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CORRELATION OF SERUM INTERLEUKIN-6 LEVELS WITH JOINT INVOLVEMENT AND THROMBOCYTOSIS IN SYSTEMIC JUVENILE RHEUMATOID ARTHRITIS FABRIZIO
DE
BENEDETTI, MARGHERITA MASSA, PIERA ROBBIONI, ANGEL0 RAVELLI, GIUSEPPE R. BURGIO, and ALBERT0 MARTINI
We measured interleukin-6 (IL-6) levels in 70 serum samples obtained from 25 patients with systemiconset juvenile rheumatoid arthritis (JRA), using the hybridoma cell line B9. Patients with systemic-onset JRA had significantly elevated serum IL-6 levels during active disease (mean f SD 92.1 2 75.1 hybridoma growth factor unitdml; P < O.OOOO1 versus healthy age-matched controls), but not during remission. Serum IL-6 levels correlated with the extent and severity of joint involvement (P < 0.001) and with platelet counts (PC 0.05). Our data suggest that IL-6 plays a significant role in the pathogenesis of systemic-onset JRA.
The systemic onset form of juvenile rheumatoid arthritis (JRA) is characterized by the presence of chronic arthritis associated with high spiking fever, and is frequently accompanied by other systemic symptoms including evanescent cutaneous rash, hepatosplenomegaly , lymphadenomegaly , and serositis (1,2). The typical fever pattern is characterized by the presence of one or two daily spikes, usually in the late afternoon or evening; most patients are afebrile in the morning. There is prominent laboratory evidence of inflammation, including marked elevation of the erythrocyte sedimentation rate (ESR) and of acute-phase From the Clinica Pediatrica, Universita’ di Pavia, IRCCS Policlinico S. Matteo, Pavia, Italy. Supported in part by IRCCS S. Matteo, Pavia, and by grant 89.02675.04 from CNR, Rome, Italy. Fabrizio de Benedetti, MD; Margherita Massa, PhD; Piera Robbioni, PhD; Angelo Ravelli, MD; Giuseppe R. Burgio, MD; Alberto Martini, MD. Address reprint requests to Alberto Martini, MD, Clinica Pediatrica, IRCCS Policlinico S. Matteo, P. le Golgi 2,27100 Pavia, Italy. Submitted for publication December 26, 1990; accepted in revised form April 18, 1991.
Arthritis and Rheumatism, Vol. 34, No. 9 (September 1991)
protein levels, thrombocytosis, and neutrophilic leukocytosis. Systemic features vary in duration; in approximately half of the patients, arthritis does not remit when fever is controlled (1,2). Interleukin-6 (IL-6) is a pleiotropic cytokine which plays an important role in the induction of immune and inflammatory responses (3). In particular, IL-6 induces fever, acute-phase protein synthesis, leukocytosis, and thrombocytosis (3,4). Many types of cells have been shown to produce IL-6 (3), including synovial cells (5,6) and chondrocytes (7). Elevated levels of IL-6 have been found in sera and synovial fluids of adult patients with rheumatoid arthritis (RA) (8). In the present study, we investigated serum 1L-6 levels in patients with systemic-onset JRA.
PATIENTS AND METHODS Patients. A total of 70 serum samples were obtained from 25 children (mean age 8.7 years, range 2-17 years) who fulfilled the American Rheumatism Association proposed criteria for the diagnosis of systemic-onset JRA (9). On at least 1 occasion during the study, all systemic-onset JRA patients had active disease, as defined by evidence of synovitis on examination. During the course of the study, the JRA went into clinical remission, as defined by the absence of active synovitis, in 14 patients. All but 1 patient were treated with nonsteroidal antiinflammatory drugs (NSAIDs) during active disease, and approximately twothirds of them were also treated with methotrexate andor low-dose steroids (on an alternate-day regimen in the majority of cases). At the time serum samples were obtained, the extent of articular involvement was assessed by the number of involved joints (defined as soft tissue swelling, or, in the case of the hips and shoulders, pain with loss of active motion). For each joint with active arthritis, swelling was graded (1 = mild, 2 = moderate, 3 = severe), and a joint swelling score
IL-6 IN JRA was calculated by summing the scores of all joints. Systemiconset JRA patients with active disease were divided into 2 groups according to the presence or absence of systemic symptoms, defined as the presence of high spiking fever. The 2 groups of patients had similar ESR and C-reactive protein (CRP) values and were comparable in terms of age at onset and disease duration. Seven children (mean age 5.5 years, range 2-11 years) who had presented with a systemic JRA-like illness, with arthralgia but without arthritis, were also investigated. All of them had high spiking fever and macular rash, and some had hepatosplenomegaly and serositis; 4 were receiving NSAIDs, and 3 NSAIDs and low-dose steroids (2 on an alternate-day regimen). Age at onset, ESR values, and CRP concentrations were comparable with those of the systemiconset JRA patients (data not shown). After a mean followup of 2.4 years (range 0.9-4.5 years), 2 of these 7 patients developed polyarthritis, 8 months and 2 years, respectively, after the onset of systemic symptoms. In all subjects (except for the 3 patients indicated below), all serum samples were obtained during the morning, when the body temperature was normal. In 3 children with active JRA who had intravenous lines present for therapeutic reasons, serial samples were obtained at periods that included the fever peak. Sera obtained from 17 age-matched healthy children (mean age 8.5 years, range 3-15 years) hospitalized for minor surgical procedures or bone marrow donation were used as controls. Permission for drawing of extra blood during routine venipuncture was obtained from parents of all children. Blood was allowed to clot for 2 hours at room temperature and centrifuged at 800g for 10 minutes. Sera were then collected, aliquoted, and stored at -20°C until use. Synovial fluids from 2 patients with systemic-onset JRA, obtained before intraarticular steroid injection, were also studied. Synovial fluids were centrifuged at 10,OOOg for 10 minutes, aliquoted, and stored at -70°C until use. IL-6 assay. Serum and synovial fluid IL-6 levels were measured using the hybridoma cell line B9, as described by Helle et al (10). This assay provides a sensitivity in the picograms-per-milliliter range. B9 cells (kindly provided by Dr. L. Aarden, Netherlands Red Cross, Amsterdam) were cultured in RPMI 1640 supplemented with 10% heatinactivated fetal calf serum, 2 mM L-glutamine, 50 pg/ml gentamicin, and 2 x lO-’M 2-mercaptoethanol (complete medium) in the presence of 0.2 ng/ml of human recombinant IL-6 (Genzyme, Boston, MA). Cells from the tenth through twenty-fifth passages were used in the assays. B9 cells (2,00O/well) were cultured for 96 hours in complete medium, in the presence of serial dilutions of heat-inactivated (56°C for 30 minutes) samples of IL-6. After a 4-hour pulse with 3H-thymidine (1 pCi/well), cultures were harvested and 3H-thymidine incorporation was measured using standard procedures. IL-6 levels were expressed in hybridoma growth factor (HGF) units/ml, where 1 HGF unit is the amount of IL-6 required to achieve half-maximal proliferation of B9 cells. In neutralization experiments, 10 sera from patients with active systemic-onset JRA were preincubated with a 1: 1,000 dilution of a goat anti-IL-6 antiserum (kindly provided by Dr. L. Aarden), or with
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Table 1. Serum levels of interleukin-6 (IL-6) in 25 patients with systemic-onset juvenile rheumatoid arthritis (JRA) grouped according to the presence of active disease or clinical remission at the time of sampling, in patients with active systemic-onset JRA grouped according to current treatment, and in healthy age-matched controls* Group (no. of samples) Controls (17) Systemic-onset JRA Active disease (25) Clinical remission (14) Active systemic-onset JRA No treatment (1) NSAID (10) NSAID + prednisone (10) NSAID + MTX (7) NSAID + MTX + prednisone (8)
IL-6 4.1 92.1 5.5
* 4.2 2 ?
75.1 6.6t
100 128.1 -+ 301.1 107.7 91.0 77.4 ? 68.6 72.1 ? 38.1
*
P vs. controls