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The effects of cytokine and mitogen stimulation on homocysteine (HCY) metabolism in different cells were investigated: in human dermal microvascular endothelial cells (HDMEC), T lymphocytes, mature and immature dendritic cells, and myelomonocytic (THP-1) and monocytic cell lines (U-937). Furthermore, the influence of supplementation of cells with folate acid, methionine and the combination of both on HCY metabolism was investigated. Unstimulated HDMEC and dendritic cells only produced very little amounts of HCY, and stimulation did not change HCY formation significantly either. However, higher HCY concentrations were detected in HDMEC and dendritic cells under supplementation with methionine and slightly less under supplementation with folate. Proliferating T lymphocytes showed an increase in HCY production on stimulation with increasing doses of mitogens; proliferative activity was associated with HCY formation. THP-1 and U-937 cells produced significantly more HCY than endothelial cells; U-937 cells produced most HCY, which was mainly due to their high proliferation rate. Stimulation of both cell lines with lipopolysaccharide and interferon-γ, respectively, showed a significant effect on HCY production of cells; in THP-1 cells, stimulation with IFN-γ and lipopolysaccharide induced neopterin formation. Methionine supplementation strongly increased and folate supplementation slightly decreased HCY formation in both cell lines. Thus, inflammation may play a role in moderate hyperhomocysteinemia.