Role of Neutrophil NADPH Oxidase in the Mechanism of Tumor Necrosis Factor-α-induced NF-κB Activation and Intercellular Adhesion Molecule-1 Expression in Endothelial Cells

Jie Fan,Randall S Frey,Arshad Rahman,Asrar B Malik

doi:10.1074/jbc.m110054200

Abstract

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.

Highlights

The pro-inflammatory cytokine TNF␣,1 released during sepsis, promotes adhesion of neutrophil (PMN) to the endothelium by inducing the expression of intercellular adhesion molecule-1
We explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-␣ (TNF␣)-induced NF-␬B activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells
TNF␣ has been shown to play an important role in the adhesion of neutrophils to endothelial cells [1, 13]

Summary

The abbreviations used are

TNF␣, tumor necrosis factor ␣; PMN, polymorphonuclear neutrophil; ICAM-1, intercellular adhesion molecule-1; TNFR, tumor necrosis factor receptor; TRADD, TNF␣-associated death domain-containing protein; TRAF2, TNFR-associated factor 2; NIK, NF-␬B-inducing kinase; MLVEC, mouse lung vascular endothelial cells; WT, wild-type; HBSS, Hanks’ balanced salt solution; GSH, reduced glutathione; i.p., intraperitoneal; ANS, anti-mouse neutrophil serum; EMSA, electrophoretic mobility shift assay; Ab, antibody; TRX, thioredoxin; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase. Signals mediating TNF␣-induced NF-␬B activation are initiated by the engagement of TNF receptor type I at the plasma membrane and relayed through specific TNFR-associated proteins. TRADD interacts in turn with two other adaptor proteins, TNFR-associated factor 2 (TRAF2) and receptor-interacting protein (RIP), and forms a complex required for NF-␬B activation [6, 7]. The cytosolic component p47phox is phosphorylated, and the entire cytosolic complex migrates to the membrane where it associates with cytochrome b558 to assemble the active oxidase [17]. Diating TNF␣-induced NF-␬B activation and ICAM-1 expression using mice genetically deficient in p47phox or gp91phox subunit of NAD(P)H oxidase and co-cultures involving mouse. NAD(P)H oxidase and thereby of oxidant generation significantly delayed the TNF␣-induced NF-␬B activation and ICAM-1 expression in both lungs and MLVEC of p47phoxϪ/Ϫ or gp91phoxϪ/Ϫ mice. Our data establish that the effects of NADPH oxidase inhibition were secondary to inhibition of NIK activation and the subsequent I␬B␣ degradation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION