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Originally published online as doi:10.1189/jlb.1202626 on August 1, 2003

Published online before print August 1, 2003
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(Journal of Leukocyte Biology. 2003;74:942-951.)
© 2003 by Society for Leukocyte Biology

Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-{kappa}B

Sumiko Iho*,1, Yukie Tanaka{dagger}, Rumiko Takauji{ddagger}, Chino Kobayashi§, Ikunobu Muramatsu, Hiromichi Iwasaki**, Kishiko Nakamura{dagger}{dagger}, Yutaka Sasaki{ddagger}{ddagger}, Kazuwa Nakao{ddagger}{ddagger} and Takayuki Takahashi§§,2

* Department of Immunology and Medical Zoology,
{dagger} Forensic Medicine,
§ Anesthesiology & Reanimatology, and
Pharmacology,
{dagger} Central Research Laboratories, and
** Division of Transfusion Medicine, Fukui Medical University;
{dagger}{dagger} College of Medical Technology, Kyoto University;
{ddagger}{ddagger} Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine; and
§§ Department of Hematology and Clinical Immunology, Kobe City General Hospital

1 Correspondence: S. Iho, Department of Immunology and Medical Zoology, Faculty of Medicine, Fukui Medical University, 23 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan. E-mail: ihosumik{at}fmsrsa.fukui-med.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL-8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil-IL-8 production in vitro; the causative role of NF-{kappa}B in its production, in association with the possible production of reactive oxygen intermediates that activate NF-{kappa}B; and the nicotinic acetylcholine receptors (nAChRs) involved in IL-8 production. Nicotine stimulated neutrophils to produce IL-8 in both time- and concentration-dependent manners with a 50% effective concentration of 1.89 mM. A degradation of I{kappa}B-{alpha}/ß proteins and an activity of NF-{kappa}B p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, n{omega}-Nitro-L-arginine methyl ester, prevented nicotine-induced IL-8 production, with an entire abrogation of DHR oxidation, I{kappa}B degradation, and NF-{kappa}B activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF-{kappa}B activity, thereby producing IL-8. The nAChRs seemed to be involved in IL-8 production. In smokers, blood IL-8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil-IL-8 production via nAChR by generating peroxynitrite and subsequent NF-{kappa}B activation, and the IL-8 appears to contribute to leukocytosis in tobacco smokers.

Key Words: chemokine • reactive oxygen intermediates • leukocytosis • tobacco smoker • I{kappa}B


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tobacco smoking exerts many effects, the majority of which are unfavorable, on almost all tissues or organs, including the respiratory, cardiovascular, nervous, endocrine, and gastrointestinal systems [1 ]. As far as the effect of smoking on the hematopoietic system is concerned, leukocytosis has been well recognized [2 3 4 ]. The leukocytosis includes neutrophilia, lymphocytosis, and, in a certain analysis [3 , 4 ], monocytosis. The cause of the leukocytosis, however, has not yet been clarified. One of the reasons may be due to the fact that it remains difficult to account for such a pattern of leukocytosis in smokers from the in vivo biologic action of a single hematopoietic cytokine. However, interleukin (IL)-8, the prototypic CXC chemokine, attracts neutrophils and T lymphocytes [5 , 6 ], and causes mild to moderate neutrophilia [7 , 8 ] and possibly lymphocytosis through enhanced movement of these leukocytes when administered to experimental animals. Regarding cigarette smoking, it has been shown, in an animal study, that an accelerated release of neutrophils from the bone marrow leads to leukocytosis [9 ]. Therefore, IL-8 seems to be a possible candidate cytokine responsible for the leukocytosis in smokers.

IL-8 is produced by leukocytic and nonleukocytic cells. Among these cells, neutrophils produce a very small quantity of IL-8, but when stimulated with IL-1, IL-15, tumor necrosis factor (TNF)-{alpha}, or lipopolysaccharide (LPS), they produce large amounts of IL-8 [10 ]. On the other hand, while smoking tobacco, it is likely that neutrophils circulating in the blood vessels of the respiratory tracts are exposed to high concentrations of nicotine, a kind of alkaloid and a major substance in tobacco smoke. If nicotine stimulates these neutrophils to produce IL-8, leukocytosis in smokers could largely be explained by this mechanism because of the largest cell population of neutrophils in the circulating white cells. However, such studies have yet to be conducted by any investigators. Therefore, in this study, we examined the effect of nicotine on neutrophil IL-8 production in vitro and investigated the mechanisms of possible nicotine-induced IL-8 production. We also examined blood IL-8 levels in smokers and nonsmokers and the relationship between IL-8 levels and the smoking habits, to explore the possible involvement of IL-8 in smokers’ leukocytosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil culture
Neutrophils were separated from citrated venous blood of healthy volunteers, with informed consent, by a two-step gradient centrifugation with specific gravities of 1.076 and 1.090 g/ml. The cell preparation contained >98% of neutrophils; and >99% of the cells were viable. Neutrophils were suspended, unless otherwise indicated, in RPMI-1640 (Sigma, St. Louis, MO) with 2% heat-inactivated fetal calf serum (FCS) (Equitech-Bio, Inc., Ingram, TX; endotoxin <0.03 ng/ml), and cultured in 96-well flat-bottom plates (Corning, Corning, NY) in triplicate at 37°C with 5% CO2, under the indicated conditions.

Reagents
The reagents added to the culture are described in the Results section. They were purchased from Sigma, except vecuronium ((+)-1-(3{alpha},17ß-diacetoxy-2ß-piperidino-5{alpha}-androstan-16ß-yl)-1-methylpiperidinium, Sankyo Pharmaceutical Company, Tokyo, Japan), mouse anti-human IL-8 monoclonal antibody (mAb) (Genzyme, Minneapolis, MN), and purified mouse myeloma immunoglobulin (Ig)G1{kappa} (ICN, Costa Mesa, CA). Nicotine was neutralized to pH 7.2 with HCl.

Detection of cytokine/chemokine, reactive oxygen intermediates (ROIs), nitric oxide (NO), and myeloperoxidase (MPO) production
Cytokine/chemokine concentrations of blood plasma and cell-free culture supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Camarillo, CA or TFB, Inc. Tokyo, Japan). Superoxide anion (O2-) was monitored by the reduction of extracellular cytochrome c [11 ]. Hydrogen peroxide (H2O2) was detected using Amplex® Red Hydrogen Peroxide Assay Kit (Molecular Probes, Eugene, OR). Oxidation of dihydrorhodamine 123 (DHR) [12 ] was measured using a flow cytometer and ONOO- production was assessed with the inhibition test using NO synthase (NOS) inhibitor [13 ]. Regarding NO, neutrophils were cultured in Dulbecco’s modified Eagle’s medium with 2% FCS, and nitrite concentrations of the culture supernatants were measured by the Salzman method [14 ]. The amount of MPO was detected using BIOXYTECH® MPO-EIA (Oxis ResearchTM, Portland, OR).

Reverse transcriptase polymerase chain reaction (RT-PCR)
One microgram of total ribonucleic acid (RNA) was subjected to one-step RT-PCR using Ready-To-Go RT-PCR Beads (Amersham, Arlington Heights, IL) with specific primers for IL-8 or ß-actin (Toyobo, Tokyo, Japan), under predetermined conditions. The sequences of the IL-8 primers were 5'-ATGACTTCCAAGCTGGCCGTGGCT3' (sense) and 5'-TCTCAGCCCTCTTCAAAAACTTCT3' (antisense), and those of ß-actin were 5'-TCACCAACTG GGACGACATGGAG3' (sense) and 5'-CTCCTTAATGTCACGCACGATTTC3' (antisense). Amplified PCR products (27 cycles) were electrophoresed on 5% polyacrylamid gels. The gels were stained with ethidium bromide and photographed.

Western blotting
Thirty micrograms of whole cell lysates were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore Co., Bedford, MA). The transblotted membranes were stained with rabbit polyclonal antibody against inhibitory nuclear factor-{kappa}B (I{kappa}B)-{alpha} or I{kappa}B-ß (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by incubation with a horseradish peroxidase-conjugated anti-rabbit Ig (Amersham); the bands were developed with enhanced chemiluminescence reagents (Amersham) and exposed to film.

Migration assay
A two-hour migration assay was conducted in a trans-well (Coster transwell, 3 µm pore) placing 100,000 neutrophils suspended in RPMI-1640 with 0.1% bovine serum albumin in the upper chamber. The cells which had migrated to the lower chamber were counted.

Measurement of the activity of nuclear factor (NF)-{kappa}B p65 and p50
Neutrophils were treated with nicotine, TNF-{alpha}, or medium alone under the conditions described in the Results section, and the nuclear extract was prepared. NF-{kappa}B activities were assayed using the TransAM NF{kappa}B p50 and p65 Kits (Active Motif, Carlsbad, CA) according to the manufactures’ instruction.

Statistical analysis
Statistical analyses were performed using Student’s t test for parametric data, the Mann-Whitney U test for nonparametric data, and Spearman’s rank correlation coefficient for analysis of correlation. The best fitting regression curve, with its correlation coefficient squared, was drawn using Microsoft® Excel 2002 analysis software. Fifty percent effective concentration (EC50) and 50% inhibiting concentration (IC50) were calculated using curve-fitting program GraphPad Prism 2.0 (GraphPad, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nicotine induces neutrophils to produce IL-8
Nicotine ([-]-1-methyl-2-[3-pyridyl] pyrrolidine) induced neutrophils from healthy non-smokers to produce IL-8 in both a dose- and time-dependent manner (Fig. 1 ). The minimum effect of nicotine was observed at 0.1 mM, and the concentrations needed to induce maximum IL-8 release ranged from 2.5 to 3.0 mM, with an EC50 of 1.89 ± 0.37 mM (mean±standard deviation (SD), n=6). The maximum amount produced by nicotine was equivalent to that produced by 10 ng/ml LPS. An obvious increase in nicotine-induced IL-8 production was observed from 3 h of culture. Nicotine-stimulated neutrophils did not produce TNF-{alpha} or IL-1ß, and the addition of polymyxin B (LPS inhibitor) did not alter the nicotine effect (data not shown), indicating that nicotine directly triggers the production of IL-8 in neutrophils. Freshly isolated monocytes were unresponsive to nicotine in producing IL-8 (data not shown).



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  		Figure 1. Nicotine induces IL-8 production by neutrophils in dose- and time-dependent manners. Neutrophils (3x106/ml) were cultured for 16 h with (A) varying concentrations of nicotine, or (B) for various periods with or without 3 mM nicotine. Values shown are representative of (A) six and (B) three independent experiments with neutrophils from different donors, and express the mean ± SD in triplicate cultures. * and **: significant at p < 0.05 and p < 0.01, respectively, compared with the respective controls.

 
Nicotine-induced autocrine IL-8 enhances chemokinetic migration
Nicotine, at ~30 µM, is chemotactic for neutrophils [15 ]. Because high concentrations of chemotactic substances may produce chemokinesis, we examined whether nicotine, at the concentrations effective for IL-8 induction, causes chemotactic or chemokinetic movement in neutrophils and whether it is dependent on autocrine IL-8. In a transwell migration assay, nicotine potently promoted neutrophil migration, which was equally observed in whichever chamber, the upper or the lower, the nicotine was placed, and the enhanced migration was completely abrogated by addition of anti-IL-8 mAb (Table 1 ). It appears that nicotine induces chemokinetic activity in neutrophils and the enhanced migration is mediated by nicotine-induced autocrine IL-8. In addition, these results suggest that nicotine-induced IL-8 production occurs within 2 h at levels undetectable by ELISA but enough for a quick response in an autocrine system.


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  		Table 1. Nicotine Enhances Chemokinetic Migration of Neutrophilsa

 
Nicotine stimulates de novo synthesis of IL-8 in neutrophils
Since neutrophils released a small quantity of IL-8 even in cultures with medium alone, we investigated whether IL-8 production induced by nicotine was ascribed to a release of a preformed product or to a de novo synthesis of IL-8. The treatment of neutrophils with actinomycine D or cyclohexamide completely abrogated the effect of the nicotine (data not shown), and the induction of mRNA for IL-8 was detected by RT-PCR in neutrophils cultured for 2 h with nicotine at 1 and 2 mM (Fig. 2 ). These results indicate that nicotine stimulates de novo synthesis of IL-8 in neutrophils. In the experiments below, nicotine was used at 2-3 mM, to induce higher neutrophil responses, which allowed the data to be analyzed more accurately.



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  		Figure 2. Nicotine induces expression of mRNA for the IL-8 gene in neutrophils. Neutrophils (5x106/ml) were cultured for 2 h with medium alone (lane 1), and 1 mM (lane 2) and 2 mM of nicotine (lane 3), and the total RNA was subjected to RT-PCR. Similar results were observed in three other donors’ neutrophils.

 
Nicotine induces the generation of ROIs, and ONOO- causes IL-8 production in neutrophils
The balance between the amounts of pro-oxidant and anti-oxidant is distorted in the blood of smokers [16 ]. The main source of blood free radicals in smokers is thought to be circulating neutrophils in the alveolar arterioles [17 ]. Since oxidative stress regulates IL-8 gene transcription [18 ], we hypothesized that nicotine induces ROI generation and this leads to IL-8 production in neutrophils. As expected, the IL-8 induction was abrogated in the presence of a thiol-containing antioxidant, N-acetyl-L-cystein (NAC) [19 ] (Fig. 3 ), indicating that ROIs are involved in nicotine-induced IL-8 production. Indeed, a rapid increase in the production of O2- was observed in nicotine-stimulated neutrophils in a 5-min culture and the amount exceeded the levels of spontaneous O2-, which was detected from 30 min after the initiation of the culture (Fig. 4 ). This indicates that the nicotine stimulation accelerates and intensifies the production of O2- in neutrophils. The effective doses of nicotine needed for the production of O2- were the same as those needed for the production of IL-8 (data not shown). Under the most biological conditions, O2- is very rapidly and spontaneously reduced to H2O2 [20 ]. In nicotine-stimulated neutrophils, twofold higher levels of H2O2 were detected (Table 2 ). However, the amounts detected at the maximum induction time, 1-2 h, were very low (<2 µM/5x106 cells/ml) compared with 10 mM of exogenous H2O2, which induced IL-8 at a comparable level to that induced by 2 mM nicotine in neutrophils (our observation).



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  		Figure 3. Antioxidants and NF-{kappa}B inhibitors suppress nicotine-induced IL-8 production. Neutrophils (2 to 5x106/ml) were cultured for 16 h with or without NAC (20 mM), L-alanine (50 mM), DMSO (1%), L-NAME (10 mM), PDTC (100 µM), MG-132 (10 µM), DEX (1 µM), or curcumin (20 µM), in the presence of medium or 2.5 mM nicotine. The effective concentrations of these reagents were determined in preliminary experiments. Values shown (mean±SD, n=3) are representative of experiments repeatedly performed with neutrophils from different donors. The inhibition of nicotine-induced IL-8 production by NAC, L-NAME, PDTC, MG-132, or DEX was complete, that of L-alanine or DMSO was partial, and that of curcumin was insignificant when statistically analyzed. *: P < 0.01, compared with respective controls.

 


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  		Figure 4. Nicotine induces neutrophils to produce O2- production. Neutrophils (5x106/ml) were exposed to 3 mM nicotine ({diamondsuit}) or to medium ({square}) for the indicated times and the abilities of the culture supernatants to reduce cytochrome c were monitored. Values are representative of three independent experiments and expressed as means ± SD (n=3). *: P < 0.01, compared with respective controls.

 

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  		Table 2. Nicotine Increases H2O2 Production and DHR Oxidation in Neutrophilsa

 
O2- and H2O2 are relatively unreactive to most biological substrates, but their generation yields a highly reactive hydroxyl radical (OH•) [20 ]. In addition, neutrophils produce MPO in the culture with medium alone (<100 ng/5x106 cells/ml in 1 h-culture supernatant: our observation). Therefore, although nicotine did not induce the MPO production, the spontaneously released MPO may give rise to the production of hypochlorous acid (HOCl) from H2O2 which is increased following nicotine stimulation, in the presence of Cl- [20 , 21 ]. However, dimethyl sulfoxide (DMSO) and L-alanine, both of which scavenge OH• [22 ] and HOCl [23 ], respectively, showed only partial effects (DMSO: 38% inhibition; L-alanine: 20% inhibition, means of three individuals), suggesting that these reactive species participate much less in nicotine-induced IL-8 production in neutrophils.

It has recently been shown that ONOO- is one of the most important initiators of IL-8 gene expression among ROIs [24 ]. Since neutrophils are capable of generating NO both spontaneously and in response to certain stimuli, the rapid induction of O2- by nicotine could possibly bring about the efficient generation of ONOO- through the reaction with NO [20 , 25 , 26 ]. We found that nicotine stimulation increases intracellular DHR oxidation in neutrophils (Table 2) : An increase in the oxidation was observed in a 25 min short-term culture and the longer culture showed a more conspicuous production. And, N{omega}-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor [24 , 25 ], prevented the nicotine-enhanced DHR oxidation (Table 2) , and entirely abrogated IL-8 production (Fig. 3) . In neutrophils, NO was detected when cultured with medium alone (<1.9 µM/4x106 cells/ml at 17 h). Nicotine did not enhance the production of NO even in the presence of SOD, but rather decreased the amount (e.g., 0.84 vs. 0.38 µM/4x106 cells/ml at 17 h), although the statistical difference could not be analyzed due to the levels close to the detection limit of 0.16 µM. These results imply that ONOO- is generated by the reaction of constitutively expressed NO with nicotine-induced O2-, and causes IL-8 production in neutrophils.

Nicotine-induced ROIs cause I{kappa}B degradation and nuclear translocation of NF-{kappa}B
IL-8 gene transcription is controlled by transcription factors such as NF-{kappa}B or activator protein 1 (AP-1) [27 28 29 30 ], and the activation of these factors is redox-regulated [18 , 27 28 29 30 31 ]. As shown in Fig. 3 , nicotine-induced IL-8 production was completely abrogated by pyrrolidinedithiocarbamate (PDTC, an antioxidant that inhibits I{kappa}B phosphorylation [28 , 32 ]), Z-Leu-Leu-Leu-al (MG-132, a proteasome inhibitor [33 ]), or dexamethasone (DEX, 9{alpha}-fluoro-16{alpha}-11ß,17{alpha},21-trihydroxy-1,4-pregnadiene-3,20-dione), which blocks the binding of NF-{kappa}B to the {kappa}B binding site [28 , 31 ]. Curcumin (1,7-bis [4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dion, an AP-1 inhibitor [34 ]), which we confirmed decreases TNF-{alpha}- and IL-1ß-induced IL-8 production in the preliminary experiments, showed no effect on the nicotine-induced IL-8.

Therefore, to examine whether NF-{kappa}B is involved in the nicotine-induced IL-8 production, both I{kappa}B degradation and NF-{kappa}B activities were tested. In untreated neutrophils, both I{kappa}B-{alpha} and I{kappa}B-ß proteins were detected and nicotine caused the degradation of both proteins (Fig. 5 ). The time at which the degradation started differed among individuals, but the start time was detected mostly within 10~30 min after the nicotine treatment. More importantly, this degradation was inhibited by L-NAME. Neutrophils expressed constitutively activated NF-{kappa}B, and nicotine increased the activity of NF-{kappa}B, which was measured by the binding of both p65 and p50 to the oligonucleotide containing the NF-{kappa}B consensus site, 5'-GGGACTTTCC -3' (Fig. 6 ). The increase of NF-{kappa}B activity was detected mostly from 90 min after the treatment, although the time needed to increase the activity shifted somewhat with the individual for both proteins. The degree of its increase was comparable to that increased by TNF-{alpha} (40 ng/ml), which was assigned as a positive signal for the activation of NF-{kappa}B. As was the degradation of I{kappa}B proteins, the activation of NF-{kappa}B was also completely inhibited by L-NAME. It appears, therefore, that, in nicotine-stimulated neutrophils, IL-8 production is controlled by ROIs, particularly ONOO-, which causes I{kappa}B degradation and subsequent activation of NF-{kappa}B. We note that, in some individuals whose neutrophils highly expressed the constitutively activated NF-{kappa}B, its expression decreased by the L-NAME-treatment (data not shown). This may be ascribed to the spontaneous production of O2- (Fig. 4) , which would generate ONOO- in unstimulated neutrophils as stated above. It should also be noted that, by contrast with the nicotine activation, the inhibition by L-NAME of TNF-{alpha}-activated NF-{kappa}B (Fig. 6) and IL-8 (data not shown) was partial.



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  		Figure 5. Nicotine-induced ROIs are involved in the degradation of I{kappa}B-{alpha}/ß in neutrophils. Neutrophils (5x106/ml) were cultured for 10 min (I{kappa}B-{alpha}) or 15 min (I{kappa}B-ß) with or without 2.5 mM nicotine in the presence or absence of 10 mM of L-NAME. This figure represents one set of data. The similar results were obtained in three other experiments with different neutrophils. The loading controls in each experiment showed no difference in the sample volumes in the different set of experiments (data not shown).

 


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  		Figure 6. Nicotine-generated ROI enhances NF-{kappa}B activities in neutrophils. Neutrophils were cultured (A) at various times or (B) 90 min with nicotine (2.5 mM), TNF-{alpha} (40 ng/ml), or medium alone in the presence or absence of 10 mM of L-NAME. The nuclear proteins (10 µg) were assayed for their binding activity to the NF-{kappa}B consensus sites using TransAM NF{kappa}B p50 and p65 Kits. Values are representative of three independent experiments and expressed as means ± SD (n=3). An asterisk shows a statistically significant difference (p<0.01) compared with the respective controls with medium. The symbols # and ## show the complete inhibition and the partial inhibition, respectively, as compared with the respective controls without L-NAME.

 
IL-8 production is mediated by the interaction of nicotine with nicotinic acetylcholine receptors (nAChRs)
Nicotine and its agonists can bind to neutrophils [35 , 36 ], but none of the particular nAChR subtypes on neutrophils have been determined. Because the EC50 of nicotine for neutrophils was 5000 times higher than that observed for central nervous systems (200~300 nM at EC50), neutrophils may have certain type(s) of nAChR that are different from the {alpha}4ß2 expressed in central nervous systems. Using nAChR agonists and antagonists, we first examined whether the nicotine-induced IL-8 production is mediated via nAChR and then determined the subunit components (Table 3 ).


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  		Table 3. Nicotine-Induced IL-8 Production in Neutrophils Is Mediated by nAChRsa

 
(±)-Epibatidine (exo-[±]-2-[6-chloro-3-pyridinyl]-7-azabicyclo[2.2.1]heptane, a potent agonist for neuronal nAChRs) [37 ], and (+)-anatoxin-a ([+]-2-acetyl-9-azabicyclo[4.2.1]non-2-ene, {alpha}3-selective) [38 ] were equipotent to nicotine in inducing IL-8. (–)-Cytisine (more potent for ß4 than for ß2) [39 , 40 ] also induced IL-8, but this agonist was less effective as compared with nicotine. Choline ([2-hydroxyethyl]trimethylammonium, an {alpha}7-selective) [41 , 42 ] showed no effect. Pilocarpine ((3S-cis)-3-ethyldihydro-4-[(1-methyl-1H-imidazol-5-yl)methyl]-2(3H)-furanone), a ligand acting selectively on muscarinic receptors, did not induce IL-8 production (data not shown).

In antagonist-inhibition tests (Table 3) , mecamylamine (N, 2, 3, 3-tetramethyl-bicyclo[2.2.1]heptan-2-amine, a channel blocker of the {alpha}3- and {alpha}4-subtypes) [40 , 43 , 44 ] inhibited the IL-8-inducing activity of nicotine, with 0.87 mM of IC50 against 3 mM nicotine. However, unresponsiveness to both antagonists, dihydro-ß-erythroidine ((3ß)-1,6-didehydro-14,17-dihydro-3-methoxy-16(15H)-oxaerythroidine-15-one)and hexamethonium (N,N,N,N’,N’,N’-hexamethyl-1,6-hexanediaminium), which act potently on {alpha}4-containing subtypes [40 , 45 ] and {alpha}3ß4 [43 ]/{alpha}4ß2 [46 ] in humans, respectively, nominated {alpha}3ß2 for a functional subtype in neutrophils, excluding the possibility of both {alpha}4-containing subtypes and {alpha}3ß4. In agreement with the inactive agonist, choline, for neutrophil-IL-8 production, an {alpha}7-specific antagonist, methyllycaconitine ([1{alpha},4(S),6ß,14{alpha},16ß]-20-ethyl-1,6,14,16-tetramethoxy-4-[[[2-(3-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]-oxy]methyl]-aconitane-7,8-diol) [42 ], did not inhibit nicotine-induced IL-8. The muscle nAChR-selective antagonist, vecuronium [47 ], inhibited the nicotine activity (1 mM of IC50).

These profiles of the agonist/antagonist effects indicate that nAChRs are involved in the nicotine-induced IL-8 production in neutrophils, and the nAChRs appear to be composed of at least subunit {alpha}3, and possibly {alpha}1, but not of subunit {alpha}4 or {alpha}7.

Plasma IL-8 levels are higher in smokers than those in nonsmokers
The possibility of nicotine-induced IL-8 production in vivo was examined by comparing plasma IL-8 levels between 20 smokers and 30 nonsmokers (male) from 20 to 49 years of age. There was no correlation between IL-8 levels and age in the nonsmokers. The IL-8 levels were significantly higher at p < 0.01 in the smokers (1.11±0.48 pg/ml, mean±SD, detection limit: 0.1 pg/ml) as compared with those of the nonsmokers (0.41±0.31 pg/ml). When analyzed with smoking habits, the plasma IL-8 concentrations were positively correlated with the duration from the initiation of smoking and the number of cigarettes smoked per day (Fig. 7 ).



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  		Figure 7. Blood IL-8 levels are positively correlated to the degree of cigarette smoking. The concentrations of plasma IL-8 in healthy smokers and nonsmokers were analyzed using ultrasensitive ELISA kit (detection limit: 0.1 pg/ml) in relation to (A) the duration of cigarette smoking, and (B) to the number of cigarettes smoked per day. The best fitting regression curve, with its correlation coefficient squared, was drawn using Microsoft® Excel 2002 analysis software. Spearman’s rank correlation coefficients for (A) and (B) were 0.865 (p<0.0001) and 0.631 (p=0.004), respectively, when analyzed using smokers’ data.

 
Possible involvement of neutrophil-derived chemokine in leukocytosis in smokers
In the data above, the nicotine-activation of NF-{kappa}B prompted us to consider the possibility that the nicotine-induced neutrophil activation is involved not only in neutrophilia but also in lymphocytosis and monocytosis observed in smokers. We then examined whether nicotine-stimulated neutrophils produce monocyte chemoattractant protein (MCP)-1 or macrophage inflammatory protein (MIP)-1{alpha}, since both require NF-{kappa}B activation for their gene transcription. The concentrations of MCP-1 protein in the culture supernatant of nicotine-stimulated neutrophils were 33.1±10.2 pg/4x106 cells/ml (mean±standard error, n=5, in 16 h-culture), which were significantly higher when compared with those in non-stimulated culture (2.4±0.9). MIP-1{alpha} was also detected with 26.8±12.0 in the nicotine-stimulated culture, but there was no statistical difference as compared with the amounts, 10.9±4.9, in the control culture. In plasma, the MCP-1 levels were higher in smokers than those in nonsmokers (529±23.3 pg/ml, 33 nonsmokers vs. 698±28.6, 21 smokers; p<0.01), and MIP-1{alpha} was not detected in either group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated, for the first time, that nicotine stimulates neutrophils to produce IL-8 in vitro. Nicotine-induced ROIs, especially ONOO-, appeared to act as a key initiator in the NF-{kappa}B activation and subsequent IL-8 production, through promoting I{kappa}-B degradation. The nicotine-induced neutrophil IL-8 production could contribute to the elevated plasma IL-8 levels in smokers, and this could be one of the causes of leukocytosis in smokers.

NF-{kappa}B and AP-1 are key transcription factors for the maximum expression of IL-8 [27 , 28 , 30 ]. In nicotine-stimulated neutrophils, IL-8 production was entirely dependent on NF-{kappa}B activation, as indicated by the abrogation of IL-8 production in the presence of the NF-{kappa}B inhibitor, DEX, rather than that of the AP-1 inhibitor, curcumin. It has been reported that the DNA binding activity of NF-{kappa}B is increased by oxidative stress [28 29 30 ]. From the profile of ROIs generated by nicotine stimulation, we expected OH•, HOCl, and/or ONOO- to directly contribute to nicotine activation of neutrophils. However, the scavengers, DMSO and L-alanine (and also L-methionine: data not shown) showed little, if any, partial effects, whereas L-NAME inhibition was complete in all of the following successive events: DHR oxidation, I{kappa}B degradation, NF-{kappa}B activation, and IL-8 production. In neutrophils, therefore, the nicotine activation appears to be ascribed to ONOO-, which we think is formed by the reaction of nicotine-induced O2- with preformed NO, supported by the facts that constitutive NO was detected in unstimulated neutrophils, and its amount tended to decrease following nicotine stimulation.

Unstimulated neutrophils expressed, to some extent, constitutively activated NF-{kappa}B, as has previously been indicated by McDonald et al. [48 ]. This might contribute to the constitutive expression of NOS, whose gene expression is NF-{kappa}B-dependent. And, this may be the reason why neutrophils express small amounts of NO and IL-8. Since neutrophils spontaneously generate O2- in vitro (Fig. 4) , the O2- -derived ONOO- would also contribute to the basal expressions of NF-{kappa}B and IL-8, as well. This speculation stems from our data that the PDTC/NAC treatment decreased the basal levels of NO, DHR oxidation, activated NF-{kappa}B (data not shown), and IL-8 (Fig. 3) in unstimulated neutrophils. The exact amount of ONOO- induced by nicotine was not calculated in our system, but it is presumed that ONOO-, even at a level undetectable by the oxidation of DHR, would cause I{kappa}B degradation because I{kappa}B degradation and its inhibition by L-NAME were observed earlier than the increase in DHR123 oxidation in some individuals. This could explain the rapid enhancement of neutrophil migration, which is mediated by an autocrine IL-8 induced by nicotine stimulation.

It has been shown that intravenous administration of IL-8 induces a neutrophil mobilization from the bone marrow [7 , 8 ]. Cigarette smoking has also been suggested to contribute to neutrophilia by stimulating the bone marrow in an animal study [9 ]. Therefore, we presumed IL-8 to be a causative cytokine for neutrophilia in smokers. Indeed, the levels of plasma IL-8, but not TNF-{alpha}, IL-1ß, IL-6, granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage CSF (data not shown), which are directly or indirectly involved in leukocytosis, were higher in smokers than in nonsmokers. The in vivo effect of smoking on IL-8 production was supported by the positive correlation between plasma IL-8 levels and heavy smoking. The in vitro data that the nicotine-enhanced neutrophil migration was mediated by IL-8 also support our hypothesis. Nicotine exerts a chemotactic activity at ~31 µM without affecting O2- production in neutrophils [15 ]. In our study, 600 µM of nicotine enhanced chemokinetic movement in association with O2- production. The different actions may be attributable to the difference of nicotine concentrations.

From our in vitro results, we submit that neutrophils contribute to the elevated levels of IL-8 in smokers. There have been reports showing that cigarette smoke induces IL-8 in human bronchial epithelial cells [49 ], and in alveolar macrophages in guinea pigs [50 ]. As another source of IL-8, therefore, cells in the respiratory system other than neutrophils should be taken into consideration. Nevertheless, because neutrophils are the largest population of cells in the circulation and are concentrated in the pulmonary capillaries [51 ] and because monocytes do not respond to nicotine in vitro (our observation and [15 ]), IL-8 produced by nicotine in these neutrophils should greatly reflect the elevated plasma-IL-8 concentration.

Regarding lymphocytosis, because IL-8 promotes lymphocyte trafficking or recruitment to the IL-8-injected site in experimental animals [5 , 6 ], it is likely that IL-8 causes lymphocytosis through the mechanism of lymphocyte mobilization as in IL-8-induced neutrophilia. Indeed, in our previous study with nude mice, subcutaneous inoculation of human carcinoma cell lines (PC-3 and HLC-1) that produce IL-8, but not IL-1, IL-6, TNF-{alpha}, or G-CSF [52 ], caused marked lymphocytosis, as well as neutrophilia (unpublished observations), although a single IL-8 injection appears not to induce significant lymphocytosis [8 ]. Furthermore, in this study, we observed that nicotine-stimulated neutrophils produced NF-{kappa}B-regulated CC chemokine, MCP-1, and the plasma MCP-1 levels were also higher in smokers as compared with those in nonsmokers. MCP-1 attracts monocytes and T lymphocytes [53 54 55 ]. Therefore, the neutrophil activation seems to contribute to smokers’ leukocytosis, by causing not only neutrophilia but also lymphocytosis and monocytosis.

In neutrophils, the conspicuous effects of nicotine were observed at concentrations above 100 µM in the induction of both ROIs and IL-8, whereas nicotine concentrations in smokers’ sera were at 25~444 nM [56 ]. It has been assumed that, in smokers, the nicotine concentrations in the tissues of the respiratory tract are as high as those in their saliva [57 ], in which nicotine concentrations reached mM levels [58 ]; therefore, neutrophils passing through the lung or residing in the tissues of the respiratory tract could be exposed to effective concentrations of nicotine. Indeed, a transient exposure, as short as 60 min, to 1 mM nicotine was equipotent in inducing IL-8 in neutrophils, compared with that induced in the presence of nicotine throughout the culture (data not shown).

In nonsmokers, a nicotine binding site has been detected with Kd around 10-9 M in neutrophil-membrane preparation [36 ], which is considered very low compared with the doses effective for IL-8 induction. In reference to the relatively high nicotine concentration (in mM) required to induce IL-8 in neutrophils, the nAChRs appeared to consist of subunits, {alpha}3, most likely combined with ß2, and {alpha}1. It has been reported that the effective concentrations of nicotine differ with the nAChR subtypes [59 60 61 ]. The nicotine concentrations equivalent to those in smokers’ blood are adequate to regulate the function of the {alpha}4ß2 subtype in the central nervous system (200~300 nM at EC50). In contrast, the muscle-type AChR or {alpha}3 AChR requires much higher doses (1 mM or higher, at maximum). Such differences in the sensitivity of the nAChR subtypes to nicotine may account for the higher concentrations needed to induce neutrophil IL-8 in vitro. The requirement of these high concentrations for nicotine effect may also be explained with respect to the cell types or their activation/differentiation status, because differentiated human macrophages or monocytic cell line, U937 cells, are significantly more sensitive to cholinergic agonists than peripheral blood mononuclear cells or neutrophils: nM to µM for the former and µM to mM for the latter [15 , 57 , 62 63 64 ]. Nevertheless, since nicotine can cross cell membranes, the involvement of the pathways independent of the nAChRs in neutrophil activation still remains to be elucidated in relation to their localization in the intact cells.

Concerning the {alpha}3-containing subtypes, the agonist/antagonist selectivity in neutrophils was discordant with those reported in the autonomic/central nervous systems [44 ]. For example, the potent activities of (+)-anatoxin-a and (±)-epibatidine in this study suggest the {alpha}3ß4 subtype involvement, but the ineffectiveness of hexamethonium, which has a high affinity to {alpha}3ß4, does not support this subtype. The unresponsiveness of neutrophils to hexamethonium is consistent with the report by Lebargy et al. [36 ]. Such responsiveness to the known agonists/antagonists suggests that the neutrophil nAChRs may differ from those expressed in the nervous systems, in terms of functional properties.

Recent studies have shown that neutrophils take part in the inflammatory and immune responses by generating a variety of cytokines/chemokines in response to specific stimuli [10 ]. In this way, because nicotine induces neutrophils to produce IL-8, as we have found in this study, chronic activation of neutrophils may possibly cause certain changes in the physiological status in smokers. One of the possibilities is that nicotine activation reduces the capacity of neutrophils to accomplish the specific immune responses. Intravenous IL-8 reduces neutrophil recruitment to sites of inflammation [65 , 66 ]. Therefore, for example, the nicotine-elevated plasma or tissue IL-8 and possibly the subsequent nonspecific movement of neutrophils in smokers would disturb their efficient locomotion to the targeting sites where they should encounter and protect the host from foreign organisms. Furthermore, tobacco smoking appears to be maleficent because overgeneration of ROIs by nicotine-stimulated neutrophils may cause tissue injury. In fact, sequestration of activated neutrophils mobilized into the lung has been reported in animal studies [50 , 67 ], and, in humans, increased release of toxic oxygen metabolites from the marginated neutrophils has been observed in smokers [68 ].

Nicotine has been shown to attenuate the LPS-induced IL-8 production through the decrease in NF-{kappa}B activity in phorbol myristate acetate-treated U937 cells [64 ]. In the differentiated human macrophages, nicotine suppresses the LPS-induced TNF-{alpha} production through a post-transcriptional mechanism [62 ]. In the activated neutrophils, the action of nicotine differs with the stimuli [15 , 57 , 63 ]. On the other hand, for the cells in the unstimulated or undifferentiated states, the nicotine effect seems restricted to neutrophils; in monocytes, nicotine is less effective in inducing IL-8 (our observation) and chemotaxis [15 ]. One of the mechanisms responsible for the diversity in nicotine effects among the cells may be related to the expression of intracellular molecules, which are regulated by the activation and/or differentiation signals and also differ with cell lineage. For example, unlike the neutrophils, U937 cells do not express constitutively activated NF-{kappa}B [69 ]; and no response to xanthine/xanthine oxidase has also been reported [70 ]. Additionally, the response to ROIs seems to be dependent on the activation status of the cells: Indeed, although the NF-{kappa}B activation is increased by oxidative stress [28 29 30 ], H2O2 has been shown to decrease LPS-induced NF-{kappa}B activation in neutrophils [71 ]. Modulation of the distinct molecules by nicotine stimulation may be responsible for the diverse effects in various types of cells. Further studies are required to elucidate the mechanisms which involve the nicotine effects in relation to the immune function in smokers.


   ACKNOWLEDGEMENTS
 
This study was supported in part by the Smoking Research Foundation, by grants from the Japan Health Science Foundation, and by the Kobe City Foundation for the Promotion of Medical Research. The authors are grateful to Drs. S. Itani, Y. Nojyo, and H. Matsuoto for their helpful comments concerning this study.


   FOOTNOTES
 
2 T. Takahashi, Department of Hematology and Clinical Immunology, Kobe City General Hospital, 4 Minatojima-Nakamachi, Chuo-ku, Kobe 650-0046, Japan Back

Received December 28, 2002; revised June 18, 2003; accepted June 20, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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