TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase
Jie Fan, … , Randall S. Frey, Asrar B. Malik
Jie Fan, … , Randall S. Frey, Asrar B. Malik
Published October 15, 2003
Citation Information: J Clin Invest. 2003;112(8):1234-1243. https://doi.org/10.1172/JCI18696.
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TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase

Abstract

Interactions of polymorphonuclear neutrophils (PMNs) with endothelial cells may contribute to the activation of endothelial cell responses involved in innate immunity. We explored a novel function of PMN NADPH oxidase in the mechanism of Toll-like receptor-2 (TLR2) upregulation induced by LPS-TLR4 signaling in endothelial cells. We showed that LPS induced TLR2 up-regulation through TLR4- and MyD88-dependent signaling. In neutropenic mice, the LPS-induced NF-kB activation and TLR2 expression were significantly reduced, and both responses were restored upon repletion by PMN obtained from WT mice but not by PMNs from NADPH oxidase gp91phox–/– mice. These findings were recapitulated in mouse lung vascular endothelial cells cocultured with PMNs, indicating that the augmented NF-kB activation and the resultant TLR2 upregulation in endothelial cells were secondary to oxidant signaling generated by PMN NADPH oxidase. The functional relevance of NADPH oxidase in mediating TLR4-induced TLR2 expression in endothelial cells was evident by markedly elevated and stable ICAM-1 expression as well as augmented PMN migration in response to sequential challenge with LPS and peptidoglycan. Thus, PMN NADPH oxidase–derived oxidant signaling is an important determinant of the cross talk between TLR4 and TLR2 and the control of endothelial cell activation.

Authors

Jie Fan, Randall S. Frey, Asrar B. Malik

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Effects of sequential challenges of LPS and PGN on MIP-2–induced PMN mig...
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, gp91phox–/–, 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 gp91phox–/– 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 gp91phox–/– 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.