Specific transcriptional inhibition of bone marrow-derived macrophage tumor necrosis factor-α gene expression and protein production using novel enantiomeric carbocyclic nucleoside analogues

Tumor necrosis factor-α (TNF-α) is a powerful macrophage-derived proinflammatory cytokine, via both direct effects on host tissues as well as indirectly through the induction of other proinflammatory mediators, including interleukin- (IL) 1β and IL-6. Activation of murine bone marrow derived macrophages (BMDMφ) with lipopolysaccharide (LPS) causes rapid expression of TNF-α, which as an autocrine factor enhances BMDMφ function through IL-1β and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDMφ TNF-α using novel enantiomeric carbocyclic nucleoside analogues, BMDMφ were derived in vitro from routine bone marrow progenitors using colony stimulating factor 1 and treated with combinations of LPS (1-100 nG/ml) and the enantiomeric carbocyclic nucleoside (10-100 μM) analogues MDL 201, 112 (9-[(1S,3R)-cis-cyclopentan-3- ol]adenine); MDL 201,451 (9-[1R,3S)-cis-cyclopentan-3-ol]adenine); MDL 201,449 (9-[(1R,3R) trans cyclopentan-3-ol]adenine) and MDL 201,484 (9- [(1S,3S)-trans cyclopentan-3-ol]adenine). Northern blot analysis showed that MDL 201,449 was the most effective agent in vitro at selectively inhibiting TNF-α. MDL 201,449 reduced TNF-α mRNA levels by nearly 50% for up to 4 hr after the simultaneous addition of LPS and the synthetic agent. In contrast, mRNA and secreted protein levels for IL-1β (measured by the D10.S bioassay) and mRNA for TNF-α receptor p60 and TNF-α receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDMφup-to 2 hr after LPS treatment and at concentrations as low as 10 μM. Regulation of BMDMφ IL-6 by carbocyclic nucleoside analogues in response to LPS appears to be both concentration and time dependent, because IL-6 mRNA and secreted protein levels were inhibited at only high drug concentrations (100 μM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that Mφ-derived proinflammatory cytokine gene expression is differentially, rather than coordinately, regulated by selective signal transduction and/or molecular pathways. Enantiomeric carbocyclic nucleoside analogues that specifically inhibit TNF-α may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-α has been shown to have an important role in initiating the early stages of disease.