Impact of interleukin-21 in the pathogenesis of primary Sjogren's syndrome: increased serum levels of interleukin-21 and its expression in the labial salivary glands

IL-21 is a pleiotropic cytokine that belongs to the common cytokine receptor γ chain (γc)-dependent cytokine family, which is produced by activated CD4+ T cells and NKT cells [1]. The IL-21 receptor (IL-21R) consists of the IL-21R α chain and the γc chain and is expressed on T cells, NK cells, NKT cells, B cells, dendritic cells (DCs) and macrophages as well as on non-hematopoietic cells, including keratinocytes and fibroblasts [2]. The activation of IL-21R by the binding of IL-21 enhances the proliferation of T cells after their prior stimulation with anti-CD3 [3]. IL-21 also controls the functional activity of effector T helper (Th) cells and the differentiation of Th17 cells, and counteracts the suppressive effects of regulatory T cells [4]. IL-21 alone is capable of directly inducing both B lymphocyte-induced maturation protein-1 (Blimp-1), which is required for plasma-cell differentiation, and Bcl-6, which is required for germinal center reactions [5]. IL-21 also promotes B-cell differentiation by synergizing with BAFF and enhancing the CD40-mediated induction of activation-induced deaminase (AID) and Blimp1 [6]. Overexpression of IL-21 in mice results in hypergammaglobulinemia and autoantibody production [7].

Primary Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by keratoconjunctivitis sicca, xerostomia, and extraglandular abnormalities [8]. At the immunological level, it is characterized by both T-cell lymphocytic infiltration of the exocrine glands and B-cell hyper-reactivity. Hypergammaglobulinemia is a common laboratory finding in primary SS. Since previous observations suggest that IL-21 and IL-21R may be associated with immunoglobulin production, autoantibody production, and B-lymphocyte hyperactivity [3, 9], it is thought that IL-21 is involved in the pathogenesis of autoimmune disease. However, the precise roles IL-21 and IL-21R in human autoimmune disease are still poorly understood. To determine whether IL-21 participates in primary SS pathogenesis, we examined whether the serum IL-21 levels of patients with primary SS correlate with various laboratory parameters. The expression of the IL-21/IL-21R cytokine/receptor pair by the salivary glands of patients with primary SS was also investigated.

In this study, we assessed the expression pattern of IL-21 and IL-21R in LSG tissues of primary SS patients. Our results encouraged the original observation demonstrating the correlation between IL-21 expression and IgG levels in association with disease severity of SS.

The role of IL-21 in the pathogenesis of SS is poorly understood, but several lines of thought support the notion that it may contribute to a pivotal role in the process of disease. First, it is known that IL-21 is involved in the proliferation and survival of B cells, promoting their differentiation into Ig-producing plasma cells [1]. The latter ability of IL-21 [7, 18] suggests that this cytokine may play important roles in B cell-mediated autoimmune diseases and allergies. Second, naïve IL-21R KO mice have diminished serum IgG1 levels and immunization of these mice with T-dependent antigen results in lower antigen-specific IgG1 levels compared to wild-type mice [3]. Third, SS patients have increased IgG levels in their serum, although the reason for this increment has not been explained. The B-cell infiltration in the LSGs from primary SS patients is less than the T-cell infiltration; however, B-cell hyperactivity is an important characteristic of primary SS. Indeed, a prominent humoral autoimmune response in patients with primary SS is the production of anti-Ro/SSA antibodies. Moreover, with regard to the IgG subclasses of the anti-Ro antibodies, IgG1 predominates [19]. In this study, we found that the serum IL-21 levels correlated with the serum globulin, IgG, and, in particular, IgG1 levels in primary SS patients. These observations are consistent with the role IL-21 plays in plasma-cell differentiation [7]. In addition, we found the IgG1 levels correlated with the serum anti-Ro/SSA antibody titers. Thus, our observations support the notion that IL-21 may promote autoantibody production by inducing IgG class switch recombination in B cells.

IL-21 regulates the activation, proliferation and survival of CD4+ T cells as well as that of B cells [20, 21]. Specifically, it up-regulates the expression of the ROR γt transcription factor, promotes Th17-cell differentiation, and induces IL-23R expression on Th17 cells, thus facilitating the ability of IL-23 to expand the Th17-cell response [22, 23]. IL-21 also regulates the functional activity of CD8+ T cells and NK cells [20, 21]. Naive CD8+ T cells express low levels of IL-21R [24] and, although IL-21 does not by itself induce CD8+ T-cell proliferation, it acts synergistically with IL-15 or IL-7 to induce the proliferation of both naïve and memory phenotype CD8+ T cells [25].

Our study showed that IL-21 correlates with the degree of lymphocytic infiltration in the LSGs of patients with primary SS. The lymphocytic foci and periductal areas of the LSGs had many IL-21 and IL-21R positive cells. In contrast, the LSGs of healthy control subjects did not express IL-21 or IL-21R. Together these data suggest that IL-21 and IL-21Rpositive cells may play an important role in the sialoadenitis suffered by patients with primary SS.

Early studies showing that two mouse models of SLE have elevated IL-21 levels suggested that IL-21 may play a role in autoimmunity. Herber et al. showed that, in the MRL-Fas^lpr mouse model of SLE, blocking IL-21 with IL-21R-Fc reduced renal disease lymphadenopathy, skin lesions, and circulating autoantibodies and IgG [26]. Similarly, administration of an IL-21R-Fc fusion protein to BXSB.B6-Yaa+/J mice, the second SLE animal model, decreased IL-21 production, lymphocyte activation and circulating IgG1 levels [27]. These data are in agreement with the results from our study, suggesting the notion that IL-21 may participate in autoimmune disease pathogenesis by influencing lymphocytic activation and IgG production. Further supporting this possibility is that the blockade of endogenous IL-21 activity by an IL-21R-Fc fusion protein in the cultured RA synovial membrane cells significantly inhibited the production of inflammatory cytokines [28]. Moreover, administration of IL-21R-Fc to collagen-induced arthritis mice and adjuvant-induced arthritis rats alleviated their clinical and histological signs of inflammation [9].

Additional lines of evidence also suggest that IL-21 may promote human autoimmune disease in general. Yuan et al. have reported that patients with SS have higher serum IL-21 levels than healthy control subjects, which correlated positively with gamma-globulin levels [29]. Moreover, by using a large sample of SLE patients and healthy controls, Sawalha et al. found that SLE is associated with two single nucleotide polymorphisms of the IL-21 gene [30]. In addition, patients with SLE have elevated IL-21 serum levels that correlate with the severity of the disease [31]. Similarly, Astrid et al. have reported that synovial fibroblasts and synovial macrophages of patients with RA express IL-21R (IL-21 was not tested) [32]. In addition, the epidermis of patients with systemic sclerosis expressed IL-21R (but not IL-21) [33]. Recently, genome-wide association studies have provided convincing evidence that the chromosomal 4g 27 region that harbors the IL-21 and IL-21 genes is associated with chronic inflammatory disorders, including SLE, inflammatory bowel disease and psoriasis [30, 34, 35].

Interestingly, two previous studies reported that they were not able to detect IL-21 in the synovium of RA patients or the skin of systemic sclerosis patients [32, 33]. This may relate to an observation made in the experimental autoimmune encephalitis (EAE) animal model; while the treatment of mice with IL-21 after EAE was induced did not affect the severity of the disease, treatment before disease induction greatly enhanced the inflammatory influx into the central nervous system and the severity of the disease [36]. The latter mice also exhibited higher levels of circulating myelin-specific antibodies and IFN-γ, but the level of IL-4 remained similar to that of control mice. Thus, the effects of IL-21 may be specific to the early stages of the autoimmune response. We found what may support this hypothesis. When we measured the serum levels of proinflammatory cytokines, such as IL-6, IL-17 and IL-23, to determine whether serum IL-21 expression correlates with systemic inflammation, we failed to detect any significant correlations (data not shown). Thus, it is possible that IL-21 is involved at the onset of autoimmune disease rather than later during the active systemic inflammation stage. In the present study, the LSG biopsies were performed for diagnostic purposes, thus the biopsy specimens are likely to be tissues from the early phase of primary SS. Since we found that IL-21 and IL-21R are expressed in these LSGs, it may be that IL-21 plays an important role in the onset and early development of primary SS.

Our data showed that IL-21 expression was prominent in infiltrating lymphocytes and ductal cells. It is not yet known whether the epithelial cells lining the duct produce IL-21. Therefore, we could not completely exclude the possibility that the high expression of IL-21 in the ductal cell is the specificity of the reagents or the artifact in the staining procedure. However, it is known that IL-21 is expressed in non-immune cells as well as in lymphocyte. IL-21 expression is enhanced at the site of the involved gut in Crohn's disease [37]. IL-21 is also expressed in neurons in the gray matter in multiple sclerosis [38]. This suggested the possibility that IL-21 can be produced in non-immune cells, including epithelial cells.

The glandular destruction in SS has been shown to be mediated mainly by primed CD4+ T cells [39]. Of the lymphocytes that infiltrate the salivary glands in patients with SS, 45 to 50% are CD4+ T cells, 20% are CD8+ T cells, and about 20% are B cells [40]. Interestingly, the confocal microscopic analyses in the present study revealed that the LSG-infiltrating cells that stained for IL-21 also stained for CXCR5. CXCR5 is a chemokine receptor that is expressed by all mature B cells as well as by a subset of antigen-experienced CD4+ T cells in lymphoid tissue [41]. CXCR5 is strongly implicated in the follicular migration of early-activated T cells and their consequent co-localization with B cells [42]. The priming of CD4+ T cells in lymphoid tissue result in their novel expression of CXCR5 [43, 44]. However, CXCR5 is rapidly lost during T-cell proliferation, which indicates that the follicular homing program is an early and transient stage in the T-cell activation and differentiation process [44, 45]. Continued CXCR5 expression after priming may reflect qualitative or quantitative aspects of this stimulation [46]. Since the majority of the T cells in the salivary glands of primary SS patients are CD4+ T cells, and the IL-21 produced by activated CD4+ T cells is known to up-regulate their expression of CXCR5 [16], IL-21 possibly promotes the co-localization of T cells and B cells in the functional ectopic germinal centers in the salivary glands of SS patients, which in turn elevates autoantibody production. Taken together, these observations suggest that high expression of IL-21 may play an important role in the pathogenesis of primary SS by affecting both T cells and B cells.

Our present study is the first to report that there is a relationship between the elevated serum levels of IL-21 and IgG, (especially IgG1), in primary SS and that the lymphocytic infiltration in the LSGs of patients with primary SS express high levels of IL-21 along with lower levels of IL-21R. Moreover, the expression of IL-21 showed a tendency to increase as more lymphocytes infiltrated into LSGs. Further investigations on the systemic and localized effects of IL-21 in primary SS would provide a basis for targeting this molecule for customized therapy.