Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay

Malononitrilamides (MNAs) are a new class of immunomodulatory drug highly e ffective in in vivo models of allo- and xenotransplantation. Knowledge of t heir effects on immune cells, however, is limited and has been derived sole ly from investigations using isolated mononuclear cells. This use of purifi ed cells to investigate drug activity is not ideal, so we have combined the analytical power of flow cytometry with our mitogen-driven, whole blood ly mphocyte activation and proliferation assays to investigate the in vitro me chanism of action of MNAs. We first show that MNAs (A77 1726, HMR1279, and HMR1715), as well as brequinar (BQR) and cyclosporine (CsA), effectively in hibit cell activation antigen expression and lymphocyte proliferation. We n ext show that the inhibitory effects of MNAs and BQR, but not CsA, are reve rsed by the addition of uridine to the culture. These results suggest that inhibition of pyrimidine biosynthesis may be a mechanism by which MNAs supp ress both lymphocyte activation and proliferation since these effects were reversed when uridine nucleotide pools were replenished. This novel finding of suppression of activation antigen expression by MNAs in whole blood exp ands our understanding of the effects of this new class of drug. (C) 1999 E lsevier Science B.V. All rights reserved.