Effects of four common stimulants on intracellular cytokines and CD62L of splenic CD8+ T cells from mice infected with Schistosoma japonicum
Li ZHENG, Yuan HU, Yan-juan WANG, Xi-bao HUANG, Yu-juan SHEN, Yu-xin XU, Wen-ci GONG, Shun-xiang CAI, Jian-ping CAO
2017, 35(3):
218-223.
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Objective To investigate the effects of four stimulants Ionomycin (I), Phorbol-12-myristate-13-acetate(P), Brefeldin A(B), and Monensin (M) on intracellular cytokines and CD62L of splenic CD8+ T cells from mice infected with Schistosoma japonicum. Methods Twenty-one C57BL/6 female mice were infected with S. japonicum using the abdominal patch method (20 ± 2 cercaria per mouse). The splenic single cell suspension was prepared at 8 and 12 weeks after infection, and were stimulated in vitro by P (50 ng/ml) + I (1 μmol/L) + M (2 μmol/L) for 4 h. Flow cytometry was performed to determine the proportion of CD8+ T cells secreting IFN-γ and detect surface expression of CD62L. Meanwhile, the splenic single cell suspension at 4 weeks after infection was stimulated by I (1 μmol/L), P (50 ng/ml), B (1 μg/ml), M (2 μmol/L), P + I, P + I + M, P + I + B, or P + I + M + B for 4 h, and the supernatant levels of IL-4, IL-17A, IFN-γ, IL-10, IL-1β and G-CSF were determined with cytometric bead array. CD62L expression on CD8+ T cells was examined by flow cytometry, and the mean fluorescence intensity (MFI) was calculated. Results After infection with S. japonicum, the proportion of IFN-γ/CD8+ T cells was(1.3 ± 0.8)% at 8 weeks, and dropped to(0.7 ± 0.2)% at 12 weeks, which were both significantly lower than(5.6 ± 0.8)% in the uninfected control group(P < 0.05). CD62L was not detected on CD8+ T cell surface in control and infection groups. The supernatant levels of IL-4 in the P + I and P + I + M groups were(177.2 ± 56.0) and (13.7 ± 2.2) pg/ml, respectively. The supernatant levels of IL-17A in the P + I and P + I + M groups were (361.8 ± 81.3) and (33.7 ± 2.9) pg/ml, respectively. The supernatant levels of IFN-γ in the P + I and P + I + M groups were (1534.0 ± 316.6) and (135.3 ± 16.1) pg/ml, respectively. The supernatant levels of IL-10 in the P + I, P and I groups were (705.5 ± 179.6), (34.8 ± 13.9) and (43.1 ± 13.9) pg/ml, respectively. The supernatant levels of G-CSF in the P and P + I groups were (44.6 ± 8.0) and (21.7 ± 2.9) pg/ml, respectively. The supernatant levels of IL-1β in the P, I, and P + I + M groups were (3.9 ± 1.0), (6.4 ± 0.2) and (3.7 ± 0.3) pg/ml, respectively. The above cytokine levels were all significantly different from the corresponding control level (P < 0.01 or P < 0.05). Results of flow cytometry showed a significant leftward shift of CD62L peak in the P + I, P + I + B, P + I + M, and P + I + B + M groups, compared with the control group, and MFIs of CD62L on CD8+ T cells were 2.7 ± 0.1, 2.6 ± 0.2, 2.5 ± 0.1 and 2.5 ± 0.1, all significantly lower than the control (P < 0.01). Conclusion In the late stage of schistosome infection(8-12 weeks), there is a lower percentage of IFN-γ-secreting CD8+ T cells among splenic cells. The P + I stimulation decreases the expression of CD62L.
