Carbonylation caused by cigarette smoke extract is associated with defective macrophage immunity

Am J Respir Cell Mol Biol. 2011 Aug;45(2):229-36. doi: 10.1165/rcmb.2010-0272OC. Epub 2010 Oct 8.


Oxidants in cigarette smoke inhibit pathogen recognition receptor function and phagocytosis, but the molecular basis of this inhibition remains obscure. We sought to identify the inhibitory mechanisms that impair alveolar macrophage function. Balb/c mice were acutely exposed to four cigarettes for 4 hours before treatment with intranasal LPS (1 μg). The mice exhibited significantly reduced airway neutrophilia and expression of TNF-α. Balb/c-derived MH-S alveolar macrophage cells exposed to cigarette smoke extract (CSE) displayed a similar inhibitory response to stimulation with LPS. The induction of inflammatory genes by recombinant (r) TNF-α (100 ng/ml) was also impaired by CSE. Because both pathways converge on NF-κB, the degradation of IκBα and the phosphorylation of p65 were assessed and shown to be blunted by CSE. CSE also blocked the activity of activator protein-1 (AP-1) by inhibiting p38 mitogen activated protein kinase (MAPK) in a reduced glutathione (GSH)-reversible manner. The induction of specific Toll-like receptor (TLR)-negative regulators (suppressor of cytokine signaling-1 [SOCS-1], interleukin-1 receptor associated kinase-M [IRAK-M], and IL-10) did not account for the impaired responses of TLRs. As free radical species are abundant in CSE and GSH restored function, a panel of oxidative/nitrosative stress markers was screened using immunocytochemistry. The panel identified protein carbonylation as the major CSE-inducible marker. Oxyblot analysis confirmed that CSE potently introduced carbonyl groups to many proteins in a dose-dependent and time-dependent manner that inversely correlated with the expression of TNF-α. The formation of pseudopodia was not prevented, but these membrane extensions were heavily carbonylated, and primary alveolar macrophages were also targeted for carbonylation. Oxidants in cigarette smoke drive a rapid, persistent, and global protein carbonylation that may represent a common pathway to altered immunity in disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Electrophoretic Mobility Shift Assay
  • Extracellular Signal-Regulated MAP Kinases / genetics
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Glutathione / pharmacology
  • Interleukin-10 / genetics
  • Interleukin-10 / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages, Alveolar / drug effects*
  • Macrophages, Alveolar / immunology*
  • Macrophages, Alveolar / metabolism
  • Mice
  • Mice, Inbred BALB C
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Phagocytosis / drug effects
  • Phosphorylation / drug effects
  • Protein Carbonylation / drug effects*
  • RNA, Messenger / genetics
  • Receptors, Interleukin-1 / genetics
  • Receptors, Interleukin-1 / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Smoking*
  • Toll-Like Receptors / genetics
  • Toll-Like Receptors / metabolism
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology*
  • p38 Mitogen-Activated Protein Kinases / genetics
  • p38 Mitogen-Activated Protein Kinases / metabolism


  • Lipopolysaccharides
  • NF-kappa B
  • RNA, Messenger
  • Receptors, Interleukin-1
  • Toll-Like Receptors
  • Transcription Factor AP-1
  • Tumor Necrosis Factor-alpha
  • Interleukin-10
  • Extracellular Signal-Regulated MAP Kinases
  • p38 Mitogen-Activated Protein Kinases
  • Glutathione