Immunobiology (2001) 203, pp. 629- 641 © 200 1 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol 1Institute for Medical Chemistry and Biochemistry, University of Innsbruck, and Boltzmann Institute for AIDS Research, Innsbruck, 2Institute for Experimental Physics, 3Medical Chemical Institute and Pregl Laboratory, University of Graz, Graz, 4Institute for Experimental Pathology, and 5Institute for Medical Biology and Human Genetics, University of Innsbruck, Innsbruck, Austria Induction of Apoptosis by 7,S-Oihydroneopterin: Involvement of Radical Formation BARBARA WIRLEITNER l , RAINER CZAPUTA 2 , KARL OETTL 3 , GUNTHER BacK 4 , BERNHARD WIDNERl, GILBERT REIBNEGGER 3 , GOTTFRIED BAIER 5 , DIETMAR FUCHS l , and GABRIELE BAIER-BITTERLICH l Received August 28, 2000 . Accepted in revised form February 28, 2001 Abstract Interferon-y is a cytokine released in large amounts during cell-mediated immune response. It induces the expression of proinflammatory cytokines and enhances macrophage capacity to secrete reactive oxy- gen intermediates and the pteridines neopterin and 7,8-dihydroneopterin. To assay the role of these pter- idines in the immune system several studies were performed. Thereby, 7,8-dihydroneopterin was found to induce apoptosis in T lymphocytes. In this study we report that caspases are involved in 7,8-dihydro- neopterin-mediated apoptosis in Jurkat T cells. In connection with this result we found that 7,8-dihydro- neopterin can increase Fas ligand expression detected in Western blot analysis and promoter reporter assays. Antioxidants potently reduced the effect of 7,S-dihydroneopterin on Fas ligand promoter activa- tion suggesting an involvement of oxidative stress. In further investigations, ESR-measurements were performed to evaluate the role of7,8-dihydroneopterin in the formation of radicals. We found that the pteridine in combination with the spin trap DMPO induces the production of DMPO-OH spin adducts. This reaction was sensitive to the presence of chelated metal ions and could completely be blocked by the addition of superoxide dismutase. These data suggest that 7,8-dihydroneopterin in aque- ous solution leads to the formation of'OH radicals via generation of superoxide anion. We hypothesize that an overproduction of radicals caused by high levels of 7,8-dihydroneopterin is likely to be respon- sible for the pro-apoptotic effects observed in cell cultures and possibly contributes to the pathogenesis of diseases involving immune activation and elevated concentrations of neopterin-derivatives. Introduction Elevated concentrations of interferon-y (IFN-y) associated with activated cell-mediated immune response lead to the expression of various proinflammatory cytokines, and enhance macrophage capacity to secrete reactive oxygen intermediates (1,2) as well as Abbreviations: DMPO = 5,5-dimethyl-1 pyrroline N-oxide; DTPA = diethylenetriamine pentaacetic acid; HB = Hepes-buffer; NAC = N-acetylcysteine; PDTC = pyrrolidine dithiocarbamate; ROI = reac- tive oxygen intermediate; SOD = superoxide dismutase. 0171-2985/011202/04-629 $ 15.00/0