Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

210

166

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G1 to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.

Section: Blockade Of Nucleotide Synthesis Inhibits Proliferation Of Pmentioning

confidence: 99%

Differential Control of Cell Cycle, Proliferation, and Survival of Primary T Lymphocytes by Purine and Pyrimidine Nucleotides

Quéméneur

Gerland

Flacher

et al. 2003

210

166

“…The antiproliferative effect of mycophenolate acid on T cells was clearly demonstrated in vitro (7,8,12,13). Methotrexate (MTX) is a folate antagonist that blocks dihydrofolate reductase and TS (14), but it can also block the first step of purine biosynthesis (15). We have previously shown that in primary T cells, MTX inhibits T cell division by specifically depleting thymidine nucleotides (8,16).…”

Section: Restriction Of De Novo Nucleotide Biosynthesis Interferes Wimentioning

confidence: 99%

Restriction of De Novo Nucleotide Biosynthesis Interferes with Clonal Expansion and Differentiation into Effector and Memory CD8 T Cells

Quéméneur

Beloeil

Michallet

et al. 2004

Nucleotide synthesis inhibitors are currently used in neoplastic diseases or as immunosuppressive agents for the prevention of acute rejection in organ transplantation and the treatment of autoimmune disorders. We have previously described that these inhibitors interfere with proliferation and survival of primary T cells in vitro. However, the precise effects of nucleotide restriction on effector and memory functions have not been elucidated. In this study, we investigated the impact of nucleotide synthesis inhibition on CD8 T cell differentiation by using TCR transgenic mice (F5) specific for the influenza virus nucleoprotein 68 peptide presented on the H-2Db molecule. Our results show that methotrexate and 5-fluorouracil prevent the acquisition of effector functions, such as IFN-γ, granzyme B expression, and cytotoxic function following antigenic stimulation of naive cells. Surprisingly, in the presence of mycophenolate mofetil, activated F5 cells are still able to produce granzyme B and to kill target cells but to a lesser extent compared with control. All three inhibitors interfere with the differentiation of naive cells into memory CD8 T cells. In contrast, the drugs are unable to inhibit the development of improved cytotoxic functions displayed by memory CD8 T cells.

“…The mechanism by which MTX mediates its immunosuppressive and an anti-inflammatory effects is still elusive. Apoptosis and clonal deletion of activated peripheral T cells (10), suppression of purine biosynthesis in mitogen-stimulated T cells (11), down-regulation of inflammatory cytokines (12)(13)(14), adenosine release (15,16), modulation of release of metalloproteases, and expression of cell surface adhesion molecules (17) have been used to explain the effects of MTX in RA.…”

mentioning

confidence: 99%

Methotrexate Suppresses NF-κB Activation Through Inhibition of IκBα Phosphorylation and Degradation

Majumdar

Aggarwal

2001

173

119

Methotrexate (MTX), a folate antagonist, is a commonly used anti-inflammatory, antiproliferative, and immunosuppressive drug whose mode of action is not fully established. Due to the central role of NF-κB in these responses, we postulated that MTX must mediate its effects through suppression of NF-κB activation. We investigated the effects of MTX on NF-κB activation induced by TNF in Jurkat cells. The treatment of these cells with MTX suppressed TNF-induced NF-κB activation with optimum effects occurring at 10 μM MTX for 60 min. These effects were not restricted to Jurkat cells because other cell types were also inhibited. Besides TNF, MTX also suppressed the NF-κB activation induced by various other inflammatory stimuli. The suppression of TNF-induced NF-κB activation by MTX correlated with inhibition of IκBα degradation, suppression of IκBα phosphorylation, abrogation of IκBα kinase activation, and inhibition of NF-κB-dependent reporter gene expression. Because ecto 5′ nucleotidase inhibitor (α,β-methylene adenosine-5′-diphosphate) blocked the effect of MTX, adenosine mimicked the effect of MTX, and adenosine A2b receptor antagonist (3,7-dimethyl-1-propargylxanthine) reversed the inhibitory effect of MTX, we suggest that MTX suppresses NF-κB activation by releasing adenosine. A partial reversal of MTX-induced NF-κB suppression by thymidine and folinic acid indicates the role of the thymidylate synthase pathway also. Overall, our results clearly demonstrate that MTX suppresses NF-κB activation through the release of adenosine, which may contribute to the role of MTX in anti-inflammatory, immunomodulatory, and antiproliferative effects.