Utilizing a multiplex assay for cytokines and inflammatory markers, we characterized the profile of these biomarkers in a well defined cohort of JIA patients and controls. We observed striking elevations of a number of cytokines. Of special interest to us were the increased serum levels of sCD154 in children with different JIA subtypes. This elevation was most pronounced in children with systemic, RF-positive polyarticular and ERA subtypes, and modest among children with the oligoarticular subtypes. To our knowledge, ours is the first study to investigate sCD154 levels in JIA. Elevated levels of sCD154 have been found in other autoimmune disorders including SLE, RA, IBD, systemic sclerosis and Sjogren's syndrome [18
]. Expression of CD40 on synovial monocytes, fibroblasts and dendritic cells in RA has been reported [29
]. After stimulation in vitro, peripheral blood T-cells from patients with RA had increased and longer expression of CD154 compared to T-cells from controls [16
]. Blocking CD40 on synovial fibroblasts from RA patients with soluble anti-CD40 antibodies prior to co-culture with synovial mononuclear cells resulted in decreased TNFα levels [16
]. Moreover, blockade of CD154 with an antibody ameliorates collagen induced arthritis in a murine model of RA [31
]. These observations suggest a major role for CD154 in the perpetuation of inflammation in RA.
The inflammation in synovial tissue of children with JIA is characterized by the presence of antigen presenting cells and activated T-cells, and is indistinguishable from the pathology observed in patients with RA [32
]. Our results suggest that sCD154 might also play a role in the pathogenesis of most of the JIA subtypes. It is conceivable that the activated CD4 T-cells in JIA synovium may be the source of sCD154, although it is possible that other cells, such as activated platelets release sCD154. Cell-bound as well as sCD154 have been shown to activate endothelial cells in vitro
, and increase production of leukemia inhibitory factor, IL6 and granulocyte-macrophage colony stimulating factors [14
]. We speculate that sCD154 could possibly interact with cells bearing CD40, possibly enhancing antigen presentation, and production of other inflammatory mediators in JIA.
Our correlation analyses showed significant association between sCD154 and the pro-inflammatory markers IL6, IL1 β, IL8 and TNFα. It has been shown that sCD154 has the potential to activate endothelial cells in vitro
, and increase production of IL6 [14
]. Elevated levels of IL6 in JIA have been reported by several authors [2
]. Indeed, levels of IL6 were strikingly higher among all JIA subtypes compared to controls in our study. IL6 was also significant in the logistic regression analysis suggesting that it likely plays a major role in concert with other cytokines in the pathogenesis of JIA. The increased levels of the pro-inflammatory cytokines IL1β and IL8 observed in our cohort have also been reported in different forms of JIA as well as RA [3
]. In agreement with earlier findings, we observed that levels of TNFα were elevated in all subtypes except those with persistent oligoarticular JIA, in which fewer joint involvement and less systemic inflammation is observed [6
]. Together these observations support a primary or secondary role for the inflammatory cytokines IL6, IL1β, IL8 and TNFα in different JIA subtypes.
The elevated levels of sIL2R and IL10 in the different subtypes of JIA are in agreement with previous studies [2
] although with some subtle differences which may be attributable to the differences in the size of the cohorts. For example, although we observed increased levels of sIL2R in a JIA subtype equivalent to ERA in a study by Mangge et al [8
], these concentrations were not significantly different from controls. Nevertheless, sIL2R levels tend to be highest in systemic or polyarticular JIA suggesting that it maybe a biomarker of JIA. Our logistic regression analysis suggests that IL10 might have a protective effect on disease, as might be anticipated by its inhibitory activity.
Some cytokines were barely detected in the serum in a majority of subjects detected in our study. For example, undetectable levels of IL2, and IFNγ have been reported by other investigators [34
]. On the other hand, some of the results described here have not previously been reported by others. For instance some authors have reported finding no significant elevations in the levels of IL1β, IL8, or TNFα [2
]. Differences in the results reported in this study and others could reflect heterogeneity of the different JIA cohorts. Other differences could be due to methodological issues, such as test specimen (serum versus plasma) and assay methodology. It is likely that some cytokines are more elevated in the synovial fluid and not in the serum, especially in children with persistently oligoarticular JIA.
Our study has several strengths. Our cohort of ~80 JIA patients and 80 pediatric controls is larger than most series. Our patients have been classified using the ILAR criteria, which results in more homogeneous subtypes compared to earlier studies. To our knowledge sCD154 has not been measured in JIA patients before. We also measured several other cytokines previously implicated in JIA. The multiplex assay used small volumes of serum to assay thirteen cytokines. This method has also been successfully used by de Jager et al[33
], but sCD154 and sIL2R were not among the cytokines included in their study. They also found that IL6 differed between controls and patients. We have used an objective partially automated multianalyte assay which we have found to reliably measure cytokines and other analytes in a variety of disorders including immune deficiency [26
], coronary artery disease [37
] and acute rheumatic fever [38
]. We believe the differences in cytokine profiles, as well as in the inflammatory marker sCD154, suggest that these might play a role in the pathogenesis of at least some subtypes of JIA.
The past decade has seen the emergence of biological therapeutic agents directed against several mediators of inflammation. Our findings of elevated TNFα is in agreement with the response seen with anti-TNF therapy in children with JIA. The elevated levels of IL1β and IL6 support the roles for biological agents targeting these cytokines as well. Finally, sCD154 could also be a potential target of biological therapy, although some studies in individuals with SLE raise the concern for pro-thrombotic effects, possibly due to effects of these agents on platelets and/or the endothelium [39
]. If the importance of the CD40–CD154 pathway in JIA is confirmed, and safety concerns could be addressed, therapy directed at inhibiting this pathway deserves further exploration in JIA.