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Human PBMC of healthy blood donors were used to investigate the interaction of cytokines in human MLC. Two-way MLC was performed because irradiation did not influence the cytokine release. We found different kinetic patterns for IL-2, IFN-gamma, and sIL-2R. Production of IFN-gamma was dependent on IL-2 release because anti-IL-2 addition resulted in more than 90% reduced IFN-gamma levels. Treatment with rIL-2 altered IFN-gamma kinetics, but not the total amount of IFN-gamma. Addition of anti-IFN-gamma led to decreased production of IL-2 and sIL-2R. A down-regulation of IL-2 and sIL-2R could also be observed after treatment with rIFN-gamma. No production of IL-4 and IL-10 was detected in MLC (detection limit 5 pg/ml). This could not be explained by IFN-gamma antagonism because IL-4 and IL-10 were not detectable even after addition of anti-IFN-gamma. Testing the TH1-TH2 cell antagonism, the addition of rIL-4 and rIL-10 resulted in IFN-gamma suppression depending on the timing of exposition. Treatment with rIL-10 inhibited IL-2 and sIL-2R release. We found no production of IL-1 alpha and IL-1 beta in MLC, whereas IL-6 and TNF-alpha release could be detected. Surprisingly, release of IL-6 and TNF-alpha could be blocked completely by addition of anti-IFN-gamma. This suggests that the release of IL-6 and TNF-alpha in MLC is dependent on IFN-gamma produced by T cells. In summary, TH1 cytokines play a central role in MLC regulation, whereas TH2 cytokines appear to be of little importance.