The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14 C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic- 14 C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 M Leflunomide. 100 M Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidinerelated effects reported for Leflunomide at 100 M and above.

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies.

Sensitive high performance liquid chromatography techniques, which differentiate between purine and pyrimidine ribonucleoside and deoxyribonucleoside triphosphates, were used to quantify pools in phytohemagglutinin-stimulated T-lymphocytes (98% CD4+ and CD8+) from healthy volunteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with 14C-radiolabeled precursors (40 microM), azaserine (20 microM; a glutamine antagonist), and ribavirin (50 microM; an IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocytes meet their metabolic requirements by salvage. Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h. These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.

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