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Published ahead of print on June 28, 2007, doi:10.1165/rcmb.2007-0130OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 617-623, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2007-0130OC

{alpha},beta-Unsaturated Aldehydes in Cigarette Smoke Release Inflammatory Mediators from Human Macrophages

Fabrizio Facchinetti1, Francesco Amadei2, Pierangelo Geppetti3, Francesca Tarantini3, Claudia Di Serio3, Alberto Dragotto4, Paolo M. Gigli4, Silvia Catinella2, Maurizio Civelli1 and Riccardo Patacchini1

1 Department of Pharmacology, and 2 Department of Analytical Chemistry, Chiesi Pharmaceuticals SpA, Parma, Italy; 3 Department of Critical Care Medicine and Surgery, and 4 Department of Thoracic Surgery, University of Florence, Florence, Italy

Correspondence and requests for reprints should be addressed to R. Patacchini, Department of Pharmacology, Chiesi Pharmaceuticals SpA, Via Palermo 26/A, 43100, Parma, Italy. E-mail: r.patacchini{at}chiesigroup.com


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Smoking cigarettes is the major risk factor for chronic obstructive pulmonary disease (COPD). COPD is a condition associated with chronic pulmonary inflammation, characterized by macrophage activation, neutrophil recruitment, and cell injury. Many substances contained in cigarette smoke, including reactive oxygen species (ROS), have been proposed to be responsible for the inflammatory process of COPD. However, this issue remains unsettled. By gas chromatography/mass spectrometry (GC/MS) we show that acrolein and crotonaldehyde, two {alpha},beta-unsaturated aldehydes, are contained in aqueous cigarette smoke extract (CSE) at micromolar concentrations and mimic CSE in evoking the release of the neutrophil chemoattractant IL-8 and of the pleiotropic inflammatory cytokine TNF-{alpha} from the human macrophagic cell line U937. In addition, acrolein (10–30 µM) released IL-8 also from cultured human alveolar macrophages and THP-1 macrophagic cells. 4-hydroxy-2-nonenal (30–100 µM), an endogenous {alpha},beta-unsaturated aldehyde that is abundant in lungs of patients with COPD, stimulated the release of IL-8 from U937 cells, whereas the saturated aldehyde, acetaldehyde, was ineffective. CSE-evoked IL-8 release was remarkably (> 80%) inhibited by N-acetyl-cysteine (0.1–3 mM) or glutathione monoethyl ester (1–3 mM). Both compounds, by forming covalent adducts (Michael adducts), completely removed unsaturated aldehydes from CSE. Our data demonstrate that {alpha},beta-unsaturated aldehydes are major mediators of cigarette smoke–induced macrophage activation, and suggest that they might contribute to pulmonary inflammation associated with cigarette smoke.

Key Words: chronic obstructive pulmonary disease • IL-8 • TNF-{alpha} • U937 cells • THP-1 cells



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study provides new insights into the identity of the substances in cigarette smoke or generated in the lung, producing chronic inflammation. {alpha},beta-Unsaturated aldehydes may become new pharmacologic targets for anti-inflammatory drugs for chronic obstructive pulmonary disease.

 
Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD), a syndrome characterized by progressive airflow limitation (1). Analysis of the inflammatory cell profile in alveoli and small airways of patients with COPD shows an increased number of neutrophils, CD8+ lymphocytes, and macrophages (2, 3). Alveolar macrophages, in particular, are thought to play a critical role as orchestrators of chronic inflammation and parenchymal tissue destruction observed in COPD (3–5). Alveolar macrophages contribute to airway inflammation in smokers and in patients with COPD by secreting a variety of inflammatory mediators, including TNF-{alpha} (6) and IL-8 (7), two factors whose levels are increased in bronchoalveolar lavage (BAL) fluid derived from smokers (8, 9) and patients with COPD (9) versus nonsmokers/healthy individuals. IL-8 is a powerful chemoattractant and activating agent for neutrophils. TNF-{alpha} is a pleiotropic cytokine released by activated macrophages, which plays a central role in early and late inflammatory processes. At pulmonary level, deletion of TNF-{alpha} receptors protects mice from smoke-induced lung inflammation and emphysema (6, 10, 11).

Cigarette smoke is a complex mixture of more than 4,700 chemical compounds (12), which includes high concentrations of free radicals and other oxidants that are thought to contribute to the oxidative damage observed in lungs of patients with COPD (3, 13, 14). Additional harmful volatile constituents of tobacco smoke are aldehydes, such as acetaldehyde, acrolein, and crotonaldehyde (15, 16). Exposure to cigarette smoke (either as gas phase or aqueous extract) evokes release IL-8, TNF-{alpha}, and other cytokines from cultured alveolar macrophages (6, 17), human peripheral blood mononuclear cells (PBMCs), and/or monocytic cells differentiated along the macrophagic pathway (18–22). In addition, cigarette smoke extract (CSE) stimulates the release of IL-8, TNF-{alpha}, and other inflammatory mediators in bronchial (23, 24) and alveolar (25) epithelial cells. Smoke-induced activation of macrophages involves NF-{kappa}B (21), activator protein-1 (AP-1) (20), and extracellular-regulated kinases 1 and 2 (ERK1/2) (18). In contrast, other studies have reported that in PBMCs (26), bronchial epithelial cells (27), or T cells (28), cigarette smoke reduces the release of inflammatory cytokines elicited by lipopolysaccharides (LPS) or other stimuli. This effect is thought to be responsible for the suppression of the immune response observed in the lungs of smokers (29, 30).

Despite the growing interest in this field, the identity and precise role of the substance(s) contained in cigarette smoke capable of regulating the release of pro-inflammatory cytokines have not yet been elucidated. Here, we have addressed the question of the effect of cigarette smoke on the release of two key pro-inflammatory cytokines, TNF-{alpha} and IL-8. Using U937 and THP-1 mononuclear cell lines differentiated into adherent macrophages, human alveolar macrophages, and aqueous CSE as the stimulus, we provide evidence that volatile {alpha},beta-unsaturated aldehydes, present in cigarette smoke or produced in the lung of patients with COPD, play a major role in the regulation of macrophage activation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Cultures
Human myeloid leukemia U937 and THP-1 cell lines were obtained from ICLC (Genoa, Italy). To induce differentiation into adherent macrophages, cells were plated in 48-well plates (105 cells/well) and incubated for 6 to 7 days (U937) or for 24 hours (THP-1) with 20 nM or 100 nM of phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO), respectively, as previously described (31, 32). Human alveolar macrophages, obtained by BAL of four different individuals (adult males) undergoing diagnostic bronchoscopy for lung cancer, were isolated in RPMI 1640, supplemented with 10% FBS, 2 mM glutamine, 100 U penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Sigma), according to standard protocols (33). None of the subjects showed signs of pulmonary infection and/or inflammation, including fever, at the day of bronchoscopy. Ethical approval of the study with human subjects was obtained from the Ethics Committee of the University of Florence. All subjects gave informed, written consent.

Production of CSE and Treatment of Cells
Aqueous CSE was generated from the combustion of four cigarettes (Marlboro Red, 12 mg tar, 0.9 mg nicotine each) bubbled through 50 ml of culture medium following a slightly modified method described previously (7, 34). To ensure standardization between experiments and batches of CSE, the absorbance was adjusted to 1.0 (optical density, OD) at 320 nm. This concentration of CSE was defined as 100%. Different concentrations of CSE diluted with the culture medium were employed, ranging from a 5-fold dilution (20%) up to a maximum of 200-fold dilution (0.5%). Cells were routinely exposed to freshly prepared CSE for 18 hours. Subsequently, supernatants were collected and stored at –80°C for subsequent determination of cytokines concentrations by enzyme-linked immunosorbent assay (ELISA). In some experiments, CSE was incubated (1 h at room temperature) in the presence of vehicle (RPMI) or of the following scavenger compounds: N-acetylcysteine (NAC), uric acid, mannitol, or GSHMEE, before the cells were challenged as described. All other inhibitors were added to cells 15 minutes before CSE. Additional details on CSE preparation are provided in the online supplement.

Vitality Assay (MTT Reduction Assay)
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay (35). Viability was expressed as percentage of the values (corresponding to 100%) of untreated cells.

Head Space Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry Analyses
Aldehydes contained in CSE were identified by head space gas chromatography/mass spectrometry (HS GC/MS). All the GC/MS analyses were performed by using an Agilent 6890 (Agilent Technologies, Palo Alto, CA) instrument, under experimental conditions described in detail in the online supplement. Detection and characterization of chemical adducts created by the reaction of glutathione (GSH) with aldehydes contained in CSE were performed by HS liquid chromatography/mass spectrometry (LC/MS) analysis. A triple quadrupole Quattro-Micro mass spectrometer (Micromass, Manchester, UK) coupled with an Alliance LC system equipped with an ultraviolet detector (Waters, Milford, CA) was used. LC/MS conditions are reported in detail in the online supplement.

ELISA
Human IL-8 and TNF-{alpha} were measured using a paired antibody quantitative ELISA kit purchased by Biosource International (Camarillo, CA).

Statistical Analysis
All values are expressed as means ± SEM of the given number (n) of experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test for multigroup comparisons. P < 0.05 was considered a level of statistical significance.

Chemicals
4-HNE was purchased from Alexis (Lausen, Switzerland); U0126 from Upstate (Charlottesville, VA), and TPCA-1 from Calbiochem (La Jolla, CA). All other compounds were from Sigma-Aldrich (St. Louis, MO). Solutions of 4-HNE (100 mM), TPCA-1 (10 mM) and U0126 (10 mM) were prepared in DMSO and then diluted in culture medium to the desired final concentration. All other compounds were dissolved directly in culture medium.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
CSE Induces the Release of IL-8 and TNF-{alpha} from U937 and THP-1 Cells
To determine the effect of CSE on IL-8 and TNF-{alpha} release, we treated U937 and THP-1 cells with different concentrations of freshly prepared CSE for 18 hours at 37°C. CSE elicited a dose-dependent increase of IL-8 and TNF-{alpha} release from both U937 (Figures 1A and 1C) and THP-1 macrophages (Figures 1B and 1D). By using QCL-1000 endotoxin kit (Lonza Bioscience, Walkersville, MD), we found that endotoxin levels in undiluted CSE (100%) were under the detection limit (< 0.05 EU/ml), thus showing that the pro-inflammatory effect of smoke extracts were not due to the presence of bacterial contamination. In THP-1 macrophagic cells, maximal effects of CSE were attained at 5% (Figures 1B and 1D), a concentration not affecting cell viability (Figure 1F). Higher concentrations of CSE (10–20%) significantly decreased cell viability, a phenomenon that was associated with a decline of IL-8 and TNF-{alpha} release (Figures 1B and 1D). In U937 cells toxicity and reduced IL-8/TNF-{alpha} release became apparent for CSE greater than 20% (data not shown).


Figure 1
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  		Figure 1. Cigarette smoke extract (CSE) induces IL-8 and TNF-{alpha} release from U937 and THP-1 cells. Effect of increasing concentrations of CSE on cytokine release in (A, C) U937 and in (B, D) THP-1 cells. E and F show the effect of CSE on U937 and THP-1 cell viability, respectively (MTT reduction assay). Each histogram is the mean ± SEM of n >= 4 independent observations. *Statistically different from basal: P < 0.01.

 
Nitrogen Bubbling Treatment Reduces CSE-Evoked IL-8 Release and CSE Content of Volatile Aldehydes
To evaluate the pro-inflammatory contribution of volatile components of cigarette smoke, we compared the efficacy of a CSE bubbled for 2 hours with nitrogen gas to that of control CSE, in U937 cells. We found that control CSE (10%) induced a significant increase of IL-8 release (547 ± 82.3% over the basal), while "bubbled" CSE (10%) exhibited a smaller effect (245 ± 21.6% over the basal, n = 6; P < 0.01). This result prompted us to investigate whether and to which extent aldehydes, which are among the most volatile compounds contained in CSE (15, 16), were decreased by nitrogen treatment. As summarized in Table 1, an elevated number of aldehydes were detected in CSE, by head space gas chromatography/mass spectrometry (HS GC/MS). The most abundant {alpha},beta-unsaturated aldehydes were crotonaldehyde, acrolein, and methacrolein. Among saturated aldehydes, the most abundant were acetaldehyde, propionaldehyde, and i-butyraldehyde (Table 1). Indeed, bubbling of nitrogen gas into CSE dramatically reduced the aldehyde content, with an average reduction of approximately 70% compared with control CSE (cf. Table 1).


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  		TABLE 1. MOST ABUNDANT ALDEHYDES IN CIGARETTE SMOKE EXTRACT, AND THEIR SENSITIVITY TO VARIOUS TREATMENTS

 
{alpha},beta-Unsaturated Aldehydes Mimic the Effect of CSE
To evaluate the potential pro-inflammatory effect of aldehydes, we exposed macrophagic cell cultures to the most abundant saturated or unsaturated aldehydes present in CSE. We found that acrolein (10–100 µM) significantly stimulated the release of both IL-8 and TNF-{alpha} from U937 (Figures 2A and 2B) and IL-8 from THP-1 cells (Figure 2C). Acrolein concentrations greater than or equal to 200 µM were found to significantly affect cell viability. In contrast, acetaldehyde lacked any stimulatory effect on IL-8 release (Figure 2F) in a wide concentration range (3–300 µM). The {alpha},beta-unsaturated aldehyde crotonaldehyde (3–100 µM) mimicked the stimulatory effect of CSE and acrolein on IL-8 release (Figure 2E), as did the acrolein analog cinnamaldehyde (Figure 2G). Concentrations of crotonaldehyde greater than 100 µM decreased cell viability (data not shown) and consequently reduced IL-8 release (Figure 2E). Similarly to acrolein, 4-hydroxy-2-nonenal (4-HNE, 30 and 100 µM), one of the most abundant and harmful endogenous products of lipid peroxidation, induced a robust release of IL-8 from U937 cells (Figure 2H). We also found that acrolein elicits IL-8 release from primary human lung alveolar macrophages (Figure 2D). This latter result provides a proof of concept that {alpha},beta-unsaturated aldehydes are capable of stimulating primary cells resident in the lung.


Figure 2
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  		Figure 2. Effect of saturated and {alpha},beta-unsaturated aldehydes on IL-8 and TNF-{alpha} release from macrophages. Effects of increasing concentrations of acrolein on (A) IL-8 and (B) TNF-{alpha} release from U937 cells. Effect of acrolein on IL-8 release from (C) THP-1 cells and from (D) human primary pulmonary macrophages. Effects of (E) crotonaldehyde, (F) acetaldehyde, (G) cinnamaldheyde, and (H) 4-hydroxy-2-nonenal (4-HNE) on IL-8 release from U937 cells. The chemical structure of each aldehyde is shown in the corresponding panel. Each histogram is the mean ± SEM of n = 3–4 independent experiments performed in sextuplicate. *Statistically different from basal: P < 0.01.

 
{alpha},beta-Unsaturated Aldehyde Scavengers, but Not Antioxidants, Blunt IL-8 Release Elicited by CSE
To quantify the contribution of unsaturated aldehydes to smoke-induced stimulatory effects, we incubated CSE with two well-recognized {alpha},beta-unsaturated aldehyde scavengers (36), NAC and glutathione monoethyl ester (GSHMEE), a cell-permeable analog of glutathione (GSH), 1 hour before challenging U937 cells. Both NAC (0.1–3 mM) and GSHMEE (1, 3 mM) almost completely reduced CSE-evoked IL-8 release (Figures 3A and 3B). NAC (1 mM) or GSHMEE (1–3 mM) also abolished IL-8 release induced by acrolein (100 MM) (Figure 3C). Neither NAC nor GSHMEE affected cell viability up to the highest concentrations tested (data not shown). We also showed that the protective effect of NAC (3 mM) was selective for CSE, as demonstrated by the inability of NAC to block LPS-mediated release of IL-8 (Figure 3D). HS GC/MS analysis showed that both NAC and GSHMEE (1 mM each) almost completely removed free acrolein from CSE, and significantly reduced crotonaldehyde and other unsaturated aldehydes, without affecting significantly the levels of saturated aldehydes (cf. Table 1). To obtain direct evidence that NAC/GSHMEE reacted with unsaturated aldehydes, we analyzed CSE for the detection of chemical adducts formed by these compounds. We were able to characterize a number of chemical adducts generated by the incubation of CSE with NAC/GSHMEE. Two of these chemical species are shown in Figure 4 and correspond to adducts of GSHMEE with acrolein or crotonaldehyde. Their chemical structures (Figure 4) show that they were generated by a Michael addition, a reaction produced by thiol group of GSHMEE and the double chemical bond present in acrolein or crotonaldehyde. Our finding is consistent with previous studies on the chemical reactivity of unsaturated aldehydes (31). No increase of the oxidized form of GSHMEE (i.e., glutathione disulfide, GSSG) was observed after incubation of GSHMEE with CSE (not shown). To assess the contribution of ROS contained in CSE to smoke-induced stimulatory effects, we incubated CSE with mannitol (1 mM) or uric acid (1 mM), two known scavengers of ROS, including peroxynitrite and superoxide anion (37). Both compounds, however, failed to affect CSE-evoked IL-8 release (CSE: 10,289 ± 342; CSE plus mannitol: 9,628 ± 567; CSE plus uric acid: 10,338 ± 395 pg/ml; n = 12–18). To determine whether oxidant compounds could stimulate IL-8 release, we tested increasing concentrations of H2O2 on U937 cells. H2O2, only at the highest concentration tested (0.6 mM), induced a significant, though very weak, stimulation of IL-8 release (Figure 3E). Higher concentrations of H2O2 caused toxic effects that affected cell viability (data not shown).


Figure 3
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  		Figure 3. Effects of scavengers of aldehydes and free radicals on CSE-induced IL-8 release in U937 cells. (A) Effect of NAC (0.1–3 mM) on CSE (5%)-induced IL-8 release. (B) Effect of glutathione monoethyl ester (GSHMEE) (1, 3 mM) on CSE-induced IL-8 release. (C) Effect of NAC (1 mM) and GSHMEE (1 and 3 mM) on acrolein (100 µM)-induced IL-8 release. (D) Failure of NAC (3 mM) to prevent LPS (1 µg/ml)-induced IL-8 release. (E) Effect of increasing concentrations of H2O2 on IL-8 release. Each histogram is the mean ± SEM of n = 2–3 independent experiments performed in sextuplicate. Statistically different from the release evoked by acrolein (#) or CSE (*) alone: P < 0.01. (n.s.) Not statistically significant.

 

Figure 4
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  		Figure 4. Chemical adducts generated by incubating CSE with GSHMEE. ES+ LC/MS reconstructed ion chromatogram (RIC) of CSE (100%) incubated 1 hour with GSHMEE 3 mM (a), untreated GSHMEE 3 mM (b), and untreated CSE (c). Ions are detected at m/z 392 (acrolein [M+H]+ adduct, left) and m/z 406 (crotonaldehyde [M+H]+ adduct, right) and their structures reported.

 
Effect of Acrolein on LPS-Stimulated U937 Cells
Because acrolein has recently been described to suppress the release of IL-8 and other cytokines from T cells activated by an inflammatory stimulus (28), we investigated the effect of acrolein on LPS-stimulated U937 cells. Acrolein (30–100 µM) not only failed to reduce, but rather increased the release of IL-8 elicited by LPS (1 µg/ml) (Figure 5A). A significant reduction in LPS-induced cytokine secretion was observed with more elevated concentrations of acrolein (200 µM) which, however, affects cell viability (Figure 5B).


Figure 5
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  		Figure 5. Effects of acrolein on LPS-stimulated U937 cells. (A) Release of IL-8 evoked by acrolein (30 and 100 µM), LPS (1 µg/ml), and their combination in U937 cells. (B) Effect of acrolein on U937 cell viability (MTT reduction assay). Each histogram is the mean ± SEM of n = 2 independent experiments performed in sextuplicate. Statistically different from basal (*) or from LPS (#) alone: P < 0.01.

 
Effect of ERK1/2 and Ikbeta Kinase-2 Inhibitors on Acrolein Effect
Previous studies indicated that the acute stimulatory effect of cigarette smoke on macrophages was mediated by ERK1/2 and Ikbeta kinase-2 (IKK-2)/NF-{kappa}B intracellular signaling pathways (18, 21). Indeed, both the selective ERK1/2 inhibitor U0126 and the IKK-2 inhibitor TPCA-1 reduced to a similar extent the amount of IL-8 that was secreted from U937 cells stimulated by acrolein and CSE (U0126 [10 µM]: 81,4 ± 6.5% and 80.3 ± 7.5%; TPCA-1 [1 µM]: 77.9 ± 8.2% and 46.7 ± 5.1%; TPCA-1 [10 µM]: 63.4 ± 5.0% and 68.4 ± 6.0% inhibition of acrolein or CSE, respectively; n = 12–18).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our study shows that {alpha},beta-unsaturated aldehydes, in particular acrolein and crotonaldehyde, play a causal role in the activation of human macrophages induced by cigarette smoke. First, according to previous results (15, 16, 28), we detected elevated (µM) concentrations of acrolein and crotonaldehyde in CSE. Second, acrolein and crotonaldehyde produced the same effect (IL-8 and TNF-{alpha} release) as CSE in cultured monocytic U937 cells differentiated along the macrophagic line. Importantly, acrolein also stimulated IL-8 release from primary cultures of human alveolar macrophages and THP-1 cells. Third, the ROS and aldehyde scavengers, NAC and GSHMEE, prevented either CSE- or acrolein-induced stimulatory effects on macrophages. In addition, we have shown that, by generating unreactive Michael adducts, both NAC and GSHMEE effectively scavenged unsaturated aldehydes from CSE. Overall, these data suggest that a cause–effect relationship exists between the reduction of the burden of unsaturated aldehydes in CSE and the anti-inflammatory action of these two compounds. Indeed, NAC is completely ineffective in preventing IL-8 release when this latter is evoked by a different stimulus (LPS). However, it may be argued that the anti-inflammatory action of NAC and GSHMEE might result (at least in part) from their ability to scavenge ROS contained in CSE. In accord with a previous report (38), we observed only a small, although significant, effect of H2O2 as compared with the robust response evoked by CSE or acrolein, in releasing IL-8. Thus, it seems unlikely that exogenously administered ROS may play a major and direct role in inducing cytokine release from macrophages, and specifically in the release evoked by CSE. Our data do not exclude the possibility that endogenously generated ROS may contribute to the overall lung inflammation orchestrated by macrophages. ROS may, indeed, either generate endogenous aldehydes in the lung by lipid peroxidation of cell membrane phospholipids, or contribute to deplete the store of the endogenous aldehyde scavenger, GSH.

Interestingly, we observed that both NAC and GSHMEE selectively blunted unsaturated, but not saturated, aldehydes in CSE, a finding consistent with the known chemical reactivity of thiols (36). In addition, the most abundant saturated aldehyde present in CSE, acetaldehyde, failed to activate cytokine secretion from macrophagic cell lines. Thus, saturated aldehydes, including acetaldehyde, do not seem to contribute to the CSE effect on macrophages.

It has been recently shown that CSE increases IL-8 and TNF-{alpha} release from MonoMac6 human macrophage–like cells (21), an effect associated with a reduction of intracellular GSH. However, unexpectedly, CSE-induced consumption of GSH was not accompanied by the formation of the oxidized form of GSH (i.e., GSSG). In light of our results, the GSH consumption reported previously (21) may be explained by a reaction of GSH with unsaturated aldehydes leading to the formation of Michael adducts, rather than by a direct oxidation of GSH by ROS. Our data obtained with CSE and acrolein on THP-1 macrophages are apparently in contrast with the results of Nordskog and coworkers (39), who showed that cigarette smoke was unable to stimulate the synthesis of several cytokines in these cells, including IL-8 and TNF-{alpha}. It is worth noting, however, that cigarette smoke condensate (i.e., the particulate component of smoke trapped in filters) was employed as a stimulus in the study by Nordskog and colleagues (39), while we employed aqueous CSE, which contains (also) the most volatile smoke components. On the basis our present findings, it is conceivable to speculate that smoke condensate lacks (or is poor of) the unsaturated aldehydes that actually account for the most of the stimulatory effect of smoke on macrophages. COPD is associated with oxidative stress, due to an increased burden of smoke-derived inhaled oxidants, and to the high amount of ROS generated by the increased number of activated macrophages and neutrophils present in alveolar spaces (3, 40). ROS can react with cell membrane phospholipids, generating oxidative by-products of polyunsaturated fatty acids. This process ultimately leads to the formation of various reactive carbonyl species, including 4-HNE, acrolein, malondialdehyde, glyoxal, and others (3, 36, 40). 4-HNE is the most abundant {alpha},beta-unsaturated aldehyde generated by lipid peroxidation. 4-HNE is capable of activating specific intracellular signaling pathways by alkylating DNA and biologically active proteins, thereby eliciting a variety of inflammatory and cytotoxic responses (36, 41–43). Importantly, 4-HNE was found elevated in lungs of patients with COPD, and the amount of chemical adducts between 4-HNE and proteins in the parenchyma was found inversely correlated with the forced expiratory volume in 1 second (FEV1), the main diagnostic parameter of COPD severity (40). Although cessation of smoke habit is the only therapeutic intervention in COPD that has been shown to slow disease progression (1), chronic airway inflammation may persist even after the patient has quit smoking (44, 45). Our data with 4-HNE suggest that inflammation might be triggered by smoke-inhaled acrolein and crotonaldehyde, while endogenous unsaturated aldehydes, such as acrolein itself and 4-HNE, might contribute to the evolution of the disease into a chronic condition that outlasts the cessation of smoking.

The mechanism(s) through which acrolein stimulates macrophages may be due to its ability to activate multiple redox-sensitive molecular targets (46). Two intracellular signaling pathways, NF-{kappa}B and ERK1/2, have recently been shown to mediate cigarette smoke–induced cytokine release from human macrophages (18, 21). Because in the present experiments two selective inhibitors of NF-{kappa}B and ERK1/2, TPCA1 (47) and U0126 (18), respectively, prevented both CSE- and acrolein-induced IL-8 release, we propose that CSE and acrolein activate the same intracellular pathways that eventually results in the release of proinflammatory cytokines. This finding offers further support to the proposal that {alpha},beta-unsaturated aldehydes mediate the CSE-evoked response.

Several studies have reported that cigarette smoke evokes inhibitory effects on activated lymphocytes (28), monocytes (26), and bronchial epithelial cells (27). In particular, acrolein and crotonaldehyde (28) have been identified as the mediators of cigarette smoke–induced inhibition of IL-8 release from anti-CD3–stimulated T cells. In the present experimental conditions, we observed an inhibitory effect on LPS-stimulated cells, but only at concentrations of acrolein that markedly affected cell viability. In addition, acrolein, at nontoxic concentrations, not only failed to reduce, but rather increased the release of IL-8 evoked by LPS from U937 cells. Therefore, it may be hypothesized that responsiveness of pulmonary macrophages to bacterial endotoxins might be modified by the level of unsaturated aldehydes in the lung. The more elevated concentration of unsaturated aldehydes, the higher the level of response of macrophages to bacterial endotoxins. Inhibitory effects of aldehydes might be associated with toxic effects evoked by elevated concentrations of these agents.

In conclusion, we have shown that {alpha},beta-unsaturated aldehydes contained in cigarette smoke, in particular acrolein and crotonaldehyde, and 4-HNE (which is generated in high concentrations in lungs of patients affected by COPD), are major pro-inflammatory stimulants for macrophages. Thus, our results shed light on the still unsettled question regarding the identity of the substances present in cigarette smoke, or generated in the lung by lipid peroxidation, responsible for initiating and maintaining the inflammation response. Acrolein and other {alpha},beta-unsaturated aldehydes are therefore potential pharmacologic targets for antinflammatory therapy in COPD, a disease in which none of the existing therapies has proven capable of halting the decline in lung function.


   Acknowledgments
 
The authors thank Ms. Paola Caruso for skillful technical assistance.


   Footnotes
 
This study was supported in part by the Italian Ministry for University and Scientific Research (MiUR), grant code: RBIP06YM29.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2007-0130OC on June 28, 2007

Conflict of Interest Statement: F.F., M.C., R.P., S.C., and F.A. are employees of Chiesi Pharmaceuticals. P.G., C.D.S., and F.T.'s institution received two grants from Chiesi Pharmaceuticals in 2005 and 2006 for the study of novel bronchodilators for COPD. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 16, 2007

Accepted in final form June 6, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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