Ethanol suppresses LPS-induced Toll-like receptor 4 clustering, reorganization of the actin cytoskeleton, and associated TNF-α production

Ethanol (EtOH) suppresses cytokine responses induced through most Toll-like receptors (TLRs), but the mechanism of action is unclear. We recently found that acute EtOH alters lipopolysaccharide (LPS)-induced partitioning of CD14, a critical component of the LPS receptor complex, within lipid raft fractions in the macrophage-like cell line RAW264.7. Here we investigated the role of receptor clustering in alteration of the responses of cells to LPS caused by EtOH both in vitro and in vivo. The cellular distribution of CD14, TLR4, actin cytoskeleton, and tumor necrosis factor-alpha (TNF-alpha) were studied by confocal microscopy following exposure of cells to LPS with or without EtOH. TLR4 and CD14 were clustered into highly colocalized patches on the cell membrane accompanied by the reorganization of the actin cytoskeleton in some of the RAW264.7 cells as well as peritoneal cells following LPS treatment. Addition of EtOH reduced the number of cells that had LPS-induced receptor patches and in which this reorganization occurred. Cells on which CD14 and TLR4 formed clusters or caps had substantially higher levels of membrane-bound TNF-alpha compared with cells without clustering or capping of these molecules. Interference with the actin cytoskeleton by cytochalasin D suppressed the production of TNF-alpha and receptor clustering, as EtOH did. These data confirm our previous observations, suggest a novel mechanism of EtOH action that involves interference with receptor clustering, and indicate a potential role of actin filaments in the formation of receptor patches, subsequent activation of macrophages by LPS, and production of TNF-alpha.