Abstract

Tumor necrosis factor-alpha (TNF-alpha) 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) 1 beta and IL-6. Activation of murine bone marrow-derived macrophages (BMDM phi) with lipopolysaccharide (LPS) causes rapid expression of TNF-alpha, which as an autocrine factor enhances BMDM phi function through IL-1 beta and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDM phi TNF-alpha using novel enantiomeric carbocyclic nucleoside analogues. BMDM phi were derived in vitro from murine 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 microM) 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-alpha. MDL 201,449 reduced TNF-alpha 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-1 beta (measured by the D10.S bioassay) and mRNA for TNF-alpha receptor p60 and TNF-alpha receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDM phi up-to 2 hr after LPS treatment and at concentrations as low as 10 microM. Regulation of BMDM phi 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 microM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that M phi-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-alpha may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-alpha has been shown to have an important role in initiating the early stages of disease.