in OA. The present study demonstrated that when chondrocytes were treated with the ROS scavenger NAC, IL-8 expression induced by the synergy between mitochondrial dysfunction and cytokines was significantly reduced, confirming that ROS production is a key step in this inflammatory pathway. However, because NAC did not completely block the inflammatory response, other mitochondrial damage–associated molecules such as ATP, mtDNA, or RNS may also be involved in this inflammatory process (11, 22, 24). In other cell types, mitochondrial dysfunction also increased the generation of ROS, resulting in potentiation of cytotoxicity or inflammatory cell accumulation (39, 41). TNF receptor type I mutant cells exhibited altered mitochondrial function with enhanced ROS generation, and pharmacologic blockade of mitochondrial ROS (i.e., with NAC) reduced inflammatory cytokine production induced by lipopolysaccharide (42). Additionally, antioxidant treatments can improve disease progression in animal models of OA (43).
Increased oxidative stress may lead to the up-regulation of redox-sensitive transcription factors, such as NF-κB, that contribute to the proinflammatory phenotypic alterations in OA tissue, including the induction of IL-8 and COX-2 expression. In our study, the pharmacologic modulation of NF-κB with BAY 11-7085, an inhibitor of NF-κB activation, significantly prevented up-regulation of IL-8 expression, suggesting that NF-κB may modulate the cytokine-induced IL-8 expression associated with mitochondrial dysfunction. Furthermore, we also found that oligomycin potentiated NF-κB activation induced by IL-1β (Vaamonde-García C, et al: unpublished observations). Other groups have found similar results, reporting enhanced sensitivity to activation of NF-κB in the setting of mitochondrial dysfunction (41, 44). However, other redox-sensitive transcription factors or other means of posttranscriptional regulation, such as modulation of mRNA degradation, could also be involved (45, 46).
Because of the critical roles of oxidative stress and inflammation in the pathology of inflammatory diseases such as OA, a debate over various therapeutic strategies using antioxidant compounds is well under way. Resveratrol, a natural compound found in high concentrations in grape skin and red wine, shows great promise as an antioxidant and antiinflammatory agent. In animal models, resveratrol protected against age-related diseases in mice and improved mitochondrial function (47, 48). In vitro, this molecule has proven to have a number of beneficial effects in several cell types, i.e., by reducing NF-κB activation, PGE2 production, and free radical formation or by inducing mitochondrial biogenesis and protecting against chondrocyte apoptosis (49). In this study, we demonstrated that supplementation with this natural antioxidant significantly reduced the IL-8 levels and the chemotaxis induced by the synergy between mitochondrial dysfunction and cytokines. Similar findings have been reported in human monocytic U937 cells, in which resveratrol inhibited phorbol 12-myristate 13-acetate–induced IL-8 production at the protein and mRNA levels (50). Our results suggest that resveratrol may be a useful treatment strategy in OA.
On the whole, we have provided additional support to the hypothesis that a decline in mitochondrial function participates in the chondrocyte inflammatory phenotype observed in OA pathology. The results of the present study verify that mitochondrial dysfunction alone may generate low-grade inflammation in chondrocytes and demonstrates, for the first time, that a decline in mitochondrial function increases chondrocyte inflammatory responsiveness to cytokines, accelerating the mechanisms that may contribute to the impairment of cartilage and joint function in OA and aging. Future studies should be undertaken to evaluate whether resveratrol is a possible treatment of OA.