In the present study, using both an in vivo ALI mouse model and the in vitro culture of AMΦ from TLR4−/− and MyD88−/− mice, we demonstrate that LPS up-regulation of TLR3 in AMΦ is dependent on TLR4-MyD88-NF-κB signaling. The functional relevance of the amplification in TLR4-induced TLR3 expression in AMΦ was demonstrated by a marked increase in the expression of the chemokines and cytokines, including macrophage inflammatory protein-2 (MIP-2), macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), in the ALI mouse model. Furthermore, we observed alveolar-capillary permeability and the induction of polymorphonuclear leukocyte (PMN) migration in response to sequential challenges with LPS and Poly I:C. Thus, TLR3 up-regulation in AMΦ is dependent on TLR4-MyD88-NF-κB signaling, which may represent an important mechanism responsible for amplifying PMN migration.