The Relation Of BLyS And HCV-associated Autoimmunity

Background:The mechanisms or pathophysiology that lead to presence of auto-antibodies in part of chronic hepatitis C patients are not yet fully understood.Cross-reactive immune response between a host antigen(GOR)and an amino acid sequence of HCV core antigen may be the cause of low titer of autoantibody but not commensurates the immune disorders in HCV patients.Host factors seem to be involved in the HCV associated autoimmunity.Another research shows that 50%-90%patients of MC and its associated glomerulonephritis are HCV-RNA positive.Antibody to Liver Kidney Microsomal type 1(LKM1)was detected in HCV infected patients, which is a sign of autoimmune hepatitis.Liver histological changes of HCV infected patients are similar to that of autoimmune hepatitis. Researchers speculate that autoimmunity may play a role in the mechanism of HCV pathogenesis.The presence of autoantibody in HCV patients may be due to HCV induced immune disorders.Objective:Immune cells are infected because of pan-tropism of HCV.Some research show T-cell subgroups are changed and the balance of the immunity system is broken in HCV infected patients.The changes in immune function of B cell and its subsets are not yet known.To this end,serum BLyS levels,expression of BLyS-mRNA in PBMC and the changes of B cell subsets were investigated in autoantibody-positive HCV patients and compared with autoantibody-negative HCV patients and healthy donors.The purpose of this research is to investigate the relation of HCV infection and autoimmunity,deeply understand the mechanism of HCV pathogenesis and provide support for patient-focused treatment.Methods:(1).Research subjects were divided into healthy control group and HCV-group,which was further divided into positive autoantibody subgroup and negative-autoantibody subgroup.(2)Serum BLyS levels were tested with ELISA assay;Conventional methods were used for detection of immunoglobulin and RF;Serum Laboratory Markers of Liver Fibrosis were detected by radioimmunoassay;HCV-positive patients were tested for cryoglobulin with qualitative detection method.(3).27 HCV-RNA positive patients fell into two groups according to the serum BLyS levels(elevated and normal),and were treated with interferon-αplus ribavirin combination therapy.Serum specimens were collected and tested for HCV-RNA in 12 consecutive weeks.The virus response was analyzed in relation to serum BLyS levels.(4).PBMCs were isolated by centrifugation of heparinized blood on a Ficoll-Hypaque density gradient. The expression of BLyS-mRNA in PBMC was detected by RT-PCR.(5) Freshly isolated PBMCs were stained for the lymphocyte surface markers of CD19~+,CD38~+ and BLyS~+ molecules then measured by flow cytometry (FACS).The percentage of BLyS~+ cell,CD19~+ cell and CD38+ cell in PBMC were calculated,and their correlations analyzed.(6).Experimental data were processed with SPSS for Windows(version 10.0).Results:58 HCV infected patients were assayed for serum BLyS levels with ELISA method and compared with a healthy control group of 40 individuals.Results demonstrate that BLyS serum levels in HCV-infected patients are markedly elevated when compared to healthy control persons. Mean serum BLyS was increased in patients with HCV infection (4.7±1.1)ng/ml compared to(3.1±0.67)ng/ml in healthy controls(P＜0.01). RF positive rate in HCV infected people was 27.6%compared to 2.5%in healthy group.BLyS levels was increased in HCV patients with positive RF compared to those with negative RF(5.4±0.98 ng/ml vs 4.4±1.0 ng/ml P＜0.01).The serum level of BLyS was found to be correlated with Serum Laboratory Markers of Liver Fibrosis(HA,C-Ⅳ,PcⅢ, LN)rather than ALT,HCV-RNA,or,IgG,IgA&IgM,(HA:r=0.599,P＜0.01; C-Ⅳ:r=0.392,P＜0.05;PcⅢ:r=0.268,P＜0.05;LN:r=0.336,P＜0.05;& ALT.r=0.23,P＞0.05;HCV-RNA r=0.19,P＞0.05;IgG:r=0.161,P＞0.05; IgA:r=0.21,P＞0.05;IgM:r=0.254,P＞0.05).We investigated the BLyS difference in responsiveness to 12-week interferon and ribavirin combination treatment in HCV patients.Result show the 12-week accumulative HCV-RNA disapearance rate was higher in BLyS normal group compared to BLyS elevated group(Log-rank test,P＜0.05).The median VR time was 6.6 weeks in patients with normal BLyS,but 10.3 weeks in patients with elevated BLyS.The median VR time is longer in patients with elevated BLyS than with normal BLyS(P＜0.05).The expression of BLyS-mRNA in peripheral blood mononuclear cells(PBMC)from patients with HCV was investigated and analyzed in relation to HCV associated autoimmunity.PBMCs of 48 patients with HCV were assayed for BLyS-mRNA by reverse transcription-PCR (RT-PCR)and compared to two control groups[25 healthy volunteers and 20 chronic HBV infection].Research subjects were also assayed for rheumatoid factor(RF)and mixed cryoglobulin(MC).Expression of BLyS-mRNA in PBMC from patients with HCV was significantly elevated compared to healthy control group and disease control group(0.54±0.22 vs 0.40±0.14 P＜0.01 and 0.54±0.22 vs 0.42±0.17 P＜0.05).BLyS-mRNA levels were higher in HCV patients with a positive RF and/or MC than those without(0.64±0.25 vs 0.49±0.20,P＜0.05).To explore the mechanism of elevated BLyS in HCV infected patients,we investigated the expression of BLyS-mRNA in PBMC after administration of INF-γ.Without stimulation of INF-γ,the expression of BLyS-mRNA at 0h,6h,12h,24h is not significantly changed in both HCV group and healthy group(HCV: F=1.86,P＞0.05 Control:F=1.17,P＞0.05)After treated with INF-γ,the expression of BLyS-mRNA in PBMC from HCV patients or healthy control increased,and peaked at 6h,followed by a decline.Mean BLyS-mRNA levels of HCV group are higher than that of control group when compared at the same time after treatment.Peripheral blood lymphocytes of forty-eight patients with HCV and thirty healthy subjects were stained for the lymphocyte surface markers of CD19~+,CD38~+ and BLyS~+ molecules,then measured by flow cytometry (FACS).Result showed that the percentage of BLyS~+ lymphocytes in PBMC from HCV-patients were significantly increased compared to healthy control(4.0±2.2 vs 2.3±0.75,P＜0.01).the percentage of CD 19~+cell, CD19~+CD38~+cell,and the ratio(%)of CD19~+CD38~+/CD38~+ are 21.6±6.0 12.1±5.8 23.8±11.9 in HCV infected patients,compared, to13.4±3.9 5.8±1.6 11.4±2.3 in heathy control.The percentage of CD19~+ cell,CD38~+cell in PBMC and the ratio of CD19~+CD38~+/CD38~+ are higher in HCV patients than in healthy controls.The percentage of CD38~+ cell is not significantly changed when compared to control group (52.0±8.4 vs 50.0±7.0 P＞0.05).The increase of BLyS~+ cells is significantly correlated with the increase of CD19~+CD38~+ lymphocyte (r=0.303,P＜0.01).CONCLUSION:1.Part of HCV patients is autoantibody-positive,which is a sign of autoimmunity.2.Higher levels of serum BlyS in HCV patients coordinated with overexpression of BlyS-mRNA in PBMC,and provided testimony of overexpression of BlyS leading to autoimmunity.3.The percentage of BlyS~+ cell and CD19~+ cell increased in HCV patients.The correlation between BlyS~+ cell and CD19~+CD38~+ cell provide further support to HCV associated immunity.BlyS leading to abnormal proliferation and disorder of B-cell is a possible mechanism.4.The correlation of BlyS with serum markers of liver cirrhosis links higher serum BLyS level to chronic HCV infection and its poor prognosis.5.Further research is needed to explore the role of BlyS in anti-virus therapy.