LPS upregulates TLR2 expression in lungs and endothelial cells through TLR4 and MyD88 signaling. (a and b) Mice were injected intraperitoneally with LPS (10 μg/10 g body wt) and whole lungs were harvested at the times indicated after LPS injection. Data are representative of three independent studies. (a) RT-PCR for TLR2 mRNA expression in the lungs. GAPDH mRNA is identified as a control for RNA loading. (b) Western blot for TLR2 protein in lungs. Corresponding actin is shown as evidence of comparable loading between lanes. (c and d) MLVECs were incubated with LPS (1 μg/ml) for 2 hours, TLR2 mRNA was detected using RT-PCR (c), and TLR2 protein level was measured by Western blotting (d). The data are representative of three independent studies.

Neutrophil NADPH oxidase signals LPS-induced TLR2 expression in lungs. (a) RT-PCR showing the effects of PMN depletion and repletion on the expression of TLR2 mRNA in lung tissue of WT mice 2 hours after intraperitoneal LPS. PMN depletion was performed with rabbit anti-mouse neutrophil serum (150 μl administered intraperitoneally) 16 hours before injection of LPS. PMN repletion was performed in neutropenic mice using 1 × 10⁶ PMNs isolated from whole blood of WT or gp91^phox–/– mice; cells in 150 μl of saline were injected into the tail vein. GAPDH mRNA was detected for normalizing the densitometry of TLR2. The graph depicts the mean and SEM from three mice. *P < 0.01 compared with LPS treatment alone (lane 2). (b) EMSA showing the effect of PMN depletion and repletion on NF-κB nuclear translocation in lungs. PMN depletion and repletion experiments are as described in a; the data are representative of three independent studies.

Increased TLR2 expression results in augmented and stable ICAM-1 expression. (a) The results show the effects of sequential challenges of LPS and PGN, the ligands for TLR4 and TLR2, respectively, on ICAM-1 expression in lungs. In WT mice, LPS (10 μg/10 g body wt), PGN (10 μg/10 g body wt), LPS plus PGN, or saline (SAL) was injected intraperitoneally at time 0, and 2 hours later, a dose of either LPS (10 μg/10 g body wt), PGN (10 μg/10 g body wt), or saline was injected. Lung tissue was harvested at the times indicated, and ICAM-1 protein was detected using Western blotting. (b) WT or TLR2^–/– mice were injected with E. coli (1 × 10⁵ cells/10 g body wt, intraperitoneally) and ICAM-1 expression in the lung was measured 4 hours later by Western blotting. The data are representative of three independent studies.

Model of PMN NADPH oxidase–derived oxidant signaling in mediating the TLR4-TLR2 cross talk in endothelial cells. LPS stimulation induces NADPH oxidase activation and production of reactive oxygen species (ROS) in PMNs as well as the initiation of MyD88-dependent NF-κB signaling in endothelial cells and the consequent expression of TLR2 and ICAM-1. Adhesion of PMNs to endothelial cells is mediated by binding of constitutive ICAM-1 to CD18 integrin and provides the appropriate coupling required for PMNs to transmit oxidant signals to endothelial cells. The oxidants augment NF-κB signaling and TLR2 expression (+), which results in the augmented response of the cell to PGN, thereby amplifying ICAM-1 expression (circled +) and promoting stable adhesion of PMNs to endothelial cells and increased PMN migration. Thus, the PMN NADPH oxidase–mediated TLR4-TLR2 cross talk activates a positive feedback signal leading to sustained and amplified endothelial activation in response to invading pathogens.

NF-κB mediates LPS-TLR4–induced TLR2 upregulation. (a) EMSA showing changes in NF-κB nuclear translocation in lung tissue of WT mice and TLR4 knockout mice after LPS intraperitoneal injection. (b and c) Confluent MLVECs were treated with LPS (1 μg/ml) in the presence or absence of the NF-κB inhibitor IKK-NBD (100 μM) for 2 hours, followed by washing three times with HBSS and cell lysis. RT-PCR was performed using total RNA extracted from the cells (b), and Western blot was performed with the cell lysates (c). The data are representative of three independent studies.

Augmented LPS induction of TLR2 in endothelial cells requires PMN NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91^phox–/– mice, respectively, and were added at a concentration of 1 × 10⁵ cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis (a) as well as Western blotting with anti-TLR2 antibody (b). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis (a and b, lane 6). (c) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47^phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable PEG-catalase (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.

Effects of sequential challenges of LPS and PGN on MIP-2–induced PMN migration in vivo and transalveolar PMN migration. (a) Data obtained using in vivo air pouch model in which MIP-2 was used to induce migration of PMNs in WT, gp91^phox–/–, and TLR4^–/– mice sequentially challenged with intraperitoneal injections of two doses of LPS or LPS followed by PGN (as described in Methods). Sequential double LPS challenge increased PMN migration to 2.8- and 2.4-fold in WT and gp91^phox–/– mice, respectively, in response to MIP-2, but did not increase PMN migration in TLR4^–/– mice. Sequential injection of PGN 2 hours after LPS, however, markedly increased PMN migration in WT mice, to 4.1-fold that measured in the saline group. In contrast, sequential treatment of LPS and PGN did not significantly increase PMN migration in gp91^phox–/– or TLR4^–/– mice compared with either single LPS or sequential double LPS groups. *P < 0.01 compared with WT animals within the same treatment group; **P < 0.01 compared with WT animals in other treatment groups (n = 3 per group). Numbers (1–7, top) indicate the different groups. (b) To address the role of TLR2 in regulating transalveolar PMN migration in the lung, E. coli was injected intraperitoneally (1 × 10⁵ cells/10 g body wt) into WT and TLR2^–/– mice, and BALF PMNs were counted 4 hours later (n = 3 per group). White bars, saline alone; gray bars, E. coli.