- Email: [email protected]
Acrolein in cigarette smoke inhibits T-cell responses
Acrolein in cigarette smoke inhibits T-cell responses Cherie Lambert, PhD,a,c Jesica McCue, PhD,a,c Mary Portas, BS,a Yanli Ouyang, MD, PhD,a JiMei Li, MD,a Thomas G. Rosano, PhD,b Alexander Lazis,a and Brian M. Freed, PhDa Denver, Colo, and Albany, NY
Background: Cigarette smoking inhibits T-cell responses in the lungs, but the immunosuppressive compounds have not been fully identified. Cigarette smoke extracts inhibit IL-2, IFN-g, and TNF-a production in stimulated lymphocytes obtained from peripheral blood, even when the extracts were diluted 100-fold to 1000-fold. Objective: The objective of these studies was to identify the immunosuppressive compounds found in cigarette smoke. Methods: Gas chromatography/mass spectroscopy and HPLC were used to identify and quantitate volatile compounds found in cigarette smoke extracts. Bioactivity was measured by viability and production of cytokine mRNA and protein levels in treated human lymphocytes. Results: The vapor phase of the cigarette smoke extract inhibited cytokine production, indicating that the immunosuppressive compounds were volatile. Among the volatile compounds identified in cigarette smoke extracts, only the a,b-unsaturated aldehydes, acrolein (inhibitory concentration of 50% [IC50] = 3 mmol/L) and crotonaldehyde (IC50 = 6 mmol/L), exhibited significant inhibition of cytokine production. Although the levels of aldehydes varied 10-fold between high-tar (Camel) and ultralow-tar (Carlton) extracts, even ultralow-tar cigarettes produced sufficient levels of acrolein (34 mmol/L) to suppress cytokine production by >95%. We determined that the cigarette smoke extract inhibited transcription of cytokine genes. The inhibitory effects of acrolein could be blocked with the thiol compound N-acetylcysteine. Conclusion: The vapor phase from cigarette smoke extracts potently suppresses cytokine production. The compound responsible for this inhibition appears to be acrolein. (J Allergy Clin Immunol 2005;116:916-22.)
Basic and clinical immunology
From athe Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado at Denver and Health Sciences Center; and bthe Department of Pathology and Laboratory Medicine, Albany Medical Center. c These two authors contributed equally to this work. Supported by grant ES05673 from the National Institute of Environmental Health Sciences, National Institutes of Health, and by a grant from Philip Morris USA Inc and Philip Morris International. Disclosure of potential conflict of interest: B. Freed has received grants/research support from Philip Morris USA Inc and Philip Morris International. Received for publication February 25, 2005; revised May 24, 2005; accepted for publication May 31, 2005. Available online August 1, 2005. Reprint requests: Brian M. Freed, PhD, Division of Allergy and Clinical Immunology, University of Colorado at Denver and Health Sciences Center, Campus Box B164, 4200 E Ninth Avenue, Denver, CO 80262. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.046
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Key words: Cigarette smoke, T cell, activation, cytokine, gene expression, acrolein
There is a strong correlation between cigarette smoking and the incidence and severity of pulmonary infections, including pneumococcal and varicella pneumonias,1-5 Legionnaires disease,6,7 influenza,7 and tuberculosis.8-10 In addition, Miguez-Burbano et al11 recently reported that smoking doubled the risk of developing Pneumocystis carinii pneumonia among HIV-infected patients. Although these phenomena may involve several physiological processes, the major factor is likely to be the profound suppression of T-cell responses observed in smokers’ lungs.12-14 We and others have shown that cigarette smoking suppresses the mitogenic responses of lung lymphocytes and the production of IL-1b, IL-6, and TNF-a by PBMCs and alveolar macrophages.15-20 The specific compounds in cigarette smoke that elicit these effects have not been identified. Recently, we demonstrated that extracts from a single high-tar (Camel Ultralights; R.J. Reynolds Tobacco Co., Winston-Salem, NC) cigarette significantly inhibited production of IL-2 and IFN-g by human peripheral blood lymphocytes, even when the extracts were diluted 100fold to 1000-fold.15 The inhibitory effect was observed even in extracts from ultralow-tar cigarettes containing >1 mg tar per cigarette, and nicotine had no effect on these responses even at millimolar concentrations. These observations suggested the presence of potent immunosuppressive compounds in cigarettes, and the purpose of this study was to identify them. The fact that cigarette smoke is composed of more than 5000 chemicals21 would appear to make identification of individual immunosuppressive compounds nearly impossible. However, identification was greatly facilitated by our discovery that the vapor from cigarette smoke extracts exhibited the same immunosuppressive activity as the whole extract. We therefore used gas chromatography/mass spectroscopy (GC/MS) and HPLC to identify the volatile immunosuppressive components in cigarette smoke extracts.
METHODS Preparation of cigarette smoke extracts and chemicals Cigarette smoke extracts (CSEs) were freshly prepared as described previously.15 Acrolein, crotonaldehyde, acetaldehyde, butyraldehyde, and propionaldehyde (Acros Organics, Pittsburgh, Pa) were prepared in PBS. Toluene, xylene, styrene, and limonene
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Abbreviations used CSE: Cigarette smoke extract GC/MS: Gas chromatography/mass spectroscopy IC50: Inhibitory concentration of 50%
(Sigma, St Louis, Mo) were prepared in absolute ethanol just before their addition to the cultures.
Human PBMC and lymphocyte culture Peripheral blood was drawn from healthy, nonsmoking adult volunteers after informed consent. PBMCs were isolated and cultured as described previously.15 T cells were isolated from PBMC by negative selection using a pan–T-cell isolation kit II (Miltenyi, Auburn, Calif). T-cell purity was monitored by a FACScan analyzer (Becton Dickinson, Mountain View, Calif) using antibodies specific for CD3, CD4, CD8, and CD45 and was consistently >90% CD31. PBMC (106/mL) or T cells (5 3 105/mL) from individual donors were cultured in 24-well tissue culture plates (Costar, Corning, NY) at 37°C in 5% CO2. The cells were pretreated for 3 hours with 1 mL CSE, bubbled through RPMI or reagent grade aldehydes as indicated, and then stimulated with 10 ng/mL anti-CD3 (Orthoclone OKT3, Orthobiotec, Raritan, NJ) plus 10 nmol/L phorbol 12-myristate 13acetate for 24 hours. Untreated lymphocytes were cultured on a plate separate from the CSE or aldehyde-treated samples to prevent carryover inhibition. Cells from 1 individual were used per experiment, and each experiment was replicated a minimum of 3 times. The University of Colorado Combined Institutional Review Board approved the human subject protocol used in this study.
GC/MS Gas chromatography/mass spectroscopy analysis of CSE vapor phase was performed with a Varian 3800 Gas Chromatograph (Varian Chromatography Systems, Walnut Creek, Calif) equipped with a Varian 1079 injector, Rtx-1MS capillary column (15 m, 0.25 mm internal diameter, 0.5 mm film thickness; Restek, Bellefonte, Pa) and an 8200 Varian autosampler fitted with a 12 position carrousel for headspace vials. Helium was used as the gas chromatography carrier at a flow rate of 1 mL/min. A column temperature program with an initial 5-minute hold at 40°C, and a temperature ramp from 40°C to 90°C (10°C/min) was used. The injector temperature was maintained at 80°C, and injections of headspace samples were performed in a split mode (1:30). Mass spectral detection was performed with a Varian Saturn 2000 mass spectrometer fitted with a SilChrom ion trap maintained at 200°C. A 10-mAmp filament current was used for electron ionization, and the mass spectrum was scanned from 35 to 250 m/z. The GC/MS system and data management were controlled by Saturn GC/MS Workstation software (version 5.2). Analytical samples (3 mL) were pipetted into 10-mL reaction vials and crimp-sealed with aluminum pressure seals fitted with polytetrafluoroethylene/silicone liners (Supelco, Bellefonte, Pa). For storage or shipment, the samples were maintained at 210°C. After ambient temperature equilibration for at least 60 minutes, 100-mL samples of the headspace over the liquid samples were analyzed by GC/MS as described.
vigorously and allowed to react for 30 minutes at room temperature, after which the reaction was stopped by the addition of 10 mL pyridine injected through the vial septum. Samples were stored at 220°C for less than 1 week. Samples were chromatographed on 3.9-mm 3 150-mm Nova-Pak C18 columns (Waters, Milford, Mass) with a Nova-Pak C18 guard column (Waters) and monitored at 360 nm with a Waters 490E UV detector. Samples of 10 mL or 100 mL were injected by using a Waters 717 autosampler and chromatographed with a flow rate of 1.5 mL/min in a mobile phase of 100% buffer A (60% water/30% acetonitrile/10% tetrahydrofuran vol/vol/vol) for 1 minute, followed by a linear gradient of 100% buffer A to 100% buffer B (40% water/60% acetonitrile vol/vol) over a period of 10 minutes. The identity of each aldehyde was confirmed by comparison of retention times to 2,4-dinitrophenylhydrazine– aldehyde standards (Waters). Concentrations of the aldehydes were determined by the area under the curve method in comparison with dilutions of the known standards using Waters Empower software (Build 1154).
HPLC analysis of cigarette aldehydes
Assays of lymphocyte function
The levels of aldehydes in CSE were determined by C18 reversephase HPLC after derivatization with 2,4-dinitrophenylhydrazine. Within 5 minutes of collection, 80 mL of the CSE were derivatized with 120 mL of 10-mmol/L 2,4-dinitrophenylhydrazine (Acros, Fairlawn, NJ) and placed in sealed vials. The samples were shaken
Cell viability was monitored by fluorescence-activated cell sorting analysis of 7-aminoactinomycin D (Molecular Probes) exclusion per company protocol.22 The levels of IL-1b, IL-2, IL-6, IL-8, IL-10, IFN-g, TNF-a, and GM-CSF in the culture supernatants were measured by Luminex bead human cytokine multiplex kit
Basic and clinical immunology
FIG 1. Inhibition of PBMC cytokine production by vapors from CSEs. CSE represents cells treated directly with 1 mL of a cigarette smoke extract prepared from 1 Camel cigarette. Data represent the means 6 SEMs of at least 3 separate experiments. *P < .05 compared with stimulated (Stim); NS, nonstimulated.
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FIG 2. GC/MS and HPLC analysis of Camel CSE. Left, GC/MS peaks eluting in the first 2 minutes were aldehydes. Right, HPLC analysis of Camel extracts after conjugation to 2,4-dinitrophenylhydrazine. Peaks were identified as (1) formaldehyde, (2) acetaldehyde, (4) acrolein, (5) acetone, (7) propionaldehyde, (8) crotonaldehyde, and (10) butyraldehyde. Peaks 3, 6, and 9 could not be identified.
(BioSource International, Camarillo, Calif) according to the manufacturer’s instructions. Standard curves with serial dilutions of human recombinant cytokine standards were performed in each assay, and data were analyzed by 5-parameter regression with StatLia Enterprise software, version 3.1 (Brendan Scientific, Carlsbad, Calif). TNF-a and IL-2 steady-state mRNA levels were measured by Quantikine mRNA quantitation assays according to the manufacturer’s instructions (R&D Systems, Inc, Minneapolis, Minn). Total RNA was isolated by a QIAGEN RNeasy mini kit (Valencia, Calif) according to the manufacturer’s instructions.
Statistical analysis The mean 6 SEM was determined for each treatment group in the individual experiments, performed a minimum of 3 times. To establish statistically significant P values for cytokine measurements (P < .05), the values obtained from the treatment groups were converted to percent control (stimulated group) and evaluated by 1-way ANOVA and Dunnett multiple comparison test. This method allowed us to account for significant variances among donors.
Basic and clinical immunology
RESULTS Inhibition of cytokine production by the vapor phase of cigarette smoke extracts As can be seen in Fig 1, direct exposure of human PBMC to 1 mL of the CSE inhibited production of IL-1b, IL-2, GM-CSF, IL-6, IFN-g, IL-8, and TNF-a human PBMC by >99%. This inhibitory effect did not require direct exposure of the cells to CSE. Rather, the inhibitory effect carried over in the vapor phase to untreated cells in neighboring wells. Wells closest to the CSE exhibited the greatest level of suppression, whereas those farther away on the plate exhibited progressively less inhibition. The CSE was toxic to PBMC when placed in direct contact, but cells >4 cm away exhibited no toxicity despite 75% to 95% inhibition of cytokine production. All of the cytokines showed similar sensitivity to the extracts except IL-8, which was noticeably more resistant to the effects of CSE. Analysis of vapor phase from cigarette smoke extracts The fact that the inhibitory effects of CSE carried over to neighboring wells indicated that the immunosuppressive compounds were highly volatile. This hypothesis was
verified by the fact that lyophilization completely removed the inhibitory activity from CSE. We therefore collected the CSE vapor phase and placed it in sealed glass vials. The vapor phase was then sampled at 37°C by using a needle and subjected to GC/MS analysis (Fig 2, left). Among the peaks identified, those in the highest concentrations were toluene, xylene, styrene, and limonene, along with numerous aldehydes in the peaks eluting in <2 minutes. Because GC/MS was not able to resolve and quantitate the aldehydes completely, CSE was reanalyzed by C18 reverse-phase HPLC after conjugation of the carbonyl groups to 2,4-dinitrophenylhydrazine, which enhanced the limit of detection to 1 to 3 mmol/L. As can be seen in Fig 2 (right), 10 peaks were detected in CSE from Camel cigarettes. Table I summarizes the major compounds measured in CSE, which include high levels of acetaldehyde, acetone, and acrolein, followed by significantly lower amounts of propionaldehyde, crotonaldehyde, and butyraldehyde.
Assessment of suppressive activity of vapor phase constituents To assess the cytokine inhibitory activity of CSE, we determined the dose of the cigarette extract compounds that inhibited production of IL-2 by 50% (IC50; Table I). Toluene, xylene, styrene, and limonene did not suppress IL-2 production in vitro, even at doses as high as 2 mmol/L. The aldehydes segregated into 2 distinct groups: the a,b-unsaturated aldehydes (acrolein and crotonaldehyde) were potent inhibitors of cytokine production, whereas acetone and the saturated aldehydes (acetaldehyde, butyraldehyde, and propionaldehyde) were essentially inactive (Table I). Acrolein and crotonaldehyde inhibited IL-2 with an IC50 of 3 and 6 mmol/L, respectively, levels well in the range of 1 cigarette. Even though acetaldehyde was present at very high levels (1352 mmol/L), it did not inhibit IL-2 production. These observations suggest that, on the basis of the levels present and the relative inhibitory effects, acrolein and crotonaldehyde are the predominant inhibitors of cytokine production in the CSE vapor. Cigarette smoke extract derived from unfiltered Carlton (Brown and Williamson Tobacco Corp, Louisville, Ky) and filtered Camel represented the range of tar content in
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TABLE I. Effects of cigarette smoke aldehydes on cytokine production Aldehyde levels (mmol/L)* Aldehyde
Camel
Acrolein CH2=CHCHO Crotonaldehyde CH3CH=CHCHO Acetone CH3COCH3 Acetaldehyde CH3CHO Propionaldehyde CH3CH2CHO Butyraldehyde CH3CH2CH2CHO
503 101 507 1352 115 49
6 6 6 6 6 6
21 12 24 63 15 8
Carlton
IC50 (mmol/L)y
6 6 6 6 6 6
3 6 500 1500 >2000 >2000
34 2 48 111 9 4
5 1 8 15 1 1
*Mean level from 10 experiments detected in extracts prepared from 1 cigarette bubbled through 10 mL PBS. IC50 is the dose that inhibited IL-2 production by 50%.
TABLE II. Aldehyde levels from CSE detected in carryover wells Acrolein (mmol/L)
Initial level Straight* 1.4 cm* 2.7 cm* 4.1 cm* 5.4 cm* 6.8 cm* 8.1 cm*
487 101 33 17 9 5 4 1
6 6 6 6 6 6 6 6
61 36 6 2 2 1 2 1
Crotonaldehyde (mmol/L)
81 24 3 1
6 6 6 6 0 0 0 0
11 5 2 1
Acetaldehyde (mmol/L)
Butyraldehyde (mmol/L)
Propionaldehyde (mmol/L)
6 6 6 6 6 0 0 0
36 6 3 661 161 0 0 0 0 0
88 6 8 24 6 24 362 0 0 0 0 0
1279 140 24 9 1
173 53 9 4 1
IL-2y
1 1 1 4 35 51 89 109
6 6 6 6 6 6 6 6
1 1 1 4 22 16 36 41
*Mean 6 SEM levels detected after 3-h exposure from 3 experiments. IL-2 levels displayed as percent stimulated.
dependent manner but had much less effect on production of IL-8 (Fig 3). Acrolein also inhibited production of GM-CSF, IL-6, IL-10, and IFN-g with dose-response curves similar to that of IL-2 (data not shown).
Effects of acrolein on T-cell responses IL-1b and IL-2 are produced by macrophages and T cells, respectively. Because IL-1b also promotes T-cell activation and IL-2 production, inhibition of the latter could have been a result of an indirect effect of the macrophages. To determine whether acrolein directly affected T-lymphocyte responses, we obtained T cells from PBMC by negative selection. As shown in Fig 4, cultures of pure T cells were as sensitive to the effects of acrolein as cultures containing macrophages. Acrolein inhibited production of IL-2, TNF-a, and GM-CSF with an IC50 of <2.5 mmol/L, whereas IL-8 was again more resistant. Treatment with 0.1-mmol/L to 5-mmol/L acrolein did not affect T-cell viability, but 10-mmol/L acrolein decreased viability to 60%. These data suggest that acrolein can inhibit T-lymphocyte cytokine production through a mechanism that does not require accessory cells. Acrolein inhibits cytokine gene expression The mechanism by which acrolein inhibits cytokine production is as yet unknown. To determine the molecular level at which acrolein inhibited cytokine production, we isolated mRNA from PBMC treated with acrolein. As can be seen in Fig 5, the acrolein dose of 1 mmol/L to 2 mmol/L inhibited the induction of IL-2 and TNF-a mRNA. Furthermore, the level of mRNA correlated with the
Basic and clinical immunology
commercial cigarettes, from <1 mg and 26 mg. Even the Carlton cigarette produced sufficient acrolein to suppress cytokine production by >95%. CSE from Marlboro (Philip Morris USA Inc, Richmond, VA) cigarettes yielded 394 mmol/L 6 29 mmol/L acrolein, which represents the average tar content of commercial cigarettes (15 mg). Extracts of Marlboro UltraLights, Camel UltraLights, Winston Lights (R.J. Reynolds Tobacco Co.), and Winston Smooth (R.J. Reynolds Tobacco Co.) cigarettes all yielded between 311 mmol/L and 370 mmol/L acrolein and corresponding levels of the other aldehydes, indicating a lack of correlation between the purported lightness of the tobacco and the level of acrolein. To confirm further which compounds were responsible for cytokine suppression, we measured the levels of aldehydes that carried over from the CSE and compared them with the inhibition of IL-2 production. Undiluted Camel CSE was placed into 1 well of a 24-well plate as described in Methods. After a 3-hour incubation, the aldehyde levels in the neighboring wells were measured by HPLC. As can be seen in Table II, there was excellent correlation between the level of acrolein present and suppression of IL-2 production. In contrast, the amount of acetaldehyde that carried over to the nearest well (24 mmol/L) was not enough to affect cytokine production (Table I). Only trace amounts (<1 mmol/L) of crotonaldehyde, propionaldehyde, and butyraldehyde could be detected at a distance of 2.7 cm from the CSE. The effects of commercial grade acrolein on cytokine production were identical to those of the cigarette smoke extracts. Acrolein inhibited IL-1b and IL-2 in a dose-
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FIG 3. Acrolein inhibits production of PBMC IL-2, IL-1b, and IL-8. The data are expressed as the means 6 SEMs of 3 separate experiments. *Values significantly different from the positive control (Stim) at P < .05. NS, Nonstimulated.
FIG 4. Inhibition of IL-2, TNF-a, IL-8, and GM-CSF by acrolein in T lymphocytes. The data are expressed as the means 6 SEMs of 3 separate experiments. *Values significantly different from the positive control (Stim) at P < .05. NS, Nonstimulated.
Basic and clinical immunology
effects on cytokine protein levels, indicating that acrolein inhibits transcription of cytokine genes.
Protection against the immunosuppressive properties of CSE by N-acetylcysteine N-acetylcysteine scavenges reactive electrophilic species through the chemical reactivity of its thiol group. Therefore, we used N-acetylcysteine to test whether CSE compounds and acrolein react with thiol groups. One-hour pretreatment of PBMC with 5-mmol/L N-acetylcysteine completely protected cells against the inhibitory effects of the CSE diluted 1:100 (Fig 6, A) or 200-mmol/L acrolein (Fig 6, B). Whereas the CSE diluted 1:100 inhibited IL-2 production by 85%, pretreatment with N-acetylcysteine restored IL-2 production. In addition, pretreatment with
J ALLERGY CLIN IMMUNOL OCTOBER 2005
FIG 5. Acrolein inhibits IL-2 and TNF-a mRNA. The results are representative of 3 separate experiments; *P < .01. NS, Nonstimulated; Stim, stimulated.
FIG 6. Protection against immunosuppressive properties of CSE by N-acetylcysteine (NAC). PBMCs were pretreated with 5-mmol/L NAC and exposed to Camel smoke extracts diluted 1:100 (A) or 200-mmol/L acrolein (B) for 3 hours, or sham-treated, then stimulated with phorbol 12-myristate 13-acetate and anti-CD3. *P < .05. NS, Nonstimulated; Stim, stimulated.
N-acetylcysteine before acrolein addition restored IL-2 production to nearly 75%. N-acetylcysteine added to cells at the same time as acrolein was equally effective as the 1-hour pretreatment, but addition of N-acetylcysteine 30 minutes after acrolein treatment offered no protection. These data strongly suggest that the inhibitory effects of the CSE and acrolein depend on reactivity with critical cellular thiols.
DISCUSSION Cigarette smoking has long been known to suppress immune responses in the lungs and render smokers more susceptible to respiratory tract infections, including pneumonia. However, the specific immunosuppressive
compounds have never been definitively identified. We have conducted an extensive analysis of the immunosuppressive effects of cigarette smoke and have identified 2 classes of inhibitory compounds. The phenolic components in the particulate phase of cigarette smoke (phenol and catechol) inhibit cell cycle progression by activated T cells.23,24 However, they have very little effect on the production of inflammatory cytokines, which appears to be a hallmark of the immunosuppressive effects of cigarette smoke both in vitro and in vivo.15-20 Our studies reported here suggest that acrolein, which is present in very high concentrations in the vapor phase of all cigarettes, is a potent inhibitor of cytokine production by both macrophages and T cells. Only the a,b-unsaturated aldehydes exhibited this inhibitory activity; saturated aldehydes were inactive even at millimolar concentrations. The presence of the C=C olefin in an aldehyde dramatically increases its reactivity with cysteine, lysine, and histidine residues. Thus, it is not surprising that the inhibitory activity of the CSE and the unsaturated aldehydes could be completely abrogated by pretreating the cells with N-acetylcysteine. More importantly, the relative susceptibility of individual smokers to acrolein may depend on the levels of glutathione present in their lungs, and variations in lung glutathione levels are believed to be an important variable in lung cancer.25 Acrolein inhibits production of a wide variety of T-cell cytokines that are thought to be critical in pulmonary immunity to infectious agents and cancer, including IL-2, IL-4, IFN-g, GM-CSF, and TNF-a.26-32 However, production of IL-8 by T cells was notably more resistant to the effects of both cigarette smoke and acrolein. In fact, CSE and acrolein have been shown to induce IL-8 production in epithelial cells and neutrophils, and inhibition of IL-8 production by T cells was seen only with toxic doses of acrolein.33,34 These findings suggest that acrolein can account for both the influx of neutrophils and suppression of T-cell responses in lungs of smokers. The potency and volatility of acrolein could also account for the apparent immunosuppressive effects of passive smoke. A recent study of 4486 infants demonstrated a strong correlation between the incidence of severe respiratory tract infections and the proximity of the child to cigarette smoke while nursing.35 In addition, children infected with Mycobacterium tuberculosis are 5 times more likely to develop active pulmonary tuberculosis if exposed to tobacco smoke.36 At a respiration rate of 2 to 4 L/min, a child could theoretically receive an immunosuppressive dose of acrolein (;10-30 mg) from a 1-hour exposure to the levels of second-hand smoke typically found in restaurants.37 Further research is therefore necessary to identify biomarkers of acrolein exposure and to assess the pharmacodynamics of acrolein exposure.
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31. Woolard MD, Hodge LM, Jones HP, Schoeb TR, Simecka JW. The upper and lower respiratory tracts differ in their requirement of IFN-g and IL-4 in controlling respiratory Mycoplasma infection and disease. J Immunol 2004;172:6875-83. 32. Wolf F, Michaud K, Anderson J, Urbansky K. Tuberculosis infection with rheumatoid arthritis and the effect of infliximab therapy. Arthritis Rheum 2004;50:372-9. 33. Finkelstein EI, Nardini M, van der Vleit A. Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease? Am J Physiol Lung Cell Mol Physiol 2001;281:L732-9. 34. Mio T, Romberger DJ, Thompson AB, Robbins RA, Heires A, Rennard SL. Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells. Am J Respir Crit Care Med 1997;155:1770-6. 35. Blizzard L, Ponsonby AL, Dwyer T, Venn A, Cochrane JA. Parental smoking and infant respiratory infection: how important is not smoking in the same room with the baby? Am J Public Health 2003;93:482-8. 36. Altet MN, Alcaide J, Plans P, Taberner JL, Salto E, Folguera LI, et al. Passive smoking and risk of pulmonary tuberculosis in children immediately following infection: a case-control study. Tuber Lung Dis 1996; 77:537-44. 37. Fischer T, Weber A, Grandjean E. Air pollution due to tobacco smoke in restaurants. Int Arch Occup Environ Health 1978;41:267-80.
Basic and clinical immunology
Background: Cigarette smoking inhibits T-cell responses in the lungs, but the immunosuppressive compounds have not been fully identified. Cigarette smoke extracts inhibit IL-2, IFN-g, and TNF-a production in stimulated lymphocytes obtained from peripheral blood, even when the extracts were diluted 100-fold to 1000-fold. Objective: The objective of these studies was to identify the immunosuppressive compounds found in cigarette smoke. Methods: Gas chromatography/mass spectroscopy and HPLC were used to identify and quantitate volatile compounds found in cigarette smoke extracts. Bioactivity was measured by viability and production of cytokine mRNA and protein levels in treated human lymphocytes. Results: The vapor phase of the cigarette smoke extract inhibited cytokine production, indicating that the immunosuppressive compounds were volatile. Among the volatile compounds identified in cigarette smoke extracts, only the a,b-unsaturated aldehydes, acrolein (inhibitory concentration of 50% [IC50] = 3 mmol/L) and crotonaldehyde (IC50 = 6 mmol/L), exhibited significant inhibition of cytokine production. Although the levels of aldehydes varied 10-fold between high-tar (Camel) and ultralow-tar (Carlton) extracts, even ultralow-tar cigarettes produced sufficient levels of acrolein (34 mmol/L) to suppress cytokine production by >95%. We determined that the cigarette smoke extract inhibited transcription of cytokine genes. The inhibitory effects of acrolein could be blocked with the thiol compound N-acetylcysteine. Conclusion: The vapor phase from cigarette smoke extracts potently suppresses cytokine production. The compound responsible for this inhibition appears to be acrolein. (J Allergy Clin Immunol 2005;116:916-22.)
Basic and clinical immunology
From athe Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado at Denver and Health Sciences Center; and bthe Department of Pathology and Laboratory Medicine, Albany Medical Center. c These two authors contributed equally to this work. Supported by grant ES05673 from the National Institute of Environmental Health Sciences, National Institutes of Health, and by a grant from Philip Morris USA Inc and Philip Morris International. Disclosure of potential conflict of interest: B. Freed has received grants/research support from Philip Morris USA Inc and Philip Morris International. Received for publication February 25, 2005; revised May 24, 2005; accepted for publication May 31, 2005. Available online August 1, 2005. Reprint requests: Brian M. Freed, PhD, Division of Allergy and Clinical Immunology, University of Colorado at Denver and Health Sciences Center, Campus Box B164, 4200 E Ninth Avenue, Denver, CO 80262. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.046
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Key words: Cigarette smoke, T cell, activation, cytokine, gene expression, acrolein
There is a strong correlation between cigarette smoking and the incidence and severity of pulmonary infections, including pneumococcal and varicella pneumonias,1-5 Legionnaires disease,6,7 influenza,7 and tuberculosis.8-10 In addition, Miguez-Burbano et al11 recently reported that smoking doubled the risk of developing Pneumocystis carinii pneumonia among HIV-infected patients. Although these phenomena may involve several physiological processes, the major factor is likely to be the profound suppression of T-cell responses observed in smokers’ lungs.12-14 We and others have shown that cigarette smoking suppresses the mitogenic responses of lung lymphocytes and the production of IL-1b, IL-6, and TNF-a by PBMCs and alveolar macrophages.15-20 The specific compounds in cigarette smoke that elicit these effects have not been identified. Recently, we demonstrated that extracts from a single high-tar (Camel Ultralights; R.J. Reynolds Tobacco Co., Winston-Salem, NC) cigarette significantly inhibited production of IL-2 and IFN-g by human peripheral blood lymphocytes, even when the extracts were diluted 100fold to 1000-fold.15 The inhibitory effect was observed even in extracts from ultralow-tar cigarettes containing >1 mg tar per cigarette, and nicotine had no effect on these responses even at millimolar concentrations. These observations suggested the presence of potent immunosuppressive compounds in cigarettes, and the purpose of this study was to identify them. The fact that cigarette smoke is composed of more than 5000 chemicals21 would appear to make identification of individual immunosuppressive compounds nearly impossible. However, identification was greatly facilitated by our discovery that the vapor from cigarette smoke extracts exhibited the same immunosuppressive activity as the whole extract. We therefore used gas chromatography/mass spectroscopy (GC/MS) and HPLC to identify the volatile immunosuppressive components in cigarette smoke extracts.
METHODS Preparation of cigarette smoke extracts and chemicals Cigarette smoke extracts (CSEs) were freshly prepared as described previously.15 Acrolein, crotonaldehyde, acetaldehyde, butyraldehyde, and propionaldehyde (Acros Organics, Pittsburgh, Pa) were prepared in PBS. Toluene, xylene, styrene, and limonene
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Abbreviations used CSE: Cigarette smoke extract GC/MS: Gas chromatography/mass spectroscopy IC50: Inhibitory concentration of 50%
(Sigma, St Louis, Mo) were prepared in absolute ethanol just before their addition to the cultures.
Human PBMC and lymphocyte culture Peripheral blood was drawn from healthy, nonsmoking adult volunteers after informed consent. PBMCs were isolated and cultured as described previously.15 T cells were isolated from PBMC by negative selection using a pan–T-cell isolation kit II (Miltenyi, Auburn, Calif). T-cell purity was monitored by a FACScan analyzer (Becton Dickinson, Mountain View, Calif) using antibodies specific for CD3, CD4, CD8, and CD45 and was consistently >90% CD31. PBMC (106/mL) or T cells (5 3 105/mL) from individual donors were cultured in 24-well tissue culture plates (Costar, Corning, NY) at 37°C in 5% CO2. The cells were pretreated for 3 hours with 1 mL CSE, bubbled through RPMI or reagent grade aldehydes as indicated, and then stimulated with 10 ng/mL anti-CD3 (Orthoclone OKT3, Orthobiotec, Raritan, NJ) plus 10 nmol/L phorbol 12-myristate 13acetate for 24 hours. Untreated lymphocytes were cultured on a plate separate from the CSE or aldehyde-treated samples to prevent carryover inhibition. Cells from 1 individual were used per experiment, and each experiment was replicated a minimum of 3 times. The University of Colorado Combined Institutional Review Board approved the human subject protocol used in this study.
GC/MS Gas chromatography/mass spectroscopy analysis of CSE vapor phase was performed with a Varian 3800 Gas Chromatograph (Varian Chromatography Systems, Walnut Creek, Calif) equipped with a Varian 1079 injector, Rtx-1MS capillary column (15 m, 0.25 mm internal diameter, 0.5 mm film thickness; Restek, Bellefonte, Pa) and an 8200 Varian autosampler fitted with a 12 position carrousel for headspace vials. Helium was used as the gas chromatography carrier at a flow rate of 1 mL/min. A column temperature program with an initial 5-minute hold at 40°C, and a temperature ramp from 40°C to 90°C (10°C/min) was used. The injector temperature was maintained at 80°C, and injections of headspace samples were performed in a split mode (1:30). Mass spectral detection was performed with a Varian Saturn 2000 mass spectrometer fitted with a SilChrom ion trap maintained at 200°C. A 10-mAmp filament current was used for electron ionization, and the mass spectrum was scanned from 35 to 250 m/z. The GC/MS system and data management were controlled by Saturn GC/MS Workstation software (version 5.2). Analytical samples (3 mL) were pipetted into 10-mL reaction vials and crimp-sealed with aluminum pressure seals fitted with polytetrafluoroethylene/silicone liners (Supelco, Bellefonte, Pa). For storage or shipment, the samples were maintained at 210°C. After ambient temperature equilibration for at least 60 minutes, 100-mL samples of the headspace over the liquid samples were analyzed by GC/MS as described.
vigorously and allowed to react for 30 minutes at room temperature, after which the reaction was stopped by the addition of 10 mL pyridine injected through the vial septum. Samples were stored at 220°C for less than 1 week. Samples were chromatographed on 3.9-mm 3 150-mm Nova-Pak C18 columns (Waters, Milford, Mass) with a Nova-Pak C18 guard column (Waters) and monitored at 360 nm with a Waters 490E UV detector. Samples of 10 mL or 100 mL were injected by using a Waters 717 autosampler and chromatographed with a flow rate of 1.5 mL/min in a mobile phase of 100% buffer A (60% water/30% acetonitrile/10% tetrahydrofuran vol/vol/vol) for 1 minute, followed by a linear gradient of 100% buffer A to 100% buffer B (40% water/60% acetonitrile vol/vol) over a period of 10 minutes. The identity of each aldehyde was confirmed by comparison of retention times to 2,4-dinitrophenylhydrazine– aldehyde standards (Waters). Concentrations of the aldehydes were determined by the area under the curve method in comparison with dilutions of the known standards using Waters Empower software (Build 1154).
HPLC analysis of cigarette aldehydes
Assays of lymphocyte function
The levels of aldehydes in CSE were determined by C18 reversephase HPLC after derivatization with 2,4-dinitrophenylhydrazine. Within 5 minutes of collection, 80 mL of the CSE were derivatized with 120 mL of 10-mmol/L 2,4-dinitrophenylhydrazine (Acros, Fairlawn, NJ) and placed in sealed vials. The samples were shaken
Cell viability was monitored by fluorescence-activated cell sorting analysis of 7-aminoactinomycin D (Molecular Probes) exclusion per company protocol.22 The levels of IL-1b, IL-2, IL-6, IL-8, IL-10, IFN-g, TNF-a, and GM-CSF in the culture supernatants were measured by Luminex bead human cytokine multiplex kit
Basic and clinical immunology
FIG 1. Inhibition of PBMC cytokine production by vapors from CSEs. CSE represents cells treated directly with 1 mL of a cigarette smoke extract prepared from 1 Camel cigarette. Data represent the means 6 SEMs of at least 3 separate experiments. *P < .05 compared with stimulated (Stim); NS, nonstimulated.
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FIG 2. GC/MS and HPLC analysis of Camel CSE. Left, GC/MS peaks eluting in the first 2 minutes were aldehydes. Right, HPLC analysis of Camel extracts after conjugation to 2,4-dinitrophenylhydrazine. Peaks were identified as (1) formaldehyde, (2) acetaldehyde, (4) acrolein, (5) acetone, (7) propionaldehyde, (8) crotonaldehyde, and (10) butyraldehyde. Peaks 3, 6, and 9 could not be identified.
(BioSource International, Camarillo, Calif) according to the manufacturer’s instructions. Standard curves with serial dilutions of human recombinant cytokine standards were performed in each assay, and data were analyzed by 5-parameter regression with StatLia Enterprise software, version 3.1 (Brendan Scientific, Carlsbad, Calif). TNF-a and IL-2 steady-state mRNA levels were measured by Quantikine mRNA quantitation assays according to the manufacturer’s instructions (R&D Systems, Inc, Minneapolis, Minn). Total RNA was isolated by a QIAGEN RNeasy mini kit (Valencia, Calif) according to the manufacturer’s instructions.
Statistical analysis The mean 6 SEM was determined for each treatment group in the individual experiments, performed a minimum of 3 times. To establish statistically significant P values for cytokine measurements (P < .05), the values obtained from the treatment groups were converted to percent control (stimulated group) and evaluated by 1-way ANOVA and Dunnett multiple comparison test. This method allowed us to account for significant variances among donors.
Basic and clinical immunology
RESULTS Inhibition of cytokine production by the vapor phase of cigarette smoke extracts As can be seen in Fig 1, direct exposure of human PBMC to 1 mL of the CSE inhibited production of IL-1b, IL-2, GM-CSF, IL-6, IFN-g, IL-8, and TNF-a human PBMC by >99%. This inhibitory effect did not require direct exposure of the cells to CSE. Rather, the inhibitory effect carried over in the vapor phase to untreated cells in neighboring wells. Wells closest to the CSE exhibited the greatest level of suppression, whereas those farther away on the plate exhibited progressively less inhibition. The CSE was toxic to PBMC when placed in direct contact, but cells >4 cm away exhibited no toxicity despite 75% to 95% inhibition of cytokine production. All of the cytokines showed similar sensitivity to the extracts except IL-8, which was noticeably more resistant to the effects of CSE. Analysis of vapor phase from cigarette smoke extracts The fact that the inhibitory effects of CSE carried over to neighboring wells indicated that the immunosuppressive compounds were highly volatile. This hypothesis was
verified by the fact that lyophilization completely removed the inhibitory activity from CSE. We therefore collected the CSE vapor phase and placed it in sealed glass vials. The vapor phase was then sampled at 37°C by using a needle and subjected to GC/MS analysis (Fig 2, left). Among the peaks identified, those in the highest concentrations were toluene, xylene, styrene, and limonene, along with numerous aldehydes in the peaks eluting in <2 minutes. Because GC/MS was not able to resolve and quantitate the aldehydes completely, CSE was reanalyzed by C18 reverse-phase HPLC after conjugation of the carbonyl groups to 2,4-dinitrophenylhydrazine, which enhanced the limit of detection to 1 to 3 mmol/L. As can be seen in Fig 2 (right), 10 peaks were detected in CSE from Camel cigarettes. Table I summarizes the major compounds measured in CSE, which include high levels of acetaldehyde, acetone, and acrolein, followed by significantly lower amounts of propionaldehyde, crotonaldehyde, and butyraldehyde.
Assessment of suppressive activity of vapor phase constituents To assess the cytokine inhibitory activity of CSE, we determined the dose of the cigarette extract compounds that inhibited production of IL-2 by 50% (IC50; Table I). Toluene, xylene, styrene, and limonene did not suppress IL-2 production in vitro, even at doses as high as 2 mmol/L. The aldehydes segregated into 2 distinct groups: the a,b-unsaturated aldehydes (acrolein and crotonaldehyde) were potent inhibitors of cytokine production, whereas acetone and the saturated aldehydes (acetaldehyde, butyraldehyde, and propionaldehyde) were essentially inactive (Table I). Acrolein and crotonaldehyde inhibited IL-2 with an IC50 of 3 and 6 mmol/L, respectively, levels well in the range of 1 cigarette. Even though acetaldehyde was present at very high levels (1352 mmol/L), it did not inhibit IL-2 production. These observations suggest that, on the basis of the levels present and the relative inhibitory effects, acrolein and crotonaldehyde are the predominant inhibitors of cytokine production in the CSE vapor. Cigarette smoke extract derived from unfiltered Carlton (Brown and Williamson Tobacco Corp, Louisville, Ky) and filtered Camel represented the range of tar content in
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TABLE I. Effects of cigarette smoke aldehydes on cytokine production Aldehyde levels (mmol/L)* Aldehyde
Camel
Acrolein CH2=CHCHO Crotonaldehyde CH3CH=CHCHO Acetone CH3COCH3 Acetaldehyde CH3CHO Propionaldehyde CH3CH2CHO Butyraldehyde CH3CH2CH2CHO
503 101 507 1352 115 49
6 6 6 6 6 6
21 12 24 63 15 8
Carlton
IC50 (mmol/L)y
6 6 6 6 6 6
3 6 500 1500 >2000 >2000
34 2 48 111 9 4
5 1 8 15 1 1
*Mean level from 10 experiments detected in extracts prepared from 1 cigarette bubbled through 10 mL PBS. IC50 is the dose that inhibited IL-2 production by 50%.
TABLE II. Aldehyde levels from CSE detected in carryover wells Acrolein (mmol/L)
Initial level Straight* 1.4 cm* 2.7 cm* 4.1 cm* 5.4 cm* 6.8 cm* 8.1 cm*
487 101 33 17 9 5 4 1
6 6 6 6 6 6 6 6
61 36 6 2 2 1 2 1
Crotonaldehyde (mmol/L)
81 24 3 1
6 6 6 6 0 0 0 0
11 5 2 1
Acetaldehyde (mmol/L)
Butyraldehyde (mmol/L)
Propionaldehyde (mmol/L)
6 6 6 6 6 0 0 0
36 6 3 661 161 0 0 0 0 0
88 6 8 24 6 24 362 0 0 0 0 0
1279 140 24 9 1
173 53 9 4 1
IL-2y
1 1 1 4 35 51 89 109
6 6 6 6 6 6 6 6
1 1 1 4 22 16 36 41
*Mean 6 SEM levels detected after 3-h exposure from 3 experiments. IL-2 levels displayed as percent stimulated.
dependent manner but had much less effect on production of IL-8 (Fig 3). Acrolein also inhibited production of GM-CSF, IL-6, IL-10, and IFN-g with dose-response curves similar to that of IL-2 (data not shown).
Effects of acrolein on T-cell responses IL-1b and IL-2 are produced by macrophages and T cells, respectively. Because IL-1b also promotes T-cell activation and IL-2 production, inhibition of the latter could have been a result of an indirect effect of the macrophages. To determine whether acrolein directly affected T-lymphocyte responses, we obtained T cells from PBMC by negative selection. As shown in Fig 4, cultures of pure T cells were as sensitive to the effects of acrolein as cultures containing macrophages. Acrolein inhibited production of IL-2, TNF-a, and GM-CSF with an IC50 of <2.5 mmol/L, whereas IL-8 was again more resistant. Treatment with 0.1-mmol/L to 5-mmol/L acrolein did not affect T-cell viability, but 10-mmol/L acrolein decreased viability to 60%. These data suggest that acrolein can inhibit T-lymphocyte cytokine production through a mechanism that does not require accessory cells. Acrolein inhibits cytokine gene expression The mechanism by which acrolein inhibits cytokine production is as yet unknown. To determine the molecular level at which acrolein inhibited cytokine production, we isolated mRNA from PBMC treated with acrolein. As can be seen in Fig 5, the acrolein dose of 1 mmol/L to 2 mmol/L inhibited the induction of IL-2 and TNF-a mRNA. Furthermore, the level of mRNA correlated with the
Basic and clinical immunology
commercial cigarettes, from <1 mg and 26 mg. Even the Carlton cigarette produced sufficient acrolein to suppress cytokine production by >95%. CSE from Marlboro (Philip Morris USA Inc, Richmond, VA) cigarettes yielded 394 mmol/L 6 29 mmol/L acrolein, which represents the average tar content of commercial cigarettes (15 mg). Extracts of Marlboro UltraLights, Camel UltraLights, Winston Lights (R.J. Reynolds Tobacco Co.), and Winston Smooth (R.J. Reynolds Tobacco Co.) cigarettes all yielded between 311 mmol/L and 370 mmol/L acrolein and corresponding levels of the other aldehydes, indicating a lack of correlation between the purported lightness of the tobacco and the level of acrolein. To confirm further which compounds were responsible for cytokine suppression, we measured the levels of aldehydes that carried over from the CSE and compared them with the inhibition of IL-2 production. Undiluted Camel CSE was placed into 1 well of a 24-well plate as described in Methods. After a 3-hour incubation, the aldehyde levels in the neighboring wells were measured by HPLC. As can be seen in Table II, there was excellent correlation between the level of acrolein present and suppression of IL-2 production. In contrast, the amount of acetaldehyde that carried over to the nearest well (24 mmol/L) was not enough to affect cytokine production (Table I). Only trace amounts (<1 mmol/L) of crotonaldehyde, propionaldehyde, and butyraldehyde could be detected at a distance of 2.7 cm from the CSE. The effects of commercial grade acrolein on cytokine production were identical to those of the cigarette smoke extracts. Acrolein inhibited IL-1b and IL-2 in a dose-
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FIG 3. Acrolein inhibits production of PBMC IL-2, IL-1b, and IL-8. The data are expressed as the means 6 SEMs of 3 separate experiments. *Values significantly different from the positive control (Stim) at P < .05. NS, Nonstimulated.
FIG 4. Inhibition of IL-2, TNF-a, IL-8, and GM-CSF by acrolein in T lymphocytes. The data are expressed as the means 6 SEMs of 3 separate experiments. *Values significantly different from the positive control (Stim) at P < .05. NS, Nonstimulated.
Basic and clinical immunology
effects on cytokine protein levels, indicating that acrolein inhibits transcription of cytokine genes.
Protection against the immunosuppressive properties of CSE by N-acetylcysteine N-acetylcysteine scavenges reactive electrophilic species through the chemical reactivity of its thiol group. Therefore, we used N-acetylcysteine to test whether CSE compounds and acrolein react with thiol groups. One-hour pretreatment of PBMC with 5-mmol/L N-acetylcysteine completely protected cells against the inhibitory effects of the CSE diluted 1:100 (Fig 6, A) or 200-mmol/L acrolein (Fig 6, B). Whereas the CSE diluted 1:100 inhibited IL-2 production by 85%, pretreatment with N-acetylcysteine restored IL-2 production. In addition, pretreatment with
J ALLERGY CLIN IMMUNOL OCTOBER 2005
FIG 5. Acrolein inhibits IL-2 and TNF-a mRNA. The results are representative of 3 separate experiments; *P < .01. NS, Nonstimulated; Stim, stimulated.
FIG 6. Protection against immunosuppressive properties of CSE by N-acetylcysteine (NAC). PBMCs were pretreated with 5-mmol/L NAC and exposed to Camel smoke extracts diluted 1:100 (A) or 200-mmol/L acrolein (B) for 3 hours, or sham-treated, then stimulated with phorbol 12-myristate 13-acetate and anti-CD3. *P < .05. NS, Nonstimulated; Stim, stimulated.
N-acetylcysteine before acrolein addition restored IL-2 production to nearly 75%. N-acetylcysteine added to cells at the same time as acrolein was equally effective as the 1-hour pretreatment, but addition of N-acetylcysteine 30 minutes after acrolein treatment offered no protection. These data strongly suggest that the inhibitory effects of the CSE and acrolein depend on reactivity with critical cellular thiols.
DISCUSSION Cigarette smoking has long been known to suppress immune responses in the lungs and render smokers more susceptible to respiratory tract infections, including pneumonia. However, the specific immunosuppressive
compounds have never been definitively identified. We have conducted an extensive analysis of the immunosuppressive effects of cigarette smoke and have identified 2 classes of inhibitory compounds. The phenolic components in the particulate phase of cigarette smoke (phenol and catechol) inhibit cell cycle progression by activated T cells.23,24 However, they have very little effect on the production of inflammatory cytokines, which appears to be a hallmark of the immunosuppressive effects of cigarette smoke both in vitro and in vivo.15-20 Our studies reported here suggest that acrolein, which is present in very high concentrations in the vapor phase of all cigarettes, is a potent inhibitor of cytokine production by both macrophages and T cells. Only the a,b-unsaturated aldehydes exhibited this inhibitory activity; saturated aldehydes were inactive even at millimolar concentrations. The presence of the C=C olefin in an aldehyde dramatically increases its reactivity with cysteine, lysine, and histidine residues. Thus, it is not surprising that the inhibitory activity of the CSE and the unsaturated aldehydes could be completely abrogated by pretreating the cells with N-acetylcysteine. More importantly, the relative susceptibility of individual smokers to acrolein may depend on the levels of glutathione present in their lungs, and variations in lung glutathione levels are believed to be an important variable in lung cancer.25 Acrolein inhibits production of a wide variety of T-cell cytokines that are thought to be critical in pulmonary immunity to infectious agents and cancer, including IL-2, IL-4, IFN-g, GM-CSF, and TNF-a.26-32 However, production of IL-8 by T cells was notably more resistant to the effects of both cigarette smoke and acrolein. In fact, CSE and acrolein have been shown to induce IL-8 production in epithelial cells and neutrophils, and inhibition of IL-8 production by T cells was seen only with toxic doses of acrolein.33,34 These findings suggest that acrolein can account for both the influx of neutrophils and suppression of T-cell responses in lungs of smokers. The potency and volatility of acrolein could also account for the apparent immunosuppressive effects of passive smoke. A recent study of 4486 infants demonstrated a strong correlation between the incidence of severe respiratory tract infections and the proximity of the child to cigarette smoke while nursing.35 In addition, children infected with Mycobacterium tuberculosis are 5 times more likely to develop active pulmonary tuberculosis if exposed to tobacco smoke.36 At a respiration rate of 2 to 4 L/min, a child could theoretically receive an immunosuppressive dose of acrolein (;10-30 mg) from a 1-hour exposure to the levels of second-hand smoke typically found in restaurants.37 Further research is therefore necessary to identify biomarkers of acrolein exposure and to assess the pharmacodynamics of acrolein exposure.
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Basic and clinical immunology