Because chemokine-dependent PMN migration and inflammatory cytokine secretion are important determinants of ALI, we next addressed the role of LPS/TLR4-mediated up-regulation of TLR3 in alveolar-capillary permeability using Evan’s blue, which binds albumin, in WT, TLR4−/−, and TLR3−/− mice. We assessed permeability at 4, 6, 8, and 10 h after LPS/Poly I:C administration. As shown in Fig. 7A, at the 8 h time point, either LPS (Group 3) or Poly I:C (Group 2) alone caused a slight increase in permeability in WT mice when compared to the saline (SAL) control (Group 1). In contrast, LPS/Poly I:C (Group 4) caused a further increase in alveolar-capillary permeability in WT mice when compared with the LPS/SAL group. In TLR4−/− mice (Group 5), alveolar-capillary permeability was markedly attenuated in response to LPS/Poly I:C challenge compared with WT mice (Group 4). The alveolar-capillary permeability was similar to the results observed for LPS alone (Group 3) when TLR3−/− mice were used (Group 6). As shown in Fig. 7B, the histopathology of lung tissues was also assessed 8 h following LPS/Poly I:C administration. Injection of LPS at time 0 followed by a saline injection (Group 3) at 4 h caused marked changes in lung histopathology at 8 h. A saline injection at time 0 and a Poly I:C injection (5 μg/g body weight, intratracheally administered) at 4 h resulted in minimal changes (Group 2). However, the sequential injection of LPS at time 0 and Poly I:C injection at 4 h (at a time when TLR3 was up-regulated) in WT mice (Group 4) led to augmented changes in histopathology, including diffuse interstitial edema and inflammatory cell infiltration, compared with the single LPS or Poly I:C challenge. In contrast, the sequential injection of Poly I:C at 4 h after LPS failed to induce augmented histopathology changes in TLR4−/− mice (Group 5) compared with WT mice. In addition, we did not observe augmented histopathology changes in TLR3 mice (Group 6) and instead observed a change similar to that observed for LPS alone (Group 3). Taken together, these data show the important role of LPS/TLR4 signaling in the up-regulation of TLR3 expression in ALI.