Juvenile rheumatoid arthritis (JRA), the most common rheumatic disease in children, is characterized by large numbers of infiltrating leukocytes in inflamed joints. Although the molecular signals that control the recruitment of leukocytes to the joints have not been fully characterized, this process is believed to be controlled by certain chemokines (1, 2). Chemokines, a superfamily of small (8–14-kd), structurally related chemotactic cytokines, have been reported to selectively recruit and activate leukocytes at sites of inflammation (1, 3, 4). These chemokines can be divided into 2 major subfamilies, the CXC and CC chemokines. CXC chemokines such as interleukin-8 (IL-8) have been implicated in acute inflammation, since they exert their function mainly on neutrophils, whereas the CC chemokines, including RANTES and monocyte chemoattractant protein 1 (MCP-1), attract and activate a variety of cells, including monocytes, macrophages, lymphocytes, eosinophils, and basophils, and have been implicated in chronic inflammatory disease (1, 3–5).
Recent data from animal models suggest that both RANTES and MCP-1 play important roles in the pathogenesis of arthritis (2, 5–8). In adjuvant-induced arthritis in the rat, increased levels of RANTES have been found in both the blood and the joint, and synovial levels of RANTES have been found to correlate with clinical symptoms of joint inflammation (5, 6). Furthermore, administration of anti-RANTES antibody has been shown to prevent the onset of arthritis and greatly ameliorate arthritis symptoms once the disease develops (6). Similarly, elevated levels of MCP-1 have been found in both the blood and the joint in animal models of arthritis (5, 7). In addition, injection of MCP-1 antagonist was shown to markedly reduce the severity of arthritis and the infiltration of monocytes, and pretreatment with this antagonist could prevent the development of arthritis (7, 8). The pivotal role of RANTES and MCP-1 in arthritis in humans is underlined by the findings of an enhanced production of these two CC chemokines in serum and/or synovial fluid (SF) of adult patients with rheumatoid arthritis (RA) (9–12).
Taken together, these data led us to hypothesize that RANTES and MCP-1 are important in the regulation of inflammation in JRA, and that a determination of RANTES and MCP-1 profiles may be useful for monitoring disease activity in JRA patients. To test this hypothesis, we longitudinally investigated serum and SF levels of RANTES and MCP-1, as well as the in vitro migration of mononuclear cells toward the SF, in patients with JRA. We also investigated correlations of these CC chemokines with subtypes of JRA and with clinical and laboratory parameters of disease activity.
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In this longitudinal study, we investigated serum and SF levels of RANTES and MCP-1 in patients with JRA, as well as the in vitro migration of PBMCs toward SF from JRA patients. Increasing evidence implicates an important role of CC chemokines, particularly RANTES and MCP-1, in the pathogenesis of chronic inflammatory diseases (1, 3–5, 18–20). Recent data from studies of animal models and adults with RA suggest that RANTES and MCP-1 are involved in the pathogenesis of chronic arthritis (2, 5–12). Boiardi et al (9) observed high levels of serum RANTES in a series of adult RA patients during the active stage of disease, and methotrexate treatment significantly lowered the serum RANTES levels. High serum levels of RANTES after 6 months of methotrexate treatment seem to be predictive of radiologic erosions after 1 year in the same patient cohort (9). To the best of our knowledge, this is the first report of a study of RANTES levels in patients with JRA.
The results of our study demonstrate that the different onset types of JRA are all associated with significantly elevated serum levels of RANTES during active disease. After treatment, we observed a significant reduction in serum RANTES levels among the 3 subtypes of JRA when clinical remission had been achieved, suggesting the association of this chemokine with clinical disease activity. This hypothesis is supported by the significant correlations between serum RANTES levels and several conventional parameters of inflammation, including the CRP concentration, hemoglobin value, WBC count, and platelet count. Of particular interest is the observation that the serum RANTES level was equally or more strongly correlated with JRA activity than was either the CRP concentration or the ESR. This was observed at the time of clinical remission in JRA patients, when CRP and ESR values were within the range of normal, but serum RANTES levels remained significantly elevated. This finding is consistent with the recent report on an experimental model of arthritis in rats (6). In that study, Barnes et al (6) described high levels of RANTES in whole blood and joints of Lewis rat during the course of adjuvant-induced arthritis, and the high blood levels of RANTES persisted several weeks after clinical recovery.
Furthermore, in our followup study of JRA patients who fulfilled the criteria for clinical remission, a relationship was found between serum RANTES levels and the duration of clinical remission, with low levels of RANTES being associated with prolonged clinical remission and high levels with shorter clinical remission. Therefore, the elevated concentrations of RANTES during clinical remission suggest immunoactivation even in the absence of clinically apparent disease, possibly indicating an imminent flare of JRA.
The course of JRA can be highly variable. Although some patients experience complete remission and require no further treatment, a subgroup of JRA patients experience a flare of disease shortly after remission, particularly when therapeutic agents are discontinued gradually. The identification of such individuals remains a challenge for pediatric rheumatologists. The monitoring of JRA is traditionally based on clinical observations and determinations of conventional parameters of inflammation, such as the CRP concentration, ESR, WBC count, hemoglobin value, platelet count, and serum ferritin level. While the CRP and ESR values are reliable biochemical indicators that are preferred for monitoring inflammatory activity in adults with RA, neither assessment is sufficient for distinguishing among JRA patients with moderately active disease, JRA patients with inactive disease, and healthy children (21). Surprisingly, the results of our study show that it is possible to predict imminent relapse in JRA patients during clinical remission by determining serum RANTES levels. Specifically, the normalization of serum RANTES levels during clinical remission was associated with a sustained remission in 82.5% of the patients, whereas a relapse of disease occurred within 6 months in 57.8% of the patients in whom high serum RANTES levels persisted during remission.
Taken together, these findings strengthen the conclusion that in patients with JRA, serum RANTES levels reflect the presence of joint inflammation better than does either the CRP concentration or the ESR. Further study is necessary to determine whether these JRA patients with persistently high serum RANTES levels would benefit from continuing aggressive therapy until the RANTES levels return to normal.
The mechanisms underlying the involvement of RANTES in the pathogenesis of JRA remain to be identified and explained. RANTES has been strongly implicated in arthritis, chiefly because of its potent effect on chemotaxis and activation of monocytes and T lymphocytes (22, 23). Current evidence on the pathogenesis of JRA suggests that it is indeed a severe immunoinflammatory reaction in which mononuclear cells are important effector cells. Although the initiating event(s) is not clear, products of mononuclear cells along with abnormal cellular and humoral responses are responsible for the cascade of events that lead to the disease process (24). In the JRA patients in our study, SF levels of RANTES were associated with the in vitro migration of PBMCs, suggesting that RANTES plays a role in the recruitment of mononuclear leukocytes into the inflamed joints. This is supported by the significant inhibition of SF chemotactic activity for PBMCs by the addition of anti-RANTES antibody in our neutralization experiments. An analogous feature was also observed in adult RA patients by Volin et al (25). The role of RANTES in T cell and monocyte recruitment to sites of inflammation is also underlined by the high levels of expression of the chemokine receptor CCR5 (receptor for RANTES, MIP-1α, and MIP-1β) on activated monocytes and T lymphocytes observed in the synovial fluid and tissue of RA patients. This activation can induce a Th1 shift in the immune-mediated response (26). Similarly, in the joints of mice with collagen-induced arthritis an up-regulation of CCR1 and CCR5 (both are receptors for RANTES) was found in the synovium (2).
Taken together, these data indicate the important role played by RANTES in the recruitment of mononuclear cells in chronic arthritis. Of note, increased RANTES expression has been associated not only with juvenile and adult RA, but also with several inflammatory disorders, including asthma, atopic dermatitis, atherosclerosis, glomerulonephritis, endometriosis, allogeneic graft rejection, some neurologic disorders, and certain malignancies (27). These observations support the assumption that RANTES is not specific to chronic arthritis. Accordingly, RANTES might be implicated in mediating the amplification and perpetuation, rather than the initiation, of the chronic synovial inflammation present in JRA patients.
Our neutralization studies indicated that MCP-1 may be one of the major chemokines that contribute to the chemoattraction and retention of mononuclear leukocytes in the inflamed joints of JRA patients. Our examination of MCP-1 also showed that serum levels of MCP-1 were significantly elevated in patients with systemic-onset JRA, similar to the findings of the cross-sectional analysis reported by De Benedetti et al (28). However, that study did not follow up on and compare MCP-1 levels during active disease and remission. We showed that the MCP-1 levels correlated with clinical disease activity, as demonstrated by the significant reduction in the serum MCP-1 levels after treatment. We also found that serum MCP-1 levels were associated with current systemic features at the time of sampling, the WBC count, and the serum ferritin level. In addition, we observed a trend toward a correlation between SF MCP-1 levels and PBMC migration in vitro toward SF from patients with systemic-onset JRA, although the small number of SF samples studied in this subgroup did not allow for statistical analysis. These findings strengthen the conclusion that serum MCP-1 levels are related to disease severity in patients with systemic-onset JRA. Furthermore, elevated MCP-1 levels were still found during periods of remission and reflected immunoactivation, despite the absence of clinical symptoms as well as normalization of laboratory parameters of inflammation. Our study also showed that serum levels of RANTES and MCP-1 varied among different subtypes of JRA. This suggests that CC chemokines have differing roles in the different JRA subtypes and likely reflect JRA subtype heterogeneity.
The reasons for the high levels of expression of RANTES and MCP-1 in JRA remain unclear. The correlation with levels of acute-phase proteins suggests that their production is in fact cytokine driven. It is well known that the production of RANTES and MCP-1 is up-regulated by the proinflammatory cytokines IL-1 and tumor necrosis factor α (TNFα) in a variety of cell types, including cells present in arthritic joints (10–12, 25, 29). The fact that proinflammatory cytokines such as IL-1, IL-6, and TNFα have been found in elevated levels in the blood and SF of patients with JRA (30) indicates that these cytokines may lead to the synthesis and release of RANTES and MCP-1 in these patients. Further study is necessary to determine the regulation of the production of CC chemokines and the expression of corresponding genes in vivo during joint inflammation in patients with JRA.
In summary, we conclude that RANTES is a key molecule in the pathogenesis of all 3 onset types of JRA, whereas MCP-1 appears to be particularly important in systemic-onset JRA. Our findings indicate that RANTES and MCP-1 may be major chemoattractants for mononuclear leukocytes into the JRA joint. Serum CC chemokine levels represent more highly sensitive markers of disease activity than the conventional parameters of inflammation. Of particular interest is that the odds ratio for the probability of a prolonged clinical remission, rather than an early relapse within 6 months, was 6.45 for children in whom serum RANTES levels normalized during remission as compared with those who had persistently high levels of RANTES during remission. It seems reasonable to suggest that RANTES and MCP-1 may represent potential therapeutic targets in the modulation of the inflammation in JRA.