The Effect Of Polysaccharide Nucleic Acid Fraction Of Bacillus Calmette Guerin On Peripheral Blood Lymphocytes Th1/Th2Differentiation In Patients With Chronic Urticaria

Objective：Chronic urticaria (CU) is one kind of skin diseases， the exactpathophysiology is not well understood. Although not life-threatening，it hasan important impact on patients quality of life as well as heart diseases.According to the American Academy of Dermatology literature latest reportsthat patients with chronic urticaria are at increased risk of cancer，especiallyhematologic malignant tumors.It is unclear that the co-existence of chronicurticaria and malignancy is a chance，or they share some pathogenesis，but itreminds us that the study of chronic urticaria can not be ignored. In addition toH1receptor antagonists， H2receptor antagonists， antihistamines，glucocorticoids，immunetherapy has become a research hotspot.There is a dynamic balance between Th1and Th2. Numerous studieshave shown that there is a differentiation bias of Th cells in patients withchronic urticaria. Some scholars believe the helper T cells play an importantrole in the pathogenesis of chronic urticaria. Polysaccharide Nucleic AcidFraction of Bacillus Calmette Guerin (BCG-PSN) is a functionimmunomodulatory substances extracted from BCG. Numerous controlledstudies as well as case reports have demonstrated that BCG-PSN can beemployed successfully in patients with chronic urtiaria，the mechanism may berelated to the modulation of immune. To reveal the possible mechanism ofusing BCG–PSN on chronic urticaria. The expressions of IL-2，IL-4，IL-10and IFN-γ as markers of effector T cell responses were examined in peripheralblood lymphocytes of patients with chronic urticaria，after treated withdifferent concentrations of BCG-PSN for96h.Provide clinical witness for theuse of BCG-PSN to treat chronic urticaria. Methods：1.Cells：The peripheral blood lymphocytes of30patients with chronicurticaria come from Department of Dermatology，Affiliated Hospital ofChengde Medical College，from February2012to July2012，inpatient andoutpatient patients are diagnosed with a clinical diagnosis of chronicurticaria.Peripheral blood lymphocytes of30healthy controls are from healthyvolunteers in our hospital.2.Experimental group and the intervention：six groups have been set up inthe experiment according to the concentration of BCG-PSN in RPMI-1640complete medium of peripheral blood lymphocytes of patients with chronicurticaria，namely0μl/ml group，5μl/ml group，10μl/ml group，20μl/ml group，40μl/ml group，80μl/ml group，set up peripheral blood lymphocytes of healthyhuman in RPMI-1640complete medium as a control group.Each groupcontains the same concentration of PHA (100μg/ml).3. Immunocytochemical staining：the expressions of IFN-γ，IL-2，IL-4and IL-10in each group were detected by Immunocytochemical staining.4.ELISA：the expressions of IFN-γ，IL-2，IL-4and IL-10in each groupwere detected by ELISA.5.The statistical analysis was performed with SPSS17.0using one-wayanalysis of variance (ANOVA).P<0.05was considered statistically significant.Correlation between the two variables were analyzed using Pearsoncorrelation analysis.Result：1. The expressions of IFN-γ，IL-2，IL-4and IL-10in peripheral bloodlymphocytes of patients with chronic urticaria： It was shown byimmunocytochemical staining that the percentages of positive cells(%)inIFN-γ and IL-2were9.786±1.926and12.023±2.381respectively，bothmuch lower than those of30.472±5.247and32.148±3.370in healthycontrol group respectively，the percentages of positive cells(%)in IL-4andIL-10were17.891±3.361，17.922±2.916respectively，both much higherthan those of10.331±2.632，10.781±2.477in healthy control group respectively，with statistical differences between each groups after ANOVA(p<0.05). The culture supernatant levels of IFN-γ，IL-2，IL-4and IL-10weredetected by ELISA，the assays showed that the expressions of IFN-γ andIL-2(pg/ml)were205.107±92.424and244.838±54.780respectively，bothmuch lower than those of428.837±138.009and340.203±60.769in healthycontrol group respectively，the expressions of IL-4and IL-10(pg/ml)were29.764±11.219and70.556±24.714respectively，both much higher thanthose of11.850±4.331and32.588±17.321in healthy control grouprespectively，with statistical differences between each groups after ANOVA(p<0.05).2.The effect of BCG-PSN on IFN-γ expression in peripheral bloodlymphocytes of patients with chronic urticaria：Different concentrations ofBCG-PSN (0μl/ml,5μl/ml,10μl/ml,20μl/ml,40μl/ml,80μl/ml)acting onperipheral blood lymphocytes of patients with chronic urticaria with theinduction by PHA，each group of lymphocytes cultured for96h，then detectedthe percentage of positive cells in IFN-γ by immunocytochemical staining，theassays showed that with the concentration of BCG-PSN increasing，thepercentage of positive cells in IFN-γ was getting higher and higher，renderinga dose-dependent relationship(r=0.804， p<0.01).As the concentration ofBCG-PSN increasing，the percentage of positive cells in IFN-γ graduallyclosed to the healthy control group.The analysis of variance indicated thedifference was significant (F=150.137，p<0.01).Mutiple comparisons，thedifferences were statistically significant (p<0.05)，in addition to10μl/ml groupvs20μl/ml group.The culture supernatant level of IFN-γ was detected byELISA， the assays showed that with the concentration of BCG-PSNincreasing，the level of IFN-γ was getting higher and higher，rendering adose-dependent relationship (r=0.450， p<0.01).As the concentration ofBCG-PSN increasing，the level of IFN-γ gradually closed to the healthycontrol group. The analysis of variance indicated the difference wassignificant (F=15.790，p<0.01). Mutiple comparisons，the differences werestatistically significant (p<0.05)，in addition to0μl/ml group vs5μl/ml group， 10μl/ml group vs20μl/ml group，20μl/ml group vs40μl/ml group，20μl/mlgroup vs80μl/ml group，20μl/ml vs the healthy control group，40μl/ml vs80μl/ml group，40μl/ml group vs the healthy control group，80μl/ml group vsthe healthy control group.3.The effect of BCG-PSN on IL-2expression in peripheral bloodlymphocytes of patients with chronic urticaria：Different concentrations ofBCG-PSN (0μl/ml，5μl/ml，10μl/ml，20μl/ml，40μl/ml，80μl/ml) acting onperipheral blood lymphocytes of patients with chronic urticaria with theinduction by PHA，each group of lymphocytes cultured for96h, then detectedthe percentage of positive cells in IL-2by immunocytochemical staining，theassays showed that with the concentration of BCG-PSN increasing，thepercentage of positive cells in IL-2was getting higher and higher，rendering adose-dependent relationship(r=0.735， p<0.01).As the concentration ofBCG-PSN increasing，the percentage of positive cells in IL-2gradually closedto the healthy control group. The analysis of variance indicated the differencewas significant (F=88.730，p<0.01). Mutiple comparisons，the differenceswere statistically significant (p<0.05)，in addition to5μl/ml group vs10μl/mlgroup，40μl/ml group vs80μl/ml group.The culture supernatant level of IL-2was detected by ELISA, the assays showed that with the concentration ofBCG-PSN increasing，the level of IL-2was getting higher and higher，rendering a dose-dependent relationship (r=0.428，p<0.01).As the concentra-tion of BCG-PSN increased，the level of IL-2gradually closed to the healthycontrol group. The analysis of variance indicated the difference wassignificant (F=10.258，p<0.01). Mutiple comparisons, the differences werestatistically significant (p<0.05)，in addition to0μl/ml group vs5μl/ml group，5μl/ml group vs10μl/ml group，10μl/ml group vs20μl/ml group，20μl/mlgroup vs40μl/ml group，20μl/ml vs the healthy control group，40μl/ml vs80μl/ml group，40μl/ml group vs the healthy control group,80μl/ml group vsthe healthy control group.4.The effect of BCG-PSN on IL-4expression in peripheral bloodlymphocytes of patients with chronic urticaria：Different concentrations of BCG-PSN (0μl/ml，5μl/ml，10μl/ml，20μl/ml，40μl/ml，80μl/ml) acting onperipheral blood lymphocytes of patients with chronic urticaria with theinduction by PHA，each group of lymphocytes cultured for96h，then detectedthe percentage of positive cells in IL-4by immunocytochemical staining，theassays showed that with the concentration of BCG-PSN increasing，thepercentage of positive cells in IL-4was getting lower and lower，rendering adose-dependent relationship(r=-0.624， p<0.01).As the concentration ofBCG-PSN increasing，the percentage of positive cells in IL-4gradually closedto the healthy control group. The analysis of variance indicated the differencewas significant (F=45.744，p<0.01). Mutiple comparisons，the differenceswere statistically significant (p<0.05)，in addition to20μl/ml group vs40μl/mlgroup，40μl/ml group vs80μl/ml group，40μl/ml group vs the healthy controlgroup，80μl/ml group vs the healthy control group.The culture supernatantlevel of IL-4was detected by ELISA，the assays showed that with theconcentration of BCG-PSN increasing，the level of IL-4was getting lower andlower，rendering a dose-dependent relationship (r=-0.415，p<0.01).As theconcentration of BCG-PSN increasing，the level of IL-4gradually closed tothe healthy control group. The analysis of variance indicated the differencewas significant (F=16.731，p<0.01). Mutiple comparisons，the differenceswere statistically significant (p<0.05),in addition to5μl/ml group vs10μl/mlgroup，10μl/ml group vs20μl/ml group，20μl/ml group vs40μl/ml group，20μl/ml vs80μl/ml group，40μl/ml vs80μl/ml group，80μl/ml group vs thehealthy control group.5. The effect of BCG-PSN on IL-10expression in peripheral bloodlymphocytes of patients with chronic urticaria：different concentrations ofBCG-PSN (0μl/ml，5μl/ml，10μl/ml，20μl/ml，40μl/ml，80μl/ml)acting onperipheral blood lymphocytes of patients with chronic urticaria with theinduction by PHA，each group of lymphocytes cultured for96h，then detectedthe percentage of positive cells in IL-10by immunocytochemical staining，theassays showed that with the concentration of BCG-PSN increasing，thepercentage of positive cells in IL-10was getting lower and lower，rendering a dose-dependent relationship (r=-0.472，p<0.01). As the concentration ofBCG-PSN increasing，the percentage of positive cells in IL-10graduallyclosed to the healthy control group. The analysis of variance indicated thedifference was significant (F=24.522，p<0.01). Mutiple comparisons，thedifferences were statistically significant (p<0.05)，in addition to the0μl/mlgroup vs5μl/ml group，5μl/ml group vs10μl/ml group，20μl/ml group vs40μl/ml group，20μl/ml group vs80μl/ml group，40μl/ml group vs80μl/mlgroup.The culture supernatant level of IL-10was detected by ELISA，theassays showed that with the concentration of BCG-PSN increasing，theexpression of IL-10was getting lower and lower，rendering a dose-dependentrelationship (r=-0.278，p<0.01).As the concentration of BCG-PSN increasing，the level of IL-10gradually closed to the healthy control group. The analysisof variance indicated the difference was significant (F=7.720， p<0.01).Mutiple comparisons，the differences were statistically significant (p<0.05)，in addition to the0μl/ml group vs5μl/ml group，5μl/ml group vs10μl/mlgroup，5μl/ml group vs20μl/ml group，10μl/ml group vs20μl/ml group，10μl/ml vs40μl/ml group，10μl/ml group vs80μl/ml group，20μl/ml group vs40μl/ml group，20μl/ml vs80μl/ml group，40μl/ml vs80μl/ml group，80μl/mlgroup vs the healthy control group.Conclusion：1. The expressions of IFN-γ and IL-2were lower and the expressions ofIL-4and IL-10were higher in peripheral blood lymphocytes of patients withchronic urticaria.There is a differentiation bias of Th cells in peripheral bloodlymphocytes of patients with chronic urticaria，Th2plays an important role inchronic urticaria.2. BCG-PSN can effectively increase the expressions of IFN-γ and IL-2，decrease the expressions of IL-4and IL-10in peripheral blood lymphocytes ofpatients with chronic urticaria,thus regulate the T cell subsets and balance ofThl/Th2.