Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF‐κB1 DNA‐binding domain

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The α,β‐unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of IL‐2, IL‐10, GM‐CSF, IFN‐γ and TNF‐α by human T cells, but did not inhibit production of IL‐8. Acrolein inhibited induction of NF‐κB DNA‐binding activity following mitogenic stimulation of T cells, but had no effect on induction of NFAT or AP‐1. Acrolein inhibited NF‐κB1 (p50) binding to the IL‐2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2,000‐fold more potent than crotonaldehyde in blocking DNA binding to an NF‐κB consensus sequence. Matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys61 and Arg307) in the DNA‐binding domain. Crotonaldehyde reacted with Cys61, but not Arg307, while the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor. Supported by NIEHS grant ES05673 and by a grant from Philip Morris Incorporated.