Indian Pediatrics 2002; 39:739-742
Cytokine Production by Peripheral Blood Mononuclear Cells From Patients With Juvenile Idiopathic Arthritis
Juvenile idiopathic arthritis (JIA) is a chronic inflammatory arthritis of childhood characterized by synovitis and systemic features. The major players in the pathogenesis are T cells, macrophages and B cells. Cytokines are glycoproteins secreted from inflammatory cells like, activated T cells and macrophages. They are essentially of two varieties, pro-inflammatory (IL-1, IL-6, TNF-alpha, IL-8) or anti-inflammatory (IL-4, IL-10, IL-1 receptor antagonist). Cytokines are instrumental in regulating the magnitude, nature and duration of inflammatory response in arthritis. In adult rheumatoid arthritis (RA) pro-inflammatory cytokines IL-6, TNF-alpha and IL-1 have a major role in pathogenesis of synovitis(1). In contrast to adult RA, JIA is a heterogeneous disease with many distinct subsets. Systemic onset disease has marked systemic manifestations like fever, rash, hepato-splenomegaly with mild arthritis whereas polyarticular disease has persistent inflammatory arthritis involving more than 4 joints with mild systemic manifestations(2). The cytokines involved in the pathogenesis of JIA as a whole and in different subsets are still being investigated. Serum IL-1 and IL-6 levels are increased in patients with JIA in majority of the studies(3-5). We evaluated the production of other pro-inflammatory cytokines from peripheral blood mononuclear cells of patients with systemic onset and polyarticular form of JIA. Oligoarticular form was not included as they have a limited systemic response.
Subjects and Methods
Children with polyarticular or systemic onset JIA(2) with active disease were included in the study. Active disease was defined in polyarticular group as presence of at least 4 swollen and tender joints along with elevated ESR or CRP. In systemic onset group active disease was defined as presence of systemic manifestations like fever, rash along with elevated ESR (>30 mm in first hour) or C-reactive protein > 0.6 mg/dL. Ten healthy individuals (controls) and 10 patients with adult rheumatoid arthritis (disease control) were also included. None of the patients or disease controls were receiving any disease modifying drugs or corticosteroids. Ten mL blood was collected in heparinized tube and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation method. After counting, the cell count was adjusted to 2.5 million/mL in RPMI-1640 supplemented with a 10% fetal calf serum. Two mL of these cells were cultured for 18 hours at 37ºC. The supernatant was harvested after centrifugation at 1500 rpm for 15 minutes. The supernatant was aliquoted and stored at –70ºC.
TNF-alpha, IL-4, IL-2, and IFN-gamma were assayed using antibodies from Genzyme Corporation USA (Duoset) and the procedure prescribed by them. IL-8 was assayed using a Predicta ELISA kit from Genzyme, USA.
As the data were not normally distributed non-parametric tests were used for inter-group comparison like Mann Whitney U test and P value of <0.05 was considered significant.
There were 17 children (10 boys/7 girls) included in the study, of them 8 had polyarticular and 9 had systemic onset disease. Their mean age was 13.5 years (3-22 years) and the mean duration of disease was 1.5 years. Most of these children were receiving non-steroidal anti-inflammatory drugs.
The median IL-2 and IFN-gamma levels were no different in the three groups (Table I). Interleukin 8 levels were higher in JIA group (1061.9 pg/mL) as compared to control group (999.5 pg/mL; P <0.001) and adult RA (851.4 pg/mL; P < 0.001) (Fig 1).
Table I- Median (Range) of Different Cytokines in Three Different Groups
The median TNF alpha levels were no different in the three groups; controls (875 pg/mL), JIA (815.2 pg/mL) and adult RA (422.6 pg/mL). IL-4 was not detectable in any of the groups. Among IL-8 and TNF-alpha, only TNF-alpha was higher among systemic onset disease (1200 pg/mL) as compared to polyarticular disease (605 pg/mL; P < 0.05) and controls ( P < 0.05).
Our results suggest that TNF-alpha and IL-8 are the major cytokines in systemic onset JIA and IL-8 in polyarticular JIA. T-cell derived cytokines like IL-2, IFN-gamma and IL-4 levels are not increased. We measured spontaneous production of these cytokines by PBMC instead of serum cytokines levels as those are affected by nonspecific binding factors like serum proteins, receptor antagonists and soluble receptors(6).
Our observation of increase in production of IL-8 is similar to that reported earlier(7) but is in variance to other workers(8) who found normal serum levels and further, no difference in the three subtypes of JIA. Recently, immune complexes present in JIA have been shown to induce production of IL-8 from peripheral blood mononuclear cells as well as from synovial monocytes(9). IL-8 may be responsible for neutrophilic leukocytosis seen in patients with systemic onset disease since it is a neutrophil chemotactic factor. A direct relationship between synovial fluid cell count and IL-8 level has been reported, suggesting a role for it in synovitis(7).
IL-2 and IFN-gamma levels have been found to be normal in most studies supporting our observation(4,5,10). Our finding of no significant increase in TNF-alpha levels in the overall JIA population is similar to that reported(10,11), but is at variance with that reported by other workers(8). These discrepancies are probably due to the use of serum/plasma/culture supernatants in different studies.
We found higher levels of TNF alpha in systemic onset disease as compared to polyarticular disease as well as controls. Rooney et al.(5) found these to be higher in systemic onset group, thereby comple-menting our results. Elevated levels of TNF receptors in systemic onset JIA indirectly imply increased TNF levels(11). Synovial fluid mononuclear cells from patients with JIA express TNF-alpha(12). The pathogenic role of TNF is further supported by an excellent response to anti-TNF therapy in systemic onset JIA(13).
We are grateful for the technical support provided by Mr. Sultan Alam in conducting the experiment.
Contributors: AA and RM planned the study and wrote the manuscript. In addition AA collected and analyzed the samples and did statistical analysis. AA will act as the guarantor for the manuscript.
Funding: This work was supported by a grant from Department of Science and Technology, New Delhi to AA.
Competing interests: None stated.