Apoptosis has been recognized to play a relevant role in cartilage damage in osteoarthritis (OA) (1), although the extent of the phenomenon varies from study to study (2, 3). Apoptosis also has secondary degradative effects due to the presence of postapoptotic cell byproducts that are not effectively removed from the tissue, such as pyrophosphate and calcium crystals (4). Furthermore, apoptotic bodies have been reported to have high contents of matrix-degrading enzymes (4, 5). Apoptotic cells have been mainly localized in two distinct zones of OA cartilage: the superficial layer of cartilage in late-stage disease and the clusters containing proliferating chondrocytes (1).
Hashimoto et al (6) have reported a linkage between chondrocyte apoptosis and cartilage degradation. It is likely that in this case, apoptosis induction occurs following the loss of chondrocyte interaction with the extracellular matrix (ECM) (7), a mechanism typical of adherent cells. Other investigators (8) have suggested that apoptosis is the counterpart of cell proliferation, which is typical of the regenerating/proliferating cartilage. There is a close link between cell proliferation and apoptosis: when a cell picks up the machinery to proliferate, it also acquires an abort pathway (9). One of the first reports of chondrocyte apoptosis identified it in the hypertrophic region of growth plates (10), and it has recently been documented that in OA cartilage, the distribution of molecules relevant to apoptotic death (Bcl-2, Bax, and Fas) is correlated with regions of larger “cloning of chondrocytes” (8). On the other hand, it has been shown that the cells of the upper layer of OA cartilage present some features that are common to growth cartilage development and mineralization, such as the formation of mineral deposits, chondrocyte hypertrophy, terminal differentiation, and apoptosis (11).
Molecular signaling via soluble factors has been shown to be crucial to cartilage homeostasis, having been implicated not only in chondrocyte differentiation, but also in normal maintenance, as well as in aging and disease (12, 13). Therefore, it is likely that it also plays a relevant role in the induction of apoptosis. Two mechanisms of chondrocyte apoptosis have been described thus far: the Fas pathway and nitric oxide (NO) (1). Although chondrocytes in the superficial and upper middle zones of cartilage were shown to express Fas antigen, it is not known whether these cells express Fas ligand in intact cartilage (14). The only source of Fas ligand that has been described is inflammatory cells in the synovial tissue and synovial fluid (1), and inflammatory cells are found only occasionally in OA (1). Furthermore, many investigators have linked the occurrence of apoptosis to increased levels of NO, which acts as second messenger for prototypical cytokines (interleukin-1 [IL-1] and tumor necrosis factor [TNF]) (15, 16). Although induction of apoptosis via NO generated from exogenous NO donors has been demonstrated, the possibility that IL-1 or TNF induces apoptosis by means of such a reactive radical is a subject of controversy (17–20). Moreover, apoptosis has been successfully induced in several immortalized chondrocyte cell lines (21), which lack significant expression of inducible NO synthase (22). Thus, although the relevance of apoptosis in OA has been acknowledged, no soluble factors have yet proved to be a convincing protagonist in its induction.
A novel autocrine loop has been reported by our group: chondrocytes express and release chemokines (IL-8, growth-related oncogene α [GROα], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1α [MIP-1α], MIP-1β, and RANTES) (23, 24) and present the specific receptors (CCR1, CCR2, CCR3, CCR5, CXCR1, and CXCR2) (25). Receptor expression tends to be enhanced in OA chondrocytes, and interaction of chemokine receptors with the corresponding ligands induces the release of matrix-degrading enzymes (matrix metalloproteinase and lysosomal glycosidase), which is also augmented in OA compared with normal chondrocytes (25). This supports the hypothesis that some of these chemokine/receptor loops are likely to contribute to the induction of the catabolic program with a potency comparable with that of IL-1. It has recently been reported that aside from the interaction with their receptors, chemokines have a second important interaction with cell surface glycosaminoglycans (GAGs), particularly sulfated GAGs (26). Furthermore, ECM GAGs might play a role in governing the local chemokine concentration, supporting the formation of solid gradients around the cells (26).
A link between chemokines and apoptosis is strongly suggested by recent reports suggesting a proapoptotic role for CXCR1, CXCR2 (27), and CXCR4 (28) as well as an antiapoptotic role for CCR2 (29) and CCR8 (30). In view of the hypothesized correlation of apoptosis and cell proliferation, it is noteworthy that the same proapoptotic receptors induce cell proliferation in other models (29, 31–34). Moreover, taking into account the linkage between apoptosis and loss of survival signals from the ECM, it is also important that chemokines have been shown to induce the production of matrix metalloproteinases by chondrocytes (25).
This study is the first to provide evidence that the GROα chemokine triggers apoptosis in chondrocytes and that this phenomenon is adhesion dependent and is associated with a marked depolarization of the cells. Apoptosis was confirmed by findings of TUNEL analysis, by evidence of DNA laddering in apoptotic cells, morphologic examination of nuclear modifications (May-Grünwald-Giemsa staining), annexin labeling, caspase 3 activation, and plasma membrane depolarization, and colocalization of TUNEL positivity, activation of caspase 3, and phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK).
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The role of chemokines in the pathophysiology of OA is now starting to come to light. This is the first study to show the ability of GROα to induce chondrocyte apoptosis.
Chondrocytes are long-lived cells that adhere to the ECM and depend on anchorage for growth and metabolism (13). Indeed, chondrocytes are sensitive to growth factors and cytokines provided that there is a proper network of interactions with the ECM. It has been reported that IL-1 signaling requires proper attachment and proximity of the receptor to focal adhesions, structural links between the ECM and the cytoskeleton, as well as specialized sites of signal transduction (42). We demonstrated here that the functional effects induced by chemokines are secondary to a complex and interdependent series of signal transduction pathways that start at the cell membrane, since GROα-induced apoptosis occurred only when the cells were left for an adequate period of time to recover and reestablish a proper network of interactions with ECM proteins (37, 38).
GROα-induced apoptosis is likely to occur through so-called anoikis, that is, death dependent on the loss of normal cell–substratum contact (43). It has been reported for epithelial cells (44) and, recently, for chondrocytes (45) that α5β1 integrin bound to fibronectin protects from apoptosis. It is possible that chondrocytes undergo apoptosis when the loss of such a contact occurs as the result of the induction of matrix-degrading enzymes (mainly, stromelysin 1, which is able to degrade fibronectin) upon chemokine stimulation (25). In keeping with such observations is the antiapoptotic effect of hyaluronan on anti-Fas–treated chondrocytes, as recently reported by our group (46), and the notion that the apoptotic index is higher in the superficial layer of OA cartilage (11), where matrix-degrading activity is enhanced compared with deeper areas (6). Furthermore, it has also been reported that apoptosis is highly increased in chondrocytes in suspension compared with whole tissue (2), possibly because detachment induced strong activation of caspases 8 and 3 (47). The results of our study further support this hypothesis, given the comparison between the estimates of the apoptotic index of adherent (annexin V–labeled; May-Grünwald-Giemsa–stained) versus suspended (TUNEL-labeled) cells.
An issue that deserves further investigation is the association between apoptosis and cell proliferation. GROα was first described as the “growth-related oncogene.” It belongs to the ELR chemokines. Chemokines are a large family of molecules containing a cluster that shares the ELR motif, which is associated with the induction of cell proliferation (48). A close link between cell proliferation and apoptosis has been reported (49), and it is probably dependent on a broad spectrum of responses to oxidative stress, ranging from a mitogenic response to growth arrest to apoptosis or necrosis, depending on the stress level encountered.
Apoptosis encompasses multiple characteristic morphologic and biochemical features, including chromatin aggregation, nuclear and cytoplasmic condensation, DNA fragmentation, alteration in membrane asymmetry, and activation of apoptotic caspases (50, 51). Furthermore, it is a dynamic process in which a characteristic morphologic or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time (50). To strengthen the sensitivity and the specificity of apoptosis detection in chondrocytes, we therefore used multiple technical approaches.
The most specific assays appear to be those based on the detection of DNA strand breaks (50). The apoptotic effect of GROα was noted not only on isolated chondrocytes, but also on cells cultured within an intact ECM and, to our knowledge, this is the first report of apoptosis induced by a cytokine upon culture of human cartilage explants. When set within an intact ECM, chondrocytes are protected from apoptosis by virtue of survival signals received through the interaction of integrins and ECM proteins. Nevertheless, this model more closely resembles in vivo conditions, and furthermore, it may also allow the identification of differential effects across the various layers of cartilage, in keeping with a functional and phenotypic heterogeneity of the tissue. By using this model, apoptosis triggered by GROα was observed in the deeper layers, along with the coordinated activation of caspase 3 and the phosphorylation of JNK/SAPK, the molecular markers of the phenomenon. Safranin O labeling experiments suggest that the threshold for apoptosis is probably related to cross-talk between signaling pathways triggered by soluble factors and signals received from the ECM.
Of particular interest is the notion that GROα stimulation of chondrocytes also leads to a rapid and irreversible depolarization of the plasma membrane. It has recently been reported that the movement of intracellular monovalent cations plays a critical role in many cellular alterations related to apoptosis. The loss of intracellular potassium and sodium contributes to cell shrinkage and intracellular activation of caspases and endonucleases (41). This phenomenon occurs very rapidly after application of the apoptotic stimulus, when visualized at the single-cell level (52). Results of our confocal microscopy experiments clearly indicated that the cells are heterogeneous with respect to their response to GROα. Given the complexity of the functional behavior of adherent cells (42), it is likely that the threshold of GROα-induced apoptosis is determined not only by the presence of the specific receptor, but also by a peculiar repertoire of adhesion molecules known to regulate this phenomenon (44) and by a defined commitment to cell proliferation. The depolarizing effect of GROα is rapid and sustained and cannot be reversed by removal of the chemokine, as has been shown to occur during anti-Fas–induced apoptosis in Jurkat cells (41) as the result of a blockage of the Na+/K+-ATPase or of the inhibition of the voltage-activated potassium channels. Many recent reports strongly suggest that cellular depolarization could act as a checkpoint in the activation of apoptosis (53).
Our group of investigators has provided the first demonstration of the presence of chemokine receptors on chondrocytes (25), but the downstream signaling pathways remain to be clarified. However, pertussis toxin sensitivity and membrane potential experiments have suggested that chemokine receptors in chondrocytes are of the ion channel–linked G protein–coupled type (54) and that receptors for RANTES and GROα are associated with anion and cation channels, respectively, given the opposite sign of potentiometric effects. This hypothesis is also consistent with an antagonism observed in the experiments performed with first passage chondrocytes, with GROα inducing chondrocyte apoptosis and RANTES resulting in no morphologically evident effect compared with unstimulated controls. It is also likely that RANTES exerts an antiapoptotic action, by virtue of its hyperpolarizing effect on the cells, as has been reported for many members of the Bcl-2 protein family (55).
The ability of GROα to induce chondrocyte apoptosis is a finding of particular relevance in light of the availability of this chemokine in the joint space. We and other investigators have previously reported that chondrocytes constitutively express and release GROα (23, 24). Furthermore, GROα is readily up-regulated by inflammatory cytokines. GROα is also one of the most abundantly secreted chemokines by chondrocytes as well as by several other resident or immigrating cells within the joint, such as monocytes (56) and fibroblasts (57) of the synovial tissue, mononuclear cells and neutrophils of the synovial fluid (57), and even osteoblasts (58) and stromal cells (59) of the subchondral bone, particularly in inflamed joints.