| BackgroundSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease of unknown origin affecting major organs,which mostly occurred in women of childbearing age. SLE is primarily caused by high levels of autoantibodies and immune complex deposition. In SLE patients, disorder cytokine production induces immunodeficiency and leads to tissue inflammation and organ damage. The progranulin protein (PGRN) is an autocrine growth factor with multiple physiological and pathological functions. PGRN is mainly expressed in epithelial cells, immune cells, neurons and chondrocytes. Several studies have revealed that PGRN plays an important role in many pathological processes, including early embryonic development, wound healing and inflammation. PGRN also functions as a regulator of cartilagedevelopment and degradation. Tang Wet al. had found that PGRN can bind to TNF receptors and is therapeutic against inflammatory arthritis in mice.Therefore, PGRN is a potential target for the treatment of autoimmune diseases. However, the expression changes of PGRN in SLE patients remains unclear. Glucocorticoid (GC) is an important drug for treatment of SLE. GC inhibits the expression and function of many cytokines though two pathways:the genomic pathway and the nongenomic pathway. However, whether GCs could exert their function through affecting the expression of PGRN is need to be studied.Materials and methodsSubjectsThirty newly diagnosed SLE patients with SLEDAI≥10were recruited in the present study. All of the patients met the American College of Rheumatology revised criteria in1997for the classification of SLE. None of them had been treated with GCs and other immunosuppressive drugs prior to first collection of specimen. All of them received prednisone1mg/kg/day for21consecutive days. Peripheral blood samples were obtained again3weeks after prednisone administration. The control group included30sex-and age-matched healthy volunteers. All subjects signed informed consent forms. Ethical approval for the research was obtained from the Medical Ethical Committee of Qilu Hospital,Shandong University.Quantitative real-time polymerase chain reaction (RT-PCR)Five millilitres of heparinized venous peripheral blood were collected from each patient and control subjects before and after the administration of prednisone. PBMCs were separated by Red Blood Cell Lysis Buffer, and the total RNA was isolated by Trizol Reagent according to the manufacturer’s instructions. RNA concentration was determined using the Eppendorf Biophotometer and normalized to1ug/ml for reverse transcription. The cDNA was reverse-transcribed using the ReverTra Ace qPCR RT Kit. Real-time quantitative PCR was performed according to manufacturer’s instruction. Relative expression of PGRN, IL-6, PR3and TNFR mRNAs was determinedELISAFive millilitres of coagulation blood were collected from each patient and control subjects before and after the administration of prednisone. The blood was centrifuged and the serum specimens were stored at-80℃. Serum levels of PGRN, IL-6, PR3,TNFR and anti-dsDNA were measured using a commercial ELISA assay kit according to the manufacturer’s instructionStatistical analysisStatistical analysis was performed using SPSS17.0.ResultsClinical characteristicsAmong the30SLE patients,23were females and7were males, with ages ranged from12to52years old (28.4±9.3). The course of disease from the presence of symptoms to the enrollment varied from1to37months (12.6±10.1). SLEDAI scores ranged from10to23(15.1±3.8) of pre-treatment and6to22(12.2±4.0) of the post-treatment.PGRN level was increased in the serum of SLE patients The levels of PGRN, IL-6, TNFR, TNF-a,PR3and dsDNA antibody in SLE patients (both pre-treatment and post-treatment) were up-regulated significantly compared with that of the normal controls(p<0.05). Therefore, the level of PGRN was changed in SLE patients compared with normal controls.PGRN mRNA levels were increased in SLE patientsThe relative expression of PGRN mRNA was increased by4.88-fold in pre-treatment SLE patients compared to normal controls (P<0.01).Prednisone treatment downregulated the level of PGRN and inflammatory factors in the SLE patientsAfter treatment, there were significant downregulation in the serum levels of PGRN (P=0.02), IL-6(P=0.022), TNFR (P=0.003), TNF-a(P=0.001) and anti-dsDNA antibody(P=0.038). However, the serum level of PR3didn’t decreased after treatment (P=0.549). Relative expression of PGRN mRNA was decreased by1.34-fold in post-treatment SLE patients compared with pre-treatment ones (P<0.05). Statistically significant downregulation of IL-6(P<0.05), TNF-a(P<0.05), and TNFR (<0.05) mRNA expressions was also detected in post-treatment SLE patients compared with pre-treatment SLE patients.Correlations of PGRN with inflammatory factors in SLE patientsResults showed that the serum concentration of PGRN was correlated with the levels of IL-6(r=0.790, P<0.01), TNFR (r=0.559, P=0.01), TNF-a (r=0.438, P=0.015) and anti-dsDNA antibody (r=0.906, P<0.01) in the serum of pre-treatment SLE patients. After treatment of prednisone for3weeks, serum concentrations of PGRN in the patients were correlated with IL-6(r=0.836, P<0.01), PR3(r=0.396,P=0.031), TNFR (r=0.533, p=0.02), TNF-a (r=0.378,P=0.039) and anti-dsDNA antibody (r=0.712, P<0.01) in post-treatment SLE patients. No correlation was found between PGRN and PR3in pre-treatment SLE patients(r=0.298,P=0.110).ConclusionsOur present study showed that the levels of PGRN, IL-6,TNFR, TNF-aand anti-dsDNA decreased after administration of large doses of prednisone1mg/kg/day for twenty-one consecutive days. In a word, the present study demonstrated that PGRN is up-regulated in both active and GC-treated SLE patients. PGRN was concerned to be correlated with the disease activity of SLE. Academic studies still needed to understand the precise mechanisms of PGRN in regulating SLE. Since TNFR signaling is involved SLE processes, antagonists of the TNF/TNFR pathway may lead to a new therapeutics for this disease. PGRN may be used as a diagnostic marker of systemic lupus erythematosus. BackgroundSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease of unknown origin.SLE affects major organs, which mostly occurred in women of childbearing ages (20-40year old). SLE is primarily characterized by high levels of autoantibodies and immune complex deposition. The pathogenesis of SLE involves complex interactions between genetic and environmental factors as well asthe adaptive and innate immune systems. The breakdown of immunologic self-tolerance results in the development of autoimmune diseases. The interleukin12(IL-12) family of heterodimeric cytokines, includingIL-12, IL-23, IL-27and IL-35, have many biological effects on the regulation of immune system. IL-12, a heterodimeric cytokine composed of p35and p40subunits, is critical in Thl differentiation. IL-23is composed of one p40subunit the same as that in IL-12and one p19subunit. IL-12and IL-23are proinflammatory cytokines regulating the development of the Thl and Th17cells, respectively. IL-27is composed of Epstein-Barr virus-induced gene3(EBI-3) and p28subunit. IL-27was initially thought to be a prostimulatory cytokine, but recent studies demonstrated that it is an immunoregulatory cytokine. IL-35, the most recently identified member of this family which is composed of EBI-3and p35, is a potent inhibitory cytokine produced by populations of thymus-derived natural regulatory T cells (nTreg cells). Glucocorticoids (GCs) are powerful anti-inflammatory and immunosuppressive agents. They are widely used in the treatment of systemic autoimmune diseases, such as SLE, dermatomyositis and other systemic diseases. The interaction of GCs and GC receptor (GR) complex inhibits the transfer of leucocyte to inflammation site and damages the function of leucocyte, fibroblast and endothelial cells through modulating gene expression. GCs reduce the synthesis of pro-inflammatory cytokines, such as IL-2, IL-6, TNF-a and prostaglandins (PGs). However, the exact roles of IL-12, IL-23, IL-27and IL-35in SLE and their correlation to disease progression remain unknown. In this study, we investigate the roles of these cytokines in SLE and the possible effects of GCs on IL-12family members in SLE patients.Materials and methodsSubjectsThirty newly diagnosed SLE patients with SLEDAI>10were recruited into the present study. All of the patients met the American College of Rheumatology revised criteria in1997for the classification of SLE [17]. None of them had been treated with GCs and other immunosuppressive drugs prior to first collection of specimen. All of them received prednisone1mg/kg/day for14consecutive days. Peripheral blood samples were obtained again2weeks after prednisone administration. Patients before and after prednisone treatment were analyzed as pre-treatment and post-treatment group respectively. The control group included30sex-and age-matched healthy volunteers (23females and7males, age ranged18-59years with median30.1years). All subjects signed informed consent forms previous to entering the study. Ethical approval for the research was obtained from the Medical Ethical Committee of Shandong University.ELISAFive millilitres of coagulation blood were collected from each patient and control subject before and after the administration of prednisone. The blood was centrifuged and the serum specimens were collected and stored at-80℃. Serum levels of IL-12, IL-23, IL-27, IL-35, IL-6and anti-dsDNA antibodies were measured using commercial ELISA assay kit according to the manufacturer’s instruction.Statistical analysisStatistical analysis was performed using SPSS17.0. Data were presented as median+IQR. The comparisons among pre-treatment, post-treatment and control group were performed by independent sample nonparametric test. The correlations between IL-12and other cytokines or anti-dsDNA antibodies were assessed by Spearman rank correlation. P<0.05was considered as statistically significant. ResultsClinical characteristicsThe clinic-pathological characteristics of the30patients enrolled in this study were first analyzed. Among the patients,23were females and7were males, with age ranged from13to52years old (28.4±9.4). The course of disease from the presence of symptoms to the enrollment varied from1to37months (12.9±10.1). The systemic lupus erythematosus disease activity index (SLEDAI) scores ranged from10to23(15.2±3.8) of pre-treatment and6to22(12.4±4.0) of the post-treatment ones.Serum levels of IL-12family cytokines were upregulated in SLE patientsAs shown in Table2, before prednisone treatment, the levels of these cytokines in SLE patients with SLEDAI≥10were significantly up-regulated compared with that of the normal controls (P<0.05). We also found that all the five cytokines and anti-dsDNA antibodies were down-regulated after prednisone treatment, resulting in the levels of IL23, IL-27, and IL-35back to the same as that of normal control.Correlations of IL-12, IL-23, IL-27, IL-35with IL-6and anti-dsDNA antibodies in SLE patients.Results showed that the serum concentration of IL-12was correlated with the levels of IL-6(r=0.897, P<0.01), and anti-dsDNA antibodies (r=0.607, P<0.01) in the serum of pre-treatment SLE patients. After treatment of prednisone for2weeks, serum concentration of IL-12was correlated with IL-6(r=0.894, P<0.01), and anti-dsDNA antibodies (r=0.651, P<0.01) in post-treatment SLE patients.Moreover, serum levels of IL-23, IL-27and IL-35were correlated with IL-6before treatment, and correlated with both IL-6and ds-DNA after treatment.ConclusionsIn a word, the present study demonstrated that all the members of IL-12family were up-regulated in both active and GC-treated SLE patients. IL-12and IL-23was found to be correlated with the disease activity in SLE. Further studies are still needed to understand the mechanisms of IL-12, IL-23, IL-27and IL-35in regulating the pathogenesis of SLE, which would lead to new therapeutic strategies for this disease. |
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