EFFECT OF 4-AMINO TETRAHYDROBIOPTERIN ON SURFACE MARKER EXPRESSION AND T-CELL STIMULATORY CAPACITY OF MURINE BONE MARROW DERIVED DENDRITIC CELLS.

Abstract

P1024 Aims: The purpose of the present study was to investigate effects of 4-amino tetrahydrobiopterin on dendritic cells in order to identify a potential immunosuppressive mechanism, with particular emphasis on a role of inducible nitric oxide (NO) synthase (iNOS). 4-amino tetrahydrobiopterin is a tetrahydrobiopterin-competitive inhibitor of NO-synthases, which in a murine cardiac allograft model prolongued allograft survival comparable to cyclosporin-A treatment. Methods: We used bone-marrow derived dendritic cells of wild-type and iNOS(−/−) C57/BL10 mice. Surface antigen expression was determinend by FACS analysis. To assess T-cell stimulatory capacity, dendritic cells were incubated for 48 h with and without LPS (10 ng/ml) and with and without pteridines (50 μM - 500 μM) or the iNOS inhibitor N-iminoethyl-L-lysine (NIL, 500μM). After washing, varying amounts of dendritic cells were cocultivated with periodate-treated T-cells of the same strain for another 48 h. T-cell proliferation was then assessed by 3H-thymidine incorporation. In a separate set of experiments, LPS and pteridines were added simultaneously with periodate-treated T-cells to the dendritic cells, and proliferation was determined as described above. Apopotosis was measured by annexin V and propidium iodide staining. Nitrite and nitrate released into the supernatants was assessed by reversed phase HPLC, with cadmium reduction of nitrate and post-column mixing with the Griess reagent. Results: Dendritic cells produced up to 100 μM nitrite and nitrate upon LPS treatment, which was inhibited to baseline levels by NIL. LPS also induced apoptosis in wild type dendritic cells, which was blocked by NIL. In iNOS(−/−) dendritic cells, however, apoptosis was independent of LPS and NIL treatment. Pteridines had little effects on NO formation and apoptosis of dendritic cells. Among 6 surface markers, only 4-amino tetrahydrobiopterin, but not the other agents, downregulated MHC II and attenuated LPS-induced decrease of ICOS-ligand surface expression in both wild-type and iNOS(−/−) dendritic cells and led to a significantly decreased T-cell stimulatory capacity. When added together with dendritic cells to T-cells, 4-amino tetrahydrobiopterin and tetrahydrobiopterin caused apoptosis of T-cells. Catalase inhibited the apoptotic action of tetrahydrobiopterin, but not of 4-amino tetrahydrobiopterin. Conclusions: These results suggest the following immunosuppressive mechanism of 4-amino tetrahydrobiopterin: When present during dendritic cell maturation, it suppresses MHC-II expression and attenuates downregulation of ICOS-ligand. This results in lower T-cell stimulatory capacity and thus in an attenuated immune response. In addition, 4-amino tetrahydrobiopterin causes apoptosis of T-cells, which is independent of hydrogen peroxide formation during autooxidation of tetrahydropteridines. Our results with the iNOS inhibitor NIL and with cells from iNOS(−/−) mice clearly indicate that these mechanisms are independent of iNOS and hence describe a novel immunosuppressive mechanism of tetrahydropteridines.