High circulating levels of interleukin-18 in patients with primary Sjögren ’ s syndrome is associated with disease activity

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binding affinity [5]. The IL-18-IL-18R complex-mediated signaling regulates T-cell immunity by promoting T-helper type 1 (Th1) and Th17 response and consequently plays an important role in infection and inflammation [6,7]. Moreover, there is an increasing body of evidence showing that IL-18 may also play a role in autoimmunity. For example, increased circulating levels of IL-18 have been observed in many autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and idiopathic thrombocytopenic purpura (ITP) [8,9,16,17]. In addition, the levels of IL-18BP and sIL-18R1 have also been shown to be increased in SLE [8] and RA, respectively [9].
Although previous results suggest a role of IL-18 in pSS, the mechanism behind the role is not clear. The elevated circulating level of IL-18 has been observed in pSS patients; the levels of IL-18BP and sIL-18R1 in pSS have not been investigated. Also, whether the elevated IL-18 levels are associated with pSS clinical phenotypes such as disease activity and disease duration is not clear. In this study we investigated the association between the circulating levels of IL-18, IL-18BP and sIL-18R1 and pSS as well as its subphenotypes.
We recruited 35 pSS patients (5 males and 30 females) from the First Affiliated Hospital of Xiamen University (Table 1). The mean age of the patients at the time of the study was 43.46  12 years. All patients were diagnosed according to the standards defined by criteria of the American-European Consensus Group in 2002 [10]. All patients underwent serological evaluations, including C3 levels, C4 levels, total IgG levels, and anti-SSA and anti-SSB autoantibodies. Histological evaluation was also performed to detect the inflammatory cell infiltration which was presented as focus score of salivary gland tissue. Disease activity was determined according to the standard of European League Against Rheumatism (EULAR) and presented as Sjögren's syndrome disease activity index (ESSDAI) [11]. ESSDAI scores of 0-7 were defined as mild disease, whereas scores of above 7 were defined as active disease. The main clinical and laboratory characteristics of pSS patients are presented in Table 1. Age-and gender-matched healthy controls Sjögren's syndrome (SS) is an autoimmune disease mainly targeting exocrine glands and manifests in dryness of mouth (xerostomia) and eyes (xerophthalmia). This disease can present alone as primary SS (pSS) or associated with other connective tissue diseases as secondary SS [1]. Besides circulating autoantibodies and inflammatory cell infiltration in exocrine glands, abnormal cytokine production is also a hallmark of pSS. Some evidences indicate that interleukin-18 (IL-18) might play a role in the pathogenesis of SS. In 2004, Bombardieri et al. reported that IL-18 level is elevated systemically and locally in pSS than that in healthy controls, providing the first link between IL-18 and pSS. The subsequent studies confirmed the finding and further suggested an association between IL-18 and the production of immunoglobulin G1 (IgG1) and IL-17 [2,3]. Furthermore, Delaleu et al. showed in the mouse model of pSS that the serum levels of IL-18 are higher in the susceptible mouse strain than those in the resistant strain [4]. Taken together, this evidence suggests a role of the IL-18 in the development of pSS.
IL-18, also known as interferon (IFN)-g inducing factor, is proinflammatory cytokine belonging to the IL-1 superfamily [1]. IL-18 functions by means of binding to the IL-18 receptor (IL-18R) complex which is composed of IL-18Ra (IL-18R1) chain as the extracellular signaling domain and IL-18b chain as an adaptor molecule. The IL-18Ra acts as the signal chain that individually binds to IL-18 with low affinity, but when recruiting the binding chain IL-18b, it can combine with IL-18 tightly. There are two circulating natural antagonists of IL-18, including IL-18-binding protein (IL-18BP) with a high affinity binding with IL-18 and soluble form of IL-18R1 (sIL-18R1) with a low IL-18 were recruited from the Xiamen University Hospital. Sera were prepared freshly from peripheral blood samples of patients and controls, and stored at  80°C until use. This study was approved by the ethics committee of Xiamen University.
We detected the serum levels of IL-18, IL18R1 and IL-18BP in the patients and control subjects using sandwich enzymelinked immunosorbent assay. The free IL-18 was calculated using the method as described previously [12]. The mean  standard deviation (SD) value of IL-18 levels in pSS patients was significantly higher than that in controls (119.73  100.06 pg/mL vs. 65.03  45.70 pg/mL, P  0.01) ( Table 1). When the cutoff level for IL-18 was set as mean  2SD of levels of the 35 control sera, the rate of samples with high IL-18 levels in pSS patients was higher than that in controls (28.6% vs. 5.7%, P  0.05). The levels of IL-18BP in pSS sera and in control sera were 21.11  10.79 and 20.33  4.44 ng/ml, respectively. Significant difference between patients and control was observed neither in the levels nor in the rate of the samples with high IL-18BP levels. Also, no significant difference in the levels of IL-18R1 was observed between pSS patients (310.53  160.00 pg/ml) and controls (262.89  123.18 pg/ml) ( Table 1). We then calculated the free IL-18 levels in controls and pSS patients. Similar to the IL-18 levels, the free IL-18 levels in pSS patients were significantly higher than those in controls (31.90  28.25 pg/ml vs. 16.83  16.83 pg/ml, P  0.01) ( Table 1). Furthermore, the rate of samples with high levels of free IL-18 was also higher in pSS patients than that in controls (20% vs. 2.86%, P  0.05).
To further investigate the role of the IL-18 in pSS, we evaluated the association of IL-18 with clinical phenotypes of pSS. We firstly investigated whether the levels of IL-18 are associated with disease activity of pSS. We considered ESSDAI scores of 0-7 and ESSDAI scores of  7 representing mild disease and active disease, respectively. As shown in Figure 1A, the serum levels of IL-18 in patients with active disease are significantly higher than those in patients with mild disease (166.12  120.20 pg/mL vs. 80.66  57.86 pg/mL, P  0.01). Such significant difference was also observed in the levels of free IL-18 (40.28  31.45 pg/mL vs. 24.85  23.85 pg/mL, P  0.05) ( Figure 1B) but not in the levels of IL-18BP or IL-18R1. We next investigated whether the level of IL-18 is associated with disease duration. We compared the levels of IL-18 in patients with disease duration of less than one year with those in patients with disease duration of more than one year. The result demonstrated that there was no significant difference in either IL-18 levels or free IL-18 levels between the two patient groups ( Figure 1C, D). We also investigated the association of IL-18 with other subphenotypes of pSS, including anti-SSA, anti-SSB, total IgG levels, C3 levels, C4 levels and focus score, but no   Our results demonstrate that the serum levels of IL-18 in pSS patients are significantly higher than those in controls. This finding is in line with previous observations, which show the elevated circulating levels of IL-18 in pSS patients [2]. In addition, we show that the levels of free IL-18, which is a biologically relevant index for IL-18 activity, are elevated in pSS patients as compared with those in controls. Previous studies showed that IL-18 is produced locally by acinar cells, intraducts and CD68_ macrophages in salivary glands [2,13] of pSS patients. Taken together, these evidences suggest that IL-18 might be involved in the pathogenesis of pSS.
Maybe the most interesting finding of the present study is that the circulating levels of IL-18 and free IL-18 are associated with disease activity of pSS, with high levels of IL-18 and free IL-18 in patients with active disease. To our knowledge, this is the first study showing the association of circulating levels of IL-18 with disease activity of pSS. The levels of IL-18 have been reported to be associated with the disease activity of other autoimmune diseases such as SLE and ITP [14,15]. Taken together, this suggests that the circulating levels of IL-18 might be associated with disease activity of autoimmune diseases in general, indicating a pathogenic role in the development of autoimmune diseases.
Disturbance of the cytokine network is one hallmark of pSS [1,16]. pSS has long been thought to be associated with Th1-related cytokine, e.g. IFN-g dysregulation, while recent studies further showed that Th17-related cytokines are also associated with pSS. Since IL-18 is able to enhance both the Th1 and Th17 responses [3,16], this might be a possible mechanism of the role of IL-18 in the pathogenesis of pSS. The pathogenic role of IL-18 has been shown in many mouse models of autoimmune diseases [17]. For example, IL-18 deficiency (IL-18 / mice) suppressed the production of the pathogenic antibody and prevented the development of disease in a murine model of myasthenia gravis [18]. This makes the IL-18 a favorite molecule of therapeutic target, and so far many approaches of neutralizing endogenous IL-18 have been developed, including neutralizing antibodies to IL-18, IL-18R-blocking antibodies, IL-18BP and caspase-1 inhibitors [16]. Those therapeutic approaches have been shown to be effective in modulating the severity of many experimental models of many autoimmune diseases [16]. The association of IL-18 with disease activity of pSS demonstrated in this study encourages that the IL-18-targeting therapeutic approaches could also be evaluated in pSS and its experimental models.
In summary, our results show that the levels of IL-18, but not those of IL-18BP or sIL-18R1 are higher in pSS patients than those in controls. Furthermore, the levels of IL-18 in patients with active disease are significantly higher than those in patients with mild disease. To our knowledge, this is the first study that shows an association between IL-18 levels and the disease activity of pSS.