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JOURNAL OF INTERFERON & CYTOKINE RESEARCH 28:509–522 (2008)© Mary Ann Liebert, Inc.DOI: 10.1089/jir.2007.0081

Cigarette Smoke Condensate Upregulates the Gene and Protein Expression of Proinflammatory Cytokines in Human

Fibroblast-Like Synoviocyte Line

Miki Shizu,1 Yuka Itoh,1 Ryohei Sunahara,1 Satomi Chujo,1 Hidetoshi Hayashi,1 Yuko Ide,1 Takemasa Takii,1 Masaya Koshiko,2 Sang Woon Chung,2 Kazuichi Hayakawa,2 Keiji Miyazawa,3

Kunitaka Hirose,4 and Kikuo Onozaki1

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce proinflammatory cytokines, which are implicated in the pathogenesis of RA. When human fibroblast-like synoviocytes line MH7A was treated with cigarette smoke condensate (CSC), either mainstream or sidestream, expression levels of inter-leukin (IL)-1α, IL-1β, IL-6, IL-8, and CYP1A1 mRNA were upregulated in both time- and dose-dependent man-ners. The upregulatory effects of CSC on these cytokines were not significantly inhibited by α-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, suggesting that the effects of CSC were independent of AhR. Cycloheximide treatment indicated that the augmenting effect of CSC on IL-1α, IL-1β and IL-8, but not IL-6 and CYP1A1, mRNA expression requires de novo protein synthesis. CSC also induced cytokines at protein levels and further augmented the effects of tumor necrosis factor α on induction of these cytokines at both mRNA and protein levels. These results support the epidemiological studies indicating a strong association between heavy cigarette smoking and pathogenesis of RA.

Introduction

Rheumatoid arthritis (RA), a chronic inflammatory condition affecting several joints, is characterized by

proliferation of synoviocytes in inflamed synovia, forma-tion of pannus, and production of proinflammatory cytok-ines and chemokines by synoviocytes (Burmester and others 1997). These cytokines are implicated in the disease pathogenesis, such as production of proteases and reactive oxygen intermediates, proliferation of synovial fibroblasts, cartilage degradation, infiltration of inflammatory cells, and angiogenesis (Arend and Dayer 1995; Szekanecz and others 1998). Fibroblast-like synoviocytes are major cells producing the cytokines. Fibroblast-like synoviocytes or SV40-transformed cell clones derived from RA patients secrete, constitutively or in response to interleukin-1 (IL-1) or tumor necrosis factor α (TNFα), proinflammatory cytok-ines, including IL-1α, IL-1β, IL-6, and IL-8. The critical role of these proinflammatory cytokines in RA has been

verified by an improvement of synovial inflammation and decreased joint destruction in RA patients following treatment with neutralizing anti-TNFα antibody (Lipsky and others 2000), soluble TNF receptor (Moreland 1998), IL-1 receptor antagonist (IL-1ra) (Daniel 2004), or neutral-izing anti-IL-6 antibody (Choy and others 2002). However, the etiology and the mechanisms responsible for cytokine induction remain unknown.

Epidemiological studies indicate an association of ciga-rette smoking with disease outcome in patients with early inflammatory polyarthritis (Harrison and others 2001) and with increase of rheumatoid factor and nodule formation in patients with RA (Tuomi and others 1990). A strong asso-ciation between heavy cigarette smoking and RA was also reported, particularly in patients without a family history of RA (Hutchinson and others 2001). Interestingly, the risk of disease from smoking is quite high in individuals with shared epitope (SE) in HLA-DR (Padyukov and others 2004).

1Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho, Nagoya, Japan.

2Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, Japan.3Central Research Laboratories, Kissei Pharmaceutical Co., Ltd, Nagano, Japan.4Molecular Cell Biology Team, Biomedical Research Laboratories, Kureha Chemical Ind. Co., Ltd., Shinjuku-ku, Tokyo, Japan.

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However, the scientific basis supporting the epidemiological studies has not been provided.

Polycyclic aromatic hydrocarbons (PAHs) such as 3- methylcholanthrene (3-MC), benzo[a]pyrene (B[a]P), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), environmental pollutants of great concern, cause untoward effects including carcinogenesis, teratogenesis, and immune system impair-ment (Schmidt and Bradfield 1996). We have previously reported that 3-MC, B[a]P, and TCDD upregulated IL-1β mRNA via aryl hydrocarbon receptor (AhR) in RA patient-derived SV40 T antigen-transformed human fibroblast-like synoviocyte line MH7A, which has been shown to possess similar characteristics as parental synoviocytes (Miyazawa and others 1998a, 1998b). Since cigarette smoke contains much PAHs, we hypothesized that PAHs may contribute to the pathogenesis of RA. In this study, we determined the effect of cigarette smoke condensate (CSC) on proinflam-matory cytokine synthesis in MH7A cells, and showed that CSC induced proinflammatory cytokines at both mRNA and protein levels.

Materials and Methods

Reagents

RPMI 1640, TCDD, α-naphthoflavone (α-NF) and polymyxin B were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS, USA). Human recombinant TNFα was provided by Dainippon Pharmaceutical Co. (Osaka, Japan). The specific activity of TNFα was 6 × 107 U/mg based on the cytotoxic assay using L929 cells cultured in the presence of actinomycin D.

Preparation of cigarette smoke condensate

Cigarette smoke condensate (CSC) was prepared as described previously (Kamiya and others 2005). A com-mon American brand of cigarette was used in this study. Each cigarette was 84 mm long, 25 mm in circumference, and had a charcoal filter that yields normally 9 mg of tar and 0.7 mg of nicotine. Both particulate matters from main-stream and sidestream smoke were collected using a ciga-rette smoke collection apparatus as described previously with several modifications (Grimmer and others 1987). Briefly, cigarette smoking was performed in a glass chamber (40 cm high × 25 cm i.d.). Cigarettes were smoked at a con-dition of 80 mL of puff volume per 5 s, once every 15 s. The mainstream smoke was collected on a glass fiber (Shibata, Tokyo, Japan, T60A20 55 mm). This filter system is effective for the collection of only the particulate matter. On the other hand, the sidestream smoke was collected at about 170 mL/s. Smoke from 700 cigarettes was collected. After the cigarettes were consumed, the weights of the filters with trapped par-ticulate matter were determined, and the particulate mat-ter was extracted by sonication with benzene/ethanol (1/3, v/v) three times for 15 min. The extract was filtered and evaporated to dryness under reduced pressure, and then the residue was redissolved in ethanol (CSC). The yield of main-stream CSC and sidestream CSC were 9.25 and 18.0 mg per cigarette, respectively.

Cell culture

MH7A is an immortalized cell line obtained by stably transfecting rheumatoid fibroblast-like synoviocyte cells with SV40 T antigen gene. The cells have reached over 150 popula-tion doublings through culture crisis, and are growing rap-idly. MH7A cells express IL-1 receptor, intercellular adhesion molecule 1 (ICAM-1), CD16, CD40, CD80, and CD95. IL-1β enhanced the production of IL-6 and stromelysin-1, and the surface expression of ICAM-1, in a manner similar to that in the parental cells (Miyazawa and others 1998a and 1998b). The cells were cultured in RPMI 1640, 100 U/mL of penicil-lin G, 100 µg/ml of streptomycin, 15 mM HEPES, and 10% heat-inactivated FBS at 37°C. The cells were stimulated in the medium with or without CSC solubilized in ethanol, which was used at lower than 0.1% and the vehicle alone did not affect the cytokine mRNA expression. Although CSC prepa-rations at the doses used in the experiment were endotoxin-free according to the Limulus amoebocyte assay (sensitivity limit of 0.1 ng/mL), the culture was performed in the pres-ence of polymyxin B (10 µg/mL) to prevent the effect of unde-tectable level of LPS.

RNA preparation and reverse tanscriptase (RT) reaction

Total RNA from cells was extracted as described previ-ously (Matsumura and others 2000). The amount of RNA was determined spectrophotometrically. The RT reaction was performed by incubating 2 µg of total RNA in 20 µL reaction volume containing 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 10 mM DTT, 0.5 mM dNTP, 40 ng Random Primer p(dN)6, 6 U ribonuclease inhibitor, and 40U M-MLV Reverse Transcriptase. The reaction mixtures were incubated at 37°C for 60 min, followed by 10 min incubation at 70°C in Program Temp control system PC-700 (ASTEC). The reaction mixtures were diluted five times with ddH2O and used as cDNA solu-tion. cDNA samples were stored at 4°C until PCR analysis.

Real-time PCR analysis

Assays were performed using an Applied Biosystems 7300 sequence detector (Applied Biosystems, Foster City, CA, USA). For the analysis of human IL-1α, IL-1β, IL-6, and CYP1A1, each amplification mixture (20 µL) was made to contain 80 ng cDNA, 10 µL Premix EX TaqTM (Perfect Real Time) (RR039A) (Takara Bio), 1 µL PCR primer (forward and reverse primer), 0.4 mL Rox Reference Dye (Takara Bio), and distilled water. The reaction mixtures were incubated at 95°C for 10 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and then cooled to 4°C. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the reaction mix-ture (20 µL) was made to contain 40 ng cDNA, 10 µL POWER SYBER Green PCR Master Mix (P/N 4367659) (Applied Biosystems), 2 µL PCR primer (forward and reverse primers) (5 µM), and distilled water. The reaction mixtures were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 58.2°C for 1 min, and then 60°C for 30 s, 95°C for 15 s and then cooled to 4°C. Data were analyzed with the ABI Sequence Detector’s software. The results were

CYTOKINE INDUCTION BY CIGARETTE SMOKE 511

expressed as mean ± SD for the relative expression levels compared with GAPDH, and minimum values of four inde-pendent experiments were performed. Primers for human IL-1α (Hs00174092), IL-1β (Hs00174097), IL-6 (Hs00174131), IL-8 (Hs00174103), and CYP1A (Hs00153120) were obtained from Applied Biosystems. Primers for human GAPDH, for-ward primer 5′-cctgcaccaccaactgctta-3′ and reverse primer, 5′-TCTTCTGGGtggcagtgatg-3′, were obtained from Japan Genetic Institute (Sendai, Japan).

Plasmids

PGL3-basic was purchased from Promega. pXRE4-tk-Luc, which contains four tandem repeats of xenobiotic response element (XRE), was provided by Dr. Y. Fujii, Tsukuba University. PCMV-β gal was provided by Dr. Jeffrey L. Wrana, Toronto University. pEF-AhR (wild type) and pEF-AhR (1–599), which lack C terminal (600–848), were provided by Dr. Gary H. Perdew, Pennsylvania University.

Transient transfection and luciferase assays

MH7A cells were cultured in the RPMI1640 medium supplemented with 10% heat-inactivated FBS. XRE reporter plasmid, expression plasmids of wild type, and deletion mutant of AhR and pCMV-β gal plasmid (for normaliza-tion of transfection efficiency) were transiently transfected into MH7A cells using the calcium phosphate–DNA copre-cipitation method. After 15 h of transfection, cells were incu-bated with CSC or TCDD for additional 24 h and harvested. Luciferase assay was performed with the luciferase reporter gene assay kit (Roche, Germany) according to the manufac-turer’s instructions. The light emission was measured by multilabel counter 1420 ARVO (Pharmacia, San Francisco, CA, USA). Luciferase activity was expressed after normal-ization with the β-galactosidase value in the same sample.

Measurement of cytokine levels

Protein levels of IL-1α, IL-1β, IL-6, and IL-8 were deter-mined by enzyme-linked immunosorbent assays (ELISA) Kit according to the manufacturer’s protocol by using Endogen human IL-1α ELISA Kit (PIERCE, No. EH2IL1A5), Human IL-1β ELISA Set, human IL-6 ELISA Set, and human IL-8 ELISA Set (BD Biosciences).

Statistic analysis

Differences between group means were assessed by t-test.

Results

CSC dose-dependently upregulates proinflammatory cytokine mRNA expression in MH7A synovial cells

To examine whether CSC upregulates mRNA levels of proin-flammatory cytokines in MH7A cells, the cells were treated with varying doses of mainstream CSC or sidestream CSC for 24 h, and then mRNA levels of IL-1α, IL-1β, IL-6, and IL-8 were determined by real-time PCR. As shown in Fig. 1, mainstream CSC at more than 2 µg/mL upregulated the expression levels

of IL-1α, IL-1β, IL-6, and IL-8 mRNA. Sidestream CSC at more than 2 µg/mL also upregulated the mRNA levels of IL-1α, IL-1β, IL-6, and IL-8, and it was more potent as compared to main stream CSC. Both mainstream CSC and sidestream CSC increased mRNA of CYP1A1, an inducible gene by PAHs in the similar dose-dependent manner.

CSC time-dependently upregulates proinflammatory cytokine mRNA expression in MH7A synovial cells

To determine the time-dependent effect of CSC on the proinflammatory cytokine gene expression in MH7A cells, the cells were treated with 20 µg/mL mainstream CSC or sidestream CSC for up to 24 h, and mRNA levels of proin-flammatory cytokines and CYP1A1 were determined. As shown in Fig. 2, mRNA levels of IL-1α, IL-1β, IL-6, and IL-8 were upregulated 2 h after stimulation, decreased or remained at 4 h, and then again increased more at 12 or 24 h. mRNA level of CYP1A1 was also upregulated as early as 2 h after stimulation, and the expression level markedly increased after 12 h. There were no significant differences in the kinetics between mainstream CSC and sidestream CSC.

CSC induces AhR-dependent transcriptional activation in MH7A cells

As CSC contains much PAHs, we determined whether CSC induces AhR-dependent transcriptional activation. MH7A cells were transfected with a reporter plasmid containing four tandem repeats of XRE (pXRE4-tk-Luc) (Fig. 3A), and the cells were pretreated with or without 5 µM α-NF, an antagonist for AhR, for 1 h, and treated for another 24 h with or without 0.2 or 2 µg/mL mainstream CSC, side-stream CSC, or 0.1–10 nM TCDD, and then luciferase activ-ity in the cell lysates was determined. As shown in Fig. 3B, mainstream CSC, sidestream CSC, and TCDD all increased luciferase activity in a dose-dependent manner, and α-NF inhibited the upregulation. To further confirm whether CSC induces AhR-dependent transcriptional activation expres-sion plasmids of wild type AhR (wt), a dominant negative form mutant AhR (1–599), and a reporter plasmid pXRE4-tk-Luc were transfected into MH7A cells, and effects of main-stream CSC, sidestream CSC, and TCDD were determined. As shown in Fig. 3C, overexpression of AhR (wt) increased the basal luciferase activity and AhR (1–599) inhibited the activity in a dose-dependent manner. TCDD, mainstream CSC, and sidestream CSC all markedly increased the luciferase activity by overexpression of AhR (wt), and the activity was inhibited by AhR (1–599). These results indicate that CSC, either mainstream or sidestream, contains PAHs and induces AhR-dependent transcriptional activation.

Effect of CSC and α-NF on the proinflammatory cytokine mRNA expression

To examine whether the augmenting effect of CSC on proinflammatory cytokine mRNA is mediated through AhR, the cells were treated for 24 h with 2 µg/mL main-stream CSC, sidestream CSC in the presence or absence of α-NF, and then mRNA levels of proinflammatory cytokines

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FIG. 1. Effects of CSC on mRNA expression levels of proinflammatory cytokines in MH7A cells. MH7A cells were treated with control (vehicles alone) and varying concentrations of CSC for 24 h. Then total RNA was extracted, and mRNA levels of (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) CYP1A1, and GAPDH were determined by real-time PCR. Relative expression levels between control and CSC samples were shown. **p < 0.01, *p < 0.05, between control and main stream or side stream. Means and standard deviations (error bar) based on triplicate is shown.

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FIG. 2. Time course of the CSC-induced mRNA expression of proinflammatory cytokines in MH7A cells. MH7A cells were treated for indicated periods with control or CSC (20 µg/mL), total RNA was extracted, and mRNA expression levels of (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) CYP1A1, and GAPDH were determined by real-time PCR. Relative expression levels between control and CSC samples at each time point were shown. **p < 0.01, *p < 0.05, between 0 h and main stream or side stream. Means and standard deviations (error bar) based on triplicate are shown.

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FIG. 3. CSC induced activation of AhR-dependent XRE transcription in MH7A cells. (A) Schematic diagram of pXRE4- tk-Luc, containing four tandem repeats of AhR/Arnt-binding site (XRE). (B) Transcriptional activation of XRE reporter gene by CSC or TCDD in transient luciferase assay. MH7A cells were transiently transfected with pXRE4-tk-Luc and pCMV-β-gal. Sixteen hours after transfection, cells were stimulated with indicated concentration of CSC or TCDD for 24 h, and luciferase activity in cell lysates was measured. Luciferase activity was normalized with β-galactosidase activity, and means and standard deviations (error bar) based on triplicate are shown. (C) Effect of an AhR mutant on the transcriptional activation of XRE reporter gene in MH7A cells. MH7A cells were transiently transfected with pXRE4-tk-Luc, wild-type AhR [AhR(wt)], with or without AhR mutant [AhR(1–599)], and pCMV-β-gal. Sixteen hours after transfection, cells were stimulated with indicated concentrations of CSC or TCDD for 24 h, and luciferase activity in cell lysates was measured. Luciferase activity was normalized with β-galactosidase activity, and means and standard deviations (error bar) based on triplicate are shown. **p < 0.01; *p < 0.05, between control and main stream or side stream, or between α-NF and α-NF + main stream or α-NF + side stream. n.s., not significant (p > 0.05).

were determined by real-time PCR. As shown in Fig. 4, the upregulatory effect of mainstream CSC and sidestream CSC on IL-1α, IL-1β, IL-6, IL-8, and CYP1A1 mRNA expres-sion was confirmed. α-NF inhibited the effect of both mainstream CSC and sidestream CSC. However, α-NF also inhibited the basal level of these cytokine and CY1A1, and

the relative augmenting effect of CSC was not inhibited by α-NF. At the concentration α-NF did not exhibit any cyto-toxic effect on MH7A cells (data not shown). These results suggest that upregulatory effect of CSC on IL-1α, IL-1β, IL-6, IL-8, and CYP1A1 mRNA expression was indepen-dent on AhR.

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FIG. 4. Effect of α-NF on CSC-induced cytokine mRNA expression. MH7A cells were pretreated with α-NF (5 µM) for 1 h and treated for another 24 h with CSC (2 µg/mL). Total RNA was extracted, and (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) CYP1A1, and GAPDH mRNA expression levels were determined by real-time PCR. **p < 0.01, between control and main stream or side stream, or between α-NF and α-NF + main stream or α-NF + side stream. Means and standard deviations (error bar) based on triplicate are shown.

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Requirement of de novo protein synthesis in the CSC-induced cytokine mRNA upregulation

To examine whether de novo protein synthesis is required for the CSC-induced cytokine mRNA expression, the cells were pretreated with cycloheximide (CHX) for 1 h and treated for 24 h with 20 µg/mL CSC, and then mRNA expression lev-els of IL-1α, IL-1β, IL-6, IL-8, and CYP1A1 were determined by real-time PCR. As shown in Fig. 5, CHX treatment inhib-ited both the mainstream CSC- and sidestream CSC-induced IL-1α, IL-1β, and IL-8 mRNA expressions. Especially IL-1β mRNA expression, either basal or CSC-induced, was mark-edly inhibited. In contrast, CHX augmented both the main-stream CSC- and sidestream CSC-induced upregulation of IL-6 and CYP1A1 mRNA. These results indicate that de novo protein synthesis is required for the effect of CSC on IL-1α, IL-1β, and IL-8 mRNA expression, and IL-6 and CYP1A1 mRNA expressions are downregulated by newly synthe-sized proteins.

CSC augments the effects of TNFα on the synthesis of proinflammatory cytokines at mRNA and protein levels

In addition to IL-1, TNFα is known to play an important role in the pathogenesis of RA. To determine whether CSC augments the effect of TNFα on the mRNA level of proin-flammatory cytokines, cells were treated with TNFα and CSC each, alone or in combination, for 24 h, and then mRNA levels of proinflammatory cytokines were determined by real-time PCR. As shown in Fig. 6, CSC and TNFα each alone augmented the expression levels of IL-1α, IL-1β, IL-6, and IL-8 mRNA, and cotreatment with CSC and TNFα aug-mented the mRNA expression more than each alone. The effects of CSC and TNFα on the production of proinflam-matory cytokines at protein level were also determined. CSA, either mainstream or sidestream, alone augmented the production of proinflammatory cytokines. TNFα also aug-mented the production of proinflammatory cytokines. CSC further augmented the effect of TNFα, especially IL-1α and IL-1β production (Fig. 7).

Discussion

In this study we first showed that CSC, either main-stream or sidestream, upregulated expression levels of mRNA and protein of IL-1α, IL-1β, IL-6, IL-8, and CYP1A1 in both dose- and time-dependent manners in a human RA patient-derived fibroblast-like synoviocyte cell line MH7A. Both mainstream CSC and sidestream CSC upregulated the mRNA at more than 2 µg/mL. Time course study indi-cates that CSC biphasically upregulated the cytokine mRNA levels. They upregulated the mRNA at 2 h, then decreased or remained at 4 h, and then again upregulated at 12 h after stimulation. CYP1A1 mRNA was also upregulated as early as 2 h after stimulation, and the expression level markedly increased after 12 h.

We have previously reported that PAHs such as 3-MC, B[a]P, and TCDD upregulated expression level of IL-1β mRNA (Tamaki and others 2004). A major signaling

pathway for PAHs consists of AhR/ARNT heterodimer and its responsive XREs in the promoter region of inducible genes (Wilhelmsson and others 1990; Okey and others 1994). We found that PAHs can induce AhR-dependent CYP1A1 and IL-1β mRNA expression in MH7A cells (Tamaki and others 2004). As CSC contains much PAHs, we determined whether CSC can upregulate cytokine mRNA expression via AhR. Reporter gene assay revealed that CSC, either mainstream or sidestream, induced transcriptional activa-tion, and the activation was inhibited by α-NF, a specific antagonist for AhR. In addition, the upregulatory effect of CS on luciferase activity was markedly augmented in AhR overexpressed cells, and the response was inhibited by a dominant negative form of AhR. Therefore, upregu-latory effect of CSC on the gene containing XRE appeared to be mediated by AhR in this cell line. α-NF inhibited all the proinflammatory cytokine mRNA as well as CYP1A1 mRNA induced by mainstream CSC or sidestream CSC. However, the inhibitory effect of α-NF was also observed in basal cytokine mRNA expression, and the relative aug-menting effect of CSC was not affected by α-NF. Therefore, we think that the upregulatory effect of CSC was indepen-dent on AhR, and cytokine mRNA induction by CSC was caused by molecules other than PAHs. However, we do not rule out the possible contribution of AHs, because the effect of PAHs may be masked by other inducing molecules. The molecule(s) responsible for the cytokine mRNA induction remain to be clarified.

CHX treatment inhibited the upregulatory effect of CSC on IL-1α, IL-1β, and IL-8 mRNA. Especially, IL-1β mRNA induction by CSC was completely inhibited. In contrast, IL-6 and CYP1A1 mRNA expression was augmented by the treatment with CHX. Therefore, CSC-induced IL-1α, IL-1β, and IL-8 mRNA expression requires de novo protein synthesis and the mechanism is different from those of IL-6 and CYP1A1 induction. At present we do not know the mechanism how these cytokine mRNA expression is upregulated by CSC. In this regard, it is reported that CSC or B[a]P activate NF-κB, a major transcription fac-tor involved in induction of proinflammatory cytokines by unknown mechanism (Shishodia and others 2003), and TCDD upregulates IL-1β mRNA expression at post-transcriptional level in human keratinocytes (Henley and others 2004).

We also found that CSC, either mainstream or sidestream, each alone induced proteins of IL-1α, IL-1β, IL-6, and IL-8. Furthermore, CSC augmented the effect of TNFα on induc-tion of these cytokines. Therefore, it is possible that CSC con-tributes to the pathogenesis of RA alone and by augmenting the effect of TNFα. It is controversial on the effect of ciga-rette smoking on cytokine production from macrophages or monocytes. Exposure to cigarette smoke (either as gas phase or aqueous extract) evokes release of IL-8, TNFα, and other cytokines from alveolar macrophages or blood monocytes (Churg and others 2002; Walters and others 2005), while cigarette smoke exposure into mouse or humans attenuates cytokine production by stimulated alveolar macrophages (Chen and others 2007; Gaschler and others 2008). These discrepancies may be due to the difference in the exposure time, acute versus chronic, or cell types.

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FIG. 5. Effect of CHX on the mRNA expression of CSC-induced cytokines in MH7A cells. MH7A cells were pretreated with CHX (1 µg/mL) for 1 h and treated for another 24 h with CSC (20 µg/mL). Then total RNA was extracted, and (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) CYP1A1, and GAPDH mRNA expression levels were determined by real-time PCR. **p < 0.01; *p < 0.05, between control and main stream or side stream, or between CHX and CHX + main stream or CHX + side stream. n.s., not significant. Means and standard deviations (error bar) based on triplicate are shown.

SHIZU ET AL.518

Our findings are quite interesting in considering the etiology of RA. Infectious agents may act as a trigger for RA, and a number of agents have been implicated including Epstein-Barr virus (Balandraud and others 2004), arvovirus (Altschuler 1999), and some bacteria such as Proteus (Ebringer and others 2003) and Mycobacteria (Lydyard and others 1990). However, the epidemiological data are inconclusive. Smoking is the solid environmental risk factor revealed by epidemiological studies. Studies indicate an association of

cigarette smoking with disease outcome in patients with early inflammatory polyarthritis (Harrison and others 2001) and with increase of rheumatoid factor and nodule formation in patients with RA (Kamiya and others 2005). A strong associa-tion between heavy cigarette smoking and RA, particularly in patients without a family history of RA, was also reported (Grimmer and others 1987). In addition, smokers carrying double SE genes have quite a high risk for RA (Padyukov and others 2004). Quite interestingly, based on document,

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FIG. 6. Effect of CSC on the TNFα-induced cytokine mRNA expression in MH7A cells. MH7A cells were pretreated with TNFα (5 U/mL) for 1 h and treated for another 24 h with CSC (20 µg/mL). Total RNA was extracted, and (A) IL-1α, (B) IL-1β, (C) IL-6, (D) IL-8, and GAPDH mRNA expression levels were determined by real-time PCR. **p < 0.01; *p < 0.05, between control and main stream or side stream, or between TNFα and TNFα + main stream or TNFα + side stream. Means and standard deviations (error bar) based on triplicate are shown.

CYTOKINE INDUCTION BY CIGARETTE SMOKE 519

excavation, examination of skeletons and paintings RA has not been found until the 17th century in old world (Europe) (Rothschild 2001). Thomas Syndenham (1624–1689) identi-fied and distinguished RA from the related disease, such as gout and rheumatic fever (Abe 2001). However, the reason why RA has not been found in old world until the 17th cen-tury is a big mystery, and RA is thought to be imported from new world (America), where RA was found in habitants who

lived 3000 to 5000 years ago (Rothschild and others 1988).

Rothschild, Turner and DeLuca included tobacco among variables that could be responsible for the appearance of RA in Europe (Rothschild and others 1988). In botanical terms, the birthplace of the tobacco plant is thought to have been in America. After the discovery of America, tobacco was introduced to France, Portugal, Spain, and England. In the 16th/17th-century tobacco smoking became very popular in

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FIG. 7. Effect of CSC on the protein production of TNFα-induced cytokines in MH7A cells. MH7A cells were pretreated with TNFα (5 U/mL) for 1 h and treated for another 48 h with CSC (20 µg/mL). Then culture supernatant fluid was collected, and (A) IL-1α, (B) IL-1β, (C) IL-6, and (D) IL-8 protein expression levels were determined by ELISA. **p < 0.01, between medium and main stream or side stream, or between TNFα and TNFα + main stream or TNFα + side stream. n.s., not significant. Means and standard deviations (error bar) based on triplicate are shown.

SHIZU ET AL.520

Europe (Borio 1997). These historical contexts support the findings of epidemiological studies that tobacco smoking, especially heavy smoking, is a risk factor for RA. It is of note that North America natives, among whom tobacco smoking is quite popular, have high prevalence rates of RA (Peschken and Esdaile 1999). In our study the yield of mainstream CSC and sidestream CSC were 9.25 and 18.0 mg per cigarette, respectively. Therefore, the dose of 2 µg/mL CSC, which is able to induce cytokine mRNA, is reachable if an individual with 60 kg body weight takes only 13 (based on mainstream CSC) or 6.7 cigarettes (based on sidestream CSC) and all the smoke was adsorbed. Actually heavy smokers take much more cigarettes in a day and the number reaches a tremen-dous level over a long time. Significant amount of CSC, espe-cially because PAHs are readily absorbed into adipose tissue, can be accumulated over a long period. Although there are no available reports on PAHs in CSC for absorption, smok-ing cigarettes results in a fast absorption of nicotine and subsequent distribution over all tissues (Benowitz and Jacob 1984). Therefore, higher dose of CSC can be readily achieved in heavy smokers and also secondary exposed persons. In conclusion, our findings are the first to provide the basis sup-porting the epidemiological studies.

Acknowledgments

This work was supported in part by Grant-in-Aids for Exploratory Research from Japan Society for the Promotion of Science and Grant-in-Aid for Scientific Research on Priority area from The Ministry of Education, Science, Sports, and Culture.

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Address reprint requests or correspondence to:Dr. Kikuo Onozaki

Department of Molecular Health SciencesGraduate School of Pharmaceutical Sciences

Nagoya City University3-1 Tanabe-dori

Mizuho-kuNagoya, Aichi

467-8603Japan

Tel. and fax: +81-52-836-3419

E-mail: konozaki@phar.nagoya-cu.ac.jp

Received 23 July 2007/Accepted 8 February 2008