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Published ahead of print on July 8, 2004, doi:10.1165/rcmb.2004-0161OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 463-469, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0161OC

Corticosteroid and Cytokines Synergistically Enhance Toll-Like Receptor 2 Expression in Respiratory Epithelial Cells

Toshiki Homma, Atsushi Kato, Noriko Hashimoto, Jonathan Batchelor, Mamoru Yoshikawa, Shosuke Imai, Hiroshi Wakiguchi, Hirohisa Saito and Kenji Matsumoto

Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo; Division of Allergy, National Center for Child Health and Development, Tokyo; Department of Otorhinolaryngology, Jikei Medical School, Tokyo; Departments of Microbiology and Pediatrics, Kochi Medical School, Kochi; Research Team for Allergy Transcriptome, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan

Address correspondence to: Kenji Matsumoto, M.D., Ph.D., Department of Allergy and Immunology, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan. E-mail: kmatsumoto{at}nch.go.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Respiratory epithelial cells play important roles not only in host defense mechanisms, but also in inflammatory responses. Inhaled corticosteroids are widely used for the treatment of patients with inflammatory lung disorders, including asthma, chronic obstructive pulmonary disease, and sarcoidosis. Corticosteroids effectively reduce the production of inflammatory mediators, such as cytokines and chemokines. Although these molecules are also essential for host defense responses, there is no convincing evidence that inhaled corticosteroids increase susceptibility to lower respiratory tract infections. To test the involvement of Toll-like receptor (TLR) family molecules in this phenomenon, we examined the effects of various cytokines and corticosteroid on the expression of TLRs in human respiratory epithelial cells. Among the TLRs tested, TLR2 expression was significantly enhanced after stimulation with a combination of tumor necrosis factor–{alpha} and interferon-{gamma}. Dexamethasone synergistically enhanced TLR2 expression in combination with tumor necrosis factor–{alpha} and interferon-{gamma} in terms of both mRNA and protein levels. Furthermore, increased cell-surface TLR2 was functional, judging from the remarkable induction of interleukin-6, interleukin-8, and ß-defensin–2 after stimulation with peptidoglycan. These results provide evidence for a novel function of corticosteroids in airway inflammatory disorders, and indicate that the use of inhaled corticosteroids in such disorders may have a beneficial role in host defense mechanisms.

Abbreviations: bacterial lipoprotein, BLP • dexamethasone, DEX • fluorescence-activated cell sorter, FACS • glucocorticoid receptor, GR • glucocorticoid response element, GRE • human ß-defensin–2, hBD2 • interferon, IFN • interleukin, IL • lipopolysaccharide, LPS • mitogen-activated protein kinase, MAPK • mean fluorescence intensity, MFI • MAPK phosphatase-1, MKP-1 • nuclear factor, NF • normal human bronchial epithelial cells, NHBE • nontypeable Haemophilus influenzae, NTHi • pathogen-associated molecular patterns, PAMPs • peripheral blood mononuclear cells, PBMC • peptidoglycan, PGN • Toll-like receptor, TLR • tumor necrosis factor, TNF • reverse transcriptase–polymerase chain reaction, RT-PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Respiratory epithelial cells act as a physical barrier and remove harmful substances and microbial pathogens from the airway by mucociliary clearance. A number of studies have shown that epithelial cells also play an important role in inflammatory responses by releasing various mediators, such as cytokines and chemokines, and by expressing enzymes and adhesion molecules (1). Such observations have been further demonstrated in vivo by the measurement of these mediators in bronchoalveolar lavage fluid or lung tissue specimens from patients with inflammatory lung diseases (2, 3). Regarding infectious diseases in the lung, respiratory epithelial cells also act as frontline effector cells, which release these mediators and keep the airway free of pathogenic organisms (4).

The mammalian host defense against environmental pathogens can be classified into two major forms of immune response: innate and acquired. Recent studies have revealed that a family of type I membrane proteins—Toll-like receptors (TLRs)—have an important role in innate immune responses and subsequent activation of acquired immune responses (5). To date, 10 distinct human TLRs have been identified and shown to mediate specific cellular responses to pathogen-associated molecular patterns (PAMPs). Various tissues and blood cells show distinct expression patterns of TLR1–10 (6).

Among the TLR family, TLR2 has been shown to recognize a wide variety of PAMPs, including bacterial lipoproteins, peptidoglycan (PGN) from Gram-positive bacterial cell wall, and lipoteichoic acids, presumably in combination with TLR1 or TLR6 (7). The importance of TLR2 in host defense responses against pathogenic microorganisms has been demonstrated using TLR2-deficient mice, which have been shown to be highly susceptible to infection by Staphylococcus aureus, Borrelia burgdorferi, Streptococcus pneumoniae, and Mycobacterium bovis bacillus Calmette-Guerin (7, 8). A polymorphism found in human TLR2 has been implicated as a risk factor for staphylococcal infection (9). It has been reported that respiratory epithelial cells express TLR1–6, and upon stimulation with lipopolysaccharide (LPS) or bacterial lipoproteins (BLP), produce proinflammatory cytokines and an antimicrobial molecule, human ß-defensin–2 (hBD2) (10, 11).

Endogenous corticosteroids have evolved to regulate the normal response of the body to physiologic stress and to prevent overreaction (12). Because exogenously administered corticosteroids show strong anti-inflammatory potential in the pathogenesis of various immune diseases, inhaled corticosteroids are used for the treatment of inflammatory lung disorders, such as asthma, chronic obstructive pulmonary disease, and pulmonary sarcoidosis (13, 14). The molecular and cellular mechanisms involved in the anti-inflammatory actions of corticosteroids are now becoming clearer. Their anti-inflammatory actions are mostly dependent on their inhibitory effects on synthesis of proinflammatory mediators, such as cytokines and chemokines, at the transcriptional level. These mediators, which orchestrate inflammatory reactions in the lung, are also considered to be essential for the host defense system (4). However, there is no convincing evidence that inhaled corticosteroids suppress innate immune responses in the lower airway or increase susceptibility to lower respiratory tract infections (15). The precise reasons for these phenomena are still unknown.

In this study, we investigated the effects of corticosteroid on the expression of TLRs in respiratory epithelial cells treated with inflammation- or asthma-associated cytokines and corticosteroid. We demonstrated that functional expression of TLR2 is greatly and synergistically enhanced by the combination of tumor necrosis factor (TNF)-{alpha}, interferon (IFN)-{gamma}, and corticosteroid. These findings provide evidence for a novel function of corticosteroids in airway inflammatory disorders and indicate that use of inhaled corticosteroids may have a beneficial role in host defense mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human TNF-{alpha} and interleukin (IL)-4 were purchased from R&D Systems (Minneapolis, MN). Recombinant human IFN-{gamma} was purchased from Genzyme (Cambridge, MA). Dexamethasone (DEX), RU-486, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO). MG-132 and SB-202190 were purchased from Calbiochem (La Jolla, CA). A TLR2 ligand, PGN, derived from S. aureus, was purchased from Wako pure chemicals (Osaka, Japan). Anti-TLR2 antibodies, TL2.1 (eBioscience, San Diego, CA), and H-175 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for flow cytometry analysis and Western blotting, respectively. Primers for reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR were synthesized at Sawady Technology Co. (Tokyo, Japan).

Cell Culture
The human epithelial lung cancer cell line, A549, and the simian virus 40 (SV-40)–transformed human bronchial epithelial cell line, BEAS-2B, were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium/F12 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal calf serum (HyClone, Logan, UT). Normal human bronchial epithelial cells (NHBE; Clonetics, Walkersville, MD) were maintained in serum-free bronchial epithelial cell basal medium (Clonetics) supplemented with 50 µg/ml bovine pituitary extract, 50 ng/ml human epidermal growth factor, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B (all materials from Clonetics). Cells below second passage were grown to ~ 80% confluence on type I collagen–coated culture plates (BD Biosciences, San Jose, CA) before stimulation with cytokines. Either type of airway epithelial cells was stimulated with 100 ng/ml TNF-{alpha}, 50 ng/ml IL-4, 100 ng/ml IFN-{gamma}, and 1 µM DEX, or a combination of these reagents, for 6 h (for real-time PCR) and 24 h (for flow cytometry and Western blotting) unless otherwise noted.

For functional analysis of TLR2, A549 cells were incubated with TNF-{alpha}, IFN-{gamma}, and DEX for 24 h followed by washing with phosphate-buffered saline and further incubated with 10 µg/ml PGN for 6 h. As a positive control for expression of the TLR family, peripheral blood mononuclear cells (PBMC) from healthy human volunteer donors were isolated by Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation.

RT-PCR and Real-Time RT-PCR
Total RNA samples were isolated from NHBE, BEAS-2B cells, and A549 cells using the RNeasy Kit (QIAGEN, Valencia, CA), and digested with RNase-free DNase I (QIAGEN) following the manufacturer's instructions. For the RT-PCR, an aliquot of 1 µg total RNA was reverse transcribed using SuperScript II (Invitrogen). cDNA generated from 50 ng of total RNA was amplified using Platinum Taq PCR SuperMix (Invitrogen) and primer sets shown in Table 1. Real-time quantitative RT-PCR analyses were performed with the ABI Prism 7,700 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green I PCR reagents (Applied Biosystems) as previously reported (16, 17). To determine exact copy numbers of the target genes, quantified concentrations of subcloned PCR fragments of TLR2, 3, 4, 5, and 6, IL-6 and -8, were serially diluted and used as standards in each experiment. Aliquots of cDNA equivalent to 5 ng of total RNA samples were used for each real-time RT-PCR. Data were normalized with glyceraldehyde-3-phosphate dehydrogenase levels in each sample.


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  		TABLE 1. Primers for RT-PCR and/or real-time RT-PCR

 
Western Blotting and Fluorescence-Activated Cell Sorter
For Western blotting and flow cytometry, BEAS-2B cells were dispersed into single cells by incubation with cell dissociation buffer (Invitrogen). Cell lysates (equivalent to 5 x 104 cells/well) were prepared by dissolving dispersed cells in Laemmli's sample buffer and loaded onto a 4–12% polyacrylamide gradient gel. Proteins were transferred onto Hybond-P (Amersham Biosciences, Arlington Heights, IL), which was blocked in Block Ace (Dainippon Seiyaku, Osaka, Japan) followed by incubation for 1 h with H-175 at a dilution of 1/1,000 in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 0.05% Tween 20. The membrane was then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig) G antibody at a dilution of 1/2,000 and subjected to development of signal by the enhanced chemiluminescence (ECL) reagents (Amersham Biosciences) according to the manufacturer's instructions. Flow cytometric analysis was performed according to the previous report (18). Briefly, cells were suspended in phosphate-buffered saline, containing 5 mg/ml human IgG (Sigma), 0.1% bovine serum albumin and 0.1% NaN3 and then incubated with either phycoerythrin (PE)-conjugated anti-human TLR2 (TL2.1) or PE-conjugated control IgG2a (eBioscience) antibody for 30 min in the dark at 4°C. Scanning and analysis were performed using FACScalibur (Becton Dickinson, San Jose, CA) and CellQuest software (Becton Dickinson). Results were expressed as the mean fluorescence intensity (MFI) ratio calculated by the MFI of cells stained with an antigen-specific antibody divided by the MFI of cells stained with isotype-matched control antibody.

Statistical Analysis
Differences between pairs of groups were tested using Mann-Whitney U-test (StatView 5.0 software, SAS Institute Inc., Cary, NC), with the level of significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA Expression of the TLR Family in Unstimulated Respiratory Epithelial Cells
First we examined spontaneous mRNA expression of the TLR family in unstimulated respiratory epithelial cells by RT-PCR. As shown in Figure 1A, NHBE, BEAS-2B cells, and A549 cells showed similar patterns of TLR family expression. In contrast to PBMC, which expressed all members of the TLR family (1–10), these respiratory cells mainly expressed TLR2–6. Next, real-time RT-PCR was used to examine the effects of individual cytokines, such as TNF-{alpha}, IL-4, and IFN-{gamma} (which are involved in airway inflammation or allergic disorders), and DEX on the expression of mRNA for TLR family in BEAS-2B cells. TNF-{alpha} strongly upregulated the expression of TLR2 (16.8-fold compared with control). IFN-{gamma} significantly upregulated expression of TLR3 and 4 (4.4- and 4.7-fold, respectively). In contrast, IL-4 did not affect the expression of mRNAs for TLR2–6 in this assay. In accord with low levels of expression for TLR1 and TLR7–10, cytokines and DEX did not alter the expression levels for those types of TLRs (data not shown). Intriguingly, there was an 8.2-fold increase in expression of TLR2 by DEX alone. Because there have been no reports about the expressional control of TLRs by cytokines and corticosteroid in human respiratory epithelial cells, the effects of these reagents in combination were examined further.



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  		Figure 1. Spontaneous expression of TLR family and effects of cytokines and DEX on the expression of TLR2–6 mRNA in human respiratory epithelial cells. (A) mRNA expression patterns of TLR1–10 in NHBE, BEAS-2B cells, and A549 cells were examined by RT-PCR. PBMC was used as a positive control. (B) The mRNA levels for TLR2–6 were examined using real-time quantitative RT-PCR. BEAS-2B cells were treated with TNF-{alpha} (100 ng/ml), IL-4 (50 ng/ml), IFN-{gamma} (100 ng/ml), or DEX (1 µM) alone for 6 h. Data are presented as the mean ± SEM of fold increase in three separate experiments. *P < 0.05, versus dimethyl sulfoxide–treated control.

 
Effect of Cytokines and DEX on TLR2 Expression in Respiratory Epithelial Cells
Expression of mRNA for TLR2 was enhanced by addition of IFN-{gamma} to TNF-{alpha}, whereas addition of IL-4 had no effect in either BEAS-2B or A549 cells (Figures 2A and 2B). The effect of IFN-{gamma} was synergistic because IFN-{gamma} alone had little effect on the expression of TLR2 (Figure 1B). Furthermore, the effects of TNF-{alpha} and IFN-{gamma} were dose-dependent, as treatment of BEAS-2B for 6 h with increasing concentrations (1, 10, and 100 ng/ml for both) of these cytokines significantly increased TLR2 mRNA expression (33, 68, and 85-fold compared with control, respectively P < 0.05). Because the maximum effect of these cytokines was observed at 100 ng/ml, we chose this condition for the further analysis. Surprisingly, the addition of DEX to these conditions greatly enhanced the expression of TLR2 ~ 7- to 10-fold in BEAS-2B cells (Figure 2A). Despite the high concentration of cytokines and the presence of DEX, the viability of BEAS-2B cells examined was not reduced significantly, even after 24 h treatment (by fluorescence-activated cell sorter [FACS] analysis after propidium iodide staining [data not shown]). Similar results were also observed in A549 cells and NHBE (Figures 2B and 2C). On the other hand, the expression of other TLRs (TLR3–6) was not significantly altered by a combination of the cytokines in our experimental system (data not shown).



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  		Figure 2. Synergistic effects of cytokines and DEX on TLR2 expression on three types of respiratory epithelial cells. BEAS-2B cells (A) and A549 cells (B) were treated with TNF-{alpha} (100 ng/ml), IL-4 (50 ng/ml), and IFN-{gamma} (100 ng/ml), either alone or in combination with dimethyl sulfoxide (open bars) or with 1 µM DEX (closed bars) for 6 h. (C) In NHBE cells, a combination of TNF-{alpha} (100 ng/ml) and IFN-{gamma} (100 ng/ml) with dimethyl sulfoxide (open bars) or with 1 µM DEX (closed bars) was examined. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05, DEX-treated versus dimethyl sulfoxide–treated control; {dagger}P < 0.05, a combination of TNF-{alpha} and IFN-{gamma} versus TNF-{alpha} alone.

 
Effects of DEX on Cytokine-Driven TLR2 Upregulation
We next examined the dosage effects of DEX on cytokine-driven TLR2 upregulation (Figure 3A). BEAS-2B cells were treated with 0.001, 0.010, 0.100, or 1.000 µM DEX in the presence of TNF-{alpha} (100 ng/ml) and IFN-{gamma} (100 ng/ml) for 6 h. A synergistic effect of DEX was observed in a dose-dependent fashion and clinically feasible concentrations of DEX (range, 0.01–1 µM) significantly enhanced expression of mRNA for TLR2 in this assay. To test whether DEX enhances the cytokine-driven TLR2 upregulation via glucocorticoid receptors (GRs), we added a GR antagonist, RU-486, at an equal concentration to DEX (1 µM) simultaneously with DEX and a combination of TNF-{alpha} and IFN-{gamma}. As shown in Figure 3B, RU-486 almost completely abrogated the synergistic effects of DEX, whereas RU-486 did not enhance TLR2 expression in combination with TNF-{alpha} and IFN-{gamma}. These results suggest that DEX acts through GR in upregulating mRNA expression for TLR2 in respiratory epithelial cells.



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  		Figure 3. Effect of DEX on the expression of TLR2 mRNA in BEAS-2B cells in a combination with TNF-{alpha} and IFN-{gamma}. (A) BEAS-2B cells were treated with different concentrations of DEX (0.001–1.000 µM) and a fixed concentration of TNF-{alpha} (100 ng/ml) and IFN-{gamma} (100 ng/ml) (*P < 0.05, versus dimethyl sulfoxide–treated control). (B) Effect of a GR antagonist RU-486 (1 µM) on the upregulation of TLR2 mRNA in BEAS-2B cells treated with a combination of TNF-{alpha}, IFN-{gamma}, and DEX (1 µM) (*P < 0.05, versus dimethyl sulfoxide–treated control; {dagger}P < 0.05, versus DEX treatment). (C) Effect of pretreatment (1 h) with a proteasome inhibitor MG-132 (1 µM) and a p38 MAPK inhibitor SB-202190 (10 µM) on the expression of TLR2 mRNA in BEAS-2B cells treated with a combination of TNF-{alpha}, IFN-{gamma}, and DEX (1 µM) ({dagger}P < 0.05, versus dimethyl sulfoxide–treated control).

 
It has been reported that DEX synergistically enhances nontypeable Haemophilus influenzae (NTHi)–induced TLR2 expression via activation of nuclear factor (NF)-{kappa}B pathway and inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway in human bronchial epithelial cells (19). The research showed that a p38 MAPK inhibitor enhanced the NTHi-induced TLR2 expression in HeLa cells to a degree similar to that seen with DEX. To examine the involvement of the NF-{kappa}B and p38 MAPK pathways in cytokine-driven TLR2 upregulation, we pre-incubated BEAS-2B cells with an NF-{kappa}B pathway inhibitor (20), MG-132 (1 µM), or a specific p38 MAPK inhibitor, SB-202190 (10 µM). As shown in Figure 3C, TLR2 expression stimulated by TNF-{alpha} and IFN-{gamma} was significantly inhibited by MG-132 both in the presence and absence of DEX. In contrast, SB-202190 failed to inhibit the upregulation of TLR2 by cytokines both in the presence or absence of DEX, although the same concentration (10 µM) of SB-202190 showed significant inhibitory effects on cytokine-induced inducible protein of 10 kD mRNA expression (data not shown). Our results suggest that a signaling pathway other than p38 MAPK may exist through which DEX exerts its synergistic effects on TNF-{alpha} and IFN-{gamma}–induced TLR2 upregulation.

Expression of TLR2 Protein in Respiratory Epithelial Cells
We further confirmed the upregulation of TLR2 expression at protein levels. In Figure 4A, ~ 96 kD bands corresponding to TLR2 were observed by Western blotting of total cell lysates from BEAS-2B cells treated with TNF-{alpha} and IFN-{gamma} in the presence or absence of DEX for 24 h. The intensity of each band was well correlated with the expressional level of TLR2 mRNA in BEAS-2B cells in each treatment. Moreover, FACS analysis revealed that the cell surface expression of TLR2 was also strongly upregulated in TNF-{alpha}, IFN-{gamma}, and DEX-treated BEAS-2B cells when compared with the control cells. These results indicate that treatment of respiratory epithelial cells with cytokines (TNF-{alpha} and IFN-{gamma}) and DEX strongly enhanced TLR2 expression at both mRNA and protein levels.



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  		Figure 4. Expression of TLR2 protein in BEAS-2B cells. (A) Western blotting analysis of TLR2 protein in BEAS-2B cells after treatment with cytokines and DEX for 24 h (lane 1, control; lane 2, TNF-{alpha} [100 ng/ml], IFN-{gamma} [100 ng/ml], and dimethyl sulfoxide; and lane 3, TNF-{alpha}, IFN-{gamma}, and DEX [1 µM]). (B) Cell-surface expression of TLR2 by flow cytometry on BEAS-2B cells after treatment with dimethyl sulfoxide (left panel), a combination of TNF-{alpha}, IFN-{gamma}, and dimethyl sulfoxide (center panel) and a combination of TNF-{alpha}, IFN-{gamma}, and DEX (right panel). Cells are stained with isotype control monoclonal antibody (dotted lines) and anti-human TLR2 monoclonal antibody (solid lines). Data are representative of three independent experiments.

 
Functional Analysis of TLR2 in Respiratory Epithelial Cells
Finally, to examine whether or not surface-expressed TLR2 is functional, we stimulated TLR2-upregulated A549 cells with PGN, a TLR2-specific ligand. As a result, mRNA for IL-6 and IL-8 were dose-dependently induced by the 6 h stimulation with PGN (Figures 5A and 5B). The expression of mRNA for both IL-6 and IL-8 was remarkably enhanced when cells were pretreated with DEX, TNF-{alpha}, and IFN-{gamma}. mRNA expression for IL-6 by PGN was also significantly enhanced in BEAS-2B and NHBE cells after treatment with a combination of TNF-{alpha}, IFN-{gamma}, and DEX (data not shown). We further tested the expression of the antimicrobial molecule, hBD2, because, according to a previous study that A549 cells express hBD2 upon stimulation with another TLR2 ligand, BLP (11). As a result, a significant induction of hBD2 mRNA was found only in TLR2-upregulated A549 cells upon stimulation with PGN by RT-PCR (Figure 5C). These results demonstrate that treatment of respiratory epithelial cells with cytokines (TNF-{alpha} and IFN-{gamma}) and DEX enhances cell-surface expression of TLR2 and may enhance responsiveness of epithelial cells to TLR2 ligands, such as PGN.



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  		Figure 5. PGN-induced IL-6, IL-8, and hBD2 mRNA in A549 cells after stimulation with cytokines and DEX. A549 cells were cultured in the presence (closed bars) or absence (open bars) of a combination of TNF-{alpha} (100 ng/ml), IFN-{gamma} (100 ng/ml), and DEX (1 µM) for 24 h followed by a washout of media. Cells were then treated with various concentrations of PGN for 6 h. Copy numbers of IL-6 (A) and IL-8 (B) were determined by real-time quantitative RT-PCR. Data are presented as the mean ± SEM of three separate experiments. *P < 0.05 (for TNF-{alpha}, IFN-{gamma}, and DEX-treated groups); {dagger}P < 0.05 (for nontreated groups) versus distilled water (vehicle of PGN)-treated control. (C) hBD2 induction was detected by RT-PCR. Data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated expression of the TLR family in respiratory epithelial cells and expressional control of several TLRs by cytokines and DEX. As shown in Figure 1, expression patterns of the TLR family in different respiratory epithelial cells were almost consistent and showed TLR2–6 as main transcripts. Compared with mixed human immune cell populations represented in PBMC, as measured by RT-PCR, expression of TLR1 and TLR7–10 by respiratory epithelial cells was very low. Judging from their characteristic pattern of TLR expression, respiratory epithelial cells may have the potential to respond to a broad range of PAMPs derived from Gram-positive and Gram-negative bacteria, viruses, and mycoplasmas. Our results are consistent with previous reports that either primary culture or cell lines of respiratory epithelium were activated in vitro by various PAMPs, such as bacterial lipopeptide (19), dsRNA (21), LPS (10), and flagellin (22).

There have been no reports on the control of expression of the TLR family in human respiratory epithelial cells by cytokines and corticosteroids. In the present study, we clearly showed that TLR2 was greatly upregulated by TNF-{alpha} alone, whereas TLR3 and TLR4 were upregulated several-fold by IFN-{gamma} alone (Figure 1B). A synergistic effect of IFN-{gamma} on TNF-{alpha} for TLR2 expression was also observed in these epithelial cells (Figures 2A and 2B). Upregulation of these TLR family genes in respiratory epithelial cells in response to inflammatory stimuli, such as TNF-{alpha} and IFN-{gamma}, may have important implications in augmenting the host defense against microbial organisms in sites of tissue inflammation. It is notable that DEX alone showed significant TLR2-induction in respiratory epithelial cells.

Because the expression of TLR2 was strongly upregulated by treatment with cytokines individually or in combination, and with or without DEX, we focused our study on TLR2 expression in respiratory epithelial cells. Regarding the effects of cytokines or PAMPs on expression of human TLR2 in other tissues or cell types, several reports have been published. TNF-{alpha}, IL-4, and IFN-{gamma} are known to downregulate TLR2 expression in human monocytes, whereas LPS, granulocyte–macrophage colony-stimulating factor, IL-1 and IL-10 upregulate cell surface expression of TLR2 (23). Kurt-Jones and colleagues also showed that granulocyte–macrophage colony-stimulating factor treatment enhances TLR2 expression in human neutrophils (24).

In contrast to human leukocytes, tissue residual cells show a different pattern of responses to cytokines in TLR2 regulation. In human vascular endothelial cells, LPS, IFN-{gamma}, and TNF-{alpha} are reported to upregulate TLR2 expression (25). In human oral epithelial cells, IFN-{gamma} treatment also upregulates TLR2 expression (26). The results obtained in our study show that the regulation of TLR2 expression in human respiratory epithelial cells resembles that seen in nonimmune cell types, in that TNF-{alpha} and IFN-{gamma} upregulate TLR2 expression.

One of the most important findings in our study is that DEX synergistically enhanced expression of TLR2 with a combination of TNF-{alpha} and IFN-{gamma} on human respiratory epithelial cells (Figures 2A–C); IL-4 did not alter the synergistic effect of a combination of DEX and TNF-{alpha}. In addition, we demonstrated that the enhanced expression of TLR2 at the protein level and the cell surface–expressed TLR2 were functional, judging from the remarkable induction of mRNA expression for IL-6 and IL-8 and the host defense molecule hBD2 after stimulation with PGN. Because these gene products are known to be important for innate immune responses and host defense reactions (4, 27), upregulated TLR2 in respiratory epithelial cells may consequently enhance the antimicrobial responses of the host.

Inhaled corticosteroids are widely used as a first-line therapeutic agent in patients with inflammatory lung diseases, such as asthma, and have beneficial effects in patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis (14, 28). However, there is no convincing evidence that inhaled corticosteroids suppress innate immune responses in the lower airway or increase susceptibility to lower respiratory tract infections (15, 29). Moreover, despite the fact that corticosteroids can suppress most immune and inflammatory responses, the adjunctive use of corticosteroids has been found to be beneficial in the treatment of infectious diseases, including Pneumocystis carinii pneumonia, tuberculosis, and NTHi infection (30, 31). Given that production of proinflammatory cytokines in the lung are evident in patients with such diseases (32), the enhanced expression of TLR2 can be expected when patients are treated with corticosteroids. Expression of TLR2 in vivo should be studied further in clinical settings to confirm the role of TLR2 in airway inflammation and host defense systems when inflammatory cytokines and corticosteroids are present. Such investigations may provide a new rationale for the beneficial use of inhaled corticosteroids in the treatment of patients with inflammatory lung diseases.

With respect to the molecular mechanisms through which DEX synergistically enhances TLR2 expression with cytokines, our data suggest the involvement of GR in this phenomenon, as RU-486 inhibited the synergistic effect of DEX (Figure 3B). The molecular mechanism of GR-mediated transactivation has been explained by the binding of two glucocorticoid-GR complexes as a homodimer to specific glucocorticoid response elements (GRE) in GR target genes (28, 33). The proximal promoter sequence of the human TLR2 gene has been reported (34), though a consensus sequence of the GRE/progesterone response element, PuGNACANNNTGTNCPy (where Pu, Py, and N are purine, pyrimidine, and nonspecific nucleotide, respectively) (35) was absent in the promoter region. We did find, however, a putative GRE/progesterone response element sequence, AGCACACAGTGTCCC (between –3,734 and –3,720 bp upstream of the intron II/exon III boundary of the TLR2 gene), in a TLR2-containing bacterial artificial clone (GenBank No. AC106865) by using a computational search (DNASIS Pro; Hitachi Software, Tokyo, Japan). Further studies are required to elucidate whether this putative GRE acts as an enhancer for TLR2 expression by corticosteroids.

It has been previously reported that the expression of TLR2 in bronchial epithelial cells is to be enhanced after stimulation with NTHi and DEX (19, 36). These reports showed that NTHi activated both NF-{kappa}B and p38 MAPK pathways, the former acting positively and the latter negatively in regulating TLR2 expression. Furthermore, DEX was shown to synergistically upregulate NTHi-induced TLR2 expression via induction of MAPK phosphatase-1, which in turn suppressed p38 MAPK activity. Our results, though not direct evidence, support the involvement of the NF-{kappa}B pathway for TLR2 induction (Figure 3C). In addition, we detected a moderate induction of MAPK phosphatase-1 mRNA in DEX-treated BEAS-2B cells using an oligonucleotide microarray system (data not shown); however, a p38 MAPK inhibitor had no effect on TLR2 upregulation by DEX in our study (Figure 3C). Taken together, these findings suggest the presence of different signaling pathways or transcriptional control mechanisms by which DEX synergistically enhanced cytokine-driven and NTHi-driven TLR2 upregulation. The precise molecular mechanisms for the enhanced expression of TLR2 by corticosteroid alone and in combination with cytokines should be studied further. This could, for example, provide safe and effective ways to upregulate TLR2 expression in the treatment of immune-compromised patients.

In conclusion, our findings provide evidence for a novel function of corticosteroids in airway inflammatory disorders and indicate that the use of inhaled corticosteroids in inflammatory lung diseases may have a beneficial role in the support of host defense mechanisms.


   Acknowledgments
 
The authors thank Dr. Shigeru Okumura and Naoko Okada for helpful discussions and technical assistance. This work was supported in part by a grant from the organization for Pharmaceutical Safety and Research and the Ministry of Health, Labor, and Welfare (the Millennium Genome Project, MPJ-5), and by a grant from RIKEN Research Center for Allergy and Immunology.


   Footnotes
 
Conflict of Interest Statement: T.H. has no declared conflicts of interest; A.K. has no declared conflicts of interest; N.H. has no declared conflicts of interest; J.B. has no declared conflicts of interest; M.Y. has no declared conflicts of interest; S.I. has no declared conflicts of interest; H.W. has no declared conflicts of interest; H.S. has no declared conflicts of interest; and K.M. has no declared conflicts of interest.

Received in original form May 12, 2004

Received in final form June 15, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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