Abstract

Tumor necrosis factor-α (TNFα) stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) by activating the transcription factor nuclear factor-κB (NF-κB) in human airway smooth muscle (ASM) cells. This study characterizes the receptor involved as well as critical downstream signaling events mediating cytokine-induced NF-κB activation and ICAM-1 expression. TNFα stimulation for 1 to 4 h induced ICAM-1 expression in human ASM cells. This rapid TNFα-induced ICAM-1 expression enhanced T-lymphocyte adhesion to ASM cells, which was inhibited by anti-ICAM-1 antibodies. Using immunostaining, we demonstrated that TNFα receptors TNFR1 and TNFR2 are expressed on native human tracheal smooth muscle. Treatment of cells with htr-9, an antibody that specifically activates TNFR1, also stimulated expression of ICAM-1 mRNA and protein. Utr-1, a blocking antibody to TNFR2, did not affect TNFα-mediated ICAM-1 expression. Both TNFα and htr-9 increased luciferase activity in ASM cells transfected with a NF-κB reporter plasmid. Overexpression of a dominant negative TNF receptor-associated factor 2 construct, lacking the NH₂-terminal RING finger, completely abrogated both TNFα- and htr-9-mediated increases in NF-κB reporter activity. Thapsigargin, an agent that depletes intracellular calcium stores, abrogated both cytokine-mediated NF-κB-dependent ICAM-1 mRNA transcription and protein expression but had no effect on IκB degradation. In addition, chelating cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester also inhibited cytokine TNFα-induced ICAM-1 expression. These data suggest that TNFR1, through a TNF receptor-associated factor 2-NF-κB signaling pathway, mediates TNFα-induced expression of ICAM-1 on ASM cells by involving a thapsigargin-sensitive signaling pathway.