Soluble components of the ECP product affect monocyte activation in vitro

Background

Extracorporeal photopheresis (ECP) is an effective therapy for various T-cell-mediated diseases. However, its mechanism of action remains largely unknown. Most studies focus on fate and function of cellular components of ECP product and prove their contribution to ECP efficacy, whereas the cell free fraction of the ECP product has not been thoroughly studied. This study further investigated the composition of soluble factors of the ECP product and their functional effect on monocytes in vitro.

Methods

Human peripheral blood mononuclear cells (PBMCs) of healthy blood donors were incubated with 200ng/ml 8-methoxypsoralen (8-MOP) for 30 min and treated with 2 J/cm2 UVA in vitro. PBMCs treated with either 8-MOP, UVA or left untreated served as controls. Up to 48 hours later, cell free cell culture supernatant (ECP-SN) was harvested. Concentrations of several cytokines within the ECP-SN were analysed using a cytometric bead-based immunoassay. To address the functional effect of the soluble ECP product on immune cells, PBMCs were exposed to ECP-SN alone for 12h, 24h and 48h or exposed to ECP-SN for 24h and then stimulated with R848 or INF-γ for 24h. Survival and activation marker expression on monocytes were analysed via flow cytometry.

Results

ECP treatment of PBMCs affects the cytokine milieu in cell culture supernatant over time compared to controls: Concentration of IL-6 and IL-1β were increased and MCP-1 concentration was decreased in ECP-SN compared to controls within 48h, whereas IL-8 IL-10 and IL-2 were not affected. Regarding the functional analysis of the ECP-SN, addition of ECP-SN to PBMCs induced increases cell death within 48 hours in non-monocytes. Moreover, ECP-SN activates monocytes reflected by increased CD38 and CD83 expression and further increases CD86 expression induced by R848 or INF-γ stimulation.

Conclusion

These data suggest that soluble factors may play a role in the mechanism of action of ECP in addition to cell-based effects described in the literature. Further studies exploring additional soluble factors within the ECP product, or investigating its functional effects on other cell types can contribute to a better understanding of the immunological effects induced by ECP therapy. Finally, this could help to improve ECP therapy and to expand its medical scope.

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