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Measurement of cytokines in the mixed lymphocyte culture (MLC) is thought to be a new and relevant parameter for bone marrow transplantation (BMT). Our experiments showed that IFN-gamma plays a central role in the cytokine network following alloantigenic recognition. IFN-gamma itself is induced by IL-2 since anti-IL-2 strongly reduced the secretion of IFN-gamma. As anti-IFN-gamma also diminished the response of IL-2 and sIL-2R, a feedback mechanism between these two cytokines is assumed. Addition of rIFN-gamma to the MLC augmented the release of sCD8 molecules, whereas sCD4 molecules were reduced, indicating that IFN-gamma led to T cell differentiation instead of IL-2 dependent proliferation. In the MLC, a feedback mechanism between TNF-alpha and IFN-gamma exists, since anti-TNF-gamma reduced the secretion of IFN-gamma and anti-IFN-gamma inhibited the release of TNF-alpha. Therefore, IFN-gamma plays a critical role in monocyte activation, T cell differentiation, and IL-2-induced cell growth. We conclude that measurement of IFN-gamma might be a new and more sensitive parameter for BMT than the established proliferation assay, since IFN-gamma directly quantifies T cell activation.