It has been proposed that the therapeutic effect of PUVA (psoralens+UVA radiation) is connected to its immunomodulative properties, and that the molecular basis of such properties is the oxygen-independent photoaddition of psoralens to DNA. We have investigated effects of preliminary photooxidized psoralens (POP) on the delayed-type hypersensitivity reaction (DTH) to sheep red blood cells and on growth of grafted T-cell lymphoma EL-4 in mice. We have shown that intravenous injection of POP at low concentrations activated, and at high concentrations suppressed, DTH. The POP products are thermolabile. They preserved their immunosuppressive activity for 3 days at room temperature and lost it in several min at 58 degrees C. Incubation of POP in the presence of Fe2+ during 2 h before intravenous injection leads to complete loss of its immunomodulative activity, suggesting a peroxidic nature of POP products. The POP-inhibited growth of grafted T-cell lymphoma independent of the mode of POP application in mice (intravenous or subcutaneous injections, oral or nasal administration). Our data suggest that photooxidative reactions of psoralens, in addition to oxygen-independent photoaddition to DNA, form the basis for biological activity of these drugs.

ConclusionsOur data show that IFN-gÀ/À mice are resistant to the development of AA, pointing toward an important role of IFN-g in the pathogenesis of AA. The absence of a perifollicular lymphocytic infiltrate in the skin and the reduced expression of T cell activation markers and costimulatory molecules in skin-draining lymph nodes demonstrate that T cells are not activated in the lymph nodes of IFN-gÀ/À mice. Therefore, we conclude that IFN-g is involved in T cell activation in AA pathogenesis of C3H/ HeJ mice.A pathogenetic role for IL-2 in AA could also be demonstrated, but the cogency of our data is limited because only heterozygous IL-2 þ /À mice were available for these experiments. But Hollä nder et al (Science 1998) demonstrated that IL-2 expression is reduced 50% in IL-2 þ /À mice compared with IL-2 þ / þ mice. Our data show that the frequency of AA development is significantly reduced in IL-2 þ /À mice. Therefore, we conclude that the magnitude of IL-2 expression influences the frequency of AA development. FACS analysis and immunohistochemistry show that AA resistance of half of the IL-2 þ /À mice is related to a reduced T cell activation in the lymph nodes and an absence of perifollicular infiltrates of CD4 þ and CD8 þ cells in the skin. These data suggest that AA development in the C3H/HeJ mouse model depends on the level of IL-2 expression and on subsequent T cell activation in the lymph nodes.Taken together, our results show that the type-1 cytokines IFN-g and IL-2 are involved in T cell activation in the development of AA in the C3H/HeJ mouse model. Methods and ResultsAfter experimental induction of AA by skin grafting, 90% of normal C3H/HeJ mice (control) developed AA, whereas none of the IFN-gÀ/À mice and only 47% of IL-2 þ /À mice developed AA.Immunohistochemistry of skin sections showed dense peri-and intrafollicular infiltrates of CD4 þ and CD8 þ cells in control mice with AA, whereas perifollicular infiltrates were absent in IFN-gÀ/À and IL-2 þ /À mice without AA.FACS analysis of skin-draining lymph node cells and skininfiltrating leukocytes of IFN-gÀ/À mice revealed a significant reduction of the T cell activation markers and of co-stimulatory molecules. In IL-2 þ /À mice the expansion of CD4 þ T cells was reduced and the upregulation of activation markers and costimulatory molecules was impaired.

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