Hemorrhagic shock renders patients susceptible to the development of acute lung injury in response to a second inflammatory stimulus, by as yet unclear mechanisms. We investigated the role of neutrophils (PMN) in alveolar macrophages (AM) priming, specifically, the role in mediating Toll-like receptor (TLR)4 and TLR2 cross-talk in AM. Using a mouse model of hemorrhagic shock followed by intratracheal administration of LPS, we explored a novel function of shock-activated PMN in the mechanism of TLR2 upregulation induced by LPS-TLR4 signaling in AM. We showed that antecedent hemorrhagic shock enhanced LPS-induced TLR2 upregulation in AM. In neutropenic mice subjected to shock, the LPS-induced TLR2 expression was significantly reduced, and the response was restored upon repletion with PMN obtained from shock-resuscitated mice but not by PMN from sham-operated mice. These findings were recapitulated in mouse AM co-cultured with PMN. The enhanced TLR2 upregulation in AM augmented the expression of macrophage inflammatory protein-2 (MIP-2), TNF, and macrophage migration inhibitory factor (MIF) in the AM in response to sequential challenges of LPS and peptidoglycan, a prototypical TLR2 ligand, and which physiologically associated with amplified AM-induced PMN migration into air pouch and lung alveoli. Thus TLR2 expression in AM, signaled by TLR4 and regulated by shock-activated PMN, is an important positive-feedback mechanism responsible for shock-primed PMN infiltration into the lung following primary PMN sequestration.