SIGNIFICANCE OF ELEVATED INTERLEUKIN-6 LEVEL IN JUVENILE RHEUMATOID ARTHRITIS PATIENTS

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1 SIGNIFICANCE OF ELEVATED INTERLEUKIN-6 LEVEL IN JUVENILE RHEUMATOID ARTHRITIS PATIENTS Essam Tewfik Attwa and S. Al-Beltagy* Rheumatology & Rehabilitation Department, Zagazig University Faculty of Medicine and Clinical Pathology Department*, Al-Azhar University Faculty of Medicine KEY WORDS: IL-6, JRA ACTIVITY PARAMETERS ABSTRACT IL-6 is supposed to be involved in the pathogenesis of anemia of chronic disease (ACD) and juvenile rheumatoid arthritis (JRA). We investigated IL-6 in the plasma and bone marrow in patients of JRA with and without ACD to study its significance during the course of the disease and its role in the pathogenesis of ACD in JRA. The level of IL-6 was measured with ELISA in the plasma and supernatant of bone marrow (BM) of 25 patients with JRA (10 patients had systemic onset, 15 polyarticular onset), 14 patients with systemic JRA and ACD and 11 patients with JRA without anemia. In addition IL-6 examined in 10 healthy children. We found that IL-6 level was significantly (p<0.001) higher in patients with systemic JRA, but not in patients with polyarticular JRA than those of healthy controls. Also, IL-6 level was significantly higher in patients with persistence of systemic symptoms than patients in remission (p<0.001). In patients with JRA and anemia the level of IL-6 was significantly higher in the bone marrow supernatant than those without anemia (p<0.001). There was a negative correlation between bone marrow IL-6 and Hb. 819

2 Significance of Elevated IL-6 Level in JRA Attwa & Beltagy Our findings suggested that the use of IL-6 as a predictive parameter for the development of ACD with JRA. We recommend the use of anti -IL-6 antibodies in the therapy of those patients. INTRODUCTION Juvenile rheumatoid arthritis is a clinically heterogenous condition currently divided in different clinical forms based on symptoms and onset (Cassidy & Petty, 1990). The systemic form is characterised by chronic arthritis associated with high spiking fever and systemic features including evanescent rash, hepato-splenomegaly, lymphadenopathy, serositis, and with prominent laboratory evidence of inflammation. The other JRA onset forms polyarticular and pauciarticular are mainly characterized by chronic joint inflammation. Although joints are primarily involved in the disease, extra-articular manifestations, such as anemia of chronic disease (ACD) frequently occurs, especially in patients with high disease activity. The pathogenesis of ACD remains uncertain (Means & Krantz, 1992), although many contributing mechanisms have been identified. Inflammatory cytokines, such as tumor necrosis factor-α and IL-6 are supposed to be involved in the pathogenesis of ACD. The relation between the production of cytokines and anemia was clearly indicated by the observation that treatment of anemic patients with rheumatoid arthritis with anti-il-6 antibodies as well as anti-tnf-α caused a significant rise of hemoglobin (Wendling et al., 1993 & Elliot et al., 1994). Aim of the Study: Our aim was to study the significance of IL-6 in the plasma & bone marrow during the course of disease and its role in the pathogenesis of ACD in juvenil rheumatoid arhritis. MATERIAL AND METHODS Twenty-five consecutive patients with JRA were enrolled in this study from the Outpatient Rheumatology Clinic at Arabian Oil Company Hospital in the period from March 1998 to January They fulfilled the American College for Rheumatology Criteria for the diagnosis of JRA (Arnett et al., 1988). The studied group included ten patients who had 820

3 systemic onset and fifteen had polyarticular onset. Laboratory studies (CBC, ESR, CRP & RF were recorded along with the clinical features. Active disease was defined according to the presence of synovitis on examination. Patients with systemic JRA (presence of fever) were divided into two groups according to the presence or absence of anemia as evidenced by: 1) Hemoglobin (Hb) <9Gm/dl and <11Gm/dl in girls and boys respectively). 2) MCV>85fl. 3) Serum ferritin level >50ug/l. 4) Stainable bone marrow iron. The first group (ACD) & the second one had no anemia. Ten apparently healthy children were also examined for IL-6 in the peripheral blood as a control group. Permission form was taken from their parents. Assessment: The demographics & disease characteristics of patients are shown in table (1). Peripheral blood and bone marrow (BM) samples were taken. Marrow aspiration was done under local infiltration with 10% zylocain. For serum ferretin, Parl s stain was used. IL-6 estimation was performed with ELISA as described by Helle et al. (1991) and Van Kooten et al., (1991) using a purified monoclonal antibody to human IL-6 (CIB Amsterdam, NL). Table (1): Demographic & Clinical data of patients with JRA with and without anemia. Parameter Patients with anemia (n:14) Patients without anemia (n:11) Age in years 6.0( ) 5.3( ) Sex (M:F) 2 :10 4 :7 Duration in months 4.8(2-18.5) 9.0(4-41.6) Morning stiffness in minutes 65(5-190) 15(0-145) Ritchie index 22(10-45) 5(0-42) Number of swollen joints 23(10-40) 9(3-24) Serum RF IU/ml 240( ) 120(12-218) Hb gm/dl 8.5( ) 12.2( ) Reticulocyte count 0.9(0.5-3) 1.6(1-2.5) ESR mm/hr 55(20-75) 25(6-70) CRP mg/l 14(2-52) 5(2-54) Number of patients Using NSAIDs 9(64.3%) 8(72.7%) Number of patients Using DMARDs 6(42%) 6(54%) 821

4 Significance of Elevated IL-6 Level in JRA Attwa & Beltagy RESULTS Plasma level of IL-6 was significantly higher in patients with systemic JRA than control group (22+11 vs. 12+7; p< 0.05) but no significant difference between patient with polyarticular JRA and control groups (17+5 vs p< 0.05) not shown in tables. The plasma level of IL- 6 was higher in patients with persistance of systemic symptoms than during remission (26+18 vs15+9; p< 0.001). There is a negative correlation between BM supernatent IL-6 level and HB, also there is a significant correlation between plasma IL-6 and ESR, CRP, platelet count &white cell count. IL-6 in plasma and BM in-patient with low HB was significantly higher than those with normal Hb (p< 0.001). Table (2): Plasma IL-6 in JRA during activity & Remission. Presentation (n:10) Systemic (n:15) Polyarticular Control (n:10) IL-6 Active Remission Active Remission Mean+SD Median (range) 86(8-760) <8(7-100) <8(8-80) <8(8-60) <8(7-55) Incidence 94.7 % 16.7% 55% 20% 20% p values <0.001 <0.05 Table (3): IL-6 levels (mean + SD) in peripheral blood & BM of patients with & without ACD. IL-6 With low Hb (n:14) With normal Hb (n:11) p values Peripheral blood Median (range) 15 (0-680) 8 (0-93) <0.001 Bone marrow Median (range) 887 (2-2518) 12 (0-910 <0.001 Table (4): Relationship between BM levels of IL-6, HB and serological disease activity variables in patients with JRA. Hb ESR CRP ESR CRP IL Table (5): Correlation coefficient (Rs) of the association of plasma IL-6 levels with laboratory variables in patients with active systemic JRA Laboratory variables IL-6 r ESR CRP White cell count Platelet count

5 DISCUSSION It was hypothesized that IL-6 may play a role in inflammatory diseases. Our study related that plasma IL-6 level was elevated in systemic JRA and was associated with the presence and severity of systemic symptoms. However, Hell et al. (1991) found that plasma IL-6 level in polyarticular JRA was comparable to that of controls; that IL-6 was not detected in the synovial fluid (SF), and that plasma IL-6 did not correlate with the extent or severity of joint involvement. Jacqueline et al. (1997) and Diamant et al. (1994) found a very high level of IL-6 in the synovial fluid and low level in the serum of patients with acute or chronic inflammatory arthritis. Thus, it may be assumed that the major source of circulating IL-6 is the inflammed joints, although other sites, such as BM, may produce IL-6 as well (Tanab et al., 1994). Our findings suggest that increased IL-6 production is a feature of systemic JRA, a disease characterized by prominent and chronic systemic inflammation. Morever, plasma IL-6 level significantly correlated with serum CRP concentration, a good indicator of disease activity in systemic JRA (Gwyther et al., 1982). A weaker correlation was also found with WBC & platelet counts. In our patients with systemic JRA (presence of fever) we examined an important issue thought to play a role in the development of ACD. IL-6 production was measured in BM & peripheral blood to evaluate the possible role of local BM IL-6 production in the pathogenesis of ACD. Our results indicated that patients with JRA with ACD differed from those without ACD, with respect to the BM production of IL-6. In previous studies, increased IL-6 correlated well with clinical and serological disease activity variables that were found in the synovial fluid and serum of patients with RA (Barreral et al., 1993). Furthermore, a tendency towards increased IL-6 level was described in patients with RA and ACD as compared to those without ACD (Vreugdenhil et al., 1992). Our results concerning the production of IL-6 in the peripheral blood are in accordance with those findings. However, comparison may be difficult because of the different methods used to obtain samples. Tanab et al. (1994) reported that, elevated level of IL-6 was also observed in BM of patients with RA. The present study adds that BM 823

6 Significance of Elevated IL-6 Level in JRA Attwa & Beltagy production of IL-6 was higher in patients with JRA who also have ACD as compared to those without anemia. There was a negative correlation between BM IL-6 and Hb, as well as tendency towards higher level of BM IL-6 in patients with greater decrease in Hb. Therefore, our results suggest that ACD in JRA is the result of inhibition of erythropoiesis by IL-6 production locally in the BM. The significant negative correlation observed between IL-6 level and Hb, ESR and CRP suggest that patients with RA with greater disease activity may develop anemia. This result comes in agreement with Asano et al. (1990) who reported that administration of IL-6 to experimental animals and humans resulted in anemia. Kobune et al. (1994) reported that IL-6 has been associated with changes in iron metabolism. Conclusion: Our study confirms that IL-6 level is elevated in patients with JRA and is associated with the presence and severity of symptoms. Also, the local production of IL-6 in the BM is associated with the development of ACD in JRA. This result recommends the use of anti-il-6 antibodies in treating JRA patients with ACD in whom IL-6 levels found to be elevated. REFERENCES Arnett FC, Edworthy SM, Bloch DA, et al. (1988):Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.arthritis Rheum; 31: Asano S, Okano A, Ozawa K, et al. (1990):In vivo effect of recombinant human IL-6 in primates: stimulated production of platelets Bl; 75: Barreral P, Boerbooms AMT, Janssen E, et al. (1993):Circulating soluble tumour necrosis factor receptors, IL-6 levels in rheumatoid artheritis Artheritis Rheum; 36: Cassidy JT and Petty R E (1990):Textbook of Pediatric Rheumatology,2 nd New york:churchill-livingstone. Diamant M, Hassen M, Rieneck K, et al. (1994):Stimulation of the B9 hybridoma cell line by soluble IL-6 resceptors J Immunol Methods: 173: Elliot M, Maini R, Feldman M, et al. (1994):Randomized double blind comparison of chimeric monoclonal antibody to tumour necrosis factor <(CA2) versus placebo in rheumatoid arthritis Lancet: 344:

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