| Objective: In order to seek the best antigen strategy for regulating immune functions of dendritic cells(DCs) in patients with tuberculosis and provide the scientific data for developing a new immune therapy, DCs of patients with initial treatment tuberculosis were cultured and stimulated by lipopolysaccharide(LPS), Bacillus Calmette Guerin(BCG) and Mycobacterium tuberculosis specific antigen Ag85b(Ag85b) respectively. The expressions of CD86, CD83, CD11 c, HLA-DR on DCs and the levels of IL-12, IL-10, IFN-γ in supernatant were measured.Methods:1 Based on the clinical guidelines-the tuberculosis section, 31 patients with initial treatment tuberculosis in the fifth hospital of Shi jia zhuang were enrolled. Twenty-six healthy people were also enrolled as control group.2 10 ml of peripheral blood was collected from patients and healthy people. The peripheral blood mononuclear cells(PBMCs) were separated by density gradient centrifugation and divided into four groups: blank group, LPS group, BCG group and Ag85 b group. The non-adherent cells were washed away after 3 hours of cultivation, and the adherent cells were cultured in 37℃ incubator with 5% CO2, moreover, the culture fluid was renewed every two days. After 6 days, LPS, BCG and Ag85 b were used to stimulate the cells, respectively, except the blank group. Finally the cells were collected after 24 hours. The morphology, size and growth state of DCs were observed.3 The expressions of CD83, CD86, HLA-DR and CD11 c on DCs were detected by flow cytometry.4 The concentrations of IL-12, IL-10 and INF-γ in the cultural supernatan-t of DCs were detected by ELISA.5 All statistical analyses were performed using SPSS13.0 software. The statistical significance was calculated from one way ANOVA analysis and level was set to 0.05. Data was expressed as means ± standard deviation( x ±s).Result: 1 Morphology of DCsComparing with the blank control group, the growth density of DCs in patients with initial treated tuberculosis was lower, and the morphology was not typical. After the stimulation of antigens, the DCs grew well and the typical dendritic structure could be observed. 2 Expressions of surface markers on DCs were detected by flow cytometry, and the results were as follows: 2.1 The expressions of CD83 in blank group, LPS group, BCG group, Ag85 b group among healthy people were(10.52±7.36)%,(15.01±9.01)%,(11.91±7.91)%,(11.72±6.88)%, respectively. The expressions of CD83 in four groups of tuberculosis patients were(4.93±2.67)%,(11.42±9.51)%,(8.25±6.7)%,(9.16±8.78)%, respectively. The expressions of CD83 in blank group of initial treated tuberculosis patients was significantly lower than that of healthy people(P=0.001). For the healthy people,the expression of CD83 in LPS group was significantly higher than the blank group(P=0.042). For the patients, the expressions of CD83 in LPS and Ag85 b group were also significantly higher than the blank group(P=0.01 and P= 0.026). 2.2 The expressions of CD86 in blank group, LPS group, BCG group, Ag85 b group of healthy people were(74.22±5.84)%,(82.98±11.57)%,(75.5±15.31)%,(81±10.69)%, respectively. The expressions of CD86 in four groups of tuberculosis patients were:(68.47±15.23)%,(81.48±13.1)%,(75.5±15.31)%,(81±10.69)%, respectively. For healthy people,the expression of CD86 in LPS group was significantly higher than the blank group(P=0.025). For the patients, the expressions of CD86 in LPS and Ag85 b group were much more increased than that of the blank group(P=0.001 and P= 0.007). 2.3 The expressions of HLA-DR in blank group, LPS group, BCG group, Ag85 b group of healthy people were(84.42±12.06)%,(88.98±8.3)%,(87.34±9.4)%,(88.22±9.86)%, respectively. The expressions of HLA-DR in four groups of tuberculosis patients were(81.77±8.37)%,(90.34±6.35)%,(85.1±7.45)%,(85.1±9.93)%, respectively. For the patients, the expression of HLA-DR in LPS group was significantly higher than that of the blank group, BCG group and Ag85 b group.(P<0.001, P =0.02 and 0.033). 2.4 The expressions of CD11 c in blank group, LPS group, BCG group, Ag85 b group of healthy people were(90.02±7.23)%,(89.61±8.26)%,(88.63±9.66)%,(88.87±10.47)%, respectively. The expressions of CD11 c in four groups of tuberculosis patients were(91.35±5.96)%,(90.54±6.34)%,(90.38±7.67)%,(91.07±6.06)%, respectively. There was no significant difference among these groups(P>0.05). 3 The concentrations of cytokines in the cultural supernatant were as follows: 3.1 The concentrations of IL-12 in blank group, LPS group, BCG group, Ag85 b group of healthy people were(39.00±36.78) pg/ml,(31.50±20.27) pg/ml,(33.79±26.65) pg/ml,(27.07±20.55) pg/ml, respectively. The concentrations of IL-12 in four groups of patients were(30.11±28.70) pg/ml,(27.8±18.21) pg/ml,(21.94±16.70) pg/ml,(22.80±16.05) pg/ml, respectively. There was no significant difference among healthy groups(P>0.05) as well as the groups of patients(P>0.05). 3.2 The concentrations of IL-10 in blank group, LPS group, BCG group, Ag85 b group of healthy people were(5.98±5.47) pg/ml,(11.37±11.27) pg/ml,(6.67±5.65) pg/ml,(6.28±5.91) pg/ml, respectively. And they were(2.13±2.01) pg/ml,(10.29±10.1) pg/ml,(1.98±1.53) pg/ml,(2.99±2.87) pg/ml in four groups of patients. The concentration of IL-10 in blank group of patients was significantly lower than that of healthy people(P=0.02). For the patients, the concentration of IL-10 in the LPS group was significantly higher than that of the blank group, BCG group and Ag85 b group(P<0.001).The concentration of IL-10 in the LPS group of healthy people was also significantly higher than that of the blank group, BCG group and Ag85 b group(P=0.003, 0.009 and 0.005). 3.3 The concentrations of IFN-γ blank group, LPS group, BCG group and Ag85 b group of healthy people were(7.02±6.41) pg/ml,(8.82±8.27) pg/ml,(11.01±10.75) pg/ml,(12.03±11.89) pg/ml, respectively. They were(6.92±5.86) pg/ml,(12.45±11.86) pg/ml,(21.08±20.51) pg/ml and(15.44±14.39) pg/ml in four groups of patients, respectively. After stimulation with three kinds of antigens, the concentrations of IFN-γ were increased in groups of patients and healthy people, and they were higher in the patient groups than those of control groups. For patients, the concentrations of IFN-γ in the BCG group and the Ag85 b group were higher than the blank group(P<0.001`and P= 0.004).Conclusions:1 The decreased immune function of initial treated tuberculosis patients may be due to the immaturation of the DCs.2 LPS could significantly improve the maturation and antigen presenting capacity of DCs in peripheral blood of initial treated tuberculosis patients, but it may also inhibit Th1 cell responses through the secretion of IL-10.3 The effects of BCG on improving the maturation and antigen presentin-g capacity of DCs in peripheral blood of initial treated tuberculosis patients is weak. However, its ability of stimulating IFN-γ secretion is much stronger.4 Ag85 b could enhance the maturation and antigen presenting capacity of DCs in peripheral blood of initial treatment of tuberculosis patients and the secretion of IFN-γ. |
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