Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin-6 (IL-6) family (1). Elevated levels of OSM can be detected in the synovial fluid, but not in the serum, of patients with rheumatoid arthritis (RA) (2). Immunohistochemical analysis of RA synovial tissue showed that synovial macrophages are the source of OSM in the inflamed joint (3). A pathologic role for OSM in RA is suspected, because OSM by itself can induce joint inflammation in animals. Injecting recombinant human OSM into the joints of goats induced the influx of polymorphonuclear cells (PMNs), followed by cells of the macrophage/monocyte lineage (4). Joint inflammation was also induced by adenoviral expression of murine OSM in mice (5, 6). The enhanced expression of adhesion molecules such as E-selectin and P-selectin (7, 8), CXC chemokines (8), and the CC chemokine monocyte chemotactic protein 1 (9) by OSM could contribute to the influx of inflammatory cells. Furthermore, synovial fibroblasts displayed a transformed phenotype under the influence of OSM (5), suggesting involvement of OSM in pannus formation.
Besides chronic joint inflammation, RA is also characterized by destruction of articular cartilage and bone. Cartilage consists of a framework of collagen fibers in which proteoglycans (PGs) are entrapped. These PGs can retain water, which enables the cartilage to resist compressive forces. Proinflammatory cytokines such as IL-1 (10, 11) are involved in cartilage degradation. Results from experiments in recent years suggest a similarly important role for OSM in the cartilage degradation of RA. OSM was shown to induce collagen release from bovine cartilage in vitro (12). It also stimulated PG release and suppressed PG synthesis in porcine articular cartilage explants (13). Injecting OSM into the joints of goats (4) decreased the cartilage PG content. In humans, OSM concentrations in synovial fluid correlate positively with levels of cartilage degradation markers (14). OSM was also the first cytokine that, in combination with IL-1α, was demonstrated to induce collagen release from human cartilage (3).
The development of joint inflammation and cartilage damage in experimental arthritis can be greatly influenced by the expression of proinflammatory cytokines and other mediators. We previously demonstrated that blocking of IL-1 could prevent inhibition of PG synthesis in experimental arthritis (15, 16). The formation of nitric oxide (NO) was shown to be involved in IL-1–induced inhibition of PG synthesis in vitro (17), and PG loss was reduced in experimental arthritis in mice deficient for the inducible NO synthase (iNOS) gene (18). Studies entailing blocking of tumor necrosis factor α (TNFα) showed involvement of TNFα in the early phase of joint inflammation (19, 20), while studies of experimental arthritis in IL-6–deficient mice showed involvement of IL-6 in the chronicity of arthritis (21). In the present study, we investigated the involvement of these proinflammatory mediators in OSM-induced joint disease. We injected an adenoviral vector expressing murine OSM into the joints of mice deficient for IL-1, IL-6, TNFα, or iNOS and studied the effects of these gene deletions on OSM-induced joint pathology.
Ubiquitous transgenic overexpression of bovine OSM has been found to be lethal for newborn mice. One mouse survived and developed growth plate disorganization, with enhanced growth of the hind legs (22). Growth plate damage (23, 24) as well as localized growth abnormalities (25) are characteristic features of juvenile idiopathic arthritis (JIA). Therefore, we studied the effects of murine OSM gene transfer not only on development of inflammation and articular cartilage damage, but also on the growth plate. Furthermore, we studied expression of OSM in the synovial fluid of patients with JIA.
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- PATIENTS AND METHODS
OSM is produced in the inflamed joints of patients with RA (3), and results of several in vitro experiments suggest that OSM could play an important role in cartilage damage in RA. OSM induced collagen release from bovine cartilage explants (12) and, in combination with IL-1, from human cartilage explants (3). Furthermore, OSM stimulated PG release and suppressed PG synthesis in porcine articular cartilage (13). In the present study, we used an adenoviral vector expressing murine OSM to investigate in vivo the influence of proinflammatory mediators, which are important for RA, on OSM-induced joint pathology.
The AdMuOSM vector induced inflammation, cartilage PG depletion, periosteal bone apposition, and chondrophyte formation in the joints of naive mice. Semiquantitative RT-PCR analysis showed increased expression of mRNA for IL-6, TNFα, IL-1β, and iNOS in the AdMuOSM-injected knee joint. A relationship with OSM-induced pathology was investigated in mice deficient for these proinflammatory factors.
OSM is a strong inducer of IL-6 gene expression. We had previously observed that AdMuOSM-induced inflammation was not inhibited by IL-6 deficiency (6). The present results demonstrate that cartilage PG depletion is also not affected in these mice. In contrast to the important role of IL-6 in experimental arthritis (21, 37), the present results do not indicate such a role for IL-6 in AdMuOSM-induced joint disease. A positive correlation between TNFα and OSM concentrations has been demonstrated in synovial fluid obtained from patients with RA (14). However, whether there is a direct relationship between these cytokines was, until now, not clear. Results of the present study show that cartilage PG depletion induced by AdMuOSM was not affected by TNFα deficiency. Although inflammation in TNFα-deficient mice tended to be reduced on day 7, by day 14 inflammation in these mice did not differ from that in wild-type mice. This is consistent with reports showing that TNFα is important during disease onset, but that TNFα deficiency or inhibition did not prevent development of severe arthritis in experimental models (38, 39). Taken together, our results do not suggest an important role for TNFα in OSM-induced joint pathology.
Nitric oxide is produced in the inflamed joints of patients with RA (40) and can contribute to IL-1–induced inhibition of PG synthesis (16, 41). We previously demonstrated that cartilage PG depletion, but not joint inflammation, is significantly reduced in iNOS-deficient mice with zymosan-induced arthritis (18). In the present experiments, PG depletion did not differ between iNOS-deficient and wild-type mice, indicating that NO formation is not essential for AdMuOSM-induced cartilage damage.
Joint inflammation and cartilage PG depletion in IL-1α/β–deficient mice were significantly reduced on day 7. This shows an important role for IL-1 in AdMuOSM-induced joint pathology. IL-1 has been shown to be a key factor in the development of experimental arthritis (10, 42). Both inhibition of PG synthesis and stimulation of PG breakdown can induce PG depletion in arthritis, and IL-1 can be involved in both processes (11, 43). In our experiments, ex vivo PG synthesis in patellae from AdMuOSM-injected joints was not inhibited, but rather was increased. This excess in PG synthesis, however, could not prevent articular cartilage PG loss. Breakdown of PGs would, therefore, be the main cause of the observed PG loss in wild-type mice. In the IL-1α/β–deficient mice, ex vivo PG synthesis was even further increased, and this could (at least in part) contribute to the reduced PG loss in these mice. The increased PG synthesis in the IL-1α/β–deficient mice furthermore indicates that in wild-type mice, IL-1 will partly inhibit the elevation of PG synthesis. We previously observed that blocking of IL-1 in experimental arthritis completely prevented inhibition of PG synthesis but did not influence inflammation-induced PG breakdown (15).
Bell et al (4) reported that coinjecting human OSM with recombinant human IL-1 receptor antagonist (IL-1Ra) into the joints of goats could not attenuate OSM-induced cartilage PG depletion. In a previous study, we observed that prolonged high concentrations of IL-1Ra were necessary to prevent IL-1–induced inhibition of PG synthesis in antigen-induced arthritis. These concentrations could be achieved with mini–osmotic pumps but not by bolus injection of IL-1Ra (15). The negative results described by Bell et al could therefore be attributable to poor pharmacokinetics of IL-1Ra in the joint. In the present study, we used IL-1α/β–deficient mice to circumvent these problems and observed clear involvement of IL-1 in the OSM-induced PG loss that occurred during the first week of inflammation.
We have previously shown that repeated injections of IL-1 induce inflammation and cartilage damage in the murine knee joint (11). In that study, the polymorphonuclear cell was the predominant cell type in the inflammatory infiltrate. A role for PMNs in cartilage damage has been shown in vitro (44) and in vivo (45). Recently, OSM was shown to selectively recruit PMNs in an in vitro flow chamber assay (46). Using NIMP-R14 staining, we could detect PMNs in the inflamed synovium of both wild-type and IL-1α/β–deficient mice (results not shown), suggesting that IL-1 is not necessary for OSM-induced PMN influx. This, however, does not exclude a relationship between IL-1 and PMNs in the observed PG depletion. Activation of PMNs might differ between wild-type and IL-1α/β–deficient mice; this requires further investigation.
During AdMuOSM-induced inflammation, irreversible damage to the PG network occurred, as demonstrated by the presence of the MMP-induced VDIPEN neoepitope. Expression of VDIPEN was shown to correlate with severe cartilage damage in murine arthritis (47). The VDIPEN neoepitope was also detected in cartilage from IL-1α/β–deficient mice, indicating that irreversible PG damage can occur independent of IL-1. Future research is needed to identify the enzyme that is responsible for this irreversible damage and its relationship to OSM.
Periosteal bone apposition and chondrophyte formation were induced in both wild-type and gene-deficient mice. Periosteal bone apposition can occur in the short tubular bones of the phalanges, metacarpals, and metatarsals, and also in the long bones during JIA (48, 49). To our knowledge, little is known about the significance of periosteal bone apposition in JIA. Bone apposition was not induced by overexpression of IL-1 or IL-17 (data not shown), which excludes the possibility that bone apposition was a general consequence of inflammation. We previously had observed that OSM could enhance in vitro the bone morphogenetic protein 2–induced differentiation of C2C12 cells toward the osteoblastic lineage (6). This suggests that OSM could play a positive regulatory role during bone formation by enhancing the activity of bone-forming factors. Chondrophyte and osteophyte formation is common in osteoarthritis (OA). Osteophytes can also develop in RA and JIA with secondary OA, but this happens less frequently.
In general, adenovirally mediated gene transfer to the joint results in a transient transgene expression (50, 51) lasting from 1 to 2 weeks. We observed that most of the changes in the murine knee joint had already developed during the first week, when OSM gene expression was demonstrated. The involvement of IL-1 provides circumstantial evidence for a relationship between transgene expression and the observed joint pathology. This was evident on day 7 but not on day 14. After day 7, OSM-induced inflammation subsided, and the growth plate PG content returned to normal levels. During the first week, periosteal activation, leading to bone apposition on day 14, also took place. Thereafter, the process of new bone formation did not proceed. Surprisingly, articular cartilage PG depletion continued after day 7. This is probably not directly related to OSM activity but could be a result of irreversible cartilage damage, delayed repair mechanisms, or morphologic changes (e.g., chondrophyte formation), which could influence cartilage integrity.
A unique finding associated with injection of AdMuOSM is that the growth plate became damaged. This was not observed with vectors expressing either IL-1 or IL-17, although both induced articular cartilage damage. Such growth plate damage has not been previously observed in experimental arthritis in mice of the same age. This process was demonstrated to be dependent on endogenous IL-1. In growth plates, there is a balance between cartilage matrix degradation, proliferation, matrix formation, and hypertrophy. Expression of IL-1 mRNA has been detected in the growth plate of developing bones in mice (52). In vitro results of studies using growth plate chondrocytes from the rat suggested that IL-1 induces resting growth cells to acquire a phenotype of growth zone cells in an autocrine manner (53). Furthermore, IL-1 could play a role in the bone and cartilage resorption processes that occur in the growth plate during the formation of new bone. OSM could either enhance or modify the autocrine effects of IL-1 on growth plate chondrocytes, thereby leading to growth plate PG loss, disorganization, and finally growth abnormalities.
Growth plate changes in patients with JIA have been reported. Magnetic resonance imaging studies have shown epiphyseal cartilage loss in the knees of patients with JIA (23), and in unilateral juvenile arthritis the femoral epiphysis of the arthritic side was observed to be enlarged (24). Although IL-6 is found in elevated concentrations in serum and synovial fluid in JIA (54), the presence of OSM has, as far as we know, not been investigated in these patients. Using ELISA techniques, we detected OSM in synovial fluid of most of the examined children, and we could also detect IL-1β in most of our OSM-positive samples. Our experiments in the cytokine-deficient mice indicated that OSM, in the presence of IL-1, could cause serious risks to the integrity of growth plate cartilage. It is possible that the combination of these cytokines is similarly involved in growth plate damage in JIA.
Both increased growth and growth retardation occur frequently in JIA (25, 55). Most of our synovial fluid samples were obtained from patients with oligoarthritis who had involvement of the knee joint. A study by Simon et al (56) showed a relationship between age at disease onset, involvement of the knee, and localized growth abnormalities in oligoarthritis (formerly called monoarticular and pauciarticular RA). In patients in whom JIA began before age 9 years, the involved side was the longer one. Disease onset after this age led to rapid premature closure of the growth plate and shortening of the involved side. Among the positive samples in our study, the highest concentrations of OSM were found in those obtained from the younger children, which could implicate a role for OSM in increased growth of the involved side. This is further supported by the finding that a mouse transgenic for bovine OSM had enlarged hind limbs (22). We recently began collaborations in order to increase the number of synovial fluid samples available for study from patients with the different forms of JIA. We hope that this will also enable us to further characterize OSM expression during the time course of the disease.
In conclusion, our results demonstrate an important role for endogenous IL-1 in AdMuOSM-induced joint pathology, but no involvement of TNFα, IL-6, or iNOS. The induction of growth plate damage in mice adds a newly recognized pathologic consequence of OSM expression that would be particularly relevant in JIA. The AdMuOSM vector provides a useful tool to further investigate this process in more detail. Our results in the cytokine-deficient mice and the detection of OSM in synovial fluid of patients with JIA suggest that the proinflammatory and cartilage-damaging effects of OSM are relevant in human arthropathies such as RA and JIA.