Centre for Paediatric and Adolescent Rheumatology, UCL, London, UK and
1 Division of Immunology and Allergy, University Hospital, Geneva, Switzerland
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Abstract |
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Methods. Serum and synovial fluid samples from 45 children were examined, 25 pauciarticular JCA, 13 polyarticular JCA and seven spondyloarthropathy. TNF, sTNFRI and sTNFRII levels were measured by EASIA and enzyme-linked immunosorbent assay (ELISA). Analysis of the results was carried out using non-parametric tests: KruskalWallis one-way analysis of variance was used to compare the three clinical subgroups; the MannWhitney U-test was used to compare group medians.
Results. Thirty-three serum samples were assayed for TNF. There was no significant difference between the three groups using the KruskalWallis analysis of variance. Analysis of synovial fluid TNF levels showed significantly lower levels in the spondyloarthropathy group compared with the pauciarticular JCA (P = 0.01) and the polyarticular group (P = 0.002). Significantly higher levels of sTNFRI were observed in the synovial fluid of the polyarticular JCA group compared with the pauciarticular JCA group (P = 0.004) and similarly for sTNFRII (P = 0.03). Molar ratios were calculated for TNF vs sTNFRI. The sTNFRI/TNF
ratio was significantly higher in the spondyloarthropathy group compared with the pauci- (P 0.003) and the polyarticular JCA subgroups (P = 0.003). The combined soluble receptor levels expressed as molar ratio to TNF again showed a significantly higher ratio in the spondyloarthropathy group compared with the pauciarticular group (P = 0.01) and compared with the polyarticular group (P = 0.05).
Conclusion. These results suggest that the increased joint destruction observed in polyarticular disease compared with the other two subtypes may be related to the lower sTNFR/TNF ratios observed.
KEY WORDS: Juvenile chronic arthritis, Synovial fluid, Cytokines, Tumour necrosis factor alpha, Soluble TNF receptors.
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Introduction |
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Pauciarticular JCA, involving four or fewer joints [1], usually runs a benign course. While clinically quite marked inflammation can be observed in individual joints, destruction of that joint is an uncommon finding long term [2, 3]. In contrast, polyarticular disease is frequently associated with joint damage and erosive changes [4, 5]. While serological indices of inflammation are more frequently raised in polyarticular disease, there is little to distinguish between the clinical findings of these two subgroups with respect to individual joints. The juvenile spondyloarthropathies are members of a different clinical group. Although the laboratory markers of inflammation can be impressive, the natural history of juvenile spondyloarthropathies is towards new bone formation, fibrosis and ankylosis rather than bone resorption [68]. This would suggest that different mechanisms of inflammation and/or repair occur in the subgroups of JCA.
The inflammatory cytokines have long been implicated in the pathogenesis of inflammation and in particular arthritis [912]. Tumour necrosis factor alpha (TNF) exhibits many biological actions both in vitro and in vivo pertinent to arthritis. It can induce an inflammatory response, collagenase production [13, 14], bone and cartilage resorption [15, 16] and cachexia [17]. TNF
is one of the primary stimulants of interleukin-1 (IL-1) in synovial cell cultures of adult rheumatoid arthritis patients [18]. This, along with its ability to induce HLA class I molecule expression on vascular endothelial cells and dermal fibroblasts [19], suggests that TNF
plays a central role in the cytokine network in rheumatoid arthritis as well as cellular immunity.
The cytokine network is essential to host defence and thus regulatory mechanisms are essential for homeostasis [20, 21]. Several mechanisms are currently known to down-regulate the pro-inflammatory cytokines including anti-inflammatory cytokines [22, 23] and cytokine antagonists, which are present in synovial fluids [24, 25]. TNF binds to two high affinity cell surface receptors, TNFRI and TNFRII, which are present on virtually all cell types [26, 27]. The extracellular domains of these receptors exist in a soluble form and have been shown to bind circulating TNF
and thus inhibit its biological activity [28]. Among the factors which may influence cellular responses induced by TNF
are the number of cell-associated receptors and the concentration of soluble TNFRI and TNFRII receptors in the extracellular fluid.
The relative proportion of TNF to its soluble receptors in biological fluids has proved to be important in a number of clinical conditions [2932]. In meningococcal septicaemia, a diminished ratio of soluble TNFRI and TNFRII to TNF
was observed in those patients with a fatal outcome compared with survivors [30].
We postulated, therefore, that levels of TNF and its soluble receptors might be different in the synovial fluid of the various subgroups of JCA thus reflecting their contrasting clinical courses.
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Patients and methods |
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Blood samples were available on 31 children. CRP was measured by nephelometry and ESR using the Westergren method. Serum samples were spun and rapidly frozen at -70°C until used.
Ethical Committee approval was obtained for this study at Great Ormond Street Hospital National Health Service Trust and patient assent and parent consent given.
Cytokine determination
TNF determination in synovial fluid and serum.
Synovial fluid and serum samples were initially assayed for TNF by EASIA (Medgenix, Fleurus, Belgium) with a limit of sensitivity of 20 pg/ml. In 60% of samples assayed, TNF
levels were below the limit of detection of the assay. These negative samples were subsequently reassayed using a high sensitivity enzyme-linked immunosorbent assay (ELISA) (R & D Systems Europe, Abingdon, Oxon) with a limit of sensitivity of 2 pg/ml. Both assays were chosen as their antibodies detected both free and complexed TNF
(as confirmed by the manufacturers). Intra-assay variation was 11.7% with an interassay variation of less than 10% for the individual assays used. There was a tendency for the high sensitivity kit to give lower values to high TNF levels than observed using the low sensitivity kit. This was probably due to the fact that the TNF
antibodies used in these kits are directed against different TNF
epitopes, the different standards used for the calibration curves, or to a different sensitivity because of interference with other proteins. Since the values obtained from these two assays were not linearly related, the results obtained from both assays were analysed separately using non-parametric tests (vide infra).
Determination of soluble TNF receptors RI and RII in synovial fluid and serum.
Initial samples were assayed by a method previously reported [25]. Subsequent samples were assayed by ELISA (R & D Systems Europe). Both inter- and intra-assay variation was less than 10% for each individual system used. Using values from the initial in-house assay for sTNFRI as internal controls, we observed that values on the same samples from the R & D System Method were consistently twice as high as the original results, and thus R & D readings were halved in order to be able to combine results from samples measured only with the R & D kit and those measured by both methods.
Statistical analysis
Since specimens were available in limited amounts only it was not possible to test them by both assays. As synovial fluid values for the TNF and TNFRI and TNFRII were obtained by different assays, non-parametric analysis by the KruskalWallis one-way analysis of variance was used to compare the three clinical subgroups. Subsequently the MannWhitney U-test was used to compare each pair of group medians. For analysis of TNF
values using the low sensitivity kit, samples below the limit of detection were given a value of 20 pg. For analysis of results obtained using the high sensitivity kit, values were censored at 33 pg, the highest value observed using this assay. The Bonferroni correction was used to adjust the P values obtained from the MannWhitney U-tests to take into account multiple comparisons.
All serum samples were assayed by the same assay for TNF, sTNFRI and sTNFRII and values were found to be normally distributed if log transformed using Shapiro and Francia's W-test. However, the assumption of constant variances were not valid. Therefore, the results were expressed as medians and analysed by the KruskalWallis one-way analysis of variance.
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Results |
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Synovial fluid TNF levels were compared in the three subgroups and the results obtained using both the low and high sensitivity assays are shown in Fig. 2a
, b
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Using the high sensitivity assay, TNF levels were again highest in the polyarticular group (median >33, 95% CI 18 to >33 pg/ml) (Fig. 2b
). Intermediate values were observed in the pauciarticular group (median 30.4, 95% CI 13.5 to >33 pg/ml), which were not significantly different than the polyarticular group, P = 0.3. The lowest levels were observed in the spondyloarthropathy group (median 9, 95% CI 5.918.6 pg/ml), which were significantly lower than both the pauci- and polyarticular groups, P = 0.01 and P = 0.002, respectively.
Significantly higher levels of sTNFRI were observed in the synovial fluid of the polyarticular group than in the pauciarticular group (median 9.2, 95% CI 7.311.4 pg/ml vs median 6.3, 95% CI 5.67.5 pg/ml) P = 0.004 (Fig. 3a). Levels were intermediate in the spondyloarthropathy group but were not significantly different from the other two groups (P = 0.4 and P = 1.0, respectively).
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Since the biological activity of TNF appears to be related to the ratio of TNF
to its soluble receptors in vivo, these were calculated for the synovial fluid samples using the results of the high sensitivity assay. Molar ratios were calculated taking the molecular weight of TNF
to be 17 kDa. Since TNF
circulates in a trimolecular complex, apparent molecular weight values for TNF
were taken to be 51. Values obtained using the high sensitivity assay were used. The molecular weights of sTNFRI and sTNFRII were taken to be 55 kDa and 75 kDa respectively, and molar concentrations of TNF
and its receptors calculated accordingly. The results are displayed in Fig. 4ac
. The TNFRI/TNF
ratio was much higher in the spondyloarthropathy group (median 815, 95% CI 5811447) than in the pauci- and polyarticular groups (median 286, 95% CI 224468; P = 0.003 and median 353, 95% CI 257447; P = 0.003; respectively). There were no significant differences between the polyarticular and pauciarticular groups P = 1.0 (Fig. 4a
). A similar trend was observed in the TNFRII/TNF
ratios, with the highest ratios being observed in the spondyloarthropathy group (median 980, 95% CI 3672837), which was greater than that observed in the polyarticular group (median 793, 95% CI 554980) and the pauciarticular group (median 535, 95% CI 392688). However, the differences were not statistically significant, P = 0.9 and P = 0.3 (Fig. 4b
).
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Synovial fluid levels of TNF were compared according to the clinical severity of joint inflammation (data not shown). While there was a trend to higher levels in patients with moderate disease activity, this was not statistically significant, P = 0.11. Clinically mild, moderate and marked degrees of inflammation were equally divided between the three disease subgroups (Fisher's exact test P = 0.36).
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Discussion |
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In recent years there has been increasing interest in the role of the soluble TNF receptors in a variety of diseases [3032]. Girardin et al. [30] noted that in children with severe meningococcal septicaemia, levels of sTNFRI and sTNFRII initially increased as TNF increased. However, at TNF levels greater than 500 ng/ml no further increase in the soluble receptors was observed and this was associated with a high mortality. Thus, the investigators concluded that an imbalance between TNF
and its naturally occurring inhibitors was implicated in the increased morbidity and mortality. Van Zee et al. [31] observed that soluble TNF receptors increased with inflammation in experimental bacteraemia and circulate at sufficient levels in critically ill patients to block TNF
cytotoxicity in vitro. This would suggest that in experimental bacteraemia, soluble TNF receptors protect against the TNF-mediated effects observed in septic shock.
The clinical situation is somewhat different in JCA when compared with acute septic episodes. First, serum TNF levels are either low or undetectable and certainly never reach the levels observed in septic shock. However, in our study synovial fluid levels in the 100 pg/ml range were not unusual and unlike acute septic episodes may remain elevated for months or years while synovitis persists.
In contrast to our findings, Madson et al. [36] observed increases in a number of inflammatory cytokines but not TNF in both the serum and synovial fluid in JCA. The explanation for this discrepancy may be a technical one, in that a number of anti-TNF antibodies used in assays do not detect complexed TNF
, its predominant form in biological fluids [37]. Cope et al. [24] and Roux-Lombard et al. [25] observed increased levels of both TNF
and its soluble receptors in the synovial fluid of patients with rheumatoid arthritis and that sTNFRs were four to five fold higher in synovial fluid than in serum levels. These results concur with our own where synovial fluid sTNFRs were on average six times greater than serum levels. Furthermore, as they observed, synovial fluid sTNFRII levels were on average 1.5 times higher than TNFRI. Interestingly, in our patients with JCA serum sTNFRII was twice as high as sTNFRI. This contrasts with the findings in sepsis where sTNFRII levels are at least three to four times higher than sTNFRI. These variations in the ratios of the sTNFRs in different disease states and biological fluids may be due to the different standards used in the individual assays employed. Alternatively, they may reflect the different mechanisms of shedding of the two receptors, in particular variations in responsiveness of TNFRI and TNFRII to TNF
stimulation [38], or the effect of proteolytic enzymes, particularly elastase or other metalloproteinases, known to be present in inflamed joints [39]. Furthermore, the cell populations within the joint are quite distinct from those observed in the peripheral blood. Thus, the observed sTNFRI and RII levels may reflect different receptor expression known to occur on different cell types [2840]. Since the vast majority of synovial fluid cells are neutrophils it is surprising that TNFRII, the most abundant polymorphonuclear leukocytes TNFR, is not higher in JCA synovial fluid when compared with serum. However, the relative increase of TNFRI might be explained by endothelial activation, where TNFRI is in greatest abundance.
In this study we observed the highest levels of synovial fluid TNF in patients with polyarticular disease, with significantly lower levels observed in the pauciarticular group. However, the lowest levels were observed in children with spondyloarthropathy. These findings are not related to the degree of knee joint inflammation observed clinically, since similar proportions of moderate and marked knee joint inflammation were present in the polyarticular and spondyloarthropathy groups.
For all children there was a tendency for the sTNFRs to rise with increasing TNF levels (data not shown) and this concurs with the findings in other inflammatory conditions [30]. However, in the spondyloarthropathy group, despite having the lowest levels of TNF
, the levels of sTNFRI and RII were similar to the other two groups. Thus, significantly higher ratios of sTNFRs/TNF
were observed in this group. High concentrations of sTNFRs have been shown to abrogate the biological activity of TNF
both in vitro and in vivo [38]. While 300500 molar excess may be required for almost complete loss of TNF
bioactivity, as little as 30 molar excess of the soluble receptors can have some inhibitory activity [31]. In our study, sTNFRs were present in between 100 and 5000-fold molar excess, thus it is conceivable that there would be considerable differences in the bioactivity of TNF
in synovial fluid with low and high sTNFR levels. Since significantly lower ratios were observed in the polyarticular group (on average 180 molar excess) when compared with the spondyloarthropathy group (molar excess 770), it could be anticipated that the resorptive effects of TNF
on bone and cartilage within the joint in the former group would be expected to be more marked when compared with the latter. This would provide at least part of the explanation for the different clinical courses of these disease subgroups. It could be argued that disease-modifying and anti-inflammatory agents such as methotrexate, sulphasalazine and steroids might in themselves alter cytokine and receptor concentrations. These drugs were taken by approximately 50% of both the spondyloarthropathy and polyarticular groups: 3/7 children with spondyloarthropathies were given sulphasalazine compound, and 6/13 children with polyarticular JCA were treated with methotrexate.
In conclusion we have observed a significant difference in the levels of TNF and the sTNFRs TNF
ratios in the different subgroups of JCA. Whilst recognizing that TNF is only one of the many cytokines, not to mention growth factors, involved, and this system of agonist/antagonist is only one of many methods of regulation which might influence bone destruction and remodelling in JCA, these findings reflect different pathophysiologies at play within the different subgroups of JCA, in particular, juvenile spondyloarthropathy.
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Acknowledgments |
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Notes |
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References |
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