Primary Sjögren's syndrome (pSS) is a common rheumatic disease characterized by dryness of mouth and eye and multiple organ dysfunction. The clinical symptoms of pSS at an early stage are relatively insidious and nonspecific, which is difficult to detect in time. It is widely accepted that the pathogenesis of pSS is closely related to the cytokines, especially for inflammatory cytokines involved in immune response and inflammatory reaction. Like other autoimmune diseases, pSS is mainly caused by the disorder of autoimmune tolerance mechanism, leading to abnormal T cell and B cell activation. With the deepening of immunological research, the relationship among toll-like receptors, interleukin, T cells, lymphocytes, and the pathogenesis of pSS has been continuously revealed. Until now, interleukin is the most intensively studied cytokines in the field of rheumatic immunity. Recent studies suggested up-regulation of interleukin (IL)-4, IL-6, and IL-10 in pSS patients,[3,4] but their association with clinical manifestations has not been reported yet.
Oxford 40 (OX40) is a kind of tumor-necrosis-factor receptor (TNFR), which is activated by its cognate ligand OX40L that functions as a T cell co-stimulator to amplify T and B cell proliferative response. In recent years, abundant studies provided strong evidences that OX40-OX40L interaction is critical for the progression of autoimmune diseases. For example, Jiang et al demonstrated that OX40 and OX40L in serum and in synovial fluid were highly expressed in patients with rheumatoid arthritis (RA) and were positively correlated with clinicopathological characteristics. Nevertheless, OX40 blockade inhibited the pro-inflammatory responses and ameliorated RA. Moreover, Cortini et al reported that OX40 knockout mice showed the decrease of splenic T follicular helper (Tfh) cells in patients with systemic lupus erythematosus (SLE). However, the clinical significance of OX40L in the pathogenesis of pSS remains unclear.
Progranulin (PGRN) is a autocrine growth factor implicated in neurons, lymphocytes, epithelial cells, and chondrocytes and immunocytes.[8,9] A large quantity of evidences has demonstrated that it plays a vital role in early embryonic development, inflammation, tissue reconstruction, and immune response.[8,10] Emerging evidences indicate that PGRN is associated with autoimmune diseases, including systemic lupus erythematous, systemic sclerosis, multiple sclerosis, and pSS. Meanwhile, PGRN was previously reported to be an endogenous antagonist of TNF-α through competitively binding to TNFR, which in turn mediated anti-inflammatory activity in various inflammatory diseases including rheumatoid arthritis (RA).[11,12] Serum PGRN is up-regulated in patients with pSS, but its association with OX40L needs more researches to confirm.
On the basis of aforementioned studies, in the present study, serum PGRN, serum soluble OX40L (sOX40L) levels in patients with pSS were determined to investigate the relationship between them, and to explore the expression differences among different types of patients.
2 Material and methods
2.1 Study population
This prospective study included a total of 68 pSS patients who were diagnosed in our hospital during January 2015 to December 2016. Diagnosis was made if any three of the four objective criteria were present (that is items 3–6):
- 1. ocular symptoms;
- 2. oral symptoms;
- 3. ocular signs;
- 4. histopathology;
- 5. salivary gland involvement; and
- 6. autoantibodies.
We subdivided pSS patients into 4 groups according to EULAR Sjögren's syndrome disease activity index (ESSDAI) values: stage I, subjects with ESSDAI 1 to 2 (n = 11); stage II, subjects with ESSDAI 3 to 5 (n = 31); stage III, subjects with ESSDAI 6 to 8 (n = 14); stage IV, subjects with ESSDAI ≥9 (n = 12). Meanwhile, all patients were likewise were randomized to 2 subgroups according to clinical characteristics: subjects with multiple system damage (n = 29) or simple exocrine gland injury (n = 39). To understand the relationships between clinical characteristics and serum PGRN or sOX40L levels, all pSS patients were grouped according to the serum levels of PGRN (high PGRN group: PGRN ≥11.98 pg/L; low PGRN group: PGRN < 11.98 pg/L) or sOX40L (high sOX40L group: PGRN ≥7.34 ng/mL; low sOX40L group: sOX40L < 7.34 ng/mL). In addition, a total of age- and gender-matched healthy subjects (n = 50) were recruited as controls. Patients who received radiation treatment or anticholinergic drugs or had acquired immunodeficiency disease syndrome (AIDS) or graft-versus-host disease (GVHD) were excluded. All experimental protocols were approved by the Ethics Committee of the Second Hospital of Hebei Medical University. Informed consent was obtained from all subjects.
2.2 Data collection
Patients’ characteristics included demographics such as age and sex and disease duration were recorded. Furthermore, fluorescence-enzyme immunoassay was carried out for serological analysis of La/SSB and Ro/SSA autoantibodies. Disease activity was measured according to the EULAR Sjögren's Syndrome Patients Reported Index (ESSPRI) and ESSDAI.
2.3 Measurement of serum cytokines
The blood collection and measurement of sOX40L, as well as other serum factors were conducted as reported previously. Briefly, peripheral blood samples (5 mL) were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) to obtain monocytes or did not contain EDTA to obtain serum within 24 h of admission. Isopaque-Ficoll gradient centrifugation was conducted to obtain the peripheral venous blood mononuclear cells and monocytes were harvested and collected in RPMI1640 medium. Non-EDTA blood was immersed in ice and the supernatant was obtained after centrifugation with 1500 × g for 10 min at 4°C and then the supernatants were harvested and stored at −70°C. Enzyme-linked immunosorbent assay (ELISA) was subsequently performed using commercial ELISA kits (BD Biosciences, San Diego, CA) to determine the soluble serum levels of PGRN, sOX40L, IL-4, IL-6, and IL-10 following the manufacturer's protocols.
2.4 Statistical analysis
Statistical analyses were conducted using SPSS version 22.0 (IBM, CA). Differences between groups were analyzed by Student's t test along with Mann–Whitney U test. Spearman's analysis was used to determine the correlation between PGRN and sOX40L. Data were expressed as statistically mean ± standard deviation (SD) or percentage, and P < .05 were considered statistically significant.
3.1 Baseline characteristics
The demographic characteristics of patients’ baseline characteristics are listed in Table 1 showing that inflammatory cytokines IL-4, IL-6, and IL-10 were observably elevated in pSS patients as relative to healthy controls (all P < .05). No significant differences were observed among other clinical parameters.
3.2 Correlation analysis of serum PGRN and sOX40L levels
As shown in Figure 1A (all P < .05), PGRN and sOX40L levels in pSS patients were significantly increased than those in healthy subjects. Furthermore, Spearman's correlation analysis disclosed pairwise positive correlation between PGRN and sOX40L in pSS patients (Fig. 1B; r2 = 0.3694, P < .001).
3.3 Relationship among PGRN and sOX40L and clinical characteristics
To further determine the association between serum PGRN and sOX40L levels and clinical outcome of patients with pSS, all subjects were divided into PGRN or sOX40L high/low group according to the median levels of serum PGRN or sOX40L. As presented in Tables 2 and 3, patients with elevated PGRN (≥11.98 pg/L) or sOX40L (≥7.34 ng/mL) exhibited higher ESSPRI and ESSDAI scores and IL-4, IL-6, and IL-10 serum levels than those with lower serum levels (all P < .05). However, there were no significant differences in age, sex, disease duration, anti-La/SSB positive rate and anti-Ro/SSA positive rate between groups. These findings suggested that elevated levels of PGRN or sOX40L in pSS patients were associated with higher disease activity.
3.4 Association between PGRN and sOX40L and disease severity in pSS patients
We further investigated the expressions of serum PGRN and sOX40L in different types of pSS. Compared with these patients with pSS stage of I to II and III to IV, PGRN, OX40, and sOX40L were markedly increased in patients with pSS stage II to IV (Fig. 2A; all P < .05). Additionally, PGRN and sOX40L were dramatically higher in patients with multiple system damage than in patients with simple exocrine gland injury (Fig. 2B; all P < .05). Collectively, our data suggested that the higher serum PGRN and sOX40L were significantly corrected with disease severity, suggesting that PGRN and sOX40L might play crucial roles in the pathogenesis and progression of pSS.
The basic pathogenesis of pSS is immune dysfunction, in which exocrine gland duct epithelial cells act as antigen presenting cells (APCs) to facilitate T-lymphocyte proliferation, which in turn produce a large number of immunoglobulin, leading to excessive humoral immune response to release a great deal of pro-inflammatory cytokines. Neurological manifestation including weakness of extremities and sensory dysfunction was present in all pSS patients. In this study, B-lymphocyte derived cytokines IL-4, IL-6, and IL-10 were significantly up-regulated in pSS patients as relative to the healthy controls. To the best of our knowledge, IL-4 is a pleiotropic lymphokine responsible for the proliferation, activation, and survival of B lymphocytes and switching isotypes from IgM and IgD to IgG1 and IgE.[18,19] IL-6 is identified as a B lymphocyte differentiation factor that is essential for B lymphocyte differentiation, whereas, IL-10 promotes the secretion of B lymphocyte stimulator by monocytes, dendritic cells, and macrophages.[20,21] Abundant studies highlighted the anti-inflammatory role of IL-10 in various diseases. For instance, Meng et al showed that the production of IL-10 exerted the anti-inflammatory and antioxidative effects in activated microglia through the inhibition of the NF-κB and STAT1 signaling pathways. However, Anaya et al found that IL-10 concentration was higher in pSS patients than in controls, and were positively correlated with titers of IgA RF, anti-Ro, and anti-La antibodies, as well as focus score. In comparison with patients with low IL-10 production (<9.5 pg/mL), patients producing high IL-10 had significantly more episodes of cutaneous vasculitis. Xu et al reported that NOD/LtJ mice with pSS showed higher levels of IL-4, IL-6, and IL-10 in spleen B lymphocytes and were associated with salivary gland epithelial cell apoptosis. Our findings confirmed that the occurrence of pSS triggered the secretion of inflammatory cytokines, which may exacerbate inflammation.
Meanwhile, some other inflammatory markers played crucial roles in pSS progression. For instance, Kim et al indicated that NOD-like receptor protein 3 (NLRP3) inflammasome was upregulated in pSS patients and were associated with the disease activity. Vakrakou et al further found that NLRP3 inflammasome was highly associated with impaired DNA degradation in pSS. Dupire et al illustrated that highly expressed high mobility group box-1 (HMGB1) levels were highly correlated with disease activity of pSS.
Compelling evidence has highlighted the potential role of PGRN in the pathogenesis of several autoimmune diseases. In a collagen-induced mouse model of RA, PGRN exerted the anti-inflammatory effects via TNF/TNFR pathway. In SLE patients, the up-regulated serum PGRN was correlated with the expression levels of pro-inflammatory cytokines including IL-6, TNF-α, and TNFR. In addition, PGRN was prominently elevated in dermal fibroblasts and serum of systemic sclerosis (SSc) patients, while PGRN knockdown exerted the pro-fibrotic effect. Under a collagen-induced arthritis model, PGRN null mice exhibit a higher incidence of arthritis. Recombinant PGRN protein attenuated the degradation of cartilage matrix and protected against osteoarthritis development. PGRN was also reported to be significantly increased in pSS patients before treatment, which was reduced after pSS treatment. In this study, higher levels of serum PGRN were observed in pSS patients as compared with the healthy subjects, which was consistent with results of a previous study.
To the best of our knowledge, sOX40L interaction is activated in response to antigen presentation on multiple APCs and plays the pathogenic role in autoimmune diseases. For instance, Jacquemin et al illustrated that OX40L was significantly up-regulated in myeloid APCs of SLE patients, and were highly correlated with disease activity. In our research, a positive correlation was also found between PGRN and sOX40L, suggesting that PGRN and sOX40L may all involve in the pathogenesis of pSS. However, there are still limitations. For example, the clinical samples are relatively small. There is no control group including other autoimmune disease. Moreover, whether serum levels of PGRN or sOX40L are associated with disease progression were not examined.
In conclusion, in the present study, our findings revealed highly expressed PGRN and sOX40L levels in serum of pSS patients. Additionally, serum PGRN or sOX40L were positively correlated with pSS disease activity indexes. Besides, the levels of serum PGRN and sOX40L were significantly higher in active pSS patients and in patients with multiple system damage. Our study might provide a new line of evidence that PGRN and sOX40L serve as an important markers of pSS.
Xuan Qi and Huifang Guo contributed the central idea, analysed most of the data, and wrote the initial draft of the paper. Hongtao Jin and the remaining authors contributed to refining the ideas, carrying out additional analyses and finalizing this paper.
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