Assessment of 8-methoxypsoralen and ultraviolet a light effects on human stroma generation and function.

Photopheresis or extracorporeal photochemotherapy (ECP) is a new immunomodulatory therapy in which a patient's leukocytes are exposed extracorporeally to 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) light. Although it is used for the treatment of cutaneous T cell lymphoma, graft-versus-host disease, and several autoimmune diseases, with efficacy and safety reported in almost all studies, the mechanisms by which ECP exerts its beneficial effects are still unclear. As cellular targets of this procedure are numerous, we investigated the effects of 8-MOP and UVA light on stromal precursors and mature stromal layers. Human bone marrow stromal cell layers were established in long-term bone marrow culture medium from normal marrow mononuclear cells. Normal marrow mononuclear cells were incubated with 8-MOP and/or exposed to UVA light (PUVA treatment) before culturing. A control without 8-MOP and UVA was also included in the study. Apoptosis induction was evaluated using annexin V following 7 days after PUVA. After 4-6 weeks of culture, stromal layers were examined under a phase-contrast microscope to identify structural differences between PUVA-treated and control stroma. To determine whether PUVA treatment affected stromal regulation of adherent hematopoietic cell survival, mature stromal layers, incubated with 8-MOP and exposed to UVA light, were cocultured with nonadherent mononuclear cells from normal marrow. After 24 h, the percentage of apoptotic hematopoietic cell precursors was quantified by flow cytometry. This study provides evidences that the in vitro exposure of human stromal cell precursors to UVA light, in the presence of 8-MOP, inhibits stromal layer generation by inducing apoptosis, as evidenced by annexin V staining following 7 days of culture. Here, we show an additional cell target for this psoralen following UVA irradiation. However, in a second set of experiments, PUVA treatment did not affect the stromal capacity to support hematopoiesis in culture. Our results can contribute to a better definition of ECP mechanisms of action for future development of experimental designs and clinical applications of this intriguing procedure.