To address the effect of LPS/TLR4-mediated activation of TLR3 in AMΦ on inflammatory cytokines, we assessed TNF-α and IL-6 in the serum and BALF, as well as the chemokines MIP-2 and MCP-1 in the BALF, following sequential intratracheal challenges with LPS and Poly I:C. In the ALI animal model, LPS was injected intratracheally at time 0, and Poly I:C was injected intratracheally at 4 h (the time point when TLR3 was up-regulated in AMΦ as described above). Enzyme-linked immunosorbent assays (ELISAs) were used to assess MIP-2 and MCP-1 in the BALF as well as IL-6 and TNF-α in both the BALF and serum at 8 h. LPS (Group 3) or Poly I:C (Group 2) alone induced a slight increase in MIP-2, MCP-1, IL-6, and TNF-α when compared with the saline control (Group 1) (Fig. 6), whereas sequential challenge with LPS and Poly I:C (Group 4) stimulated a marked increase in MIP-2, MCP-1, IL-6, and TNF-α expression. However, the sequential challenge with LPS and Poly I:C did not significantly increase MIP-2, MCP-1, IL-6, and TNF-α in TLR4−/− mice (Group 5) when compared to WT mice (Group 4). The sequential challenge with LPS and Poly I:C also led to a similar increase in TLR3−/− mice (Group 6) when compared to the LPS alone group (Group 3) (Fig. 6).