Effect of UVA and 8-methoxypsoralen, 4,6,4-trimethylangelicin or chlorpromazine on apoptosis of lymphocytes and their recognition by monocytes

Photopheresis (ECP) is an immunomodulatory therapy that involves extracorporeal exposure of isolated peripheral blood leukocytes to UVA irradiation in the presence of 8-methoxypsoralen (8-MOP) followed by their reinfusion to the patient. However, the underlying mechanism of ECP is not well understood yet. We selected 8-methoxypsoralen (8-MOP), chlorpromazine (CPZ) and 4,6,4'-trimethylangelicine (TMA) because of differences in their ability to induce immune suppression in rats in vivo. In this study, we investigated the role of UVA irradiation of lymphocytes in the presence of TMA, CPZ or 8-MOP on cell apoptosis, and their impact on adhesion of lymphocytes to monocytes in vitro. Apoptosis of lymphocytes and their sub-populations (lymphocytes T and B, NK cells) were determined by a flow cytometry, using AnnexinV-FITC, TUNEL assay and DNA content analysis and antibodies CD3, CD56, CD19. Mitochondrial potential was measured using CMXRos staining and the interaction of monocytes with lymphocytes was monitored by PKH26 Red Sigma staining of lymphocytes and subsequent use of flow cytometry. Our results show a significant increase of apoptosis of the photochemically treated lymphocytes and a decrease of their mitochondrial potential that depended on the dose and time after the treatment. Our data also reveal an increased recognition of apoptotic lymphocytes by freshly isolated monocytes.