To address the physiological relevance of LPS/TLR4 activation and TLR3 expression in AMΦ, we assessed MIP-2 expression in the lungs using sequential challenges of LPS and Poly I:C. Injection of LPS at time 0 followed by a saline injection (Group 3) at 4 h resulted in a marked increase in MIP-2 expression by 6 h after LPS challenge, which was further increased at 8 h and then returned to the basal level by 10 h after LPS challenge (Fig. 4A). Saline injection at time 0 and Poly I:C injection (5 μg/g body weight, intratracheally administered) (Group 2) at 4 h caused a very small increase in MIP-2 expression. 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) (Group 4) stimulated augmented MIP-2 expression compared with single LPS (Group 3) or Poly I:C (Group 2) challenge (Fig. 4A). In contrast, the sequential injection of Poly I:C at 4 h after LPS failed to induce increased MIP-2 expression in TLR4−/− mice (Group 5) compared with single Poly I:C challenge (Group 2) (Fig. 4A), and sequential challenge with Poly I:C after LPS caused only a similar increase as that observed for LPS alone in TLR3−/− mice (Group 6). The changes in the mRNA expression of MIP-2 shown in Fig. 4B paralleled those observed for protein expression.