Effect Of Anisomycin On The Behavior Of Lymphocytes And The Allogenetic Skin Transplantation In Mice

Objective: To investigate the effects of anisomycin on the behaviors of lymphocytes and the allogenetic skin transplantation in the mice. Methods: A model to evaluate lymphocyte proliferation stimulated with a polyclonal activator, concanavalin A (ConA), was established by vital dye carboxyl fluorescin diacetate succinmidyl ester (CFSE) labeling technique. Effects of the different doses of anisomycin on the lymphocyte proliferation were estimated by flow cytometry and MTT methods. The propidium iodide labeling technique was applied to assay the effect of the different doses of anisomycin on changes of the lymphocyte cell-cycle stimulated by ConA or by phorbol ester (PDB) plus Ionomycin (Ion). The percentage of the expression level of CD69 and CD25 on the activated lymphocytes was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique. The reactivity of the lymphocytes in unilateral or bilateral mixed lymphocyte reaction (MLR), the effect of the lymphocytes to kill rat liver cancer cell lines (7919) and the cytotoxicity of anisomycin to the lymphocytes were detected by MTT assay. A model of allogenetic skin transplantation in mice was established to observe the effects of anisomycin on survival time of transplanted skin, delayed type hypersensitivity (DTH) and the reactivity of the lymphocytes in mice after skin transplantation. Results: CFSE staining showed that the promoting effect of ConA on the lymphocyte proliferation was stronger and stronger under the doses of anisomycin from 0.01 ng/ml to 0.5 ng/ml. At the concentration of 0.5 ng/ml, the promoting effect was the most significant; but at the dose of 1.0～25.0 ng/ml, the promoting effect was weaker and weaker. The analysis of the cell-cycle distribution revealed that 0.01～0.5 ng/ml anisomycin could promote G2/M phase entry stimulated by ConA or promote S phase entry stimulated by PDB plus Ion and that 1.0～25.0 ng/ml anisomycin led to G0/G1 arrest and blocked S phase entry stimulated by ConA or by PDB plus Ion. Under the dose of 10.0 ng/ml, anisomycin could definitely down-regulate the expression level of CD69 and CD25 on the lymphocyte surface (P ＜0.01). At the same dose, anisomycin could also inhibit the reactivity of lymphocytes in both unilateral and bilateral MLR, and there was no difference of the inhibitory effect between anisomycin and Dexamethasone (P＞0.05). The effect of the lymphocytes to kill the rat liver cancer cells was weaker and weaker under the dose of anisomycin from 0.01ng/ml to 100.0 ng/m. At the concentration of 10.0ng/ml and 100.0ng/ml, the inhibitory effect of anisomycin was the most significant, but there was no indirect cytotoxicity of anisomycin to the mouse primary lymphocytes or to 7919 cells. In vivo, 15.0mg/kg anisomycin could prolong the survival time of transplanted skin, inhibit the development of DTH and inhibit the reactivity of the lymphocytes in mice after skin transplantation., and the effects of anisomycin against allogeneic skin transplantation rejection were stronger than cyclosporine A (CsA) (P＜0.01 or P＜0.05). Conclusion: The results suggest that anisomycin can inhibit lymphocyte behaviors such as proliferation, cell-cycle, activation, reactivity and killing effect and inhibit the rejection of allogeneic skin transplantation in mice. They provide immportant theoretical and experimental evidences for the research and development of anisomycin as a new immunosuppressant.