The Influence Of Nuclear Factor Kappa B To The Subsets Of T Lymphocytes Of Asthma

OBJECTIVE: To set up the technique of detection of intracellular cytokines by three-color flow cytometry and investigate the role of nuclear factor-KB in the signal pathway of regulating the expression of Th1/Th2 cytokines, interferon gamma and interleukin 4, by T lymphocyte in asthma.METHODS: 1. Cytokines in human T lymphocytes were assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure after stimulated withphorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. The effects of choosing different monoclonal antibodies and stimulating time to the result were explored. 2. The blood samples were taken from 20 asthmatic patients and 20 normal adults. The blood samples taken from normal adults were stimulated with PMA. The peripheral blood cells taken from asthmatic patients were divided into 2 groups: cells of 1st group were stimulated with PMA, cells of 2nd group were stimulated with PMA and pyrrolidine dithiocarbamate (PDTC). The intracellular cytokines were detected by three-color flow cytometry.RESULTS: 1. The time of stimulation and different monoclonal antibodies have great effects on the results. 2. The asthmatic subsets of Th1, Th2 and Th1/Th2 which stimulated with PMA were different with that stimulated with PMA and PDTC simultaneously and the normal adults. The asthmatic subset of Th1 and Th1/Th2 were lower than the normal adults but Th2 was higher than the normal adults. The asthmatic subset of Th1 and Th1/Th2 stimulated with PMA and PDTC were higher than those stimulated with PMA, but Th2 was lower.CONCLUSION: 1. Flow cytometry is a good tool to detect the intracellular cytokines at the single cell level. 2. There are many influence factors in the method.It is noted to select the appropriate conditions at the analysis. 3. The active of asthmatic T lymphocyte changing the expression of interferon gamma and interleukin 4 may be mediated by activating NF-kB, the activation of NF-kB signal pathway of T lymphocyte may play an important role in the pathogenesis of asthma.