Serum Interleukin 2 Levels in Patients with Rheumatoid Arthritis and Correlation with Insulin Sensitivity

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1 The Journal of International Medical Research 2002; 30: Serum Interleukin 2 Levels in Patients with Rheumatoid Arthritis and Correlation with Insulin Sensitivity O ÖNCÜL 1, C TOP 2, S ÖZKAN 3, Ş ÇAVUŞLU 1 AND M DANACI 2 1 Department of Infectious Diseases; 2 Department of Internal Medicine; and 3 Department of Anaesthesiology and Rehabilitation, Gülhane Military Medical Academy, Haydarpaşa Training Hospital, Istanbul, Turkey Interleukin 2 (IL-2), a Th1 lymphocytederived cytokine, is thought to play an important role in the pathogenesis of type 2 diabetes and rheumatoid arthritis (RA). The aim of our study was to evaluate changes in serum IL-2 levels and their correlation with glucose metabolism abnormalities, such as insulin resistance, in patients with RA. Thirty-six subjects with varying degrees of disease activity and 20 healthy age-, sex- and body mass indexmatched control individuals were evaluated. Patients with any causes of peripheral insulin resistance were excluded. After a 12-h overnight fast, fasting insulin levels, homeostatic model assessment-insulin resistance (HOMA-IR) estimated insulin sensitivity, and serum IL-2 levels were significantly higher in all patients with RA than in the control individuals. Fasting insulin, HOMA-IR scores and IL-2 levels were correlated in the RA group. This study showed that patients with RA have altered IL-2 regulation, and that there was a significant correlation between serum IL-2 levels and insulin sensitivity. KEY WORDS: INTERLEUKIN 2; INSULIN RESISTANCE; RHEUMATOID ARTHRITIS; TYPE 2 DIABETES Introduction Insulin resistance is defined clinically as the inability of a known quantity of exogenous or endogenous insulin to increase glucose uptake and utilization in an individual to the same extent as in the normal population. 1 It is now well established that insulin resistance is a strong predictor of type 2 diabetes. 2,3 Interleukin (IL) 2, a Th1 lymphocyte-derived cytokine, is the principal autocrine growth factor that promotes T-cell activation and proliferation. 4 Currently, IL-2 is considered to play an important role in the pathogenesis of type 1 diabetes. 5 The production of cytokines by activated T cells in synovial tissue plays an important role in the pathophysiology of rheumatoid arthritis (RA). 6 Type II collagen-reactive T lymphocytes selectively accumulate in the inflamed joint of patients with RA, and they may play an important role in the pathogenesis of RA. 7 Therapeutic agents for the treatment of RA that will target components of the immune activation and co-stimulatory pathways are currently in development. These include antibodies against the IL-2 receptor and blockers of the 386

2 CD28 and CD40 co-stimulatory pathways. Continuing research into the pathogenesis of RA will undoubtedly identify even more effective therapeutic approaches for the management of this disease in the future. 8 The aim of our study was to evaluate changes in serum IL-2 levels and their correlation with glucose metabolism abnormalities, such as insulin resistance, in patients with RA. Patients and methods Thirty-six patients with RA (14 males and 22 females; mean age, 51.5 ± 17.1 years [range, years]; mean body mass index [BMI], 25.8 ± 2.9 kg/m 2 ) and 20 age-, sex- and BMImatched healthy controls (eight males and 12 females; mean age, 46.6 ± 13.9 years [range, years]; mean BMI, 25.2 ± 3.2 kg/m 2 ) were recruited to the study. Patients with any other chronic inflammatory diseases were excluded. All subjects gave informed consent to participate in the study. No one was taking any medication that altered insulin action. None reported any alteration of disease activity for at least 3 months before the study. All patients underwent routine investigations for accompanying metabolic, hepatic and renal diseases. No patient had any features of recent acute illness or clinical evidence of cardiovascular, kidney, liver or endocrine diseases. Patients with type 2 diabetes and other concomitant renal, hepatic or cardiac disease were excluded from the study. The diagnosis of RA was based on the American Rheumatism Association 1987 Revised Criteria for the Classification of Rheumatoid Arthritis. 9 Fasting blood samples from all participants were collected via an intravenous catheter in ethylenediaminetetraacetate-coated (EDTA) venepuncture tubes, promptly centrifuged, separated and stored at 70 C until IL and insulin assays were performed. All subjects underwent a diagnostic protocol that included measurement of fasting IL-2 levels by enzyme-linked immunosorbent assay (Enzyme Immunoassay kit, Immunotech, Marseille, France). The inter- and intra-assay coefficients of variation were 2.4% and 3.0%, respectively. Glucose was measured using a glucose oxidase method (Hitachi 736 Auto Analyzer, Hitachi Co., Tokyo, Japan). Immunoreactive insulin was determined by double-antibody radioimmunoassay (Coat-a- Count, Diagnostics Products Corp., LA, USA). Homeostatic model assessment-insulin resistance (HOMA-IR) estimated insulin sensitivity was calculated using the formula defined by Matthews et al: 10 Fasting insulin (µiu/ml) HOMA-IR = fasting glucose (mmol/l) 22.5 With this method, a high HOMA-IR score is indicative of low insulin sensitivity. STATISTICAL ANALYSIS All data were presented as means ± SDs. For comparison of study and control data, the Mann Whitney U-test was used. Pearson correlation analysis was used to estimate the relation between test parameters. P-values < 0.05 were considered significant. Results Fasting insulin levels were significantly higher in all patients with RA than in controls (P < 0.001; Table 1). HOMA-IR scores and serum IL-2 levels were also significantly higher in patients with RA than in controls (P < and P < 0.001, respectively; Table 1). Fasting insulin, HOMA-IR and IL-2 were correlated in the whole group. There was a positive significant correlation between serum 387

3 TABLE 1: Statistical differences (according to Mann Whitney U-test) between test parameters for patients with rheumatoid arthritis (study group) and healthy individuals (control group) Study group Control group Variable (n = 36) (n = 20) P-values Sex (male/female) 14/22 8/12 Age (years) 51.5 ± ± 13.9 NS Body mass index (kg/m 2 ) 25.8 ± ± 3.2 NS Fasting glucose (mmol/l) 5.1 ± ± 0.4 NS Fasting insulin (µiu/ml) 14.7 ± ± 1.9 < Serum interleukin 2 (pg/ml) ± ± 46.8 < HOMA-IR 3.3 ± ± 0.5 < NS, not significant; P < 0.05 was considered to be statistically significant. HOMA-IR, homeostatic model assessment-insulin resistance estimated insulin sensitivity. IL-2 level and fasting insulin level, and between serum IL-2 level and HOMA-IR score in patients with RA (r = 0.31, P < 0.05 and r = 0.30, P < 0.05, respectively). Discussion Rheumatoid arthritis is an inflammatory polyarthritis genetically linked to human leukocyte antigen (HLA)-DR4 and related haplotypes. RA synovial tissue is characterized by T-cell infiltration and activation of macrophage-like cells, strongly implicating a T- cell antigen-presenting cell interaction in RA pathogenesis. 11 Brok et al. 12 investigated the prophylactic and therapeutic effects of a humanized monoclonal antibody, daclizumab, against the α-chain of the IL-2 receptor, CD25, and concluded that daclizumab has clinical potential for the treatment of RA during periods of active inflammation and for suppression of joint tissue destruction. Insulin signalling in target tissues results in a large array of biological outcomes. These events are essential for normal growth and development, and for normal homeostasis of glucose, fat and protein metabolism. 13 Insulin resistance is a common pathological state, in which target cells fail to respond to ordinary levels of circulating insulin. It is frequently associated with a number of diseases, including chronic infection, obesity and type 2 diabetes. 14 Penttinen 15 postulated that low-density lipoproteins, infections and smoking are also closely associated with insulin resistance, and that each of these factors may cause insulin resistance by the same mechanism. The first step in the cascade of events leading to insulin resistance is increased production of IL-2. This is followed by secretion of other cytokines, activation of macrophages and increased production of nitric oxide from L-arginine. The reduced amount of plasma L-arginine leads to decreased secretion of growth hormone and insulin-like growth (ILG) factor-1, leading to insulin resistance. High plasma IL-2 also causes decreased bioavailability of ILG factor-1 by reducing the production of androgenic hormones. 15 The vast and growing array of cytokines is the subject of intense research for those with potential to ameliorate diseases ranging from autoimmune disorders to cancer and 388

4 beyond. 16 Diabetes is thought to be an autoimmune disease caused by destruction of β-cells in pancreatic islets. IL-2 can enhance immune function by stimulating formation of cytolytic T cells. 17 Cytokines function at the cellular, microenvironmental level, but human cytokine assessment is most commonly performed at a macro level by measurement of serum cytokines. Jason et al. 18 investigated the relationships between serum and cellular cytokines, and reported that serum cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, interferon-γ and tumour necrosis factor-α) may reflect peripheral blood cell cytokine production and balances. In this study, we also measured serum IL-2 levels to determine peripheral blood T-lymphocyte IL-2 production. The present study clearly demonstrated that fasting insulin levels and HOMA-IR scores were significantly higher in all patients with RA than in controls. As far as we can ascertain from an extensive search of the literature, this study is the first to evaluate the importance of serum IL-2 levels in the development of insulin resistance in patients with RA. The study data showed a positive correlation between fasting insulin levels, HOMA-IR scores and serum IL-2 levels in patients with RA. Such a relationship may prove the pathogenic role of IL-2 in insulin resistance. Insulin resistance therefore seems to be the main metabolic abnormality that alters glucose metabolism, and decreases the sensitivity of peripheral tissues to insulin in patients with RA. Received for publication 14 December 2001 Accepted 22 December Cambridge Medical Publications References 1 Lebovitz HE: Insulin resistance: definition and consequences. Exp Clin Endocrinol Diabetes 2001; 109 (Suppl 2): Martin BC, Warran TH, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR: Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 1992; 340: Lillioja S, Mott DM, Spraul M, Ferraro R, Foley JE, Ravussin E, et al: Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus: prospective studies of Pima Indians. N Engl J Med 1993; 329: Norris MS, McConnell TJ, Mannie MD: Interleukin-2 promotes antigenic reactivity of rested T cells but prolongs the postactivational refractory phase of activated T cells. Cell Immunol 2001; 211: Kretowski A, Mysliwiec J, Szelachowska M, Brzozowski C, Pietruczuk M, Kinalska I: In vitro secretion of interleukin 2 and expression of IL- 2 receptor in peripheral blood lymphocytes in high risk of insulin-dependent diabetes mellitus subjects. Arch Immunol Ther Exp 1999; 47: Steiner G, Tohidast-Akrad M, Witzmann G, Vesely M, Studnicka-Benke A, Gal A, et al: Cytokine production by synovial T cells in rheumatoid arthritis. Rheumatology 1999; 38: He X, Kang AH, Stuart JM: Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis. J Rheumatol 2000; 27: Simon LS, Yocum D: New and future drug therapies for rheumatoid arthritis. Rheumatology 2000; 39 (Suppl 1): Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al: The American Rheumatism Association 1987 Revised Criteria for the Classification of Rheumatoid Arthritis. Arthritis Rheum 1988; 31: Matthews DR, Hosker JP, Turner RC: Homeostasis Model Assessment: Insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: Fang Q, Sun YY, Cai W, Dodge GR, Lotke PA, Williams WV: Cartilage-reactive T cells in rheumatoid synovium. Int Immunol 2000; 12: Brok HP, Tekoppele JM, Hakimi J, Kerwin JA, Nijenhuis EM, De Groot CW, et al: Prophylactic and therapeutic effects of a humanized monoclonal antibody against the IL-2 receptor (daclizumab) on collagen-induced arthritis (CIA) in rhesus monkeys. Clin Exp Immunol 2001; 124: Le Roith D, Zick Y: Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care 2001; 24:

5 14 Virkamaki A, Veki K, Kahn CR: Protein protein interaction in insulin signalling and the molecular mechanisms of insulin resistance. J Clin Invest 1999; 103: Penttinen J: High plasma interleukin-2: a potential cause of insulin resistance. Med Hypotheses 1995; 45: Beutler BA: The role of tumor necrosis factor in health and disease. J Rheumatol 1999; 26: Whitehead RP, Hauschild A, Christophers E, Figlin R: Diabetes mellitus in cancer patients treated with combination interleukin 2 and alpha-interferon. Cancer Biother 1995; 10: Jason J, Archibald LK, Nwanyanwu OC, Byrd MG, Kazembe PN, Dobbie H, et al: Comparison of serum and cell-specific cytokines in humans. Clin Diagn Lab Immunol 2001; 8: Address for correspondence Dr O Öncül Department of Infectious Diseases, Gülhane Military Medical Academy, Haydarpaşa Training Hospital, Üsküdar-Istanbul, Turkey. gataheh@hotmail.com 390