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Published ahead of print on May 13, 2004, doi:10.1165/rcmb.2003-0438OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 330-336, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0438OC

A20 Inhibits Toll-Like Receptor 2– and 4–Mediated Interleukin-8 Synthesis in Airway Epithelial Cells

Yasuhiro Gon, Yasukiyo Asai, Shu Hashimoto, Kenji Mizumura, Itsuro Jibiki, Tatsuya Machino, Chisei Ra and Takashi Horie

First Department of Internal Medicine, Nihon University School of Medicine, and Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, Tokyo, Japan

Address correspondence to: Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku, Tokyo 173–8610, Japan. E-mail: shuh{at}med.nihon-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of nuclear factor (NF)-{kappa}B–dependent gene expression induced by different stimuli, including tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1ß (IL-1ß). Toll-like receptor 2 (TLR2) and TLR4 have been found to transduce, respectively, peptidoglycan (PGN) and lipopolysaccharide (LPS) signals for the activation of NF-{kappa}B and the production of inflammatory cytokines. Here, we have examined the role of A20 in TLR-mediated NF-{kappa}B–dependent gene expression in human airway epithelial cells (AECs). Stimulation with LPS and PGN resulted in a significant increase in the level of A20 mRNA in primary cultured AECs and in NCI-H292 AECs. LPS and PGN induced activation of the IL-8 promoter both in NCI-H292 AECs and in HEK293 cells expressing either TLR2 or TLR4 plus MD-2. Dominant-negative myeloid differentiation protein and a mutant form of I{kappa}B{alpha} attenuated this PGN- or LPS-induced activation of the IL-8 promoter. Furthermore, overexpression of A20 inhibited activation of both NF-{kappa}B and the IL-8 promoter by PGN or LPS in these cells. Taken together, our results suggest that A20 may function as a negative regulator of TLR-mediated inflammatory responses in the airway, thereby protecting the host against harmful overresponses to pathogens.

Abbreviations: airway epithelial cells, AECs • dominant-negative form, DN • fluorescence-activated cell sorter, FACS • interleukin, IL • IL-1 receptor –associated kinase-1, IRAK1 • lipopolysaccharide, LPS • myeloid differentiation protein, MyD88 • nuclear factor-{kappa}B, NF-{kappa}B • pathogen-associated molecular patterns, PAMPs • peptidoglycan, PGN • suppressor of cytokine-signaling-1, SOCS1 • Toll receptor–IL-1R domain–containing adapter protein, TIRAP • Toll-like receptor, TLR • tumor-necrosis factor-{alpha}, TNF-{alpha} • TNF receptor–associated factor, TRAF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
As a primary interface between pathogens and the airway, epithelial cells lining the airway form a crucial site for the innate immune response. Rather than a passive barrier, the airway epithelium is an active participant in the mucosal immune response through its expression of proinflammatory genes, its secretion of inflammatory cytokines, and its recruitment of inflammatory cells in response to pathogenic bacteria and their products (1, 2).

Toll-like receptors (TLRs) are a family of transmembrane receptors that are homologous to the Drosophila Toll protein (3). These receptors are termed "pattern-recognition receptors" because they recognize repetitive patterns, termed "pathogen-associated molecular patterns" (PAMPs), that are present on diverse microbes including Gram-positive and Gram-negative bacteria, fungi, and mycobacteria. The interaction of TLR with its PAMP results in the activation of multiple intracellular signaling events through its Toll/interleukin (IL)-1R domain, including the activation of nuclear factor (NF)-{kappa}B and the production of cytokines such as tumor necrosis factor (TNF)-{alpha} and IL-6 (3, 4). TLR4 is required for the recognition of lipopolysaccharide (LPS), and mutations or deletion of TLR4 render animals unresponsive to LPS (5). By contrast, TLR2 is required for the recognition of Gram-positive and mycobacterial PAMPs including bacterial lipopeptide, lipoteichoic acid, peptidoglycan (PGN) and soluble tuberculosis factor (3, 6). Thus, TLR signaling represents a key component of the innate immune response to bacterial infection.

It is known that the dysfunction of innate immunity may result in recurrent airway infections, as is seen in cystic fibrosis (7). It is also likely that overresponses to pathogens result in acute lung injury, such as acute respiratory distress syndrome (8–10). It has been suggested that the mechanism that regulates inflammatory responses induced by innate immune function in airway epithelium may play an important role in controlling inflammatory lung disease.

The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of NF-{kappa}B–dependent gene expression that is induced by different stimuli including TNF-{alpha}, IL-1ß, and tetradecanoic phorbol acetate (TPA) (11), activation of the B cell surface receptor CD40 (12), adhesion to extracellular matrix proteins, and bacterial LPS (13). The expression of A20 is itself under the control of NF-{kappa}B (14), suggesting that A20 is involved in a negative feedback regulation of NF-{kappa}B activation. It is therefore possible that A20 may be a negative regulator of TLR-triggered inflammatory responses. At present, however, the role of A20 in TLR-mediated inflammation is unclear.

Here we show that expression of A20 is induced by the TLR4 ligand LPS and the TLR2 ligand PGN in airway epithelial cells (AECs). Furthermore, we demonstrate that A20 inhibits TLR2- and TLR4-induced NF-{kappa}B–dependent production of IL-8 by interfering with a TLR-mediated signaling pathway. Thus, A20 might play a critical role in limiting inflammation by terminating TLR-induced NF-{kappa}B responses in the airway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
LPS (Escherichia coli serotype 055:B5) was obtained from Sigma (St. Louis, MO). PGN from Staphylococcus aureus was from Fluka Biochemica (Steinheim, Switzerland). Anti-TLR2 monoclonal antibody (mAb) TL2.1 and anti-TLR4 mAb HTA125 were purchased from eBioscience (San Diego, CA). A negative control antibody (Ab) (immunoglobulin G2a [IgG2a]) was purchased from BD PharMingen (Oslo, Norway).

Cells and Cell Culture
Primary cultured normal human AECs were purchased from Clonetics (San Diego, CA). Primary cultured normal human AECs were maintained in serum-free modified LHC-9 medium (Clonetics) supplemented with epidermal growth factor (0.5 ng/ml) and bovine pituitary extract (50 µg/ml); the cells were used on their second and third passages. The AEC line NCI-H292 and the human embryonic kidney epithelial cell line HEK293 were originally obtained from ATCC and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells were trypsinized, transferred into 12-well plates, and allowed to recover overnight. The adherent cells were washed three times with serum-free Dulbecco's modified Eagle's medium.

Flow Cytometric Analysis of TLR2 and TLR4 Expression
Normal human AECs and NCI-H292 AECs were plated in 12-well tissue-culture plates at a density of 3 x 105 cells/well in an appropriate selective medium and incubated overnight at 37°C under a 5% CO2 atmosphere. Thereafter, the cells were washed three times with phosphate-buffered saline, detached from the plastic with trypsin plus EDTA, and examined by fluorescence-activated cell sorter (FACS) analysis. Expression of human TLR4 was measured by incubating the cells for 30 min on ice with Abs specific for TLR2 or TLR4 or a control Ab (5 µg/ml each), washing twice with phosphate-buffered saline, and incubating for 30 min with a secondary fluorescein isothiocyanate–labeled anti-mouse IgG (5 µg/ml). Thereafter, the cells were subjected to flow cytometric analysis on a FACSCalibur analyzer (BD Biosciences, Mountain View, CA).

Reverse Transcriptase–Polymerase Chain Reaction Analysis of A20 Expressions
Total RNA was extracted from wild-type human AECs and NCI-H292 AECs by using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. First-strand cDNA was generated from 3 µg of total RNA with random hexamer primers and a First-Strand cDNA Synthesis kit (Pharmacia, Uppsala, Sweden). For polymerase chain reaction (PCR) quantification, 0.5 µl of cDNA reaction was amplified in a 20-µl standard PCR reaction. For the detection of A20, 28 cycles were performed. PCR was performed using primers for A20 (5'-CATCAGTGCCACTTCTCAGT and 5'-GCTGCTATAGCCGAGAACAA), and species-nonspecific GAPDH (5'-AGT ATG ACT CCA CTC ACG GCA A and 5'-TCT CGC TCC TGG AAG ATG GT).

Measurement of IL-8
NCH-H292 AECs were stimulated with LPS or PGN in the presence of 10% fetal calf serum, as a source of soluble CD14. To determine the IL-8 concentration, the culture media were harvested and centrifuged after 1, 3, and 6 h of cultivation, and the supernatants were collected, filtrated with a millipore filter, and stored at –80°C until assay. The concentration of IL-8 in the culture supernatants was measured by a commercially available enzyme-linked immunosorbent assay kit (Amersham Corp., Arlington Heights, IL), used in accordance with the manufacturer's instructions. All samples were assayed in duplicate.

Plasmids
The human A20 cDNA was a kind gift from Nancy Raab-Traub and has been described elsewhere (15). Constructs encoding human TLR2 and TLR4 were kind gifts from S. Akira (University of Osaka, Osaka, Japan). The human MD-2 expression plasmid (16) was a kind gift from K. Kawasaki (National Institute of Infectious Diseases, Tokyo, Japan). Constructs encoding wild-type (wt) and a dominant-negative form (DN) of MyD88 were a kind gift from Marta Muzio (Mario Negri Institute, Milan, Italy). Constructs encoding wt of IL-1 receptor–associated kinase-1 (IRAK1) were a kind gift from Tularik, Inc. (San Francisco, CA). The construct encoding the I{kappa}B{alpha}, MAD-3 double-point mutant (AT positions 32 and 36) (17) was a kind gift from Patrick A. Baeuerle (Tularik, Inc.).

Transfection and Luciferase Assays
NCI-H292 and HEK293 cells were cultured at 0.5 x 106 /ml in 12-well plates (1 ml/well) for 16–20 h and were transfected with the amount of DNA indicated in the figure legends by using Fugene6 (Roche Diagnostic Systems, Somerville, NJ). Duplicate or triplicate wells were analyzed for each group. An NF-{kappa}B–luciferase plasmid, containing five copies of the consensus NF-{kappa}B site linked to a minimal promoter-luciferase reporter gene, was purchased from Stratagene (La Jolla, CA). Construction of a reporter construct containing 1,521 bp (nucleotides –1481 to +40) of the promoter region of the gene encoding human IL-8 was a gift from H. Takizawa (University of Tokyo, Tokyo, Japan) and has been described previously (18). Cells were allowed to recover for 24 h and then were left unstimulated or were stimulated with LPS and PGN as described in the figure legends. Six hours after treatment, cell lysates were prepared and were assayed by the Promega Luciferase Assay System (Promega Biotech, Madison, WI), used in accordance with the manufacturer's instructions. In some experiments, pRL-Tk (Promega) was co-transfected as internal control to normalize differences in the transfection efficiency. Lysates from these cells were quantified for luciferase using the Dual-Luciferase Reporter Assay System (Promega).

Statistics
Statistical significance was assessed by ANOVA. A P value of < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TLR2 and TLR4 on AECs
Initially, we wanted to determine whether wild-type AECs and NCI-H292 AECs express TLR2 or TLR4. To address this issue, the expression of TLR2 and TLR4 on primary cultured AECs and NCI-H292 AECs was assessed by FACS analysis. FACS analysis with an anti-TLR4/MD-2 (HTA125) mAb showed that both types of cell expressed TLR4 and MD-2 on the cell surface. Cell-surface expression of TLR2 was also detectable in these cells by FACS using an anti-TLR2 mAb (TL2.1) (Figure 1).



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  		Figure 1. Expression of TLR2 and TLR4 on AECs. Primary cultured normal AECs and NCI-H292 AECs were incubated with anti-TLR2 mAb (TL2.1) and anti-TLR4/MD2 mAb (HTA-125), and TLR2 and TLR4/MD2 expression was analyzed by FACS. The results are representative of two different experiments.

 
PGN and LPS Increase IL-8 Protein Levels
To confirm the functional expression of TLR2 and TLR4 on AECs, NCI-H292 AECs were cultured in the presence of LPS or PGN, and the concentration of IL-8 in the culture supernatants was determined by enzyme-linked immunosorbent assay at 1, 3, and 6 h after cultivation. As shown in Figure 2A, the concentration of IL-8 protein was increased in a time-dependent manner by LPS and PGN stimulation. NCI-H292 cells were pretreated with anti-TLR2 inhibitory antibody, TL2.1, and anti-TLR4/MD2 inhibitory antibody, HTA-125, before addition of PGN or LPS. Results in Figure 2B show that TL2.1 inhibited PGN-induced IL-8 protein from AECs, but it had no inhibiting effect on the LPS-induced IL-8. We also found that HTA-125 inhibited LPS-induced IL-8, but not PGN-induced IL-8. Our results show that stimulation of TLR2 or TLR4 in NCI-H292 AECs induces the production of IL-8.



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  		Figure 2. Production of IL-8 by LPS- and PGN-stimulated AECs. (A) NCI-H292 AECs were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 1, 3, and 6 h, and the concentration of IL-8 in the culture supernatants was determined. The results are the mean ± SD of five different experiments. *P < 0.01 compared with PGN- and LPS-induced IL-8 production. **P < 0.05 compared with PGN- and LPS-induced IL-8 production. *P < 0.01 and **P < 0.05 compared with unstimulated cells. (B) NCI-H292 cells were pretreated without antibody (1) or with 10 µg/ml of TLR2.1 (2) and HTA125 mAb (3) for 1 h before stimulation of PGN (left) or LPS (right). The cells were incubated for an additional 6 h before bioactive IL-8 in supernatants was assayed. The results are the mean ± SD of five different experiments. *P < 0.01 compared with PGN- or LPS-stimulated cells without antibodies.

 
Transfection of a Dominant-Negative Form of MyD88 Attenuates Activation of the IL-8 Promoter
We next examined the endogenous expression of TLR2 and TLR4 in HEK293 cells by reverse transcriptase (RT)-PCR. HEK293 cells expressed no or little endogenous TLR2 and TLR4 (data not shown); therefore, HEK293 cells cannot respond to PGN or LPS. To evaluate the signal transduction pathway used by the TLRs to induce IL-8 production, we transfected each TLR expression plasmid into HEK293 cells, and tested the effect of PGN- or LPS-induced activation of the IL-8 promoter by luciferase assay. We found that HEK293 cells transfected with TLR2 responded to PGN, as measured by IL-8 promoter activation (Figure 3A, left). HEK293 cells transfected with TLR4 plus MD-2, which is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses, also responded to LPS (Figure 3A, right). Overexpression of MyD88-DN inhibited activation of the IL-8 promoter by PGN in TLR2-transfected HEK293 cells (Figure 3A, left). MyD88-DN also inhibited activation of the IL-8 promoter by LPS in HEK293 cells transfected with TLR4 plus MD-2 (Figure 3A, right). Similar results were obtained in NCI-H292 AECs stimulated with PGN or LPS (Figure 3B), indicating that TLR-mediated activation of the IL-8 promoter is regulated by MyD88-dependent signaling in AECs, as well as in other types of cell.



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  		Figure 3. Dominant-negative MyD88 inhibits LPS- or PGN-induced activation of the IL-8 promoter in AECs. (A) HEK293 cells transfected with TLR2 (left) or TLR4 plus MD-2 (right) were grown on 12-well plates and co-transfected with MyD88-DN and IL-8 promoter-luciferase reporter plasmids for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. (B) NCI-H292 AECs grown on 12-well plates were co-transfected with MyD88-DN and IL-8 promoter-luciferase reporter plasmids for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. Luciferase activity in the cytoplasmic extracts was measured, and the results are given as the fold induction of mean luciferase light units with reference to unstimulated cells. *P < 0.01, compared with PGN- or LPS-stimulated empty vector-transfected cells.

 
Transfection of an I{kappa}B Mutant Attenuates Activation of the IL-8 Promoter
The IL-8 promoter has a binding site for NF-{kappa}B, one of the main transcription factors involved in regulating genes during inflammatory and immune responses. Among the many proteins exhibiting I{kappa}B function, I{kappa}B{alpha} is the only inhibitor that, in response to cell stimulation, dissociates from the NF-{kappa}B heterodimer complex, with kinetics matching that of the translocation of NF-{kappa}B to the nucleus. It has been therefore suggested that the inducible activation of NF-{kappa}B is regulated mainly by the dissociation of NF-{kappa}B and I{kappa}B{alpha} (17, 19). To examine the role of LPS- or PGN-mediated NF-{kappa}B signaling in regulating the IL-8 gene, we transfected TLR2 or TLR4 plus MD-2 expression plasmids into HEK293 cells expressing a mutated I{kappa}B{alpha} gene, in which two serines in the N-terminal domain of I{kappa}B{alpha}, Ser-32 and Ser-36, that are critical for I{kappa}B{alpha} stability have been replaced with alanine to render I{kappa}B{alpha} undegradable by cellular activators. We then assayed the activation of the IL-8 promoter by luciferase assay.

The I{kappa}B{alpha} mutant blocked the inducible activation of NF-{kappa}B by inhibiting the dissociation of NF-{kappa}B and I{kappa}B{alpha}. As shown Figure 4A, mutant I{kappa}B{alpha} completely inhibited activation of the IL-8 promoter by LPS and PGN in TLR-expressing HEK293 cells. Similar to the results of overexpressing the I{kappa}B{alpha} mutant in the NCI-H292 line, mutant I{kappa}B{alpha} also inhibited activation of the IL-8 promoter by LPS and PGN, suggesting that NF-{kappa}B is required for the induction of IL-8 production by LPS and PGN in NCI-H292 AECs (Figure 4B).



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  		Figure 4. Mutant I{kappa}B{alpha} inhibits LPS- or PGN-induced activation of the IL-8 promoter in AECs. (A) HEK293 cells transfected with TLR2 (left) or TLR4 plus MD-2 (right) were grown on 12-well plates and cotransfected with mutant I{kappa}B{alpha} (I{kappa}B-M) and IL-8 promoter-luciferase reporter plasmids for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. (B) NCI-H292 AECs grown on 12-well plates were co-transfected with mutant I{kappa}B{alpha} and IL-8 promoter-luciferase reporter plasmids for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. One representative experiment of five is shown. *P < 0.01, compared with PGN- or LPS-stimulated empty vector–transfected cells.

 
PGN and LPS Induce A20 mRNA in AECs
To study the effect of TLR signaling on the regulation of A20 in AECs, we used RT-PCR to examine whether the TLR ligands PGN and LPS could induce the expression of A20 mRNA. Primary cultured human AECs and NCI-H292 AECs in serum-containing medium were stimulated with LPS and PGN for 6 and 12 h, and harvested for the analysis of A20 mRNA by RT-PCR. As shown in Figure 2, stimulation with LPS and PGN resulted in a significant increase in the level of A20 mRNA at both 6 and 12 h, whereas LPS and PGN did not affect levels of the control GAPDH transcript (Figure 5).



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  		Figure 5. Regulation of A20 mRNA expression in AECs in response to PGN and LPS. Primary cultured human AECs (A) and NCI-H292 AECs (B) were stimulated with 100 µg/ml PGN or 1 µg/ml LPS for 6 or 12 h. Total RNA was prepared and A20 gene expression was examined by RT-PCR. Expression of GAPDH was monitored as a control. Similar results were observed in three independent experiments.

 
A20 Inhibits TLR2- and TLR4-Mediated Activation of NF-{kappa}B
The expression of A20 has been reported to involve the negative feedback regulation of TNF-{alpha}–induced NF-{kappa}B–dependent gene expression (14). We therefore hypothesized that the TLR-induced induction of A20 may function as a negative feedback loop of TLR as well as TNF-{alpha} signaling. To address this issue, we studied the effect of overexpressing A20 on TLR-mediated NF-{kappa}B–dependent gene expression by luciferase assay.

Expression of Myc-tagged human A20 was confirmed by Western blot analysis with anti-Myc Ab, and A20 protein was detected in HEK293 cells and NCI-H292 cells (Figure 6A). As shown Figure 6A, in HEK293 cells transfected with TLR2 or TLR4 plus MD2, overexpression of A20 significantly inhibited NF-{kappa}B–dependent gene expression induced by various doses of LPS or PGN. Overexpression of wild-type MyD88 or wild-type IRAK1 leads to strong induction of NF-{kappa}B–dependent gene expression. However, overexpression of A20 inhibited the effects of both wild-type MyD88 and wild-type IRAK1 (Figure 6B). This inhibition was dependent on the dose of plasmid that was used for A20 transfection. Taken together, these results imply that A20 inhibits TLR-mediated NF-{kappa}B activation.



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  		Figure 6. A20 inhibits LPS- or PGN-induced NF-{kappa}B-dependent gene expression. (A) HEK293 cells transfected with TLR2 (left) or TLR4 plus MD-2 (right) were cells grown on 12-well plates and cotransfected with A20 and NF-{kappa}B luciferase reporter plasmids for 24 h. The cells were treated with PGN and LPS for 6 h. NF-{kappa}B luciferase activity was measured by luciferase assay. *P < 0.01, compared with PGN- or LPS-stimulated empty vector–transfected cells. (B) HEK293 cells were transiently co-transfected with an NF-{kappa}B luciferase reporter plasmid and a plasmid encoding wild-type (wt) MyD88 or IRAK1, together with A20 or a pcDNA3 control vector for 24 h. The amount of total DNA was kept constant by addition of pcDNA3. One representative experiment of two with similar qualitative results is shown.

 
A20 Inhibits TLR2- and TLR4-Mediated Activation of the IL-8 Promoter
We next studied the effect of A20 overexpression on TLR-mediated IL-8 gene expression by luciferase assay. To this end, we cotransfected the IL-8 reporter gene and A20 expression plasmids into TLR-expressing HEK293 cells and NCI-H292 AECs. Expression of Myc-tagged human A20 was confirmed by Western blot analysis with anti-Myc Ab, and A20 protein was detected in NCI-H292 AECs as well as in HEK293 cells (Figure 7A). We found that PGN induced activation of the IL-8 promoter in HEK293 cells transfected with TLR2. Overexpression of MyD88-DN blocked this induction. In a similar fashion to MyD88-DN, overexpression of A20 inhibited the PGN-induced activation of the IL-8 promoter (Figure 7A, LPS). Similar results were obtained in HEK293 cells transfected with TLR4 plus MD-2 (Figure 7A, PGN). In NCI-H292 AECs, overexpression of A20 significantly inhibited activation of the IL-8 promoter by LPS and PGN (Figure 7B).



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  		Figure 7. A20 inhibits LPS- or PGN-induced activation of the IL-8 promoter in AECs. (A) Detection of MyC-tagged A20 expression in HEK293 and NCI-H292 cells. MyC-tagged A20 proteins were detected by Western blot analysis using Myc-specific antibody. (B) HEK293 cells transfected with TLR2 (left) or TLR4 plus MD-2 (right) were cells grown on 12-well plates and cotransfected with an IL-8 promoter-luciferase reporter plasmid, together with plasmids encoding A20 or MyD88-DN for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. (C) NCI-H292 AECs grown on 12-well plates were cotransfected with an IL-8 promoter-luciferase reporter plasmid together with A20 or MyD88-DN for 24 h. The cells were stimulated with PGN (100 µg/ml) or LPS (1 µg/ml) for 6 h. *P < 0.01, compared with PGN- or LPS-stimulated empty vector–transfected cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-8, a proinflammatory cytokine, is known to play a role in the production of airway inflammation when it is produced in excess. Therefore, modulating the overproduction of IL-8 may attenuate airway inflammation (20). In the present study, we have shown that A20 attenuates TLR2- and TLR4-mediated activation of the NF-{kappa}B promoter, as well as NF-{kappa}B–dependent activation of the IL-8 promoter in NCI-H292 cells. These results indicate that A20 functions as a negative regulator of the production of airway inflammation by counteracting the activation of TLR2 by PGN and the activation of TLR4 by LPS.

The expression of TLR in mammalian cells has been well documented; however, little is known about TLR expression in AECs. Becker and coworkers demonstrated that mRNA for the six published human TLR sequences is expressed in human AEC cells. They also found that human TLR2 and TLR4 were present in total RNA at levels detectable by Northern blot analysis (21). In the present study, we detected the expression of human TLR2 and TLR4 protein on primary cultured AECs and NCI-H292 AECs.

Although LPS appears to be a major signal for expression of the IL-8 gene in airway epithelia, the intracellular mechanisms of this response are unknown. Furthermore, the effects of PGN on the inflammatory response of AECs have not been examined. In this study, we showed that the overexpression of I{kappa}B{alpha} inhibits the activation of IL-8 transcription induced by PGN or LPS in NCI-H292 AECs. These results indicate that PGN- or LPS-stimulated expression of the IL-8 gene in AECs is dependent on NF-{kappa}B, as a nonfunctional mutant of I{kappa}B{alpha} blocked the inducible activation of NF-{kappa}B by inhibiting the dissociation of NF-{kappa}B and I{kappa}B{alpha}. In addition, we showed that transfecting MyD88-DN into AECs inhibits LPS- or PGN-induced activation of the IL-8 promoter. Although we completely rule out the effect of multiple genes transfection on the results, these studies strongly suggest that a TLR2/MyD88/NF-{kappa}B– or a TLR4/MyD88/NF-{kappa}B–dependent pathway regulates, respectively, TLR2- or TLR4-induced expression of the IL-8 gene in AECs.

We have also shown that in AECs, A20 expression is significantly increased by the ligation of TLR2 with PGN or the ligation of TLR4 with LPS. A20 was originally identified as a TNF-responsive gene in human endothelial cells (22). Expression of A20 itself is regulated by NF-{kappa}B, which is mediated by two adjacent NF-{kappa}B–binding sites in the A20 promoter (23). LPS has been reported to induce expression of A20 in microvascular endothelial cells (13). Our current results show that LPS induces the expression of A20 mRNA in primary cultured human AECs and NCI-H292 AECs. Furthermore, we found that TLR2 activation by PGN, as well as TLR4 activation by LPS, induces the expression of A20 in these cells. The anti-inflammatory effects of A20 have been explored. A20 has the ability to inhibit the TNF-{alpha}– and IL-1ß–induced activation of NF-{kappa}B (11, 24). O'Reilly and colleagues have reported that A20 was shown to abolish TLR-4 activation of NF-{kappa}B in HEK293 cells cotransfected with TLR4 (25). We have demonstrated here that A20 significantly inhibited PGN-induced NF-{kappa}B–dependent gene expression in HEK293 cells expressing TLR2, as well as LPS-induced NF-{kappa}B–dependent gene expression in TLR4- plus MD-2–expressing cells. Furthermore, we showed that A20 inhibited TLR2- and TLR4-mediated NF-{kappa}B activation in AECs. These results indicate that A20 may be a common negative regulator of TLRs signaling, and A20 can modulate the TLR2- and TLR4-mediated NF-{kappa}B–dependent expression of proinflammatory genes in AECs by inhibiting the activation of NF-{kappa}B. In support of our results, A20–/– mice have been reported to be highly susceptible to LPS, although it is not clear whether they have a primary or secondary response to this stimulus (26). We also found that A20 completely inhibits TLR2- or TLR4-induced transcription of IL-8 in NCI-H292 AECs, suggesting that A20 may regulate TLR-induced airway inflammation.

A20 has been reported to associate with various proteins (27). Zhang and coworkers have shown that A20 also binds inhibitor of NF-{kappa}B kinase-{gamma} (I{kappa}K-{gamma}), which was identified as a component of the purified I{kappa}K complex and is essential for activation of NF-{kappa}B (28). A20 also binds to TNF receptor–associated factors (TRAFs), including TRAF2 and TRAF6 (29, 30). The I{kappa}K complex and TRAF6 are known to function downstream of MyD88 in the TLR-signal transduction pathway, we suggest that an interaction between these molecules and A20 might be a pivotal step in the TLR-induced activation of NF-{kappa}B.

Negative regulators of TLR signaling have been reported. Tollip, Suppressor of cytokine-signaling-1 (SOCS1), and IRAK-M have been reported to act as negative regulators of TLR signaling. The adaptor protein Tollip mediates its inhibitory effects by potently suppressing the activity of IRAK after TLR activation (31). Tollip has been reported to associate directly with TLR2 and TLR4, and plays an inhibitory role in TLR-mediated cell activation. IRAK-M is induced upon TLR stimulation and negatively regulates TLR signaling (32). IRAK-M prevents the dissociation of IRAK and IRAK-4 from MyD88 and the formation of IRAK-TRAF6 complexes. SOCS1/JAB has been reported to be rapidly induced by LPS and negatively regulates LPS signaling, suggesting the suppression of TRAF6 (33). These regulators may contribute to limit the production of proinflammatory mediators during inflammation and infection. Our results suggest that A20 acts as a negative regulator of signals triggered by the TLR/MyD88 pathway, leading to transcriptionally controlled, negative regulation of innate immune responses. We therefore suggest that TLR-mediated induction of A20 may act as an important negative regulator of airway inflammation and may contribute to maintaining innate immune homeostasis in the airway.

Our results may have several important implications. First, understanding the signaling mechanisms involved in TLR-induced synthesis of IL-8 may bring new insight into the molecular pathogenesis of inflammatory airway disease, because little is known about TLR signaling in AECs. Second, our findings imply that A20 may function as a negative regulator of TLR signaling and may regulate innate immune homeostasis in the airway. Finally, our findings may lead to the development of novel therapeutic strategies for inflammatory diseases of the airway.

Received in original form December 7, 2003

Received in final form April 23, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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