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The Journal of Immunology, 2003, 171: 6820-6826.
Copyright © 2003 by The American Association of Immunologists

Activation of Toll-Like Receptor 2 on Human Tracheobronchial Epithelial Cells Induces the Antimicrobial Peptide Human {beta} Defensin-2 1

Cheryl J. Hertz*, Qi Wu§, Edith Martin Porter{dagger}, Yan J. Zhang§, Karl-Heinz Weismüller, Paul J. Godowski||, Tomas Ganz{dagger}, Scott H. Randell§ and Robert L. Modlin*,{ddagger}

* Division of Dermatology, {dagger} Will Rogers Institute Pulmonary Research Laboratories, {ddagger} Department of Microbiology and Immunology and Molecular Biology Institute, University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA 90095; § Department of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599; EMC Microcollections GmbH, Tubingen, Germany; and || Genentech Incorporated, South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As pattern recognition receptors capable of eliciting responses to a diverse array of microbial products, Toll-like receptors (TLRs) participate in the activation of host defense mechanisms that protect against infectious pathogens. Given that epithelial cells lie at the interface between the host and its environment, we designed experiments to determine whether human airway epithelial cells express TLRs and respond to TLR agonists. Immunohistochemical labeling of TLR2 in normal human airways revealed TLR2 expression throughout the epithelium, with an apparently higher level of expression on noncolumnar basal epithelial cells. Two-color immunofluorescent labeling of TLR2 and cytokeratins 8 and 15 revealed that TLR2 is coexpressed with the epithelial cell markers. In addition, airway epithelial cells grown at air-liquid interface responded to bacterial lipopeptide in a TLR2-dependent manner with induction of mRNA and protein of the antimicrobial peptide human {beta} defensin-2. Stimulation of epithelial cell cultures with lipopeptide resulted in a small and variable reduction of bacteria on the apical surface. Together, these data suggest that TLRs monitor epithelial surfaces to enhance host defense by inducing the production of an antimicrobial peptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The innate immune system plays a key role in recognizing pathogens and stimulating the production of cytokines and other proinflammatory mediators that in turn activate cellular immune responses. Besides this indirect role, the innate immune system can also directly affect host defense through the induction of antimicrobial molecules. Critical to these indirect and direct activities are pattern recognition receptors, such as the Toll-like receptors (TLRs), 3 which monitor the host for the presence of pathogens. Human TLRs are homologues of Drosophila Toll (1). In Drosophila, stimulation of Toll or the related molecule 18-wheeler leads to induction of the dorsal/Dif signaling cascade resulting in production of antimicrobial peptides that are important components of innate host defense (2, 3). The dorsal/Dif signaling pathway is highly similar to the NF-{kappa}B signaling pathway that is activated via TLRs in mammals (Ref.4 ; reviewed in Ref.5). Bacterial lipopeptide (BLP)-activation of TLR2 on human and murine monocytes results in NF-{kappa}B activation and confers antimycobacterial activity through NO-dependent and -independent pathways (6, 7). In addition, activation with TLR ligands has been associated with the induction of NF-{kappa}B and the antimicrobial peptide human {beta} defensin-2 (HBD-2) (8, 9). However, a direct connection between mammalian TLR expression, TLR activation, and antimicrobial peptide production has not been established in primary cells.

Mammalian airways have a vast epithelial surface at the interface with the environment and thus require robust mechanisms of innate defense to protect against infection by inhaled pathogens. Primary human airway epithelial cells can be cultured to form polarized cell layers with properties resembling intact epithelium, and previous studies of such epithelial cell cultures have demonstrated the presence of mRNA for TLRs 1–6 in these cells (9). We sought to examine the role of TLR2 activation of epithelial cells in host defense using polarized cultures of airway epithelial cells as a model. Given that bacterial lipoproteins are common to both Gram-positive and Gram-negative bacteria and that they are recognized by TLR2, we examined TLR2-dependent production of proinflammatory cytokine and antimicrobial pathways in epithelial cells using IL-8 and HBD-2 as markers of these responses. In addition, we examined the functional consequences of TLR2-dependent HBD-2 production by comparing bacterial growth on the surface of unactivated and lipopeptide-activated cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunohistochemistry

Immunohistochemical labeling of 4–7 µM acetone-fixed cryostat sections of normal donor airway tissue was achieved using mAbs specific for TLR2 (clone 2392; Ref.10), cytokeratin (CK) 8 (clone 35{beta}H11; DAKO, Carpinteria, CA), and CK15 (clone LHK15; Novocastra Laboratories, distributed by Vector Laboratories, Burlingame, CA), or CD14 (clone RPA-MI; Zymed Laboratories, South San Francisco, CA) using methods previously described (11). Isotype-matched Abs with irrelevant specificities (Sigma-Aldrich, St. Louis, MO) were used as negative controls. TLR2 was visualized using biotinylated horse anti-mouse IgG followed by avidin-conjugated peroxidase (ABC Elite System; Vector Laboratories) and an appropriate substrate or with streptavidin-conjugated FITC for immunofluorescence. Double immunofluorescence was performed by serially labeling sections for TLR2, followed by primary Abs specific for CK8, CK15, or CD14, and an isotype-specific tetramethylrhodamine isothiocyanate-conjugated Ab. Sections were examined and acquired using a Leica TCS-SP inverted confocal laser-scanning microscope (Leica, Deerfield, IL) fitted with appropriate filters.

Cell culture and reagents

Excess airway tissue and excised lungs from nondiseased lung donors were obtained at the time of lung transplantation under an Institutional Review Board-approved protocol at the University of North Carolina, Chapel Hill. Airway epithelial cells collected from this donor tissue were cultured as previously described (12). Briefly, epithelial cells were removed from the lower trachea and mainstem bronchi by protease XIV (Sigma-Aldrich) digestion and cells were plated in bronchial epithelial growth medium on collagen-coated dishes. After two passages, cells were cultured in endotoxin-free air-liquid interface medium on Millicell CM inserts (0.4-µm pore size; Millipore, Bedford, MA) or Transwell clear membranes (0.4-µm pore size; Costar, Cambridge, MA) coated with type VI collagen (Sigma-Aldrich). The medium was periodically tested for endotoxin levels with the Limulus Amebocyte Lysate Assay (BioWhittaker; Walkersville, MD) and endotoxin levels were below 100 pg/ml. An air-liquid interface was established at confluence on days 5–7 and the cells were grown on the porous support for up to 24 days. PAM3CysSerLys4 is an endotoxin-free synthetic BLP (EMC Microcollections, Tubingen, Germany). The anti-TLR2-neutralizing Ab (clone 2392) has been described elsewhere (10).

IL-8 ELISA

IL-8 in culture supernatant fluids was quantitated using a standard sandwich ELISA. Microtiter plates (Costar) were coated with an unconjugated anti-IL-8 capture Ab, and detection was achieved using a biotinylated Ab (clones G265-5 and G265-8, respectively, 1 µg/ml; BD PharMingen, San Diego, CA). The plate was developed using Immunopure HRP-conjugated streptavidin (Pierce, Rockford, IL) and a ABTS Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The absorbance at 405 nm was read using a microtiter plate reader and concentrations of IL-8 were calculated from a standard curve of recombinant human IL-8 (BD PharMingen).

Western blot analysis for HBD-2 protein

Cationic material in the basolateral medium was adsorbed to a carboxymethyl matrix (Bio-Rad, Hercules, CA; 50% slurry in 25 mM ammonium acetate, pH 6.4) at a ratio of 10:1 by overnight incubation under constant agitation. Following three washes in 25 mM ammonium acetate (pH 6), cationic material was eluted from the matrix with acetic acid, lyophilized, resuspended in Laemmli buffer, and subjected to SDS-PAGE (13). Basolateral medium used for Western blot analysis was from tissue culture wells containing equivalent numbers of cells, and equivalent amounts of material from each well were loaded onto the gel. Proteins were transferred to MilliPore PSQ membrane and fixed in Formalin vapor for 10 min, blocked for 30 min at 37°C in 0.75% nonfat milk powder in PBS, and probed with polyclonal rabbit anti-HBD-2 (1/1000 dilution) overnight (14). The membrane was washed with 0.1% BSA in PBS and detection was achieved with alkaline phosphatase-conjugated goat anti-rabbit IgG (1/2000, ImmunoPure; Pierce) and a nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate. Estimates of HBD-2 concentrations in basolateral supernatant fluids were made by visually comparing band intensity to that of a known amount of purified recombinant HBD-2 on the same blot.

Quantitation of HBD-2 mRNA

Relative levels of HBD-2 mRNA were determined using real-time PCR. Total RNA was isolated from cultured cells by guanidium isothiocyanate solubilization and chloroform:phenol extraction. RNA samples were digested with RQ1 RNase-free DNase in the presence of RNasin RNase inhibitor and purified with an RNeasy Mini Prep kit (Qiagen, Valencia, CA). RNA was quantified by spectrophotometry and integrity was verified on formaldehyde:agarose gels stained with SyberGold (Molecular Probes, Eugene, OR). Total RNA (200 ng) was reverse transcribed with 200 U of Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA) using 200 ng of random primers. Real-time PCR was performed using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Indianapolis, IN) in a LightCycler using 20 ng of reverse-transcribed total RNA and 500 nM HBD-2-specific primers. Cyclophilin-specific primers were used as reference. Primer sequences were as follows: HBD-2 forward, ggt gaa gct ccc agc cat c; HBD-2 reverse, aca tgt cgc acg tct ctg atg; cyclophilin forward, ccg tgt tct tcg aca ttg cc; and cyclophilin reverse, aca cca cat gct tgc cat cc. Sequence-specific standard curves were generated using 10-fold serial dilutions (0.5–0.0005 pg) of specific DNA standards. The relative concentration of each sample transcript was determined with the aid of LightCycler software.

Antimicrobial assays

A Pseudomonas aeruginosa isolate from a patient with cystic fibrosis and Escherichia coli ML35p were previously shown to be sensitive to recombinant HBD-2 and used for these studies (14, 15). To assess the antimicrobial activity on the apical surfaces of epithelial cell cultures, cultures were activated for 24 h with IL-1{beta} (1–100 ng/ml) or BLP (10–25 µg/ml). A small amount of basolateral medium was removed to confirm activation by IL-8 ELISA. Bacteria were grown to mid-log phase in trypticase soy broth, then washed with sterile, endotoxin-free water (E. coli) or 10 mM phosphate buffer (pH 7.4) containing 0.03% trypticase soy broth with 100 mM NaCl (P. aeruginosa). Based on the OD600, bacteria were diluted to 1–4 x 103 CFU/ml. Five microliters (5–20 CFU) of this suspension was then applied to the apical surface of epithelial cell cultures and the cultures were incubated at 37°C for 4–6 h. The surface of the cell cultures was then washed three times with 100 µl of PBS and serial dilutions were plated onto trypticase soy agar plates. The plates were incubated overnight at 37°C and the number of CFU were counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TLR2 on airway epithelial cells

Airways represent a major site of interaction between the host and the environment, and host defense at this interface is critical in fighting infection. Given that BLPs are common to both Gram-negative and Gram-positive bacteria and that they activate cells via TLR2, we sought to characterize TLR2 expression in normal human airway tissue. Immunoperoxidase labeling with an anti-TLR2 mAb revealed that TLR2 was expressed on several cell types within normal airway tissue. Most epithelial cells along the basement membrane, as well as columnar epithelial cells, showed positive labeling for TLR2 relative to an IgG1 isotype control Ab (Fig. 1, A and B). Connective tissue underlying the basement membrane contained inflammatory cells that expressed relatively higher levels of TLR2 than epithelial cells; occasionally, some of these cells could be found infiltrating the epithelium.



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  		FIGURE 1. Immunolabeling of TLR2 in normal human airways and airway epithelial cells cultured at air-liquid interface. Samples of normal airways (A and B) or human airway epithelial cells grown on a solid support (C and D) were frozen, sectioned, and labeled with an anti-TLR2 mAb. Visualization was achieved using a biotinylated secondary Ab, followed by streptavidin-conjugated peroxidase and an appropriate substrate. Cells shown in D are from a patient with cystic fibrosis and the membrane support is visible; all others are from normal healthy lung donors.

 
Although the location within the tissue of cells expressing lower levels of TLR2 appeared consistent with epithelial cells, we used two-color immunofluorescence and confocal scanning laser microscopy to compare the expression of TLR2 with specific epithelial cell markers. Tissue sections were serially incubated with mAbs specific for TLR2, followed by CK8 or CK15 in conjunction with isotype-specific fluorochrome-conjugated secondary Abs. The anti-CK8 Ab labeled cells throughout the epithelial layer (Fig. 2A, red), while anti-CK15 labeled undifferentiated basal epithelial cells along the basement membrane of normal airway sections (Fig. 2B, red). Anti-TLR2 labeled the epithelium as well as some cells in the underlying connective tissue. Superimposition of these images revealed that all CK8- and CK15-expressing cells (red) also express TLR2 (green), although each marker localized to different parts of the cell. In contrast, TLR2-positive cells in the underlying connective tissue had a large, ovoid histological appearance and did not express CK15. Double labeling of tissue with anti-CD14 identified these ovoid cells and the intensely labeled TLR2-positive cells in the epithelium, consistent with them being macrophages, while cells forming the epithelium did not express detectable CD14 (Fig. 2C). Based on the colabeling and histology of the TLR2-expressing cells, we conclude that airway epithelial cells along with macrophages in airway tissue express TLR2.



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  		FIGURE 2. TLR2 is coexpressed with CK8, CK15, and CD14 in normal human airway tissue. Samples of normal airways were frozen, sectioned, and labeled with an anti-TLR2 mAb. Visualization was achieved using a biotinylated secondary Ab, followed by streptavidin-conjugated FITC. CK8, CK15, and CD14 were labeled with mAb visualized with an isotype-specific tetramethylrhodamine isothiocyanate-conjugated Ab.

 
Responsiveness of airway epithelial cells to TLR2 agonists

Human tracheobronchial epithelial cells cultured at air-liquid interface form tight junctions, develop cilia, and produce mucus, making them an ideal model of human airway epithelium. Previous studies have shown that such cells express TLR2 mRNA and respond to high concentrations of the TLR4 agonist LPS with production of IL-8 and HBD-2 (9). Because TLR2 is expressed in normal human airways, we sought to determine whether these airway epithelial cells in culture express TLR2 protein and respond to TLR2 agonists. To examine expression of TLR2 in these cells, permeable membranes supporting airway epithelial cells grown for 21 days at air-liquid interface were imbedded in OCT medium, sectioned, and labeled with anti-TLR2. Cells throughout the culture were labeled by the TLR2 Ab, but not an isotype control Ab (Fig. 1, C and D).

We next examined airway epithelial cells for responsiveness to TLR2 agonists. Synthetic peptides with palmitoyl modifications act as exclusive TLR2 agonists and have biological activity resembling natural TLR2 agonists purified from Gram-positive bacteria and mycobacteria (16). In monocytic cells, activation via TLR2 results in IL-8 secretion (17) and previous studies with airway epithelial cells found that IL-8 is produced following LPS stimulation (9). Thus, we examined levels of IL-8 production as an indicator of epithelial cell responsiveness to TLR2 agonists. PAM3CysSerLys4, a synthetic BLP, was used to stimulate 14-day cultures of polarized airway epithelial cells. Airway epithelial cells produced IL-8 when stimulated with lipopeptide and these responses were dose dependent (Fig. 3A). Although a standard concentration of lipopeptide (5 µg/ml) did induce IL-8 release, we found more optimal responses with 25 µg/ml. This may be explained by the relatively lower expression of TLR2 and CD14 on the airway epithelium compared with monocytes. Airway epithelial cells from three different donors stimulated with 25 µg/ml lipopeptide also produced high levels of IL-8 (Fig. 3B).



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  		FIGURE 3. Airway epithelial cells produce IL-8 and HBD-2 in response to BLP stimulation. Normal airway epithelial cells were cultured at air-liquid interface on Transwell supports for 14 days, then incubated for 24 h in medium alone ({square}), with various doses of BLP (A, {blacksquare}) or with 25 µg/ml BLP (B and C). Basolateral culture medium was collected and analyzed for IL-8 by sandwich ELISA (A and B) or subjected to cationic extraction and Western blot analysis for HBD-2 (C).

 
Induction of HBD-2

Antimicrobial peptides are a component of mammalian innate immunity found in the granules of polymorphonuclear cells and Paneth cells of the small intestine and on epithelial cell surfaces (reviewed in Ref.18). Although these peptides are typically constitutively expressed, HBD-2 is remarkable in that it is inducible in a variety of epithelial cell types including those of the airways, skin, oral mucosa, kidney, and gastrointestinal tract (14, 19, 20, 21, 22, 23, 24, 25, 26). The promoter region of HBD-2 contains four NF-{kappa}B binding sites (27), and given that TLR2 stimulation results in activation of NF-{kappa}B, we examined induction of HBD-2 in response to lipopeptide stimulation. After airway epithelial cells were stimulated for 24 h with lipopeptide, the apical surface was washed with water, and the cationic fraction of this wash as well as the basolateral supernatant fluids was isolated and subjected to SDS-PAGE and Western blot analysis. In airway epithelial cells from three of three donors tested, stimulation with lipopeptide resulted in increased HBD-2 secretion into both the basolateral medium (~2.5- to 8-fold; Fig. 3C) and onto the apical surface (~2-fold; data not shown). These increases were estimated to result in 4–8 ng/ml in the basolateral medium and 141–880 ng/ml HBD-2 in the fluid on the apical surface of the cultured airway epithelial cells, based on a titration of the cationic material and visual comparison to purified HBD-2. Therefore, airway epithelial cells respond to TLR2 agonists by production of HBD-2.

Airway epithelial cell responsiveness to BLP is TLR2 dependent

We next sought to determine whether responsiveness of airway epithelial cells to lipopeptide was dependent upon TLR2. Fourteen-day airway epithelial cells were preincubated for 2 h with an anti-TLR2-neutralizing Ab (16) or an IgG1 isotype control Ab, then stimulated for 24 h with a suboptimal dose of lipopeptide. Pretreatment with anti-TLR2, but not the isotype control Ab, blocked lipopeptide-induced production of IL-8 (Fig. 4A). To determine whether HBD-2 induction was also blocked in the presence of anti-TLR2 Abs, mRNA and the basolateral medium were collected from the cells and analyzed for levels of HBD-2 mRNA and HBD-2 protein, respectively. In three of three donors tested, pretreatment with anti-TLR2, but not an IgG1 isotype control Ab, blocked the lipopeptide-induced increase in HBD-2 mRNA, as determined by real-time PCR (Fig. 4B, p < 0.05). In addition, pretreatment of airway epithelial cells with anti-TLR2 blocked lipopeptide-induced increases in HBD-2 protein secreted into the basolateral medium (Fig. 4C). Therefore, activation of airway epithelial cells via TLR2 leads to induction of the antimicrobial peptide HBD-2 and suggests that TLRs may participate in enhancing host defense mechanisms on epithelial surfaces.



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  		FIGURE 4. Responsiveness of airway epithelial cells to BLP is TLR2 dependent. Airway epithelial cells at day 14 of air-liquid interface culture were preincubated with 10 µg/ml anti-TLR2 or an IgG1 isotype control Ab for 2 h. A suboptimal dose of lipopeptide (BLP, 5 µg/ml) was incubated with the cells for 24 h. Basolateral medium was analyzed for IL-8 by ELISA (A) or for HBD-2 by Western blot (C). RNA was prepared from cells and used for real-time PCR quantitation of HBD-2 transcripts (B). Representative data from three different donors in three independent experiments is shown. *, p < 0.012.

 
Induction of HBD-2 results in a decrease in bacteria on the surface of airway epithelial cell cultures

Activation of TLR2 on polarized cultures of airway epithelial cells induced HBD-2 protein secretion into the basolateral medium and onto the apical surface at concentrations approaching those that have previously been shown to be antimicrobial (14). We hypothesized that lipopeptide-activated epithelial cell cultures, as a result of higher levels of HBD-2 production, would show more inherent antimicrobial activity than unactivated cultures. Because IL-1 potently induces HBD-2 in primary keratinocyte and nasal epithelial cell cultures (14, 20, 21) and activates NF-{kappa}B in our primary airway epithelial cell cultures (9), we first compared the growth of bacteria on the apical surface of untreated and IL-1{beta}-treated epithelial cell cultures.

Polarized cultures of airway epithelial cells were stimulated for 24 h and activation was confirmed by measuring the levels of IL-8 in the basolateral medium, which parallels the production of HBD-2. A HBD-2-sensitive strain of P. aeruginosa was added directly to the apical surface of epithelial cell cultures and incubated for an additional 4 or 6 h at 37°C, and the number of CFU on the apical surface of each culture was determined. Relative to the untreated cultures, IL-1{beta}-treated cultures had ~60% fewer bacteria per well than unstimulated cultures (Fig. 5, A and B). Thus, activation through the IL-1R results in a decrease in bacterial growth on the surface of airway epithelial cell cultures.



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  		FIGURE 5. Activation of airway epithelial cells with IL-1{beta} or lipopeptide reduces bacterial growth on the apical surface. A and B, Airway epithelial cells cultured at air-liquid interface were stimulated with IL-1{beta} and incubated with 5 CFU P. aeurginosa/well for 4 h (A) or 10 CFU/well for 6 h (B). The apical surface was washed with water and the bacteria in this wash were enumerated. C and D, Epithelial cell cultures were stimulated with IL-1{beta}, lipopeptide (BLP, 10 µg/ml in C; 25 µg/ml in D), inoculated with 10 CFU/well of P. aeurginosa (C) or 20 CFU/well of E. coli (D), followed by incubation at 37°C for 4 h and enumeration of bacteria.

 
We next examined whether activation with lipopeptide would have a similar effect on bacterial growth. Activation with lipopeptide resulted in almost 40% fewer P. aeruginosa on the surface of cell cultures after 4 h (Fig. 5C). A similar experiment performed with a HBD-2-sensitive strain of E. coli resulted in an ~75% reduction in the number of bacteria in lipopeptide-stimulated cultures as compared with unstimulated cultures (Fig. 5D). Together, these data indicate that activation of primary airway epithelial cells via TLR2 results in an increase in HBD-2 production and antimicrobial activity against Gram-negative bacteria.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study links mammalian TLR activation and the induction of antimicrobial peptides using a human epithelial cell model system. Immunolabeling of airway tissue revealed that epithelial cells lining airways express TLR2 and suggests that these cells may be capable of responding to TLR2 agonists such as bacterial lipoproteins in vivo. Responsiveness of airway epithelial cells in culture was tested using primary human airway epithelial cells, which were found to produce increased amounts of IL-8 and HBD- 2 in response to BLP via a TLR2-dependent mechanism. Furthermore, epithelial cell cultures activated with IL-1{beta} or lipopeptide restricted the growth of bacteria on the apical surface. In whole, this work suggests that TLR2 mediates responsiveness of airway epithelial cells to bacterial ligands and contributes to host defense via activation of indirect and direct immune pathways.

Previously, it has been reported that primary airway epithelial cells contain transcripts for TLRs 1–6 that are detectable using PCR (9). Our data provide evidence that TLR2 protein is expressed on airway epithelial cells in situ. In contrast to the high levels of TLR2 detected on professional immune cells of monocytic origin, we found TLR2 expression to be lower on airway epithelial cells. However, the level of TLR2 expressed on airway epithelial cells is sufficient for responsiveness to TLR2 ligands.

In this study, we have shown TLR2-dependent production of HBD-2 in airway epithelial cells stimulated with BLP. HBD-2 is one of a class of defensin molecules that is known for its antimicrobial activity against a broad range of bacterial, fungal, and viral pathogens (reviewed in Ref.28); this antimicrobial activity has also been demonstrated for HBD-2 (14). Although the levels of HBD-2 found in our culture system are at the lower end of in vitro antimicrobial concentrations, we have shown that lipopeptide-activated cultures are able to restrict the growth of P. aeruginosa and E. coli by as much as 75%. This suggests that up-regulation of an antimicrobial peptide via a mammalian TLR contributes to innate host defense. However, there was some variability in the IL-1{beta}- and lipopeptide-induced bacterial killing, but when an antimicrobial effect was observed with IL-1{beta} stimulation, we also saw an antimicrobial effect in lipopeptide-stimulated cultures. We speculate that the small and somewhat variable antimicrobial effects of our in vitro culture system may be due to differences in cell density or the structural integrity of the polarized epithelial cultures that may result in lower concentrations of HBD-2 on the apical surface. Alternatively, in vivo HBD-2 may act in concert with other antimicrobial molecules such as lysozyme (29).

TLR-dependent production of HBD-2 is likely not unique to airway epithelial cells, but rather a host defense mechanism common to many epithelial cell sites in the body. Expression of TLRs and HBD-2 has been demonstrated in various epithelial cell types. For example, primary gingival epithelial cells are activated when exposed to bacterial fimbriae (30) and exposure to LPS, which is known to activate monocytic cells via TLR4 (31), results in increased HBD-2 mRNA in gingival keratinocytes (22). In addition, primary keratinocytes expressing TLR2 mRNA and protein have been shown to respond to peptidoglycan, a TLR2 agonist (32), by up-regulation of HBD-2 mRNA and IL-8 protein (33), although TLR2-dependence was not demonstrated. Overall, it seems likely that TLR engagement and HBD-2 production will prove to be linked in these and other cell types.

The discovery that TLR activation directly leads to antimicrobial peptide induction in humans has important implications for understanding the evolutionary role of the TLR family. This study reveals that the conservation of insect and mammalian Tolls across species extends not only to the specificity with which they recognize ligands, the structure of their signaling domains, and the signaling pathways that they activate (1, 4), but also to the types of effector molecules induced, namely, antimicrobial peptides. However, the mammalian immune system has also evolved with other TLR-dependent antimicrobial pathways. Engagement of TLR2 on monocytes infected with Mycobacterium tuberculosis reduces the number of CFU via NO production in murine monocytes and an as of yet unidentified antimycobacterial mechanism in human monocytes (7). In both cases, TLR2 is required for the antimycobacterial activity, suggesting a direct role for TLRs in mammalian host defense.

TLR2-dependent activation of epithelial cells may also play an indirect role in host defense through its induction of the proinflammatory cytokine IL-8, as well as HBD-2. IL-8 is a neutrophil chemoattractant, and recruitment of these cells with their potent antimicrobial machinery to the site of pathogen exposure contributes to the elimination of the infection. In addition, HBD-2 acts via the chemokine receptor CCR6 to attract immature dendritic cells and memory T cells to the site of infection (34) and can stimulate dendritic cell maturation via TLR4 (35). These processes may facilitate the development of adaptive immune responses at the site of infection. Our preliminary studies suggest that TLR2 activation in airway epithelial cells leads to phosphorylation and degration of IL-1R-associated kinase 1 followed by mitogen-activated protein kinase phosphorylation and IKB{alpha} degradation, leading to activation of the AP-1 and NF-{kappa}B transcription factors, respectively (Q. Wu, C. J. Hertz, M. W. Verghese, P. J. Godowoski, K.-H. Weismueller, R. L. Modlin, and S H. Randall, manuscript in preparation). However, it remains to be determined what signaling pathways are responsible for TLR2-dependent IL-8 and HBD-2 production and what other proinflammatory pathways are activated by engagement of TLRs on airway epithelial cells.

Evidence is mounting that TLR activation is essential for appropriate innate immune responses. In Drosophila, Toll deficiencies result in a lack of induction of the antifungal peptide drosomycin that correlates with exquisite susceptibility to infection with Aspergillus fumigatus (2). Polymorphisms in mammalian TLR family members that render these molecules hyporesponsive to their typical stimuli have been identified in mice as well as humans (36, 37, 38). It remains to be determined whether altered TLR expression on epithelial cells compromises innate host defense. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the body’s own defenses by stimulation of TLR-dependent pathways.


   Acknowledgments
 
We thank M. Kelly Plonk, Annaliza Legaspi, Li Li, Amie Chen, and Drs. Matt Schibler and Maria-Teresa Ochoa for technical assistance.


   Footnotes
 
1 This work was supported in part by Grants AI22553, AI47868, and AI 07118 from the National Institutes of Health (to R.L.M.) and RO1 HL 46809 (to T.G.) and grants from the Cystic Fibrosis Foundation (to S.H.R.). C.J.H. is the recipient of a Research Fellowship Award from the Dermatology Foundation sponsored by Medicis Pharmaceutical Corporation and is a Fellow on the Clinical Immunology and Allergy Training Grant to University of California, Los Angeles (T32 AI07126-24). Back

2 Address correspondence and reprint requests to Dr. Robert Modlin, Division of Dermatology, University of California, Los Angeles, 52-121 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: rmodlin{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: TLR, Toll-like receptor; BLP, bacterial lipopeptide, HBD-2, human {beta} defensin-2, CK, cytokeratin. Back

Received for publication June 9, 2003. Accepted for publication October 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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