Modulation of Human β-Defensin-2 Transcription in Pulmonary Epithelial Cells by Lipopolysaccharide-Stimulated Mononuclear Phagocytes Via This information is current as Proinflammatory Production of September 28, 2021. Yuko Tsutsumi-Ishii and Isao Nagaoka J Immunol 2003; 170:4226-4236; ; doi: 10.4049/jimmunol.170.8.4226 http://www.jimmunol.org/content/170/8/4226 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Modulation of Human ␤-Defensin-2 Transcription in Pulmonary Epithelial Cells by Lipopolysaccharide-Stimulated Mononuclear Phagocytes Via Proinflammatory Cytokine Production1

Yuko Tsutsumi-Ishii and Isao Nagaoka2

Human ␤-defensin (hBD)-2, a cationic antimicrobial peptide primarily induced in epithelial cells in response to inflammatory stimuli, plays an important role in host defense. To elucidate the expression mechanism of hBD-2 in the lung, we investigated the modulation of hBD-2 transcription in pulmonary epithelial cells by mononuclear phagocytes stimulated with LPS. Coculture of A549 pulmonary epithelial cells with Mono-Mac-6 monocytic cells in the presence of Escherichia coli LPS markedly up-regulated hBD-2 promoter activity, whereas A549 alone did not respond to LPS to activate the hBD-2 promoter. Furthermore, IL-1␤ and TNF-␣ in the culture supernatants from LPS-stimulated monocytic cells activated the hBD-2 promoter in A549 cells. Of note, Downloaded from IL-1␤ was more potent than TNF-␣ in this effect. In addition, a mutation of the NF-␬B site at ؊200 (p␬B1 site) completely abolished this IL-1␤- and TNF-␣-induced hBD-2 promoter activation, whereas NF-␬B inhibitors (MG-132 and helenalin) strongly suppressed it. Moreover, electrophoretic mobility shift assay suggested that NF-␬B, consisting of p65-p50 heterodimer, could bind to the p␬B1 site in cytokine-stimulated A549 cells. Interestingly, flow cytometric analysis revealed that A549 cells expressed CD14 but lacked Toll-like receptor 4, which may account for the hyporesponsiveness of A549 cells to LPS. Taken together, these results ␬ ␤ ␣ suggest that hBD-2 expression in pulmonary epithelial cells is modulated by NF- B via the actions of IL-1 and TNF- produced http://www.jimmunol.org/ by LPS-stimulated mononuclear phagocytes. The Journal of Immunology, 2003, 170: 4226Ð4236.

ulmonary epithelial cells and are in direct ␪-defensin, isolated from macaque leukocytes, exhibits a unique contact with the ambient environment. The early recogni- circular structure (11, 12). In humans, the ␣-defensins are mainly P tion by these cells of inhaled pathogens and their products expressed in neutrophils (human neutrophil peptides 1Ð4) and in- is critical to the innate immune system, the first line of host defense testinal Paneth epithelial cells (human defensins 5 and 6) (9, 10). against invading microorganisms. These cells recognize bacteria In contrast, hBDs, four of which (hBD-1 to -4) have been identi- and their products, and kill bacteria by producing antibacterial fied and characterized to date, are largely expressed in various molecules (1Ð3). Pulmonary epithelial cells and macrophages also epithelial tissues, including lung and skin (7, 13Ð20). Interestingly, by guest on September 28, 2021 generate various immune effectors such as , , hBD-2 to -4 are highly inducible, whereas expression of hBD-1 is and antimicrobial peptides in response to inflammatory stimuli, constitutive (7, 11, 19). In this context, hBD-2 peptide, initially and regulate the activation and recruitment of professional phago- purified from psoriatic scales, is detected in airway surface fluid cytes (neutrophils and macrophages) and immune cells (T cells from patients with infectious lung diseases but not normal volunteers and dendritic cells) (3Ð6). Among the antimicrobial peptides, hu- (21, 22). Likewise, mRNAs for hBD-3 and -4 are up-regulated upon man ␤-defensins (hBDs)3 play a unique role linking innate and bacterial exposure in lung epithelial cells (17Ð19). Thus, hBD-2 to -4 acquired immunities (3, 7, 8). likely play a crucial role in host defense against bacterial infection as The defensin family of antimicrobial peptides is widely distrib- inducible components in the epithelial barrier (7, 10). uted among various species from plants to mammals, and exhibits In addition, hBD-1 to -3 possess chemotactic activities for im- a broad spectrum of microbicidal activities against bacteria, fungi, mature dendritic cells and memory T cells, while hBD-3 and -4 and viruses (9, 10). Mammalian defensins are divided into three elicit chemotaxis (16Ð19, 23). These characteristics sug- subfamilies: ␣- and ␤-defensins differ in the placement and con- gest that hBDs may also contribute to adaptive immunity through nectivity of their six conserved cysteine residues, while the third, the induction of immune cell migration (8, 10). Moreover, we re- cently revealed that hBD-2 activates mast cells to induce chemo-

Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan taxis, histamine release and PGD2 production, suggesting the in- volvement of hBD-2 in allergic reactions (24, 25). Received for publication June 27, 2002. Accepted for publication January 23, 2003. Given the intriguing functionality of hBDs, it is important to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance elucidate the molecular mechanisms regulating their expression. with 18 U.S.C. Section 1734 solely to indicate this fact. To date, the expression of inducible hBDs has been reported to be 1 This work was supported in part by a grant from the Promotion and Mutual Aid modulated by both microbial products and cytokines (3, 7). In Corporation for Private Schools of Japan, the Atopy (Allergy) Research Center and current thinking, it is accepted that the recognition of bacterial High Technology Research Center (Juntendo University), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and components, as well as signal transduction in this process, are Technology (Japan). achieved through pattern recognition receptors (PRRs) such as 2 Address correspondence and reprint requests to Dr. Isao Nagaoka, Department of Toll-like receptors (TLRs) and CD14. In this regard, mononuclear Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, phagocytes, which abundantly express PRRs, can respond to low Tokyo 113-8421, Japan. E-mail address: [email protected] doses of bacteria-derived components (26Ð29). In contrast, pul- 3 Abbreviations used in this paper: hBD, human ␤-defensin; PRR, pattern recognition receptor; TLR, Toll-like receptor; MDM␾, monocyte-derived ; MM6, monary epithelial cells require high doses of bacterial components Mono-Mac-6; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase. for cell activation, despite the expression of TLR1 to -6 and CD14

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 4227 mRNAs (3, 7, 30Ð32). Interestingly, pulmonary epithelial cells The human monocytic cell line MM6 was purchased from German Col- readily respond to cytokines and are triggered at a lower threshold lection of Microorganisms and Cell Cultures (Braunschweig, Germany) and ϫ (3, 4, 7). Because /macrophages are highly sensitive to cultured in RPMI 1640 supplemented with 10% FCS, 1 nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 9 ␮g/ml bovine in- bacterial stimuli and function as a principal source of proinflam- sulin (Sigma-Aldrich), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. matory cytokines, we considered that they may play a role in the Human PBMC from healthy donors were obtained by density gradient induction of hBDs in lung epithelial cells. However, little infor- centrifugation with Lymphoprep (Nycomed, Oslo, Norway). PBMC were mation is available regarding the effect of mononuclear phagocyte- allowed to adhere to plastic culture plates. After removal of nonadherent cells, adherent monocytes were collected and plated at 2 ϫ 105 cells/ml in epithelial cell interactions on the regulation of hBD expression in RPMI 1640 containing 10% human AB serum. The purity of monocytes/ epithelial cells. macrophages (Ͼ90%) was confirmed by flow cytometry as CD14- and In the present study, we focused on the inducible expression of CD64-positive cells. The cells were then cultured for 7 days to differentiate hBD-2, a well-characterized hBD, by investigating the transcrip- to macrophages. tional regulation of hBD-2 in lung epithelial cells by mononuclear For preparation of culture supernatants from monocyte-derived macro- phages (MDM␾) and monocytic cell lines (MM6 and U937), cells (2 ϫ 105 phagocytes following LPS stimulation, using A549 pulmonary ep- cells/ml) were incubated with or without 1 ␮g/ml E. coli LPS for 16 h at ithelial cells and monocytic Mono-Mac-6 (MM6) and U937 cells. 37¡C. Likewise, confluent A549 cells (1 ϫ 106 cells/35 mm plate) were Results showed that proinflammatory cytokines (IL-1␤ and incubated in the presence or absence of 10 ␮g/ml E. coli LPS or TNF-␣ (2, TNF-␣) produced from LPS-stimulated mononuclear cells up-reg- 10, and 20 ng/ml) for 16 h. Culture supernatants were collected and stored at Ϫ80¡C. They were used for stimulation of epithelial cells and measure- ulated the expression of endogenous hBD-2 mRNA and also the ment of cytokines without dilution. hBD-2 promoter activity analyzed by luciferase reporter constructs in A549 cells. Further, this cytokine-mediated hBD-2 promoter Plasmid construction Downloaded from activation in A549 cells required binding of NF-␬B p65-p50 het- A 2.3-kbp fragment of human hBD-2 promoter (Ϫ2274 to ϩ50) was am- erodimer to a proximal NF-␬B element at Ϫ200 in the hBD-2 plified from PstI-digested genomic DNA by PCR using specific primers promoter. Finally, evaluation of the surface expression of PRRs designed based on a published sequence (35), and subcloned into the pro- moterless firefly luciferase vector pGL3-Basic (Promega, Madison, WI) to revealed that CD14 was expressed but that TLR4 was hardly ex- generate hBD 2/Luc plasmid. A series of 5Ј deletion fragments were pre- pressed on A549 cells. pared by digestion with appropriate restriction enzymes or by PCR using

appropriate primers as described previously (36), and the constructs were http://www.jimmunol.org/ Materials and Methods named ⌬Ϫ2187/Luc, ⌬Ϫ1050/Luc, ⌬Ϫ577/Luc, ⌬Ϫ412/Luc, ⌬Ϫ188/Luc, ⌬Ϫ ⌬Ϫ Reagents 167/Luc, and 106/Luc, respectively (Fig. 5B). For generation of mu- tated NF-␬B constructs, individual NF-␬B sequences in the hBD-2 promoter, LPS from Escherichia coli O55:B5 and O111:B4 were obtained from d␬B(Ϫ2193 to Ϫ2182), p␬B2 (Ϫ596 to Ϫ572), and p␬B1 (Ϫ205 to Ϫ186), Sigma-Aldrich (St. Louis, MO). Biotinylation of E. coli LPS (O111:B4) were mutagenized by PCR using the hBD 2/Luc and mutant NF-␬B primers, was described before (33). A specific inhibitor of the NF-␬B p65 subunit, and the hBD-2 promoters containing mutated NF-␬B sequences were intro- helenalin (4-hydroxy-5,8-dimethyl-3-methylene-3a,4a,5,8,9,9a-hesahydro- duced to the pGL3 vector plasmid as described previously (Ref. 36 and Table 3H,4H-azuleno(6,5-b)furan-2,6-dione) was obtained from Biomol Research II). The resulting mutated NF-␬B constructs were termed d␬Bmut/Luc, Laboratories (Plymouth Meeting, PA) (34). The proteasome inhibitor MG-132 p␬B2mut/Luc, p␬B1mut/Luc, and p␬B1ϩ2mut/Luc (mutations with both the was purchased from Calbiochem (San Diego, CA). Helenalin and p␬B1 and p␬B2 sites) (Fig. 5C). by guest on September 28, 2021 MG-132 were dissolved in DMSO and further diluted in PBS to ensure Transfection and luciferase assay a DMSO concentration not exceeding 0.1%. Human recombinant TNF-␣, IL-1␤, and IFN-␥ were obtained from Pep- A549 cells (5 ϫ 105 cells/plate) were seeded into 35-mm plates 1 day roTech (Rocky Hill, NJ). Chinese hamster ovary cell-derived recombinant before transfection. Cells were transfected with 1.5 ␮goffirefly luciferase human IL-6 was from Genzyme (Cambridge, MA). Neutralizing mAb expression construct containing wild-type or mutated hBD-2 promoter and against human TNF-␣ (clone 1825.121) or IL-1␤ (clone 8516.311), and 50 ng of Renilla luciferase expression vector pRL-SV40 (Promega) using goat anti-human IL-1␤ polyclonal Ab were purchased from Genzyme- FuGENE 6 (Roche Diagnostics, Indianapolis, IN) according to the manu- Techne (Minneapolis, MN). Anti-human CD14 mAb (clone MY4) was facturer’s protocol. Following transfection, cells were cultured for 19 h, obtained from Beckman Coulter (Tokyo, Japan), and clone 61D3 was from then stimulated for 5 h with various stimuli as follows: E. coli LPS, cy- eBioscience (San Diego, CA). Anti-human TLR4 mAb (clone HTA125) tokines (TNF-␣, IL-1␤, IL-6, and IFN-␥), and monocytic cell-derived cul- was obtained from MBL (Nagoya, Japan). Anti-human TNF-␣ mAbs ture supernatants. In some experiments, cells were treated for 30 min be- (clones mAb1 and mAb11), anti-human CD14 mAb (clone LeuM3), and fore stimulation with neutralizing Abs against TNF-␣ and IL-1␤,orNF-␬B anti-human CD11b mAb (clone 44) were obtained from BD PharMingen inhibitors (helenalin and MG-132). (San Diego, CA). Anti-human TLR2 mAb (clone TL2.1) was the kind gift For coculture experiments, confluent A549 monolayers in 35-mm plates of Genentech (South San Francisco, CA). Rabbit polyclonal Abs against (1 ϫ 106 cells) were transfected, incubated for 8 h, and cocultured with 1 ϫ NF-␬B p65 (sc-109X) and NF-␬B p50 (sc-114X) were obtained from Santa 105 MM6 cells in RPMI 1640 containing 10% FCS in the presence or Cruz Biotechnology (Santa Cruz, CA). Mouse purified-IgG was from Sig- absence of 1 ␮g/ml LPS for 16 h. ma-Aldrich, IgG1 was from Beckman Coulter, and IgG2a and IgG2b were After stimulation, cells were harvested and lysed in 200 ␮l of PicaGene from eBioscience. Streptavidin-conjugated Alexa Fluor 594 was from Mo- Dual Lysis reagent (Toyo Ink, Tokyo, Japan). Firefly and lecular Probes (Eugene, OR), streptavidin-conjugated PE was from eBio- Renilla luciferase activities were measured using a PicaGene Dual SeaPansy science, and streptavidin-conjugated HRP was from Zymed Laboratories Luminescence kit (Toyo Ink) and Lumat LB9501 luminometer (Berthold, To- (South San Francisco, CA). kyo, Japan). Protein concentrations of cell extracts were determined with a BCA protein assay kit (Pierce, Rockford, IL). Promoter activities are expressed Cell lines and culture as relative light units, normalized to Renilla luciferase activity. The human lung alveolar type II epithelial cell line A549 and human mono- RT-PCR analysis cytic cell line U937 (CCL-185 and CCL-1593.2, respectively) were ob- tained from the American Type Culture Collection (Manassas, VA). A549 Confluent A549 cells (1 ϫ 106 cells/35-mm plate) were incubated with or cells were maintained in F12K (Invitrogen, Carlsbad, CA) supplemented without E. coli LPS, cytokines (TNF-␣, IL-1␤, IL-6, and IFN-␥), or mono- with 10% FCS (Sanko Junyaku, Tokyo, Japan), 100 U/ml penicillin, and cytic cell-derived supernatants for 16 h. Total RNA was then purified using

0.1 mg/ml streptomycin (Sigma-Aldrich) at 37¡Cin5%CO2. FCS con- an RNeasy mini kit (Qiagen, Valencia, CA) and treated with RNase-free tained Ͻ5 pg/100 ml LPS as certified by the manufacturer. U937 cells were DNase set (Qiagen) to remove contaminated DNA according to the man- cultured in RPMI 1640 (Sigma-Aldrich) with 10% FCS, 100 U/ml peni- ufacturer’s protocols. RT-PCR was performed using a ReverTra Dash RT- cillin, and 0.1 mg/ml streptomycin. For monocytic differentiation, U937 PCR kit (Toyobo, Osaka, Japan). In brief, cDNA was synthesized by re- cells were cultured in the presence of 10 nM PMA for 2 days, and adherent verse transcription of DNase-treated total RNA (1 ␮g) using ReverTra Ace cells were used as PMA-differentiated U937. Differentiation of cells was reverse transcriptase and random primers. PCR amplification was per- confirmed by the expression of CD11b and CD14 analyzed using flow formed with KOD dash Taq polymerase in a Thermal Cycler (PerkinElmer/ cytometry. Cetus, Norwalk, CT) for 35 cycles (for hBD-2 and CD14) or 28 cycles (for 4228 REGULATION OF hBD-2 EXPRESSION IN PULMONARY EPITHELIAL CELLS

G3PDH) of 10 s at 98¡C,10sat60¡C, and 30 s at 74¡C. To discriminate binds to a functional domain of membrane CD14, and blocks the binding mRNA-derived PCR products from genomic DNA-derived products, the of LPS to CD14. In contrast, 61D3 does not inhibit the binding of LPS to following intron-spanning PCR primers were used: hBD-2, 5Ј-CCAGCC CD14. Likewise, LeuM3 hardly blocks LPS binding (38Ð40). ATCAGCCATGAGGGT-3Ј and 5Ј-GGAGCCATCAGCCATGAGGGT-3Ј) LPS binding to A549 cells was determined by flow cytometric technique (15); hCD14, 5Ј-ACTTATCGACCATGGAGC-3Ј and 5Ј-AGGCATG (33). A549 cells (2 ϫ 105 cells) were incubated with 100 ng/ml biotinyl- GTGCCGGTTA-3Ј (37); and G3PDH, 5Ј-ACCACAGTCCATGCCAT ated E. coli LPS in the presence of 10% FCS for 15 min at 37¡C, with CAC-3Ј and 5Ј-TCCACCACCCTGTTGCTGTA-3Ј (Toyobo). All PCR gentle agitation. For blocking experiments, cells were treated with 2.5 ␮g products were resolved by 2% agarose gel electrophoresis. of neutralizing anti-CD14 mAb (MY4) or control mouse IgG for 15 min at 37¡C before addition of LPS. Subsequently, cells were incubated with Measurement of TNF-␣ and IL-1␤ in culture supernatants streptavidin-conjugated PE for 30 min at 4¡C. Cells were washed twice with PBS and analyzed with a FACSVantage. TNF-␣ and IL-1␤ in culture supernatants collected from LPS-treated A549 cells and monocytic cells (MM6 and PMA-differentiated U937 cells) were Preparation of nuclear extracts quantified by a sandwich ELISA technique. Likewise, IL-1␤ production from TNF-␣-stimulated A549 cells was measured by ELISA. A549 cells (108 cells) were incubated for 2 h with or without 1 ng/ml Microtiter plates (Corning, Acton, MA) were coated with mAb to hu- IL-1␤, 10 ng/ml TNF-␣, or monocytic cell-derived culture supernatants at man TNF-␣ (mAb1) or IL-1␤ (8516.311) overnight at 4¡C. Plates were 37¡C, and nuclear extracts were prepared from stimulated or resting cells blocked with BlockAce (Dainippon Pharmaceutical, Tokyo, Japan), and as described previously (36, 41). In brief, A549 monolayers were washed culture supernatants were added into the wells and incubated for2hat twice with PBS and harvested using a rubber policeman. Cells were lysed room temperature. After washing with PBS containing 0.05% Tween 20, in lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.2 mM EDTA, 1.5 ␣ ␤ ␮ TNF- or IL-1 was detected by development with a combination of bi- mM MgCl2, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 10 g/ml otinylated Ab (anti-TNF-␣ mAb11 mAb or anti-IL-1␤ polyclonal Ab) and leupeptin, and 10 ␮g/ml pepstatin) on ice for 10 min. Nuclei were washed

streptavidin-conjugated HRP. The detection limits of these ELISAs were in the same buffer except for Nonidet P-40, pelleted, and resuspended in Downloaded from 15 pg/ml for IL-1␤ and 31 pg/ml for TNF-␣. Flow cytometry A549 and PMA-differentiated U937 cells were detached with PBS con- taining 0.05% EDTA. These detached cells or MM6 cells (5 ϫ 105 cells) were blocked with normal mouse serum, then incubated with FITC-, PE-,

or biotin-conjugated mouse mAbs against TLR2, TLR4, or CD14, or iso- http://www.jimmunol.org/ type control (IgG1, IgG2a, or IgG2b) for 30 min at 4¡C. For detection of biotin-labeled primary Ab, cells were further incubated with streptavidin- conjugated Alexa Fluor 594 for 15 min at 4¡C. Cells were washed twice with PBS containing 1% BSA and 0.01% NaN3, and analyzed with a FACSVantage (BD Biosciences, San Jose, CA). For detection of CD14 molecules, we used three kinds of anti-CD14 mAbs (MY4, LeuM3, and 61D3). These mAbs recognize different epitopes of CD14 molecules. MY4 by guest on September 28, 2021

FIGURE 2. Effect of neutralizing Abs on hBD-2 promoter activation induced by monocytic cell-derived culture supernatants. A549 cells were transfected with the hBD 2/Luc plasmid and incubated with the culture FIGURE 1. Induction of hBD-2 promoter activity in A549 cells by co- supernatants from monocytic cells (PMA-differentiated U937 (A) and culture with LPS-treated monocytic cells. A549 cells were transfected with MM6 (B)) for 5 h. For neutralization assay, the supernatants were pre- luciferase-expression plasmid containing hBD-2 promoter (hBD 2/Luc), treated with neutralizing mAb (anti-IL-1␤ mAb, anti-TNF-␣ mAb, or both) and incubated with E. coli LPS, human IFN-␥, or both for5h(A), or or mouse IgG for 30 min before addition to A549 cells. Luciferase activ- cocultured with MM6 in the presence or absence of E. coli LPS, IFN-␥,or ities are expressed as fold activation in cells treated with LPS-stimulated both for 16 h (B). Luciferase activities are expressed as fold activation monocytic cell-derived supernatants relative to those treated with unstimu- relative to the unstimulated control (Resting). Values are mean Ϯ SD of lated monocytic cell-derived supernatants (Resting). Values are mean Ϯ five to seven independent experiments. Values were compared between SD of five to seven independent experiments. Values were compared between .p Ͻ 0.0001 ,ء .p Ͻ 0.0001. LPS-stimulated cell supernatants without and with mAbs ,ء .control (Resting) and stimulated cells The Journal of Immunology 4229

Table I. Concentrations of IL-1␤ and TNF-␣ in culture supernatants from A549, PMA-differentiated U937, and MM6 cellsa

Supernatant IL-1␤ (pg/ml) TNF-␣ (pg/ml)

PMA-U937 Resting 19.8 Ϯ 22.9 ND 1 ␮g/ml LPS 194.2 Ϯ 49.4 1886.9 Ϯ 653.2 MM6 Resting 30.0 Ϯ 26.0 ND 1 ␮g/ml LPS 177.6 Ϯ 44.3 232.2 Ϯ 18.9 A549 Resting ND ND 10 ␮g/ml LPS ND ND 2Ð20 ng/ml TNF-␣ ND NT

a PMA-differentiated U937 (PMA-U937) and MM6 cells (2 ϫ 105 cells/ml) were incubated for 16 h in the presence or absence of 1 ␮g/ml LPS. Likewise, A549 cells (1 ϫ 106 cells/35-mm plate) were incubated for 16 h in the presence or absence of 10 ␮g/ml LPS or 2Ð20 ng/ml TNF-␣, and the supernatants were collected. Data represent mean Ϯ SD from five to seven independent experiments. Background levels with FIGURE 3. Effect of monocytic cell-derived culture supernatants on the medium alone were under the detection limit in each cytokine assay. NT, Not tested. ND, Not detectable.

expression of endogenous hBD-2 mRNA in A549 cells. MDM␾, PMA- Downloaded from differentiated U937 (PMA-U937), and MM6 were treated with or without E. coli LPS (100 ng/ml for MDM␾,1␮g/ml for U937 and MM6) for 16 h, and culture supernatants were collected. A549 cells were exposed to the Results supernatants, 10 ␮g/ml LPS, or 250 U/ml IFN-␥ for 16 h, and subjected to Activation of hBD-2 promoter in A549 epithelial cells by RNA isolation as described in Materials and Methods. hBD-2 (255 bp)- LPS-stimulated monocytic cells and G3PDH (447 bp)-specific RT-PCR products were amplified using their

intron-spanning primer pairs and total RNA from A549 cells. Inflammatory stimuli such as LPS and proinflammatory cytokines http://www.jimmunol.org/ have been reported to induce the expression of hBD-2 in A549 alveolar type II epithelial cells at mRNA and peptide levels (42). However, in our experiments, hBD-2 promoter activity was not induced in A549 cells by even high doses of E. coli LPS (up to 100 extraction buffer (10 mM HEPES (pH 7.9), 420 mM NaCl, 0.2 mM EDTA, ␮ 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, 1 mM PMSF, 10 g/ml leu- peptin, and 10 ␮g/ml pepstatin). After incubation at 4¡C for 20 min with gentle rocking, the nuclei were removed by centrifugation at 12,000 ϫ g for 20 min at 4¡C. The resultant supernatants were collected and stored at Ϫ80¡C until used for gel EMSA. Protein concentrations in nuclear extracts by guest on September 28, 2021 were measured with a BCA protein assay kit.

EMSA Nuclear extracts (4 ␮g) were mixed with [32P]DNA probe (7 ϫ 104 cpm) in 15 ␮l of a binding buffer containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 1 mM EDTA, 6% glycerol, 1 mM DTT, 1 mM PMSF, 0.25 mg/ml BSA, and 2 ␮g of poly(dI-dC)⅐poly(dI-dC) (Amersham Biosciences, Pis- cataway, NJ) for 20 min at room temperature. The reaction mixtures were applied to a native 6% polyacrylamide gel in 0.25ϫ TBE (22.3 mM Tris, 22.3 mM boric acid, and 0.5 mM EDTA (pH 8.3)) at 130 V for 70 min at 4¡C. The gels were dried and exposed to Fuji RX-U x-ray film (Fuji Photo Film, Tokyo, Japan) at Ϫ80¡C. For competition assay, a 25-fold molar excess of unlabeled oligonucleotide was incubated with nuclear extracts in the reaction mixture for 15 min at room temperature before addition of the probe. For supershift experiments, 1 ␮g of polyclonal Abs against NF-␬B p65 (sc-109X), NF-␬B p50 (sc-114X), or normal rabbit IgG was added to the reaction mixture 20 min before incubation with the probe. Synthetic oligonucleotides were designed to generate a single 5Ј-G over- hang at each end after annealing with their complementary oligonucleo- tides. Oligonucleotide for the hBD-2 p␬B1 site corresponds to the sequence from Ϫ208 to Ϫ183 in the promoter (p␬B1 oligo, 5Ј-GGAAGGGATTT TCTGGGGTTCCTGAC-3Ј). NF-␬B and NF-IL-6 (C/EBP) consensus oli- gonucleotides (␬B oligo, 5Ј-GGTTGAGGGGACTTTCCCAGGC-3Ј, and EBP oligo, 5Ј-GTGCAGATTGCGCAATCTGCAC-3Ј, respectively) were synthesized based on the sequences of TransCruz Gel Shift Oligonucleo- FIGURE 4. hBD-2 response to recombinant cytokines in A549 cells. A, tides (Santa Cruz Biotechnology). The double-stranded p␬B1 oligonucle- A549 cells transfected with the hBD 2/Luc plasmid were incubated in the ␣ 32 otides were labeled by filling in the cohesive ends with [ - P]dCTP (ICN absence or presence of human recombinant cytokines (TNF-␣, IL-1␤, and Biomedicals, Costa Mesa, CA) and a Klenow fragment (New England IL-6). Luciferase activities are expressed as fold activation relative to the Biolabs, Beverly, MA) and used as a probe. unstimulated control (Resting). Values are mean Ϯ SD of four independent experiments. Values were compared between control (Resting) and cyto- -p Ͻ 0.01. B, Expression of en ,ءء ;p Ͻ 0.0001 ,ء .Statistical analysis kine-stimulated cells The data were expressed as the mean Ϯ SD, and analyzed for significant dogenous hBD-2 mRNA in A549 cells in response to cytokines. Confluent difference by a one-way ANOVA and a post-hoc test using the StatView cells were incubated with or without TNF-␣, IL-1␤, or IL-6 for 16 h, and program (SAS Institute, Cary, NC). Differences were considered statisti- subjected to isolation of total RNA. The expression of hBD-2 mRNA was cally significant if p Ͻ 0.05. analyzed by RT-PCR. 4230 REGULATION OF hBD-2 EXPRESSION IN PULMONARY EPITHELIAL CELLS

FIGURE 5. Deletion and mutation analyses of the hBD-2 promoter. A, Schematic representation of the hBD-2 promoter depicting putative cis-ele- ments: black boxes, NF-␬B sites; gray boxes, STAT sites; gray ovals, NF-IL-6 sites; open oval, AP-1 family site; and open boxes, TATA and CCAAT boxes. Hatched box shows exon 1 of hBD-2 gene. The transcription start site is num- bered ϩ1. NF-␬B binding sites are termed d␬B (position Ϫ2193/Ϫ2182), p␬B2 (Ϫ596/Ϫ572), and p␬B1 (Ϫ205/ Ϫ186). B, A549 cells were transfected with wild-type (hBD 2/Luc) or a series of 5Ј truncated promoter constructs, and in- cubated with or without 10 ng/ml TNF-␣ Downloaded from or 1 ng/ml IL-1␤ for 5 h. Luciferase ac- tivities are expressed as fold activation relative to unstimulated control (Resting) in each reporter construct. C, A549 cells were transfected with wild-type (hBD 2/Luc) or mutant NF-␬B construct

(d␬Bmut/Luc, p␬B2mut/Luc, p␬B1mut/ http://www.jimmunol.org/ Luc, or p␬B1ϩ2mut/Luc), and treated as described in B. Values are mean Ϯ SD of five to eight independent experiments. Values were compared between wild- p Ͻ ,ء .type and its derivative constructs .p Ͻ 0.001 ,ءء ;0.0001 by guest on September 28, 2021

␮g/ml) (Fig. 1A). Furthermore, 250 U/ml IFN-␥, which activates (47), further increased LPS-induced hBD-2 promoter activity (Fig. lung epithelial cells (43), did not induce activation of the hBD-2 1B), although IFN-␥ alone did not induce activation above the promoter (Fig. 1A). Likewise, this promoter was not activated by resting level (data not shown). These results suggest that LPS- coincubation with 250 U/ml IFN-␥ and 10 ␮g/ml LPS (data not stimulated MM6 cells may activate hBD-2 promoter in A549 cells shown). In the lungs, mononuclear phagocytes such as alveolar by producing some bioactive effector(s) for the epithelial cells. macrophages respond to bacteria and their products, and activate ␤ epithelial cells by producing various cytokines and chemokines (1, Activation of hBD-2 promoter in A549 cells by IL-1 and ␣ 3, 44, 45). Therefore, we tested whether the coculture of A549 TNF- produced by LPS-stimulated monocytic cells cells with the MM6 monocytic cell line affects hBD-2 promoter To identify more closely the monocytic cell-derived soluble me- activity following LPS stimulation. In preliminary experiments, diators that activate the hBD-2 promoter in A549 cells, we exam- we confirmed that MM6 cells express membrane CD14 (46) and ined the effect of culture supernatants from PMA-differentiated TLR4 (data not shown), and are highly sensitive to LPS, as pre- U937 and MM6 cells on hBD-2 promoter activation. As shown in viously reported (46). Interestingly, hBD-2 promoter activation Fig. 2, supernatants from LPS-treated monocytic cells (PMA-dif- was remarkably induced in A549 cells by coculture with MM6 ferentiated U937 and MM6 cells) markedly up-regulated hBD-2 cells in the presence of LPS (Fig. 1B). The addition of IFN-␥, promoter activity in A549 cells, whereas those from unstimulated which synergistically activates monocytes/macrophages with LPS cells (resting cells) did not.

Table II. Nucleotide sequences of NF-␬B binding sites used for wild-type and mutated hBD-2 promoter constructsa

NF-␬B Sites Wild-Type Sequences Mutated Sequences

d␬B(Ϫ2193/Ϫ2182) 5Ј-TGGGACTTCCCC-3Ј 5Ј-TACGCGTTCCCC-3Ј p␬B2 (Ϫ596/Ϫ572) 5Ј-TGGGGAGTTTCAGGGGAACTTTCAC-3Ј 5Ј-TGCTAGCTTTCAGACGCGTTTTCAC-3Ј p␬B1 (Ϫ205/Ϫ186) 5Ј-AGGGATTTTCTGGGGTTTCC-3Ј 5Ј-AGGACGCGTCTGGTACCTCC-3Ј

a Individual NF-␬B sites are termed d␬B (positions Ϫ2193/Ϫ2182), p␬B2 (Ϫ596/Ϫ572), and p␬B1 (Ϫ205/Ϫ186). Consensus sequences for NF-␬B are indicated by horizontal lines above and below the nucleotide sequences. Of note, NF-␬B binding motifs are tandemly arrayed in the p␬B1 and p␬B2 sites. Mutant NF-␬B promoter constructs (Fig. 5C) were generated by PCR using the mutated sequence primers shown in the table. Mutated bases are indicated by bold letters. The Journal of Immunology 4231 Downloaded from FIGURE 6. Binding of NF-␬Btop␬B1 site in hBD-2 promoter. Top, Nuclear extracts were prepared from untreated A549 cells (Rest) or A549 cells incubated with the following stimuli: 10 ng/ml TNF-␣, 1 ng/ml IL-1␤, or culture supernatants from resting or 1 ␮g/ml LPS-stimulated monocytic cells (PMA-differentiated U937 and MM6). Nuclear extracts (4 ␮g) were incubated with 32P-labeled p␬B1 oligonucleotide. For the competition experiment, a 25 molar excess of unlabeled competitor oligonucleotides was added to the reaction mixture: p␬B1 oligo (␬B1), NF-␬B consensus oligonucleotide (␬B), and NF-IL-6 (C/EBP␤) consensus oligonucleotide (EBP). Supershift assay was performed using 1 ␮g of anti-NF-␬B p65 Ab (p65), anti-NF-␬B p50 Ab (p50), and rabbit IgG as a negative control (IgG). Closed arrowhead (•) indicates specific binding to the probe. Positions of supershifted complexes are denoted by open arrowheads (“). Bottom, Sequence of p␬B1 oligonucleotides used in EMSA. The sequence spans from Ϫ208 to Ϫ183 in the hBD-2 http://www.jimmunol.org/ promoter. Tandemly repeated NF-␬B motifs are indicated by brackets.

Furthermore, we examined whether the promoter assay using TNF-␣ level was 10-fold higher than that of IL-1␤ in the super- the luciferase reporter gene is relevant to the expression of endog- natant from PMA-differentiated U937 cells, whereas levels were enous hBD-2 mRNA in A549 cells. Consistent with the results of almost equivalent in MM6-derived supernatant. Interestingly, it luciferase analysis, hBD-2 mRNA in A549 cells was significantly has been reported that TNF-␣ induces the production of IL-1 from increased by treatment with the supernatants from LPS-stimulated human keratinocytes and colonic epithelial cells (49, 50). Thus, we MDM␾ and monocytic cells, but not LPS alone, IFN-␥ alone, or tested the possibility that TNF-␣ might elicit the production of by guest on September 28, 2021 the supernatants from unstimulated cells (Fig. 3). These results IL-1␤ from A549 cells. As shown in Table I, 2Ð20 ng/ml TNF-␣ suggest that luciferase assay is reflecting the expression of hBD-2 failed to stimulate A549 cells to produce detectable amounts of mRNA, possibly the transcription of the hBD-2 gene. IL-1␤. Furthermore, we confirmed that LPS stimulation did not in- Among biological mediators released from LPS-stimulated duce the production of IL-1␤ and TNF-␣ from A549 cell (Table I). monocytes/macrophages, IL-1␤ and TNF-␣ are known as pivotal These findings (Fig. 2 and Table I) suggest that monocytic cell- inflammatory inducers that act on many cell types, including pul- derived IL-1␤ is likely to have more potent activity than TNF-␣ in monary epithelial cells (4, 43, 48). Thus, to define the contribution hBD-2 activation in A549 cells, although IL-1␤ is less released of IL-1␤ and TNF-␣ to hBD-2 promoter activation by culture su- than TNF-␣, especially from U937 cells. Therefore, we evaluated pernatants from LPS-treated monocytic cells, we assessed the ef- the response of hBD-2 promoter in A549 cells to IL-1␤ and TNF-␣ fect of neutralizing mAbs against these cytokines on the hBD-2 stimulation using recombinant proteins. As expected, both IL-1␤ promoter activity in A549 cells. Interestingly, neutralizing anti- and TNF-␣ substantially elevated hBD-2 promoter activity (Fig. IL-1␤ or anti-TNF-␣ mAb suppressed hBD-2 promoter activity in 4A). Of note, IL-1␤ was more potent than TNF-␣ in the activation A549 cells induced by PMA-differentiated U937-derived superna- of hBD-2 promoter even at the concentrations (0.2 ng/ml IL-1␤ tants, by 90% or only 50%, respectively. Addition of both Abs and 2 ng/ml TNF-␣, respectively) present in the culture superna- further reduced promoter activity in A549 cells (Fig. 2A). In con- tants from LPS-stimulated monocytic cells (Table I). Furthermore, trast, hBD-2 promoter activity induced by MM6-derived superna- we observed that 20 ng/ml IL-6, another proinflammatory cytokine tant was reduced 75% by addition of anti-IL-1␤ mAb, but not produced from activated monocytes/macrophages, failed to en- significantly decreased by anti-TNF-␣ mAb ( p Ͼ 0.1) (Fig. 2B). hance hBD-2 promoter activity in A549 cells ( p Ͼ 0.1) (Fig. 4A). Addition of control IgG to the monocytic cell-derived supernatants Similarly, we confirmed that the endogenous hBD-2 mRNA was did not affect hBD-2 promoter activity ( p Ͼ 0.1) (Fig. 2). These strongly induced by 0.2 ng/ml IL-1␤ and moderately by 10 ng/ml results suggest that IL-1␤ and TNF-␣ secreted from monocytic cells TNF-␣, but not by 20 ng/ml IL-6 in A549 cells (Fig. 4B). Taken are involved in the activation of hBD-2 promoter in A549 cells. In together, these results suggest that, among monocytic cell-derived particular, the greater inhibitory effect of anti-IL-1␤ mAb indicates a cytokines, IL-1␤ is likely to act as a principal modulator of hBD-2 more important role for IL-1␤ in the activation of hBD-2 promoter. transcription in A549 cells. Accordingly, we quantified the amounts of IL-1␤ and TNF-␣ in culture supernatants from PMA-differentiated U937 and MM6 cells by ELISA (Table I). IL-1␤ was detected at the lower limit in Determination of cis-elements in the hBD-2 promoter responsive ␤ ␣ the supernatants from resting cells, and significantly increased af- to IL-1 or TNF- stimulation ter LPS stimulation. Levels of TNF-␣ were also elevated in the IL-1␤ and TNF-␣ initiate NF-␬B signaling and induce the tran- culture supernatants from LPS-stimulated cells. Interestingly, scription of several genes involved in innate immune responses (4, 4232 REGULATION OF hBD-2 EXPRESSION IN PULMONARY EPITHELIAL CELLS

analysis showed that deletion of hBD-2 promoters to Ϫ412 (⌬Ϫ2187/Luc, ⌬Ϫ1050/Luc, ⌬Ϫ577/Luc, and ⌬Ϫ412/Luc) did not essentially reduce responsiveness to both IL-1␤ and TNF-␣ ( p Ͼ 0.05), although the responsiveness of ⌬Ϫ2187/Luc was slightly in- creased to IL-1␤. In contrast, further deletion of the p␬B1 site (⌬Ϫ188/Luc) resulted in a complete loss of cytokine responsiveness (Fig. 5B). These results suggest that the NF-␬B binding site, p␬B1 (Ϫ205 to Ϫ186) mainly contributes to the cytokine-induced hBD-2 transcription in A549 cells. To further elucidate the role of p␬B1 site in the transcriptional regulation of hBD-2 gene, we generated four luciferase expression constructs containing mutated NF-␬B promot- ers, termed d␬Bmut/Luc, p␬B2mut/Luc, p␬B1mut/Luc, and p␬B1ϩ2mut/Luc (Table II). Consistent with the results of deletion analysis, mutation of the p␬B1 site from Ϫ205 to Ϫ186, as well as double mutation of the p␬B1 and p␬B2 sites, completely abolished the up-regulation of promoter activity by IL-1␤ and TNF-␣ (Fig. 5C). On the contrary, mutation of d␬B(Ϫ2193 to Ϫ2182) and p␬B2 (Ϫ596 to Ϫ572) had little effect on IL-1␤- and TNF-␣-induced

hBD-2 promoter activation ( p Ͼ 0.1). Taken together, our results Downloaded from clearly demonstrate that the NF-␬B binding site p␬B1 is essential to the response of hBD-2 promoter to IL-1␤ and TNF-␣, and that bind- ing sequences for other transcription factors do not seem to be im- portant to regulation of the hBD-2 gene.

Identification of NF-␬B family members bound to the p␬B1 site http://www.jimmunol.org/ in the hBD-2 promoter NF-␬B is composed of homo- or heterodimeric subunits of the NF-␬B/Rel family members, and different combinations of NF-␬B subunits contribute to the cell type- and stimulant-specific tran- scriptional activation (51, 53). Therefore, we determined NF-␬B family members bound to the p␬B1 site (Ϫ205 to Ϫ186) in the hBD-2 promoter using EMSA.

As shown in Fig. 6, no specific DNA-protein complex was de- by guest on September 28, 2021 tected when the p␬B1 oligonucleotide probe was incubated with nuclear extracts from unstimulated A549 cells (Rest). Similarly, no specific binding was observed using nuclear extracts from A549 cells treated with resting monocytic cell-derived supernatants (Rest U937 and Rest MM6). In contrast, a marked single band with the same mobility was detected using nuclear extracts from A549 FIGURE 7. Effects of NF-␬B inhibitors on hBD-2 promoter activation cells incubated with IL-1␤, TNF-␣, or culture supernatants from induced by cytokines and culture supernatants. A549 cells transfected with LPS-stimulated U937 and MM6 cells. The specific binding com- hBD 2/Luc plasmid were incubated without (Resting) or with TNF-␣ (A), peted with excess amounts of unlabeled p␬B1 and NF-␬B consen- ␤ IL-1 (B), or the monocytic cell-derived culture supernatants (PMA-U937 sus oligonucleotides, but not with unrelated NF-IL-6 consensus (C) and MM6 (D)) for 5 h. For inhibition assay, A549 cells were incubated oligonucleotide. Furthermore, the specific complex was super- with NF-␬B inhibitors (MG-132 and helenalin) for 30 min before stimu- shifted by addition of anti-p65 Ab as well as anti-p50 Ab. lation. Luciferase activities are expressed as fold activation relative to the ␬ unstimulated control (Resting). Values are mean Ϯ SD of three indepen- These observations suggest that NF- B consisting of the p65- ␬ dent experiments. Percent inhibition of promoter activation by NF-␬B in- p50 heterodimer can bind to the p B1 site in A549 cells stimulated hibitors is indicated. Luciferase activities of cells treated with cytokines or with cytokines. LPS-stimulated culture supernatants in the absence and presence of inhibitors p Ͻ 0.001. Effect of NF-␬B inhibitors on IL-1␤- and TNF-␣-induced hBD-2 ,ءء .p Ͻ 0.0001 ,ء .MG-132 and helenalin) were compared) promoter activation 51, 52). In addition, these cytokines are reported to activate a tran- We further addressed the involvement of NF-␬B in the transcriptional scription factor, AP-1, by mediating the p38 mitogen-activated regulation of hBD-2 in A549 cells using NF-␬B inhibitors. A549 cells protein (MAP) kinase and stress-activated protein kinase/c-Jun N- were treated with different types of NF-␬B inhibitors, and then stim- terminal kinase (JNK) signaling pathways (4, 52). As shown in ulated with cytokines or culture supernatants. As shown in Fig. 7, A Fig. 5A and Table II, the hBD-2 promoter contains three NF-␬B and B,20␮M MG-132, a proteasome inhibitor, remarkably inhibited binding motifs, a single distal site at Ϫ2193 to Ϫ2182 (d␬B) and IL-1␤- or TNF-␣-mediated activation of hBD-2 promoter. Helenalin, two proximal sites at Ϫ596 to Ϫ572 and Ϫ205 to Ϫ186 (p␬B2 and which specifically inhibits binding of the NF-␬B p65 subunit to DNA, p␬B1, respectively), as well as several putative binding sequences also substantially reduced the cytokine-induced hBD-2 promoter ac- for AP-1, NF-IL-6, and STATs (36). tivity. Likewise, both inhibitors significantly inhibited the activation To substantiate the cis-elements of hBD-2 promoter responsive of hBD-2 promoter by culture supernatants from LPS-stimulated to IL-1␤ and TNF-␣, we performed the reporter assay using a monocytic cells (Fig. 7, C and D). These results suggest that the series of 5Ј truncated promoter constructs (Fig. 5B). Luciferase activation of hBD-2 promoter in A549 cells by IL-1␤ and TNF-␣ The Journal of Immunology 4233

FIGURE 8. Evaluation of the expres- sion of TLRs and CD14 on A549 and PMA-differentiated U937 cells. A549 cells were incubated with or without 10 ␮g/ml LPS for 16 h before harvest. U937 cells were cultured with 10 nM PMA for 2 days to induce monocytic differentia- tion. A549 cells and PMA-differentiated U937 cells were incubated with mAbs against TLR4 (HTA125 (A and E)), TLR2 (TLR2.1 (B and F)), and CD14 (MY4 (C and G)), or isotype controls as described in Materials and Methods. Downloaded from Shown are expression profiles of each re- ceptor in A549 cells (AÐC) and in PMA- differentiated U937 cells (EÐG). D, LPS- binding capacity of CD14 on A549 cells. Biotin-labeled E. coli LPS was incubated with A549 cells in the presence or ab- sence of neutralizing anti-CD14 mAb http://www.jimmunol.org/ (MY4) or control mouse IgG. The bind- ing of LPS to A549 cells was visualized by further incubation with streptavidin- PE. H, Expression of endogenous CD14 mRNA in A549 cells. Total RNA was isolated from A549 cells, MM6, and PMA-differentiated U937 cells. The size of RT-PCR product from A549 cells

(410 bp) was the same as that from by guest on September 28, 2021 monocytic cells, but not from genomic DNA (498 bp; not shown).

produced from LPS-stimulated monocytic cells is induced through the flow cytometry whether LPS could bind to CD14 molecules on NF-␬B-dependent pathway. A549 cells. As expected, binding of LPS to A549 cells was de- tected using a combination of biotinylated LPS and streptavi- Expression of PRRs on A549 cells din-PE (Fig. 8D). Notably, addition of neutralizing anti-CD14 Increasing evidence suggests that CD14 and TLR4 play a central role mAb (MY4), which directs a functional domain of CD14 mole- in the recognition and signal transduction of LPS (26Ð28). Therefore, cules, almost completely inhibited LPS binding, whereas control we assessed the surface expression of TLRs and CD14 on A549 cells mouse IgG could not. These results indicate that A549 cells ex- using flow cytometry. Importantly, neither TLR4 nor TLR2 protein press functional CD14 molecules with LPS-binding capacity. was detected on A549 epithelial cells (Fig. 8, A and B). Intriguingly, Finally, we evaluated whether LPS could modulate the expres- surface expression of CD14 on A549 cells was observed using anti- sion of these PRRs on A549 cells. Following LPS stimulation, CD14 mAb MY4 (Fig. 8C). However, other anti-CD14 mAbs, expression of TLR4 and TLR2 was still undetectable, and the ex- namely 61D3 and LeuM3, hardly detected CD14 on A549 cells (data not shown). We also confirmed that CD14 molecules on MM6 cells pression level of CD14 did not change (Fig. 8, AÐC). Similar re- could be detected by MY4, but not LeuM3 and 61D3 (data not sults were obtained using A549 cells treated with cytokine mixture ␤ ␣ shown), as previously reported (46). The expression of CD14 in (1 ng/ml IL-1 and 10 ng/ml TNF- ) or culture supernatants from A549 cells was further verified by RT-PCR analysis using DNase- LPS-stimulated monocytic cells (data not shown). In contrast to treated total RNA and CD14-specific intron-spanning primers. As A549 cells, PMA-differentiated U937 cells, which are susceptible shown in Fig. 8H, a 410-bp product of CD14 cDNA was specif- to LPS, apparently expressed CD14 and TLR4 as well as TLR2 ically amplified from A549 cells as well as monocytic cells (MM6 (Fig. 8, EÐG). Likewise, MM6 cells expressed all the PRRs ana- and PMA-differentiated U937 cells). Moreover, we tested using lyzed (data not shown). 4234 REGULATION OF hBD-2 EXPRESSION IN PULMONARY EPITHELIAL CELLS

These results suggest that A549 epithelial cells consistently ex- of NF-␬B via the degradation of I␬B, as well as that of AP-1, press CD14 molecules with LPS-binding capacity, but lack TLR4, which is mediated via the stress-activated protein kinase/JNK and a critical molecule for LPS signaling. p38 MAP kinase pathways (4, 52). More recently, Krisanaprakorn- kit et al. (56) reported that hBD-2 transcription is mediated in Discussion gingival epithelial cells by Fusobacterium nucleatum dependent The innate defense system plays a critical role in protecting the on MAP (p38 and JNK) kinase pathways but not NF-␬B. These lungs, which are in direct contact with exogenous air, from mi- observations raise the possibility that, in addition to NF-␬B, AP-1 crobial infection. Among various biological effectors, much recent and other transcription factors may participate in cytokine-induced attention has focused on hBD-2, an antimicrobial peptide with mi- hBD-2 transcription in A549 cells. However, deletional analysis of crobicidal activity as well as modulatory activity on adaptive im- hBD-2 promoter revealed that the p␬B1 site (Ϫ205 to Ϫ186) was munity (1Ð3, 5Ð8, 10). Because the induction of hBD-2, which is critical for the transcriptional regulation of the hBD-2 gene by primarily expressed in epithelial cells, is controlled at the tran- IL-1␤ and TNF-␣ in A549 cells, but none of the binding sequences scriptional level, the recognition and signal transduction of endog- for AP-1, NF-IL-6, and STATs was essential (Fig. 5B). Further- enous and exogenous stimuli via surface PRRs is crucial to hBD-2 more, both IL-1␤- and TNF-␣-induced hBD-2 transcription was production by epithelial cells (7). completely lost on mutation of the NF-␬B site p␬B1 (Fig. 5C). We In this study, we examined the transcriptional regulation of also noted that IL-6 and IFN-␥, which activate NF-IL-6 and STAT hBD-2 in epithelial cells by mononuclear phagocytes using lung transcription factors, did not induce hBD-2 mRNA expression and epithelia-like A549 cells and monocytic-cell lines U937 and MM6, promoter activation in A549 cells (Figs. 1, 3, and 4). and in particular demonstrated the role of proinflammatory cyto- Taken together, our observations indicate that NF-␬B mediates Downloaded from kines produced by LPS-stimulated mononuclear phagocytes, using IL-1␤- or TNF-␣-induced hBD-2 transcription in A549 cells via RT-PCR and luciferase assay. binding to the p␬B1 site, and that other transcription factors are Consistent with previous reports that A549 cells were hypore- unlikely to be involved in this transcriptional regulation. sponsive to LPS (31, 32, 45), treatment of these cells with LPS (up Furthermore, we suggested that NF-␬B p65-p50 heterodimer to 100 ␮g/ml) alone did not induce hBD-2 promoter activation could bind to the p␬B1 site in A549 cells stimulated with either

(Fig. 1A). However, coculture with monocytic cells showed a sig- IL-1␤, TNF-␣, or LPS-activated monocytic cell-derived superna- http://www.jimmunol.org/ nificant induction of hBD-2 promoter activity in these cells fol- tants (Fig. 6). The p65-p50 heterodimer is the most abundant form lowing LPS stimulation (Fig. 1B). Similar results were obtained of the NF-␬B family, and acts as a strong transactivator (53, 57, using culture supernatants from LPS-stimulated monocytic cells 58). In contrast, the p65 homodimer is reported to selectively bind (Fig. 2). Furthermore, we confirmed that the endogenous expres- to the NF-␬B site (p␬B1) in gastrointestinal cell lines exposed to sion of hBD-2 mRNA in A549 cells was induced following stim- Helicobacter pylori or flagella filament protein from Salmonella ulation with LPS-activated monocytic cell supernatants, but not enteritidis (54, 55). However, in a previous study using macro- with LPS alone or unstimulated cell supernatants (Fig. 3). These phage-like RAW264.7 cells, we showed that the p65-p50 het- findings suggest that soluble factors from LPS-stimulated mono- erodimer could bind to this site on stimulation of the cells with cytic cells are involved in the regulation of hBD-2 gene transcrip- LPS (36). Thus, the transcriptional activation of hBD-2 gene may by guest on September 28, 2021 tion in A549 cells. Using neutralizing mAbs and recombinant cy- be regulated by a combination of NF-␬B subunits (p65 and p50) in tokines and measuring cytokines in the culture supernatants, we a cell type-, stimulus-, or both-specific fashion. showed in this study that IL-1␤ and TNF-␣ released from mono- In addition, we confirmed the involvement of NF-␬B in hBD-2 cytic cells are responsible for the up-regulation of hBD-2 promoter transcription by using two NF-␬B inhibitors with different phar- activity in A549 cells (Figs. 2 and 4A; Table I). Importantly, IL-1␤ macological actions. MG-132 blocks the release of NF-␬B from showed greater potency than TNF-␣ in this effect, although IL-1␤ I␬B/NF-␬B complex by inhibiting proteasome-mediated I␬B deg- secretion from monocytic cells (MM6 and PMA-differentiated radation, whereas helenalin interferes with the binding of U937 cells) was less than that of TNF-␣ (Fig. 4A and Table I). In NF-␬B p65 subunit to DNA (34). Both inhibitors significantly fact, hBD-2 promoter activity was up-regulated by IL-1␤ in A549 reduced the hBD-2 transcription induced by IL-1␤, TNF-␣, and cells at a minimum concentration of 100 pg/ml, and reached a LPS-stimulated monocytic cell-derived supernatants (Fig. 7). plateau at 1 ng/ml, whereas Ͼ1 ng/ml TNF-␣ was required to However, these inhibitory effects were not complete. As a pos- activate the promoter (data not shown). Consistent with the pro- sible explanation for this, we speculate that one or both cyto- moter analysis, the expression of hBD-2 mRNA in A549 cells was kines may mediate a second pathway leading to NF-␬B activa- strongly up-regulated by 200 pg/ml IL-1␤, but to a lesser extent by tion, which is different from the signaling mediated by I␬B 10 ng/ml TNF-␣ (Fig. 4B). This greater contribution of IL-1␤ to degradation and insensitive to MG-132 and helenalin. Supporting hBD-2 transcription was further supported by the finding that anti- this possibility, Bergmann et al. (59) suggest that NF-␬B activation is IL-1␤ mAb alone suppressed hBD-2 promoter activation by LPS- partially regulated by phosphatidylcholine-specific phospholipase C stimulated monocytic cell-derived supernatants, by 75Ð90%, and protein kinase C isoforms. whereas combination with anti-TNF-␣ mAb resulted in minimal To limit microbial infection, mammalian host cells use PRRs additional decrease (Fig. 2). Moreover, it was shown that two other such as TLRs and CD14, which effectively recognize bacterial potent cytokines, IL-6 and IFN-␥, were unlikely to be involved in the conserved molecules including LPS and peptidoglycans (26Ð28). regulation of hBD-2 expression in A549 cells (Figs. 1, 3, and 4). Among these receptors, CD14, a GPI-anchoring receptor which Taking these results together, we conclude that hBD-2 expres- lacks a cytoplasmic domain, is required for the detection and cap- sion in A549 cells is modulated by mononuclear phagocytes ture of LPS by interacting with a transmembrane receptor, TLR4, through the production of IL-1␤ and TNF-␣ upon LPS stimulation. whereas TLR4 possesses a conserved cytoplasmic domain and acts In particular, IL-1␤ seems to be a major activator of hBD-2 ex- as a signal transducer molecule to initiate the LPS signaling (27, pression in these cells. 28, 48, 60). Actually, PMA-differentiated U937 (Fig. 8, E and G) It has been reported that the transcriptional activation of hBD-2 and MM6 cells (data not shown) expressed both TLR4 and CD14, by bacteria and their products is regulated in an NF-␬B-dependent and could produce IL-1␤ and TNF-␣ in response to LPS (Table I). manner (36, 54, 55). Both IL-1␤ and TNF-␣ induce the activation Of importance, using RT-PCR analysis and flow cytometry, we The Journal of Immunology 4235 observed that A549 cells express CD14 mRNA and protein with References LPS binding activity (Fig. 8, C, D, and H). 1. Zhang, P., W. R. Summer, G. J. Bagby, and S. Nelson. 2000. Innate immunity Furthermore, it is noteworthy that the expression of TLR4 on and pulmonary host defense. Immunol. Rev. 173:39. 2. Diamond, G., D. Legarda, and L. K. Ryan. 2000. The innate immune response of A549 cells could not be detected by flow cytometry, even after the respiratory epithelium. Immunol. 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