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Rheumatology Advance Access originally published online on July 10, 2008
Rheumatology 2008 47(9):1317-1322; doi:10.1093/rheumatology/ken259
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A role for the aryl hydrocarbon receptor and the dioxin TCDD in rheumatoid arthritis

S. Kobayashi1,2, H. Okamoto1, T. Iwamoto1,2, Y. Toyama2, T. Tomatsu1, H. Yamanaka1 and S. Momohara1

1Institute of Rheumatology, Tokyo Women's Medical University and 2Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan.

Correspondence to: H. Okamoto, Institute of Rheumatology, Tokyo Women's Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo 162-0054, Japan. E-mail: hokamoto{at}ior.twmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective. Environmental factors are involved in RA pathogenesis and epidemiological studies have suggested that smoking is an environmental risk factor for RA. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the major toxic components in cigarettes. To clarify the biological effects of smoking in RA, we investigated the role of TCDD in RA pathogenesis.

Methods. Human synovial tissue was obtained from RA and OA patients and aryl hydrocarbon receptor (AhR) expression in these tissues was evaluated using immunohistochemistry and real-time PCR. Expression of various cytokines was measured by real-time PCR following stimulation of RA synoviocytes with different concentrations of TCDD. To study the role of AhR, we treated RA synoviocytes with {alpha}-naphthoflavone, a known AhR antagonist. To evaluate which signal transduction pathways were stimulated by the TCDD–AhR interaction, we used inhibitors of nuclear factor-{kappa}B (NF-{kappa}B) and extra-cellular stimulus-activated kinase (ERK).

Results. Higher AhR mRNA and protein levels were observed in RA synovial tissue than in OA tissue. TCDD up-regulated the expression of IL-1β, IL-6 and IL-8 through binding to AhR, and this effect was transmitted via the NF-{kappa}B and ERK signalling cascades. AhR expression in synovial cells was up-regulated by TNF-{alpha}.

Conclusion. TNF-{alpha} activates AhR expression in RA synovial tissue, and that cigarette smoking and exposure to TCDD enhances RA inflammatory processes. TCDD induces inflammatory cytokines via its association with AhR, resulting in stimulation of the NF-{kappa}B and ERK signalling cascades. Thus TCDD exposure, such as smoking exacerbates RA pathophysiology.

KEY WORDS: Rheumatoid arthritis, Aryl hydrocarbon receptor, 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Smoking, Nuclear factor-kB, Extra-cellular stimulus-activated kinase, Interleukin-1, Interleukin-6, Interleukin-8


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
RA is the most common systemic autoimmune disease, affecting 0.5–1.0% of the population. This condition is characterized by chronic inflammation of synovial joint tissue, which causes progressive joint destruction and disability [1, 2]. Although the precise pathogenesis of RA remains unknown, it is widely believed that both genetic and environmental factors play a role [3, 4]. A number of epidemiological studies have suggested that smoking is an environmental risk factor for RA. Smoking has been reported to be clinically associated with RF positivity, rheumatoid nodules, functional disability and disease activity [5–8]. Recent studies have also found that smoking has a particular impact on the risk of RA in patients with the HLA-DRB1 genotype [9]. Further, some studies have suggested that smoking influences RA disease susceptibility and severity; however, this issue remains controversial, and the precise mechanism through which smoking affects RA is unclear [5–7, 10, 11].

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) are major toxic components in cigarettes. While the association between PAHs and RA is not known, PAHs have been reported to cause untoward adverse effects, including carcinogenesis, teratogenesis and immune system impairment [12]. The majority of these PAH-associated effects are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor [13]. Once TCDD binds to AhR, the complex translocates to the nucleus and the ligand-bound receptor forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT) [14]. This heterodimer then binds to specific DNA elements, termed dioxin-response elements (DREs), in the regulatory regions of target genes, such as cytochrome P450 1A1 (CYP1A1), which leads to gene expression changes and ultimately result in various toxic and biochemical responses [15]. PAHs have been reported to regulate the expression of several pro-inflammatory cytokines, such as IL-1β, in various cell types, including macrophages, B cells, keratinocytes, splenocytes and human A549 cells [16–22]. However, this may not occur entirely through DREs, as some of these genes (e.g. the human IL-1β gene) do not contain putative DREs in their promoter regions [23].

Based on these findings, we speculated that PAHs and AhR may be key molecular components underlying the association between smoking and RA. Herein, we investigate the role of one of the major dioxin components, TCDD, and its receptor, AhR, on RA synoviocytes. First, we examined the effects of TCDD on cytokine expression in RA synoviocytes and its dependence on AhR, as well as the signalling pathways involved in these interactions. Next, to study the role of AhR in RA synovial tissue, we measured AhR expression in RA synoviocytes following stimulation with a variety of cytokines. Finally, we investigated the role of TCDD and AhR in RA synoviocytes, and discuss whether or not smoking is one of the precipitating factors for RA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Preparation of articular synovial tissue
Human synovial specimens were obtained from OA and RA patients who were undergoing total knee replacement at Tokyo Women's Medical University, Tokyo, Japan. OA was diagnosed by physical examination along with radiographic findings, and RA patients had disease that met the 1987 criteria of the ACR [24]. Samples were obtained following informed consent from all patients. All experiments were approved by the Ethical Committee of Tokyo Women's Medical University.

Isolation and culture of fibroblast-like synoviocytes
Tissues were obtained under aseptic conditions and were finely minced. Synoviocytes were isolated by digestion with 1 mg/ml collagenase (Sigma Chemical Co., St Louis, MO, USA) for 3 h at 37°C in DMEM (Nikken Bio Medical Laboratory, Kyoto, Japan) with antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; GIBCO BRL, Grand Island, NY, USA). Digested tissue was briefly subjected to centrifugation, and resulting pellets were washed three times in PBS. Isolated fibroblast-like synoviocytes (FLSs) were seeded at high density in tissue culture flasks and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS; Tissue Culture Biologicals, Tulare, CA, USA) at 37°C in a humidified atmosphere of 5% CO2/95% air. The culture medium was changed every 3–5 days, and non-adherent lymphoid cells were removed. At confluence, synoviocytes were detached and passaged once, then seeded at high density and allowed to grow in DMEM supplemented as described earlier. Only the third through seventh passages were used for experiments. In some cases, synovial tissue slices were obtained for immunohistochemical analysis.

Measurement of cytokine mRNA expression by quantitative real-time PCR
Human synoviocytes from RA patients were cultured in DMEM supplemented with 10% FBS in 12-well culture plates. At confluence, the culture medium was replaced with serum-free DMEM. After 12 h, synoviocytes were incubated for an additional 6 h in the absence or presence of 2,3,7,8-TCDD (Cambridge Isotope Laboratories, Inc., Andover, MA, USA) at 0.01–100 nM. Total RNA was harvested from synoviocytes using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Chatsworth, CA, USA). Complementary DNA (cDNA) was synthesized from 0.3 mg total RNA in a 20 ml reaction using TaqMan Reverse Transcription Reagents (Applied Biosystems, Tokyo, Japan). TaqMan quantitative real-time PCR was performed using the ABI Prism 7900HT sequence detection system and TaqMan PCR Master Mix according to the manufacturer's protocol (Applied Biosystems). Primers and probes for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and various human cytokines were purchased from Applied Biosystems. The following primer sets were used: GAPDH, sense, 5'-GGGAAGGTGAAGGTCGGA-3'; antisense, 5'-GCAGCCCTGGTGACCAG-3' (PCR product = 62 bp). RNA samples lacking reverse transcriptase were used along with each real-time PCR experiment to verify the absence of genomic DNA. Incubation was initiated at 50°C for 2 min and 95°C for 10 min, which was followed by 40 cycles at 95°C for 15 s and 65°C for 1 min. Samples were compared using the comparative threshold cycle (Ct) method to determine cytokine expression relative to the time-matched vehicle-treated control. The parameter Ct is the PCR cycle number at which the fluorescence generated by cleavage of the probe reaches a fixed threshold above baseline. For each sample, the cytokines’ Ct value was normalized using DCt = cytokines Ct–GAPDH Ct. To determine relative expression levels, the following formula was used: DDCt = sample DCt–time-matched control DCt, and the value used to plot the relative cytokine expression of each sample was calculated using the expression 2–DDCt.

Measurement of cytokine levels
Cytokine concentration in FLS culture supernatants were determined using cytokine-specific ELISA kits for IL-6 (IL-6 ELISA KIT, HU, Invitrogen, CA, USA), IL-8 (IL-8 ELISA KIT, HU, Invitrogen) and IL-1β (Human IL-1β Quantikine HS Colorimetric Sandwich ELISA, R&D Systems, Minneapolis, MN, USA). Assays were performed according to the manufacturers’ instructions. Experiments were performed three times with each of the three independent cultures.

Measurement of AhR mRNA expression in synovial tissue
Synovial tissue samples from RA and OA patients (RA, n = 8; OA, n = 4) were stored for later RNA extraction (Ambion, Applied Biosystems) at –20°C until use. All the patients were non-smokers. Tissues were frozen in liquid nitrogen and finely ground by cryo-press, and total RNA was isolated using the TRIzol method (Invitrogen). Complementary DNA was synthesized from total RNA, and AhR gene expression was measured by real-time PCR as described above. AhR gene-specific primers were obtained from Applied Biosystems (Hs00169233). Experiments were performed three times for each tissue sample.

Immunohistochemistry
Immunohistochemical staining was performed using the avidin–biotin–peroxidase complex method. Specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5-mm thick) of paraffin-embedded specimens were deparaffinized and endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min. The sections were washed three times with Tris-buffered saline (TBS) and incubated for 10 min with 5% BSA in TBS to block non-specific binding. Tissue sections were then incubated with anti-AhR (H-211) (1 mg/ml) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at 37°C. Negative controls included the use of a non-specific rabbit immunoglobulin as the primary antibody (1 mg/ml) (Sigma Chemical Co.). Positive controls included the use of monoclonal antibody against CXCR3 (C6473, Sigma Chemical Co.) or CCR2 (C5848, Sigma Chemical Co.) [25]. Each slide was washed three times in TBS and incubated with biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) in a humid chamber for 30 min at 37°C, followed by washing and detection using HRP-conjugated streptavidin (Dako Cytomation Corporation, Kyoto, Japan). 3'3-Diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen (Liquid DAB K3468, Dako Cytomation Corporation). Slides were counterstained briefly with Mayer's haematoxylin and mounted. Stained tissues were examined by light microscopy.

Western blot analysis
Western blot analysis was performed by standard methods. FLSs were treated with various concentrations of TNF-{alpha} (0–100 nM) for 16 h, and the protein expression of AhR was analysed by western blot using 10 µg of total cell lysate. All incubations with antibodies were for 1 h at room temperature. An anti-AhR, antibody (sc-5579; Santa Cruz Biotechnology) was used for detection of AhR. A rabbit polyclonal anti-GAPDH antibody (Santa Cruz Biotechnology) and anti-rabbit IgG-HRP (Santa Cruz Biotechnology) was used for the detection of human GAPDH as a control.

Effect of cytokines on AhR expression in human synoviocytes
After the medium was replaced with serum-free DMEM for 12 h, synoviocytes were incubated for an additional 6 h in the absence or presence of various recombinant human cytokines, including TNF-{alpha} (rHuTNF-{alpha}: 0–100 ng/ml; R&D Systems), IL-1β (rHuIL-1β: 0–100 ng/ml; R&D Systems), IL-4 (rHuIL-4: 0–100 ng/ml; R&D Systems), IL-6 (rHuIL-6: 0–100 ng/ml; R&D Systems), IL-8 (rHuIL-8: 0–100 ng/ml; R&D Systems). AhR expression was measured by real-time PCR as described earlier. Experiments were performed three times with each of the three independent RA synoviocyte samples.

Statistical methods
Data are presented as the mean ± S.D. Statistical comparisons were performed using paired t-test and Mann–Whitney's U-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
TCDD increases IL-1β, IL-6 and IL-8 mRNA levels in RA synoviocytes
To examine the effect of TCDD on cytokine expression in FLSs, cells were treated with various concentrations of TCDD (0.01–100 nM), and cytokine mRNA levels were measured using a real-time PCR assay. As presented in Fig. 1, mRNA expression levels of IL-1β, IL-6 and IL-8, but not of TNF-{alpha} or MMP3, were up-regulated following treatment with TCDD in a dose-dependent manner. Protein expressions of these cytokines were also up-regulated by TCDD in a dose-dependent manner. Among these cytokines, IL-1β was the most remarkably up-regulated by TCDD (Fig. 1A).


Figure 1
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  		FIG. 1. TCDD increases IL-1β, IL-6 and IL-8 mRNA levels in RA synoviocytes. FLSs were stimulated with various concentrations of TCDD (0–100 nM) for 4–6 h and were harvested, followed by RNA isolation and real-time PCR studies. (A) Real-time PCR analysis of IL-1β mRNA levels and ELISA to evaluate the levels of IL-1β protein. (B) Real-time PCR analysis of IL-6 and IL-8 mRNA levels and ELISA to evaluate the levels of IL-6 and IL-8 proteins. (C) Real-time PCR analysis of TNF-{alpha} and MMP-3 mRNA levels. Experiments were performed in triplicate for each of the five independent cultures. *P < 0.05, **P < 0.01 vs none by paired t-test.

 
Expression of AhR in synovial tissue
Next, we investigated AhR expression in RA synovial tissue using an immunohistochemical analysis. We found that AhR expression was significantly higher in synovial tissue from RA patients than in that from OA patients (Fig. 2A). To see whether AhR expresses in synovial cells, we also performed immunohistochemical analysis with monoclonal antibody against CXCR3 and CCR2, these are known to be expressed in synovial cells (Fig. 2A) [25]. To confirm this result at the RNA level, we next performed real-time PCR to measure AhR gene expression in synovial tissue from RA and OA patients. Expression of AhR mRNA was also higher in tissue from RA patients than in that from OA patients (Fig. 2B).


Figure 2
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  		FIG. 2. AhR expression in synovial tissue from RA and OA patients. (A) Immunohistochemical analysis using an anti-human AhR goat polyclonal antibody. Negative control: non-specific rabbit immunoglobulin. Positive controls: anti-CXCR3 and anti-CCR2 antibody. (B) Total RNA was harvested from synovial tissue from eight RA and four OA patients. AhR mRNA levels were analysed by real-time PCR. Experiments were performed in triplicate for each of the samples.

 
TCDD increases IL-1β mRNA levels via AhR
To examine whether the effect of TCDD on IL-1β expression is mediated by its receptor, AhR, FLSs were treated with or without TCDD in the presence or absence of {alpha}-naphthoflavone ({alpha}-NF), an AhR antagonist [26], and IL-1β mRNA levels were determined by real-time PCR. As shown in Fig. 3, β-NF alone did not appear to influence IL-1β mRNA expression, but the TCDD-induced IL-1β up-regulation was completely abolished by {alpha}-NF, indicating that this effect is mediated through AhR.


Figure 3
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  		FIG. 3. TCDD increases IL-1β mRNA levels via AhR in FLSs. FLSs were treated with 100 nM TCDD, 5 nM {alpha}-naphthoflavone ({alpha}-NF) or both (100 nM TCDD, 5 nM {alpha}-NF) for 6 h, and IL-1β expression was analysed by real-time PCR. Experiments were performed in triplicate for each of the two independent cultures.

 
TCDD-stimulated IL-1β expression is dependent on the nuclear factor-{kappa}B and extra-cellular stimulus-activated kinase pathways
To determine which signalling pathways are responsible for the effects of TCDD in synoviocytes, we next treated FLSs with or without TCDD in the presence or absence of Bay 11–7082, which is known to prevent nuclear factor-{kappa}B (NF-{kappa}B) activation through inhibition of I {kappa}B-{alpha}phosphorylation [27], and U0126, an inhibitor of extra-cellular stimulus-activated kinase (ERK) activation. Both of these inhibitors significantly decreased IL-1β mRNA expression (Fig. 4). These results indicate that both the NF- {kappa}B and ERK signal transduction pathways are involved in TCDD–AhR-induced IL-1β expression.


Figure 4
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  		FIG. 4. Analysis of the involvement of the NF-{kappa}B and ERK signalling pathways in TCDD-stimulated IL-1β expression in FLSs. (A) FLSs were treated with 100 nM TCDD, 10 nM Bay 11-7082 or both (100 nM TCDD, 10 nM Bay 11-7082) for 6 h, and IL-1β expression was analysed by real-time PCR. (B) FLSs were treated with 100 nM TCDD, 10 nM U0126 or both (100 nM TCDD, 10 nM U0126) for 6 h, and IL-1β expression was analysed by real-time PCR. Experiments were performed in triplicate for each of the two independent cultures.

 
AhR expression is up-regulated by TNF-{alpha}
As presented earlier, we observed that AhR is highly expressed in synovial tissue of RA patients. To study as to which factors are responsible for this increased AhR expression, FLSs were treated with various cytokines—TNF-{alpha}, IL-1β, IFN-{gamma}, IL-4, IL-6 and IL-8—that have been reported to be involved in RA pathogenesis, and AhR mRNA levels were evaluated by real-time PCR. As shown in Fig. 5A, TNF-{alpha} inducedï expression of AhR mRNA in time- and dose-dependent manner. However, IL-1β, IL-4, IL-6, IL-8 and IFN-{gamma} did not induce the expression of AhR mRNA (data not shown). To confirm the role of TNF-{alpha} on AhR expression, FLSs were treated with TNF- {alpha} in the presence or absence of infliximab, a chimeric monoclonal anti-TNF-{alpha} antibody. To further explain the high expression of AhR in RA than OA, we performed western blot using polyclonal antibody against AhR and found that AhR protein expression was up-regulated in the presence of TNF-{alpha}, which mimic the high TNF-{alpha} environment in RA joints (Fig. 5B). As shown in Fig. 5C, infliximab abolished TNF-{alpha}-induced AhR expression. These results indicate that AhR expression is up-regulated by TNF-{alpha}.


Figure 5
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  		FIG. 5. AhR expression is up-regulated by TNF-{alpha}. (A) FLSs were treated with TNF-{alpha} (10 nM) for 0–24 h, and expression of AhR was analysed by real-time PCR. And FLSs were treated with various concentrations of TNF-{alpha} (0–100 nM) for 6 h, and expression of AhR was analysed by real-time PCR. (B) FLSs were treated with various concentrations of TNF-{alpha} (0–100 nM) for 16 h, and the protein expression of AhR was analysed by western blot. (C) FLSs were treated with TNF-{alpha} in the presence or absence of infliximab for 6 h, and AhR expression was analysed by real-time PCR. Experiments were performed in triplicate for each of the two independent cultures. *P < 0.05, **P < 0.01 vs none by paired t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Although the precise pathogenesis of RA remains unknown, a number of epidemiological studies have suggested that smoking is an environmental risk factor for this disease. To confirm these studies and to further explore the association between smoking and RA pathogenesis, we investigated the role of TCDD and AhR in RA synovial tissue. First, we noted high AhR expression in RA synovial tissue. Next, we studied the effects of TCDD on FLSs by measuring cytokine expression levels. Although PAHs have been previously shown to regulate the expression of several cytokines, particularly the pro-inflammatory cytokines IL-1β, IFN-{gamma} and IL-8, in various cell types, it is not known whether this also occurs in RA synovial tissue. We found that expression of IL-1β, IL-6 and IL-8 in FLSs was increased following TCDD treatment both at mRNA and protein levels. Among these cytokines, IL-1 β mRNA was most dramatically up-regulated (15-fold at the mRNA level). To study the role of AhR in FLS cytokine expression, we treated cells with TCDD in the presence or absence of the AhR antagonist {alpha}-NF. TCDD-induced up-regulation of IL-1 β mRNA expression was inhibited by {alpha}-NF, indicating that any TCDD-induced effects were mediated by the association between TCDD and AhR.

To further reveal which signalling pathway(s) are involved in TCDD-induced cytokine expression in FLSs, we next studied the contribution of the NF-{kappa}B and ERK cascades, as PAHs have been shown to activate these signalling pathways in the lung epithelial cell line A549 [28], vascular smooth muscle cells [29] and macrophages [17]. FLSs were treated with TCDD with or without Bay 11–7082, an inhibitor of I{kappa}B-{alpha} phosphorylation, and U0126, an inhibitor of ERK activation. Although these inhibitors alone did not affect FLS IL-1β expression, they significantly decreased TCDD-induced IL-1β mRNA up-regulation. These data indicate that TCDD-stimulated IL-1β expression in RA synoviocytes is dependent on both NF-{kappa}B- and ERK-mediated signalling.

We also found that AhR expression levels in RA synovial tissue were higher than those in OA synovial tissue. To determine as to which factors are responsible for this high AhR expression, we measured AhR expression in FLSs treated with various cytokines, and found that TNF-{alpha} up-regulated both mRNA and protein of AhR. To confirm this, FLSs were treated with the humanized monoclonal anti-TNF-{alpha} antibody infliximab, which resulted in decreased AhR expression. These findings indicated that AhR expression in RA synovial tissue was stimulated in the presence of high TNF-{alpha} levels, thereby explaining the high sensitivity to TCDD: TCDD exposure induces up-regulation of inflammatory cytokines such as IL-1β, IL-6, IL-8, leading to RA exacerbation.

The data presented herein indicate that TCDD, a major component of cigarette smoke, enhances the RA inflammatory process. TCDD induces inflammatory cytokines via its association with AhR, resulting in the stimulation of the NF-{kappa}B and ERK signalling cascades. In RA synoviocytes, high AhR expression occurs as a result of TNF-{alpha} over-production, a well-known pathological feature of RA. TCDD exposure induces the release of additional pro-inflammatory cytokines, resulting in RA exacerbation as illustrated in Fig. 6. Therefore, similar to other cigarette components that affect the immune system, sex hormone balance and vasculature in the pathogenesis of RA, TCDD also has a role in RA pathogenesis. This hypothesis is consistent with the epidemiological reports that have cited smoking as an exacerbated factor in RA patients. For example, Mattey et al. [10] reported that disease outcome in female RA patients with a history of smoking is significantly worse than in those who have never smoked, and Criswell et al. [30] reported that cigarette smoking leads to an approximately 2-fold increased risk of RA among post-menopausal women. Furthermore, Masdottir et al. [6] reported that heavy smoking was associated with rheumatoid nodules, a higher HAQ score and a lower grip strength, and that any level of smoking was associated with higher radiological joint damage and RF levels. In addition, Nyhall-Wahlin et al. [8] reported a strong association between smoking and rheumatoid nodules in early RA patients with positive RF. Further, Glossop et al. [31] reported that TNF-{alpha} release by stimulated T lymphocytes was significantly higher in patients with a history of smoking than in those who had never smoked, and these authors demonstrated a relationship between smoking duration and intensity (P ≤ 0.009), and that the ratio of TNF-{alpha}/sTNFR released from T lymphocytes was higher in past and current smokers and was associated with the extent of smoking. In addition, Pedersen et al. [32] have demonstrated strong combined gene-environment effects, with markedly increased risks of anti-CCP-positive RA in shared-epitope homozygotes who were heavy smokers (odds ratio 52.6; 95% CI 18.0–154), compared with SE non-carriers who were not exposed to these environmental risk factors. Thus, smoking plays an important role not only in RA progression, but also in RA pathogenesis. However, a recent report by Finckh et al. [33] demonstrated that radiographic joint damage progressed at a similar rate in current smokers and non-smokers, and that smoking intensity was associated with a significant inverse dose–response: heavy-smokers (>1 pack-day) progressed at a significantly slower rate than non-smokers or moderate smokers. However, the majority of research indicates that smoking is not beneficial for RA treatment, despite this controversial data. Further studies are needed to draw a clear picture of precisely how smoking influences RA pathophysiology.


Figure 6
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  		FIG. 6. Schematic representation of the role of AhR in RA. TCDD, a major component of cigarette smoke, enhances the RA inflammatory process. In RA joints, high AhR expression occurs as a result of TNF-{alpha} over-production, a well-known pathological feature of RA. TCDD induces inflammatory cytokines (IL-1β, IL-6 and IL-8) via its association with AhR, resulting in the stimulation of the NF-{kappa}B and ERK signalling cascades. And this positive feedback of cytokine storm causes RA exacerbation.

 
Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The expert technical help of Yukiko Katagiri is gratefully acknowledged.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Gabriel SE. The epidemiology of rheumatoid arthritis. Rheum Dis Clin North Am (2001) 27:269–81.[CrossRef][Web of Science][Medline]
  2. Simonsson M, Bergman S, Jacobsson LT, et al. The prevalence of rheumatoid arthritis in Sweden. Scand J Rheumatol (1999) 28:340–3.[CrossRef][Web of Science][Medline]
  3. Linn-Rasker SP, van der Helm-van Mil AH, van Gaalen FA, et al. Smoking is a risk factor for anti-CCP antibodies only in rheumatoid arthritis patients who carry HLA-DRB1 shared epitope alleles. Ann Rheum Dis (2006) 65:366–71.[Abstract/Free Full Text]
  4. Klareskog L, Stolt P, Lundberg K, et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum (2006) 54:38–46.[CrossRef][Web of Science][Medline]
  5. Saag KG, Cerhan JR, Kolluri S, et al. Cigarette smoking and rheumatoid arthritis severity. Ann Rheum Dis (1997) 56:463–9.[Abstract/Free Full Text]
  6. Masdottir B, Jonsson T, Manfredsdottir V, et al. Smoking, rheumatoid factor isotypes and severity of rheumatoid arthritis. Rheumatology (2000) 39:1202–5.[Abstract/Free Full Text]
  7. Papadopoulos NG, Alamanos Y, Voulgari PV, et al. Does cigarette smoking influence disease expression, activity and severity in early rheumatoid arthritis patients? Clin Exp Rheumatol (2005) 23:861–6.[Web of Science][Medline]
  8. Nyhall-Wahlin BM, Jacobsson LT, Petersson IF, et al. Smoking is a strong risk factor for rheumatoid nodules in early rheumatoid arthritis. Ann Rheum Dis (2006) 65:601–6.[Abstract/Free Full Text]
  9. Padyukov L, Silva C, Stolt P, et al. A gene–environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum (2004) 50:3085–92.[CrossRef][Web of Science][Medline]
  10. Mattey DL, Hutchinson D, Dawes PT, et al. Smoking and disease severity in rheumatoid arthritis: association with polymorphism at the glutathione S-transferase M1 locus. Arthritis Rheum (2002) 46:640–6.[CrossRef][Web of Science][Medline]
  11. Wolfe F. The effect of smoking on clinical, laboratory, and radiographic status in rheumatoid arthritis. J Rheumatol (2000) 27:630–7.[Web of Science][Medline]
  12. Schmidt JV, Bradfield CA. Ah receptor signaling pathways. Annu Rev Cell Dev Biol (1996) 12:55–89.[CrossRef][Web of Science][Medline]
  13. Okey AB, Riddick DS, Harper PA. Molecular biology of the aromatic hydrocarbon (dioxin) receptor. Trends Pharmacol Sci (1994) 15:226–32.[CrossRef][Medline]
  14. Denison MS, Phelan D, Winter GM, et al. Carbaryl, a carbamate insecticide, is a ligand for the hepatic Ah (dioxin) receptor. Toxicol Appl Pharmacol (1998) 152:406–14.[CrossRef][Web of Science][Medline]
  15. Hankinson O. The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol (1995) 35:307–40.[CrossRef][Web of Science][Medline]
  16. Lyte M, Bick PH. Modulation of interleukin-1 production by macrophages following benzo(a)pyrene exposure. Int J Immunopharmacol (1986) 8:377–81.[CrossRef][Web of Science][Medline]
  17. Lecureur V, Ferrec EL, N’diaye M, et al. ERK-dependent induction of TNFalpha expression by the environmental contaminant benzo(a)pyrene in primary human macrophages. FEBS Lett (2005) 579:1904–10.[CrossRef][Web of Science][Medline]
  18. Tanaka G, Kanaji S, Hirano A, et al. Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells. Int Immunol (2005) 17:797–805.[Abstract/Free Full Text]
  19. Henley DV, Bellone CJ, Williams DA, et al. Aryl hydrocarbon receptor-mediated posttranscriptional regulation of IL-1beta. Arch Biochem Biophys (2004) 422:42–51.[CrossRef][Web of Science][Medline]
  20. Henley DV, Bellone CJ, Williams DA, et al. MAPK signaling pathways modulate IL-1beta expression in human keratinocytes. Arch Biochem Biophys (2004) 424:112–8.[CrossRef][Web of Science][Medline]
  21. Vandebriel RJ, Meredith C, Scott MP, et al. Effects of in vivo exposure to bis(tri-n-butyltin)oxide, hexachlorobenzene, and benzo(a)pyrene on cytokine (receptor) mRNA levels in cultured rat splenocytes and on IL-2 receptor protein levels. Toxicol Appl Pharmacol (1998) 148:126–36.[CrossRef][Web of Science][Medline]
  22. Pei XH, Nakanishi Y, Inoue H, et al. Polycyclic aromatic hydrocarbons induce IL-8 expression through nuclear factor kappaB activation in A549 cell line. Cytokine (2002) 19:236–41.[CrossRef][Web of Science][Medline]
  23. Tamaki A, Hayashi H, Nakajima H, et al. Polycyclic aromatic hydrocarbon increases mRNA level for interleukin 1 beta in human fibroblast-like synoviocyte line via aryl hydrocarbon receptor. Biol Pharm Bull (2004) 27:407–10.[CrossRef][Web of Science][Medline]
  24. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum (1988) 31:315–24.[Web of Science][Medline]
  25. García-Vicuña R, Gómez-Gaviro MV, Domínguez-Luis MJ, et al. CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. Arthritis Rheum (2004) 50:3866–77.[CrossRef][Web of Science][Medline]
  26. Blank JA, Tucker AN, Sweatlock J, et al. alpha-Naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced murine lymphocyte ethoxyresorufin-O-deethylase activity and immunosuppression. Mol Pharmacol (1987) 32:169–72.[Abstract]
  27. Pierce JW, Schoenleber R, Jesmok G, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem (1997) 272:21096–103.[Abstract/Free Full Text]
  28. Pei XH, Nakanishi Y, Takayama K, et al. Benzo[a]pyrene activates the human p53 gene through induction of nuclear factor kappaB activity. J Biol Chem (1999) 274:35240–6.[Abstract/Free Full Text]
  29. Yan Z, Subbaramaiah K, Camilli T, et al. Benzo[a]pyrene induces the transcription of cyclooxygenase-2 in vascular smooth muscle cells. Evidence for the involvement of extracellular signal-regulated kinase and NF-kappaB. J Biol Chem (2000) 275:4949–55.[Abstract/Free Full Text]
  30. Criswell LA, Merlino LA, Cerhan JR, et al. Cigarette smoking and the risk of rheumatoid arthritis among postmenopausal women: results from the Iowa Women's Health Study. Am J Med (2002) 112:465–71.[CrossRef][Web of Science][Medline]
  31. Glossop JR, Dawes PT, Mattey DL. Association between cigarette smoking and release of tumour necrosis factor alpha and its soluble receptors by peripheral blood mononuclear cells in patients with rheumatoid arthritis. Rheumatology (2006) 45:1223–9.[Abstract/Free Full Text]
  32. Pedersen M, Jacobsen S, Garred P, et al. Strong combined gene-environment effects in anti-cyclic citrullinated peptide-positive rheumatoid arthritis: a nationwide case-control study in Denmark. Arthritis Rheum (2007) 56:1446–53.[CrossRef][Web of Science][Medline]
  33. Finckh A, Dehler S, Costenbader KH, et al. Cigarette smoking and radiographic progression in rheumatoid arthritis. Ann Rheum Dis (2007) 66:1066–71.[Abstract/Free Full Text]
Submitted 8 January 2008; revised version accepted 16 June 2008.
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