Effects of Photopheresis on Mononuclear Cells from Healthy Individuals.

Background: Extracorporeal photopheresis (ECP) has been proposed as an alternative therapy for immune-mediated disease including graft-versus-host-disease (GVHD) and transplant rejection (Photopheresis for the treatment of refractory renal graft rejection, Transplantation2005;79(1):123–5, Kumlien G et al). The mechanisms of photopheresis are still poorly understood but induction of tolerogenic dendritic cells and regulatory T cells as well as a shift of cytokine profile from Th1 to Th2 have been observed in patients treated with ECP. We wanted to investigate some basic effects of ECP on mononuclear cells from healthy individuals unaffected by pathogenic mechanisms and immunosuppressive drugs. Method: Mononuclear cells (MNCs) were collected from six healthy volunteer apheresis donors using CobeSpectra apheresis machine (Gambro BCT, Lakewood, CO, USA) and peripheral blood was drawn immediately before apheresis. Apheresis products were thoroughly mixed and divided into three equal parts. One part was left untreated, one part was irradiated with UVA 365 nm, 2J/cm2 (BioGenic, Vilber Lourmat, Marne-la-Vallée, France) and one part was treated with psoralen (8-methoxypsoralen 200 ng/ml) plus UVA-irradiation (PUVA). Peripheral MNCs and aliquots of apheresis MNCs were analysed simultaneously regarding incorporation of 3H Thymidine after stimulation with mitogens (PHA, ConA, SpA) and production of cytokines correlated with GVHD pathogenesis (IL-2, IL-6, IL-12, TNFα, IFNγ) after stimulation with antigens (BG, PHA, LPS) using commercial ELISA-kits (Quantikine, R&D Systems, Minneapolis, MN, USA). Statitical analysis was performed using a nonparametric method (one-sided ANOVA). Results: Mitogenic capacity was reduced to baselevel after PUVA-treatment but not affected by the apheresis procedure or UVA-irradiation alone. Table 1. Statistically significant decrease of cytokine production was seen only after PUVA-treatment but production of IL-2 and IL-6 was reduced after both apheresis and UVA-irradiation alone. Small amounts of IL-2, IL-6, TNFα and IFNγ were produced even after PUVA-treatment. Conclusion: Significant reduction of mitogenic capacity only after PUVA-treatment was the expected finding. Apheresis can theoretically effect cells through mechanic damage (centrifugation) and/or interaction with apheresis harness and UVA is known to have immunomodulatory effects which can explain the reduction of cytokine production. Cytokines detected after PUVA-treatment may be explained by release from apoptotic cells or by production in cells that escape apoptosis. Lymphocyte proliferation assay ConA PHA SpA MNCs = mononuclear cells peripheral vs apheresis MNCs ns ns ns peripheral vs UVA-irradiated MNCs ns ns ns apheresis vs UVA-irradiated MNCs ns ns ns peripheral vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05 apheresis vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05 UVA -irradiated vs PUVA-treated MNCs p<0.05 p<0.05 p<0.05