Macrophages produce TNFalpha when infected by bacteria, a response that follows recognition of microbial components by members of the Toll-like receptor (TLR) family. Cells that lack functional TLR4 are known to have markedly diminished responses to Gram-negative lipopolysaccharide. We demonstrate in the present work that peritoneal macrophages derived from strains of mice that carry a spontaneous, inactivating mutation in TLR4 also have impaired production of TNFalpha in response to peptidoglycan, a ligand for TLR2. This impairment is at a step of biosynthesis subsequent to the generation of mRNA. TLR4-activated signals act at this step to enhance peptidoglycan-induced TNFa production in wild-type mice. Based on these observations, we conclude that macrophages from wild-type mice are primed by chronically acting TLR4 signals, probably resulting from exposure to environmental lipopolysaccharide. These signals are required for optimal production of TNFalpha in response to TLR2 stimulation, and are absent in macrophages from TLR4 mutant animals. (C) 2004 Elsevier Inc. All rights reserved.