The Expression And Significances Of PGRN In The Primary Sjogren’s Syndrome Patients

BackgroundSjogren’s syndrome is a common chronic auto-immune disease characterized by lymphocytic infiltration glands and by ocular and oral dryness, which primarily affects the salivary and lacrimal glands. This syndrome may occur as a primary Sjogren’s syndrome (pSS) or in association with other systemic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythem-atosus (SLE). The syndrome may manifest within a wide spectrum of diseases, ranging from a limited, organ-specific autoimmune exocrinopathy to a systemic disease with widespread autoimmune manifestations and pronounced immunologic features. pSS is characterized by polyclonal B cell activation, leading to chronic hypergammaglobulinemia, increased levels of β2microglobulinemia and concomitant presence of a variety of autoantibodies. Multiple factors, including viral infection, hormonal balance, and genetic background, are involved in the pathogenesis of pSS. The presence of T cells and B cells, macrophagocyte and dendritic cells varies according to the severity of the lesion. The influence of abnormal cytokine production in this disease has attracted considerable attention. Progranulin is called granulin-epithelin precursor(GEP), proepithelin, prostatic cancer-cell derived growth factor (PCDGF).The progranulin protein (PGRN) is an autocrine growth factor with multiple physiological and pathological functions. PGRN is mainly expressed in neurons, lymphocyte, epithelial cells, chondrocytes and immune cells. In addition, PGRN were also found in the high level expression in a variety of tumor cells.Several studies have revealed that PGRN plays an important role in many pathological processes, including early embryonic development, inflammation, wound healing, tissue reconstitution and immunoreaction. Many studies have found that PGRN is related to breast cancer, ovarian cancer and multiple myeloma and other tumor pathogenesis. Other studies have found that the mutation of PGRN gene can induce PGRN protein expression is missing, resulting in frontotemporal dementia. Therefore, PGRN is also an important neurotrophic factor. PGRN also functions as a regulator of cartilage development and degradation. Tang W et al had found that PGRN can bind to TNF receptors and is therapeutic against inflammatory arthritis in mice. Qiufeng et al fond that PGRN was concerned to be correlated with the disease activity of SLE. Therefore, PGRN is a potential target for the treatment of autoimmune diseases. However, the expression changes of PGRN in pSS patients remains unclear.Based on the above idea, we detected the expression of IL-6and PGRN in the peripheral blood of pSS patients and healthy control group to explore the role of PGRN in pSS patients and its correlation with disease activity.Materials and methodsSubjectsTwenty-six newly diagnosed pSS patients were recruited in the present study. All of the patients met the international revised criteria in2002for the classification of pSS. None of them had been treated with GCs and other immunosuppressive drugs prior to first collection of specimen. All of them received prednisone10mg/day for21consecutive days. Peripheral blood samples were obtained again3weeks after prednisone administration. The control group included26sex-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.ELISAFive 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-6antibodys were measured using a commercial ELISA assay kit according to the manufacturer’s instruction.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 and IL-6mRNAs was determined.Western BlotFive 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, total cellular protein was extracted. The expression of PGRN was detected by Western Blot method.Statistical analysisStatistical analysis was performed using SPSS17.0.ResultsClinical characteristicsAmong the26pSS patients,25were females and1were males, with ages ranged from24to65years old (44.8±10.96). The course of disease from the presence of symptoms to the enrollment varied from2to98months (20.4±22.0). Among the control group,23were females and3were males, with ages ranged from23to65years old, average32.3years old.PGRN level was increased in the serum of pSS patientsThe levels of PGRN, IL-6in pSS 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 pSS patients compared with normal controls.PGRN mRNA and protein levels were increased in pSS patientsThe relative expression of PGRN mRNA was increased by3.45-fold in pre-treatment pSS patients compared to normal controls (P<0.05). Following treatment with prednisone, the relative expression of PGRN mRNA showed only a1.6-fold increase when compared with the healthy controls (P<0.05). The difference in the expression levels before and after treatment with prednisone was statistically significant (P<0.05). The PGRN protein level is higher in pre-treatment pSS patients compared to normal controls (P<0.05). The PGRN protein level is higher in pre-treatment pSS patients compared to post-treatment pSS (P<0.05).Prednisone treatment downregulated the level of PGRN and inflammatory factors in the pSS patients After treatment, there were significant downregulation in the serum levels of PGRN (10.39±7.47vs14.57±7.93, P<0.05),IL-6(1.05±0.79vs1.81±1.03, P<0.05). Relative expression of PGRN mRNA was significantly decreased in post-treatment pSS patients compared with pre-treatment ones (P<0.05). And statistically significant downregulation of PGRN protein level (P<0.05) was also detected in post-treatment pSS patients compared with pre-treatment pSS patients.Correlations of PGRN with inflammatory factors in pSS patientsResults showed that the serum concentration of PGRN was correlated with the levels of IL-6(r=0.617, P=0.001) in the serum of pre-treatment pSS patients. Meanwhile, after treatment of prednisone for3weeks, serum concentrations of PGRN in the patients were correlated with IL-6(r=0.583, P<0.01).ConclusionsOur present study showed that the levels of PGRN, IL-6decreased after administration of doses of prednisone10mg/day for twenty-one consecutive days. In a word, the present study demonstrated that PGRN is up-regulated in both pre-treatment and GC-treated pSS patients. PGRN was concerned to be correlated with the disease activity of pSS. PGRN levels are correlated with inflammatory factor IL-6in pSS patients. Glucocorticoid could significantly reduce the expression of PGRN in patients with pSS. 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 as the adaptive and innate immune systems. The breakdown of immunologic self-tolerance results in the development of autoimmune diseases. 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 Glucocorticoids (GCs) are a mainstay and the most effective treatment for patients with systemic lupus erythematosus (SLE). However, GC resistance has been demonstrated in SLE patients even managed with high dosages of GCs. Considering GCs can cause a wide range of side effects such as Cushing features, osteoporosis, and other toxicities, it is important to avoid treatment with GCs in those patients who will not benefit from its use. Thus, an insight into the molecular mechanisms underlying GC resistance becomes a key to the effective management of patients with SLE. The biological effects of GC are mainly mediated through the activation of glucocorticoid receptors (GR), forming a GC-GR complex that binds DNA to regulate gene transcription. This leads to a reduction of the cellular pool of transcription factors, such as nuclear factor κB (NF-κB) and activator protein-1(AP-1), as well as the decreased expression of genes contributing to the autoreactivity of T and B cells. To date, different GR isoforms have been identified-GRα, GRβ, GRγ, GRp, GRA, and GRB-of which GRa is thought to be the primary mediator of GC action. However, GRβ does not bind steroids and is unable to activate GC-responsive genes, behaving as an inhibitor of GRa activity. Similarly, the imbalance of GRa, GRP, or GRa/GRβ ratio was found in several diseases related to GC resistance. Heat shock protein90(HSP90) is an important molecular chaperone for the GR and is supposed to be the key factor in regulating GC effects. Whether GC resistance in SLE patients is associated with the expression of GR isoforms and HSP90is still unknown. The goal of the present study was to investigate the correlation between GRa, GRβ, HSP90expression and GC resistance in SLE.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. 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 for56consecutive days. Peripheral blood samples were obtained again2weeks after prednisone administration. Patients according to the response to GC were divided into GC sensitive group (GCS) and GC resistant group (GCR). The control group included30sex-and age-matched healthy volunteers. 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.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 GRα、GRβ and HSP90mRNAs was determined.Statistical analysisStatistical analysis was performed using SPSS17.0. Data were presented as median±IQR The comparisons among GC sensitive group, GC resistant group and control group were performed by independent sample nonparametric test. P<0.05was considered as statistically significant. ResultsClinical characteristicsThe clinic-pathological characteristics of the30patients enrolled in this study were first analyzed. Among the GCS patients,27were females and4were males, with age ranged from14to55years old (25.6±8.2). The course of disease from the presence of symptoms to the enrollment varied from2to35months (10.3±5.1). The systemic lupus erythematosus disease activity index (SLEDAI) scores ranged from5to15(9.2±3.1) of pre-treatment and0to6(2.4±1.6) of the post-treatment ones. Among the GCR patients,21were females and9were males, with age ranged from13to52years old (22.6±9.6). The course of disease from the presence of symptoms to the enrollment varied from3to34months (12.3±6.4). The systemic lupus erythematosus disease activity index (SLEDAI) scores ranged from8to22(15.2±3.8) of pre-treatment and6to13(7.1±3.4) of the post-treatment ones.The expression of GRa mRNA、GRβ、mRNA and HSP90mRNA in PBMC in SLE and the controlAs shown in Table3, the expression of GRa mRNA、GRβ mRNA and HSP90mRNA in PBMC showed among the group, and there were no significant differences between the SLE and the control group. The level of HSP90mRNA in SLE patients was significantly up-regulated compared with that of the normal controls (P<0.05). The level of GRβ mRNA was low, even not detected in some SLE and the normal control.The expression of GRa mRNA、GRβ mRNA and HSP90mRNA in PBMC in GCS and GCR groupResults showed that the level of GRa mRNA in the GC sensitive group of SLE was significantly higher than that in the GC resistant group (P<0.05). The level of GRβ mRNA in the GC resistant group of SLE was significantly higher than that in the GC sensitive group (P<0.05). We also found that the expression of HSP90in the GC resistant group was higher, but no statistical significance.ConclusionsIn a word, the expression of GRa mRNA、GRβ mRNA and HSP90mRNA in PBMC showed among the group. The expression of HSP90mRNA in the SLE was significantly higher than that in the control group. The expression of GRβ mRNA in the GC resistant group of SLE was significantly higher than that in the GC sensitive group. The expression of GRα mRNA in the GC sensitive group of SLE was significantly higher than that in the GC resistant group.