Correction: Suppression of GATA-3 Nuclear Import and Phosphorylation: A Novel Mechanism of Corticosteroid Action in Allergic Disease.

[This corrects the article DOI: 10.1371/journal.pmed.1000076.].

A comprehensive analysis of oxidative stress in the ozone-induced lung inflammation mouse model.

Ozone is an oxidizing environmental pollutant that contributes significantly to respiratory health. Exposure to increased levels of ozone has been associated with worsening of symptoms of patients with asthma and COPD (chronic obstructive pulmonary disease). In the present study, we investigated the acute and chronic effects of ozone exposure-induced oxidative stress-related inflammation mechanics in mouse lung. In particular, we investigated the oxidative stress-induced effects on HDAC2 (histone deacetylase 2) modification and activation of the Nrf2 (nuclear factor erythroid-related factor 2) and HIF-1α (hypoxia-inducible factor-1α) signalling pathways. Male C57BL/6 mice were exposed to ozone (3 p.p.m.) for 3 h a day, twice a week for a period of 1, 3 or 6 weeks. Control mice were exposed to normal air. After the last exposure, mice were killed for BAL (bronchoalveolar lavage) fluid and lung tissue collection. BAL total cell counts were elevated at all of the time points studied. This was associated with increased levels of chemokines and cytokines in all ozone-exposed groups, indicating the presence of a persistent inflammatory environment in the lung. Increased inflammation and Lm (mean linear intercept) scores were observed in chronic exposed mice, indicating emphysematous changes were present in lungs of chronic exposed mice. The antioxidative stress response was active (indicated by increased Nrf2 activity and protein) after 1 week of ozone exposure, but this ability was lost after 3 and 6 weeks of ozone exposure. The transcription factor HIF-1α was elevated in 3- and 6-week ozone-exposed mice and this was associated with increased gene expression levels of several HIF-1α target genes including Hdac2 (histone deacetylase 2), Vegf (vascular endothelial growth factor), Keap1 (kelch-like ECH-associated protein 1) and Mif (macrophage migration inhibitory factor). HDAC2 protein was found to be phosphorylated and carbonylated in nuclear and cytoplasm fractions, respectively, and was associated with a decrease in DNA-binding activity and protein expression of HDAC2. Decreased HDAC2 activity, most likely a direct result of protein modification, in combination with the loss of the antioxidative stress response and activation of the HIF-1α pathway, contribute to the inflammatory response and emphysema observed in ozone-exposed mice.

Inhaled long-acting ß2 agonists enhance glucocorticoid receptor nuclear translocation and efficacy in sputum macrophages in COPD.

BACKGROUND: Combination inhaled therapy with long-acting ß2 agonists (LABAs) and corticosteroids is beneficial in treating asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE: In asthma, LABAs enhance glucocorticoid receptor (GR) nuclear translocation in the presence of corticosteroids. Whether this biological mechanism occurs in COPD, a relatively corticosteroid-resistant disease, is uncertain. METHODS: Eight patients with mild/moderate COPD participated in a double-blind, placebo-controlled, crossover study and inhaled single doses of fluticasone propionate (FP) 100 µg, FP 500 µg, salmeterol xinafoate (SLM) 50 µg, and combination FP 100 µg + SLM 50 µg. One hour postinhalation, sputum was induced, nuclear proteins isolated from purified macrophages, and levels of activated nuclear GR quantified by using a GR-glucocorticoid response element ELISA-based assay. RESULTS: Nuclear GR significantly increased after the inhalation of FP 500 µg (P < .01), but not after the inhalation of FP 100 µg or SLM 50 µg, compared with placebo. Interestingly, SLM in combination with FP 100 µg increased nuclear GR levels equivalent to those of FP 500 µg alone. This was significantly greater than either FP 100 µg (P < .05) or SLM 50 µg (P < .01) alone. In vitro in a human macrophage cell line, SLM (10(-8) mol/L) enhanced FP (10(-9) mol/L)-induced mitogen-activated protein kinase phosphatase-1 mRNA (5.8 ± 0.6 vs 8.4 ± 1.1 × 10(-6) copies, P < .05) and 2 × glucocorticoid response element-luciferase reporter gene activity (250.1 ± 15.6 vs 103.1 ± 23.6-fold induction, P < .001). Addition of SLM (10(-9) mol/L) to FP (10(-11) mol/L) significantly enhanced FP-mediated suppression of IL-1ß-induced CXCL8 (P < .05). CONCLUSIONS: Addition of SLM 50 µg to FP 100 µg enhanced GR nuclear translocation equivalent to that seen with a 5-fold higher dose of FP in sputum macrophages from patients with COPD. This may account for the superior clinical effects of combination LABA/corticosteroid treatment compared with either as monotherapy observed in COPD.

Coordinated regulation of IL-4 and IL-13 expression in human T cells: 3C analysis for DNA looping.

Asthma is a chronic allergic disorder characterised by chronic inflammation. The balance of type I and type II (CD4+) T helper cells is of critical importance. In asthma there is an overexpression of T(H)2 cytokines, such as IL-4, IL-5 and IL-13. The genes encoding these cytokines are located together the same chromosomal region, 5q31.1 in humans. Here we confirm a central role for the transcription factors NFAT and GATA3 in the regulation of human IL-4 and IL-13. Chromatin Conformation Capture (3C) demonstrated the formation of specific ligation products containing spliced IL-4 and IL-13 DNA sequences in human T(H)2 polarised HuT-78 cells. This suggests that co-ordinate expression of T(H)2 cytokines, under the control of GATA3 and NFAT1 is due to the formation of a chromatin hub by DNA looping.

Unbalanced oxidant-induced DNA damage and repair in COPD: a link towards lung cancer.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by oxidative stress and increased risk of lung carcinoma. Oxidative stress causes DNA damage which can be repaired by DNA-dependent protein kinase complex. OBJECTIVES: To investigate DNA damage/repair balance and DNA-dependent protein kinase complex in COPD lung and in an animal model of smoking-induced lung damage and to evaluate the effects of oxidative stress on Ku expression and function in human bronchial epithelial cells. METHODS: Protein expression was quantified using immunohistochemistry and/or western blotting. DNA damage/repair was measured using colorimetric assays. RESULTS: 8-OH-dG, a marker of oxidant-induced DNA damage, was statistically significantly increased in the peripheral lung of smokers (with and without COPD) compared with non-smokers, while the number of apurinic/apyrimidinic (AP) sites (DNA damage and repair) was increased in smokers compared with non-smokers (p = 0.0012) and patients with COPD (p < 0.0148). Nuclear expression of Ku86, but not of DNA-PKcs, phospho-DNA-PKcs, Ku70 or γ-H2AFX, was reduced in bronchiolar epithelial cells from patients with COPD compared with normal smokers and non-smokers (p < 0.039). Loss of Ku86 expression was also observed in a smoking mouse model (p < 0.012) and prevented by antioxidants. Oxidants reduced (p < 0.0112) Ku86 expression in human bronchial epithelial cells and Ku86 knock down modified AP sites in response to oxidative stress. CONCLUSIONS: Ineffective DNA repair rather than strand breakage per se accounts for the reduced AP sites observed in COPD and this is correlated with a selective decrease of the expression of Ku86 in the bronchiolar epithelium. DNA damage/repair imbalance may contribute to increased risk of lung carcinoma in COPD.

Synergistic induction of endothelin-1 by tumor necrosis factor alpha and interferon gamma is due to enhanced NF-kappaB binding and histone acetylation at specific kappaB sites.

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFalpha induced a rapid and prolonged pattern of nuclear factor (NF)-kappaB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNgamma induced a delayed activation of interferon regulatory factor-1 without any effect on NF-kappaB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct kappaB sites in the preproET-1 promoter after stimulation with both TNFalpha and IFNgamma. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.

Suppression of GATA-3 nuclear import and phosphorylation: a novel mechanism of corticosteroid action in allergic disease.

BACKGROUND: GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo. METHODS AND FINDINGS: In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-kappaB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-alpha. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo. CONCLUSIONS: Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.

Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice.

RATIONALE: There is an increasing prevalence of reduced responsiveness to glucocorticoid therapy in severe asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanism of this remains unknown. Recent studies have shown that histone deacetylase activity, which is critical to glucocorticoid function, is altered by oxidant stress and may be involved in the development of glucocorticoid insensitivity. OBJECTIVES: To determine the role of phosphoinositol-3-kinase (PI3K) in the development of cigarette smoke-induced glucocorticoid insensitivity. METHODS: Wild-type, PI3Kgamma knock-out and PI3Kdelta kinase dead knock-in transgenic mice were used in a model of cigarette smoke-induced glucocorticoid insensitivity. Peripheral lung tissue was obtained from six healthy nonsmokers, nine smokers with normal lung function, and eight patients with COPD. MEASUREMENTS AND MAIN RESULTS: In vitro oxidative stress activates PI3K and induced a relative glucocorticoid resistance, which is restored by PI3K inhibition. In vivo, cigarette smoke exposure in mice increased tyrosine nitration of histone deacetylase 2 in the lung, correlating with reduced histone deacetylase 2 activity and reduced glucocorticoid function. Histone deacetylase 2 activity and the antiinflammatory effects of glucocorticoids were restored in PI3Kdelta kinase dead knock-in but not PI3Kgamma knock-out smoke-exposed mice compared with wild type mice, correlating with reduced histone deacetylase 2 tyrosine nitration. Glucocorticoid receptor expression was significantly reduced in smoke-exposed mice, in smokers with normal lung function, and in patients with COPD. CONCLUSIONS: These data show that therapeutic inhibition of PI3Kdelta may restore glucocorticoid function in oxidative stress-induced glucocorticoid insensitivity.

Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphorylation of GATA-3.

GATA-3 plays a critical role in allergic diseases by regulating the release of cytokines from Th2 lymphocytes. However, the molecular mechanisms involved in the regulation of GATA-3 in human T lymphocytes are not yet understood. Using small interfering RNA to knock down GATA-3, we have demonstrated its critical role in regulating IL-4, IL-5, and IL-13 release from a human T cell line. Specific stimulation of T lymphocytes by costimulation of CD3 and CD28 to mimic activation by APCs induces translocation of GATA-3 from the cytoplasm to the nucleus, with binding to the promoter region of Th2 cytokine genes, as determined by chromatin immunoprecipitation. GATA-3 nuclear translocation is dependent on its phosphorylation on serine residues by p38 MAPK, which facilitates interaction with the nuclear transporter protein importin-alpha. This provides a means whereby allergen exposure leads to the expression of Th2 cytokines, and this novel mechanism may provide new approaches to treating allergic diseases.

IL-1 beta and TNF-alpha regulation of the adenosine receptor (A2A) expression: differential requirement for NF-kappa B binding to the proximal promoter.

Adenosine is a potent endogenous regulator of airway inflammation that acts through specific receptor subtypes that can either cause constriction (A1R, A2BR, and A3R) or relaxation (A2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A2AR in a lung epithelial cell line (A549). IL-1beta and TNF-alpha increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A2AR gene expression. IL-1beta and TNF-alpha rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1beta- and TNF-alpha-stimulated A2AR expression was inhibited by the IkappaB kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1beta can enhance p65 association with putative kappaB binding sites in the A2AR promoter in a temporal manner. In contrast, TNF-alpha failed to enhance p65 binding to these putative sites. Functionally, the two most 5' kappaB sites were important for IL-1beta-, but not TNF-alpha-, induced A2AR promoter reporter gene activity. Finally, neither TNF-alpha nor Il-1beta had any effect on A2AR mRNA transcript degradation. These results directly implicate a major role for NF-kappaB in the regulation of A2AR gene transcription by IL-1beta and TNF-alpha but suggest that the effects of TNF-alpha on A2AR gene transcription are not mediated through the proximal promoter.

NF-kappaB and activator protein 1 response elements and the role of histone modifications in IL-1beta-induced TGF-beta1 gene transcription.

Abnormal expression of TGF-beta1 is believed to play an important role in the pathogenesis of a number of chronic inflammatory and immune lung diseases, including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. Gene activation in eukaryotes requires coordinated use of specific cell signals, chromatin modifications, and chromatin remodeling. We studied the roles of the ubiquitous inflammatory transcription factors, NF-kappaB and AP-1, in activation of the TGF-beta1 gene and histone acetylation at the TGF-beta1 promoter. IL-1beta-induced TGF-beta1 protein secretion and mRNA expression were prevented by actinomycin D and were attenuated by the inhibitor of kappaB kinase 2 inhibitor AS602868 and the JNK inhibitor SP600125, suggesting a degree of transcriptional regulation mediated by the NF-kappaB and AP-1 pathways. We demonstrated that IL-1beta activated the p65 subunit of NF-kappaB and the c-Jun subunit of AP-1. Using chromatin immunoprecipitation assays, we observed a sequential recruitment of p65 and c-Jun, accompanying ordered elevation of the levels of histone H4 and H3 acetylation and recruitment of RNA polymerase II at distinct regions in the native TGF-beta1 promoter. The specific NF-kappaB and AP-1 binding sites in the TGF-beta1 promoter were confirmed by an ELISA-based binding assay, and evidence for histone hyperacetylation in TGF-beta1 induction was supported by the observation that the histone deacetylase inhibitor trichostatin A enhanced basal and IL-1beta-induced TGF-beta1 mRNA expression. Our results suggest that IL-1beta-stimulated transcription of TGF-beta1 is temporally regulated by NF-kappaB and AP-1 and involves histone hyperacetylation at distinct promoter sites.

Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages.

Chronic obstructive pulmonary disease (COPD) is a common chronic inflammatory disease of the lungs with little or no response to glucocorticoids and a high level of oxidative stress. Histone deacetylase (HDAC) activity is reduced in cells of cigarette smokers, and low concentrations of theophylline can increase HDAC activity. We measured the effect of theophylline on HDAC activity and inflammatory gene expression in alveolar macrophages (AM) from patients with COPD. AM from normal smokers showed a decrease in HDAC activity compared with normal control subjects, and this was further reduced in COPD patients (51% decrease, P < 0.01). COPD AMs also showed increased basal release of IL-8 and TNF-alpha, which was poorly suppressed by dexamethasone. Theophylline induced a sixfold increase in HDAC activity in COPD AM lysates and significantly enhanced dexamethasone suppression of induced IL-8 release, an effect that was blocked by the HDAC inhibitor trichostatin A. Therefore, theophylline might restore steroid responsiveness in COPD patients.

Histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma.

Histone acetylation status is a key factor in the regulation of inflammatory gene transcription. We investigated the activity of histone acetylases (HAT) and deacetylases (HDAC), and the effect of glucocorticoids in alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) from subjects with asthma. Bronchoalveolar lavage was performed in 10 patients with intermittent asthma, 8 with persistent asthma, and 10 healthy control subjects. PBMCs and granulocytes were isolated from six patients with mild and severe asthma, before and after a 7-day course of prednisolone (30 mg/day). AMs were isolated for HDAC assay or incubated with dexamethasone (1 microM). HAT activity was increased (1.43 +/- 0.1 vs. 1.01 +/- 0.1 standard units/10 microg, p < 0.05), and HDAC activity was reduced (3031 +/- 243 vs. 5004 +/- 164 arbitrary fluorescence units/10 microg, p < 0.001) in AMs of subjects with asthma compared with control subjects. Dexamethasone suppressed LPS-induced granulocyte macrophage-colony stimulating factor, tumor necrosis factor-alpha, and interleukin-8 release by 83 +/- 1%, 51 +/- 7% and 20 +/- 9% (p < 0.001), respectively. Similar effects were seen on nuclear factor-kappaB inhibition, and interleukin-8 release was further reduced by the HDAC enhancer, theophylline (37 +/- 6%). Prednisolone increased HDAC activity in PBMCs from subjects with mild asthma. The increased inflammatory response in asthma may be due to reduced HDAC and enhanced HAT activity. Glucocorticoids and theophylline may downregulate the inflammatory response by modulating HAT and HDAC activity, and nuclear factor-kappaB activation.

Mitogen-activated protein kinase modulation of nuclear factor-kappaB-induced granulocyte macrophage-colony-stimulating factor release from human alveolar macrophages.

Granulocyte macrophage-colony-stimulating factor (GM-CSF), released from alveolar macrophages (AM), is an important regulator of eosinophil, T cell, and macrophage function and survival. We determined the mechanisms of GM-CSF regulation in AM from normal volunteers activated by lipopolysaccharide (LPS) by examining the role of nuclear factor-kappaB (NF-kappaB), and of p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK-1). PD 098059 (10 microM), an inhibitor of upstream activator of MKK-1, inhibited GM-CSF expression, but the expression of GM-CSF was not inhibited by SB 203580 (10 microM), an inhibitor of p38-MAP kinase. Phosphorylation of extracellular signal-regulated kinase-1 (ERK-1), ERK-2, and p38 MAP kinase by LPS were demonstrated on Western blot analysis. LPS increased NF-kappaB:DNA binding as examined by electrophoretic mobility shift assay, but this was not suppressed by PD 098059 or by SB 203580. LPS induced an increase in NF-kappaB activation as examined by p50 translocation assay without suppression by PD 098059 or by SB 203580. SN50 (100 microM), an inhibitor of NF-kappaB translocation and the specific IKK-2-Inhibitor (AS602868; 10 microM), also prevented GM-CSF expression and release induced by LPS, indicating that GM-CSF release is NF-kappaB-dependent. PD 098059, but not SB 203580, inhibited LPS-induced histone acetyltransferase (HAT) activity, indicating chromatin modification. Furthermore, AS602868 and SN 50 suppressed LPS-induced HAT activity. TSA (10 ng/ml), an inhibitor of histone deacetylase (HDAC), reversed the inhibitory effect of PD 098059, SB 203580, SN 50 and AS602868 on GM-CSF release. GM-CSF expression and release in AM is controlled by NF-kappaB activation, and this is modulated by phosphorylation of MKK-1 and p38 MAP kinase acting on histone acetylation.

Effect of acute and chronic inflammatory stimuli on expression of protease-activated receptors 1 and 2 in alveolar macrophages.

BACKGROUND: Protease-activated receptors 1 and 2 (PAR-1 and PAR-2) are 7-transmembrane G protein-coupled receptors activated by serine proteases in many cell types, including monocytes-macrophages, leading to the production of pro-inflammatory mediators, cytokines, and growth factors. OBJECTIVE: We determined the influence of chronic smoking and asthma on the expression of PAR-1 and PAR-2 receptors on alveolar macrophages (AMs). METHODS: We used RT-PCR and immunocytochemistry with confocal microscopy to determine mRNA and protein expression of PAR-1 and PAR-2 in AMs obtained from healthy smokers, asthmatic patients, and healthy subjects. In addition, we examined the effect of IL-1beta and LPS. RESULTS: PAR1 mRNA was decreased, whereas PAR2 mRNA was increased in 24-hour cultured AMs from smokers when compared with values in AMs from healthy subjects. Paradoxically, there was a higher degree of PAR-1 protein staining in AMs from smokers, whereas PAR-2 staining was similar in smokers and healthy subjects. PAR-1 and PAR-2 mRNA and protein expression were similar in asthmatic patients and control subjects. IL-1beta and LPS had no effect on PAR1 and PAR2 gene expression by AMs. CONCLUSIONS: There is a dissociation between gene and protein expression of PAR-1 and PAR-2. PAR-1 protein overexpression in AMs from smokers might be important in the pathophysiology of chronic airways disease.

The transcriptional co-activators CREB-binding protein (CBP) and p300 play a critical role in cardiac hypertrophy that is dependent on their histone acetyltransferase activity.

The CBP and p300 proteins are transcriptional co-activators that are involved in a variety of transcriptional pathways in development and in response to specific signaling pathways. We have previously demonstrated that the ability of both these factors to stimulate transcription is greatly enhanced by treatment of cardiac cells with the hypertrophic agent phenylephrine (PE). Here, we show that inhibition of either CBP or p300 with antisense or dominant negative mutant constructs inhibits PE-induced hypertrophy as assayed by atrial naturetic protein production, cardiac cell protein:DNA ratio and cell size. Furthermore, we show that overexpression of CBP or p300 can induce hypertrophy and that this effect requires their histone acetyltransferase (HAT) activity. Moreover, we show that PE can directly enhance CBP HAT activity and that artificial enhancement of HAT activity is sufficient to induce hypertrophy. Hence, CBP and p300 play an essential role in hypertrophy induced by PE, and this effect is mediated via PE-induced enhancement of their HAT activity. This is the first time a role for these factors, and their HAT activity, in hypertrophy has been directly demonstrated.

Increased p21(CIP1/WAF1) and B cell lymphoma leukemia-x(L) expression and reduced apoptosis in alveolar macrophages from smokers.

Alveolar macrophages (AMs) are the predominant defense cells in the airway, and their numbers are increased in smokers and subjects with chronic obstructive pulmonary disease. This increase may result from increased recruitment, increased proliferation, or reduced cell death. Apoptosis regulates inflammatory cell survival, and p21(CIP1/WAF1) is an important inhibitory regulator of cycle progression after oxidative stress. We have investigated whether chronic smoke exposure influences the expression and localization of cell cycle and apoptotic proteins in AM and bronchial epithelial cells in vivo. The increased numbers of AMs seen in smokers were only partially due to enhanced proliferation. p21(CIP1/WAF1) protein expression was increased in AMs and biopsies isolated from smokers and was found predominantly within the cytoplasm. In addition, B cell lymphoma leukemia (Bcl)-x(L), an antiapoptotic regulator, was also highly expressed in macrophages from smokers compared with nonsmokers and subjects with asthma. Hydrogen peroxide, an oxidative stress, induced cytoplasmic expression of p21(CIP1/WAF1) and failed to induce apoptosis in an in vitro model. These results suggested that AM and bronchial epithelial cells from smokers, in contrast to those from normal subjects and subjects with asthma, have reduced cell death. Thus, oxidative stress induced by cigarette smoking may contribute to the chronicity of inflammation in the airway, through a reduction of apoptosis.