THE ANTILYMPHOCYTIC ACTIVITY OF BREQUINAR SODIUM AND ITS POTENTIATIONBY CYTIDINE - EFFECTS ON LYMPHOCYTE-PROLIFERATION AND CYTOKINE PRODUCTION

Based on its capacity to inhibit de novo pyrimidine biosynthesis by bl ocking dihydroorotate dehydrogenase activity, the antitumor agent breq uinar sodium (BQR) has emerged as a new immunosuppressive agent. Since BQR is known to prevent the synthesis of nucleotides during cell prol iferation, we hypothesized that it would be highly effective in contro lling strong lymphocyte proliferative responses but might be less effe ctive in controlling comparatively weak responses that do not necessar ily involve new nucleotide synthesis. We addressed this question by cu lturing murine spleen cells with different types of stimuli, including Con A, phorbol myristate acetate +/- ionomycin, anti-CD3, and anti-Ig s.Addition of BQR (0.001 mug/ml to 10 mug/ml) at the start of a 72-hr culture period caused dose-dependent inhibition of strong proliferativ e responses, induced either by Con A (5 mug/ml) or PMA + ionomycin. A residual degree of proliferation persisted, however, even at the highe st BQR concentrations. In contrast, no impairment of low-concentration Con A (0.5 or 0.1 mug/ml), anti-CD3, or anti-Igs responses was observ ed. In order to ascertain its role in arresting nucleotide synthesis, we attempted to reverse the inhibitory effect of BQR by adding exogeno us uridine or cytidine to lymphocyte cultures. BQR's inhibitory activi ty was reversed completely by adding uridine at 0.1 mM. In contrast, c ombination of BQR and cytidine (0.1 mM) potentiated BQR's activity and abrogated anti-CD3 or anti-Igs-induced lymphocyte proliferation in a dose-dependent manner. A synergistic inhibitory action between BQR and cytidine was observed when the BQR concentration was higher than 0.1 mug/ml and with cytidine at 0.1 mM. Production of interleukin-2 and IL -4 was only slightly affected by BQR, but was significantly suppressed by coadministration of BQR and cytidine. Neither BQR (5 mug/ml) on it s own, however, nor combination of BQR with cytidine affected producti on of mRNA for IL-2, IL-4, or interferon-gamma, as determined by rever se-transcription polymerase chain reaction. Our observations suggest t hat BQR may not only affect dihydroorotate dehydrogenase activity, but may also inhibit the enzyme cytidine deaminase, which converts cytidi ne to uridine. These antimetabolic effects of BQR complement the well- known cytokine synthesis inhibitory actions of FK506 or CsA. The combi nation of BQR and cytidine, however, offers a further possibility for inhibition of both cytokine production and T and B cell proliferation, and may have potential for the control of graft rejection.