Effects Of Monocyte-Derived Macrophages On The Balance Of Th1/Th2 In Asthmatic Children And Modulatory Effects Of Theophylline In Vitro

Asthma is a serious global disease which etiological factor and pathogenesis are uncertain. The effective therapy has not been found yet, and the clinical effect is not satisfied in all patients. Immunological researches have showed that the balance of Th1/Th2 is disordered, and the exaggerated Th2-biased immune response plays a crucial role in the onset and development of asthma. Macrophages are important cells in immunological system, having many functions such as presenting antigen, eliminating foreign body and secreting various cytokines, etc. They also have notable effects on activating and regulating T cells by co-stimulatory molecules and cytokines signal pathway. Under some specific condition, macrophages can efficiently induce the migration of Th1/Th2. In this experiment, we mainly study the effects of monocyte-derived macrophages on intracellular cytokines expression of Th1/Th2 in asthmatic children and the modulatory effects of theophylline.Objective: (1) To induce and cultivate human monocyte-derived macrophages with rhGM-CSF in vitro .The cells was identified and the biological function was examined. (2) To observe the effects of monocyte-derived macrophages on intracellular cytokines expression of Th1/Th2 in asthmatic children and the modulatory effects of theophylline.Method: (1) Mononuclear cells isolated from human peripheral blood were incubated in RPMI1640 medium supplemented with 10% heat-inactivated FBS in the presence of rhGM-CSF for 7 days. The adherent cells were estimated to be macrophage according to morphology, phagocytosis of chicken erythrocytes and the expression of cell surface antigen. (2) The monocyte-derived macrophages of asthmatic children were cultured with or without aminophylline. After stimulated with antigen, macrophages and lymphocytes were cocultured for 3 days. The intracellular expressions of IFN-γand IL-4 in CD4+T cell were measured by flowcytometry, and the mRNA expressions of T-bet and GATA-3 were detected by RT-PCR.Result: (1)The adherent cells displayed the morphological characteristic and immunophenotype of macrophage, had biological function.(2) In contrast with own control group and antigen presentation group, the level of intracellular IL-4 and the expression of GATA-3 mRNA in lymphocytes decreased significantly(P<0.05), and the ratio of IFN-γ/IL-4 and T-bet/GATA-3 increased significantly(P<0.05) in aminophylline added group. There was no significant difference between own control group and antigen presentation group.Conclusion: (1) This method was simple and feasible for induction, cultivation and identification of human monocyte-derived macrophage. (2) Theophylline can effectively down regulation the mRNA expression of GATA-3, inhibit the secretion of IL-4, and regulate the balance of Th1/Th2 in asthmatic children. This effect may be mediated by macrophages.