IL-6 is a pleiotropic cytokine implicated in the pathogenesis of disorders such as sepsis and cancer. We noted that human monocytes are excellent producers of IL-6 as compared with monocyte-derived macrophages. Because macrophages from molecule containing ankyrin repeats induced by LPS (MAIL) knockout animals have suppressed IL-6 production, we hypothesized that regulation of MAIL is key to IL-6 production in humans and may explain the differences between human monocytes and macrophages. To test this hypothesis fresh human monocytes and monocyte-derived macrophages were compared for MAIL expression in response to LPS. LPS-induced monocyte MAIL expression was highly inducible and transient. Importantly for our hypothesis MAIL protein expression was suppressed during differentiation of monocytes to macrophages. Of note, the human MAIL protein detected was the 80 kDa MAIL-L form and human MAIL showed nuclear localization. Human MAIL-L bound to p50 subunit of the NF-kappaB and increased IL-6 luciferase promoter activity in a cEBPbeta, NF-kappaB, and AP-1-dependent fashion. Like the differences in MAIL induction, monocytes produced 6-fold more IL-6 compared with macrophages (81.7 +/- 29.7 vs 12.6 +/- 6.8 ng/ml). Furthermore, suppression of MAIL by small interfering RNA decreased the production of IL-6 significantly in both THP-1 cells and in primary monocytes. Costimulation of monocytes with LPS and muramyl dipeptide induced an enhanced IL-6 response that was suppressed by siMAIL. Our data suggests that MAIL is a key regulator of IL-6 production in human monocytes and plays an important role in both TLR and NOD-like receptor ligand induced inflammation.