Large Intestinal Epithelial Cells Response to Toll-Like Receptor 5

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Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 1 in Oxidative Burst Response to Toll-Like Receptor 5 Signaling in Large Intestinal Epithelial Cells This information is current as of September 27, 2021. Tsukasa Kawahara, Yuki Kuwano, Shigetada Teshima-Kondo, Ryu Takeya, Hideki Sumimoto, Kyoichi Kishi, Shohko Tsunawaki, Toshiya Hirayama and Kazuhito Rokutan

J Immunol 2004; 172:3051-3058; ; Downloaded from doi: 10.4049/jimmunol.172.5.3051 http://www.jimmunol.org/content/172/5/3051 http://www.jimmunol.org/ References This article cites 32 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/172/5/3051.full#ref-list-1

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 1 in Oxidative Burst Response to Toll-Like Receptor 5 Signaling in Large Intestinal Epithelial Cells1

Tsukasa Kawahara,* Yuki Kuwano,* Shigetada Teshima-Kondo,* Ryu Takeya,† Hideki Sumimoto,† Kyoichi Kishi,* Shohko Tsunawaki,‡ Toshiya Hirayama,§ and Kazuhito Rokutan2*

The NADPH oxidase 1 (Nox1) is a gp91phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released su- ؊ phox phox phox peroxide anion (O2 ) of 160 nmol/mg protein/h and expressed the Nox1, p22 , p67 , and Rac1 mRNAs, but not the gp91 , Nox4, p47phox, p40phox, and Rac2 mRNAs. They also expressed novel homologues of p47phox and p67phox (p41nox and p51nox, Downloaded from respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22phox, p51nox, and Rac1 mRNAs, but not ؊ nox the other NADPH component mRNAs, and secreted only small amounts of O2 (<2 nmol/mg protein/h). Cotransfection of p41 nox ؊ and p51 cDNAs in T84 cells enhanced PMA-stimulated O2 release 5-fold. Treatment of the transfected T84 cells with recom- ؊ binant ﬂagellin (rFliC) from Salmonella enteritidis further augmented the O2 release in association with the induction of Nox1 ؊ nox nox protein. The enhanced O2 production by cotransfection of p41 and p51 vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-␤-activated kinase http://www.jimmunol.org/ 1 and TGF-␤-activated kinase 1-binding protein 1. A potent inhibitor for NF-␬B (pyrrolidine dithiocarbamate) signiﬁcantly ؊ nox nox blocked the rFliC-primed increase in O2 production and induction of Nox1 protein. These results suggest that p41 and p51 are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense. The Journal of Immunology, 2004, 172: 3051–3058.

eactive oxygen species (ROS),3 notably superoxide anion Recently, two families of gp91phox homologues have been identi- Ϫ (O2 ) and hydrogen peroxide, operate on a variety of ﬁed: NADPH oxidase (Nox) and dual oxidase (Duox) families (5).

physiological processes, including host defense, gene ex- The Nox family comprises Nox1 (initially termed Mox1 or NOH- by guest on September 27, 2021 R phox pression, oxygen sensing, regulation of vascular tone, bone resorp- 1), Nox2 (renamed gp91 ), Nox3, Nox4 (Renox), and Nox5 tion, apoptosis, cell growth, and transformation (for reviews, see (6–10). These homologues conserve binding sites for heme, FAD, Ϫ Refs. 1–3). The best-known O2 -producing enzyme is the phago- and NADPH of Nox2 (5), and are preferentially expressed in cyte respiratory burst oxidase that plays a crucial role in a process nonphagocytic cells. The Duox family members are Duox1 and of killing microorganisms. The catalytic core of this oxidase is the Duox2 (initially termed ThOX1 and ThOX2, respectively), which phox membrane-integrated flavocytochrome b558 composed of p22 have a peroxidase homology domain plus two EF-hand motifs, as and gp91phox subunits, the latter having binding sites for heme, well as binding sites for heme, FAD, and NADPH (5). Of these flavin adenine dinucleotide (FAD), and NADPH, and transfers an family members, the Nox1 mRNA is predominantly expressed in Ϫ electron from NADPH to molecular oxygen to generate O2 (4). human colon tissue and a carcinoma cell line, Caco2 cells (6, 7). However, physiological roles of Nox1 in large intestinal epithelial *Department of Nutrition, School of Medicine, University of Tokushima, Tokushima, cells (LIEC) are not fully understood. Japan; †Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; ‡De- We previously reported that primary cultures of guinea pig gas- partment of Infectious Diseases, National Research Institute for Child Health and Ϫ Development, Tokyo, Japan; and §Department of Bacteriology, Institute of Tropical tric pit cells expressed Nox1 and spontaneously secreted O2 (11, Medicine, Nagasaki University, Nagasaki, Japan 12). ROS derived from Nox1 in the pit cells were essential for their Received for publication June 12, 2003. Accepted for publication December 22, 2003. growth at least in vitro (12). Furthermore, Helicobacter pylori LPS The costs of publication of this article were defrayed in part by the payment of page stimulated the Toll-like receptor (TLR) 4 signaling and activated charges. This article must therefore be hereby marked advertisement in accordance Ϫ Nox1 (13, 14). The increased O2 production and enhanced acti- with 18 U.S.C. Section 1734 solely to indicate this fact. vation of NF-␬B resulted in the induction of the TNF-␣ and cy- 1 This study was supported by a grant-in-aid for scientific research from Japan Society clooxygenase II mRNAs in pit cells themselves (12), suggesting a for the Promotion of Science (14370184), and Japan Society for the Promotion of Science Research Fellowships for Young Scientists (04127). potential role of Nox1 in inflammatory and immune responses 2 Address correspondence and reprint requests to Dr. Kazuhito Rokutan, Department against H. pylori. phox of Nutrition, School of Medicine, University of Tokushima, 3-18-15 Kuramoto-cho, The flavocytochrome b558 requires cytosolic proteins, p67 , Tokushima 770-8503, Japan. E-mail address: [email protected] p47phox, and a small GTPase Rac for electron-transfer reactions to 3 Abbreviations used in this paper: ROS, reactive oxygen species; DF, DMEM-Ham’s form O Ϫ (4). P40phox associates with p67phox and enhances mem- F-12; Duox, dual oxidase; FAD, flavin adenine dinucleotide; LIEC, large intestinal 2 Ϫ phox phox epithelial cell; NBT, nitroblue tetrazolium; Nox, NADPH oxidase; O2 , superoxide brane translocation of p67 and p47 in stimulated phago- anion; PAS, periodic acid-Schiff; PDTC, pyrrolidine dithiocarbamate; PVDF, poly- cytes (15). However, it remains to be elucidated whether these vinylidene difluoride filter; rFliC, recombinant structural protein of flagella filament; SOD, superoxide dismutase; TAB1, TAK1-binding protein 1; TAK1, TGF-␤-acti- cytosolic factors are necessary for activation of the other Nox and vated kinase 1; TLR, Toll-like receptor. Duox family members.

In this study, we have established primary cultures of guinea pig dues 1–15), and recombinant human p67phox were provided, as previously LIEC with 90% purity of surface mucous cells and have found that described (13). Membrane, cytosolic, and whole cell fractions were pre- these cells also produce O Ϫ even at a higher rate than that of pared from cultured cells, as previously described (11). Each sample of 20 2 ␮g protein per lane was separated by SDS-PAGE using an 8% polyacryl- gastric pit cells. Guinea pig LIEC expressed novel genes encoding amide gel and transferred to a polyvinylidene difluoride filter (PVDF). nox phox nox phox p41 (a p47 homologue) and p51 (a p67 homologue), After blocking nonspecific binding sites with 4% purified milk casein, the which have recently cloned in mouse and human, being named as PVDF was incubated for1hatroom temperature with one of the above the NOX organizer 1 and NOX activator 1 (16–18), respectively. primary Abs at a 1/1000 dilution. After being washed with PBS containing 0.05% Tween 20, bound Abs were detected by an ECL Western blotting Cotransfection of these two homologues was shown to up-regulate Ϫ detection system (Amersham Pharmacia Biotech). Bound Abs were then O2 -producing capability of Nox1, and in situ hybridization dem- removed, and the PVDF was reblotted with a mAb against ␤-actin (On- onstrated that the p41nox and p51nox transcripts were expressed in cogene Research Products, Cambridge, MA). Phosphorylation of TGF-␤- epithelial cells of mouse colonic mucosa (16–18), suggesting their activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) was as- crucial roles for Nox1 activity. Using guinea pig LIEC and human sayed, as previously described (14). colon cancer cell lines, we molecularly and functionally charac- Cytochemical and immunohistochemical stainings terized Nox1 expressed in LIEC. Mucous granule-containing cells were visualized by the periodic acid- Materials and Methods Schiff (PAS) reaction. For immunohistochemical analysis, growing LIEC Reagents on the dishes were fixed with 4% paraformaldehyde in PBS for 20 min. They were then incubated in PBS containing 0.03% Triton X-100 for 2 min A recombinant structural protein of flagella filament (rFliC) of Salmonella on ice and blocked with 4% purified milk casein. These cells were incu- enteritidis was prepared, as previously described (19). Staphylococcus au- bated with a 1/500 dilution of anti-Nox1-C1 Ab for 1 h. After washing, Downloaded from reus peptidoglycan and LPS from Escherichia coli K-235 were purchased they were treated with a 1/500 dilution of biotin-linked goat Ab against from Fluka Chemie AG (Buchs, Switzerland) and Sigma-Aldrich (St. rabbit IgG (Amersham Pharmacia Biotech) for 1 h, and were then incu- Louis, MO), respectively. Pyrrolidine dithiocarbamate (PDTC) was ob- bated with a 1/500 dilution of streptavidin-conjugated FITC probe for 30 tained from Calbiochem (San Diego, CA). Phosphorothioate-stabilized min at room temperature. The cells were mounted with Vectashield mount- CpG oligodeoxynucleotide (CpG DNA) (TCCATGACGTTCCTGAT ing medium (Vector Laboratories, Burlingame, CA). Fluorescence was GCT) (20) was purchased from Hokkaido System Science (Sapporo, viewed using a confocal laser-scanning microscopy (model Axiovert 25CFL; Leica, Heiderberg, Germany). Fibroblasts and macrophages were Japan). http://www.jimmunol.org/ immunocytochemically identified with mAbs against vimentin (Santa Cruz Preparation and culture of cells Biotechnology, Santa Cruz, CA) and a macrophage Ag (HAM56 clone); vimentin-positive fibroblasts and HAM56-positive macrophages were vi- The present study was approved by the Animal Care Committee of Uni- ϳ sualized by diaminobenzidine streptavidin-biotin HRP and Vector red al- versity of Tokushima. Male guinea pigs weighing 250 g were purchased kaline phosphatase methods (Vector Laboratories), respectively. from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Under gen- Paraffin-embedded guinea pig colon tissues were cut into 3-␮m-thick- eral anesthesia with pentobarbital, ascending to sigmoid portions of the ness sections and deparaffinized. After blocking nonspecific binding sites guinea pig colon were resected and extensively washed with PBS. Colonic with 4% purified milk casein, they were incubated with a 1/1000 dilution mucosa was scraped with a sterile glass slide and finely minced with sterile of anti-Nox1-C1 Ab in TBS containing 0.1% Tween 20 and 2% BSA surgical blades. The minced pieces were then incubated in DMEM-Ham’s overnight at 4°C. After washing, bound Abs were visualized using Vector F-12 (1:1) (DF) medium (Life Technologies, Grand Island, NY) containing red alkaline phosphatase kit (Vector Laboratories). Finally, specimens by guest on September 27, 2021 0.03% collagenase S-1 (Nitta Gelatin, Osaka, Japan) and 0.2% BSA for 30 were counterstained with hematoxylin and mounted with Aquatex (Merck, min at 37°C. The digested tissues were next washed three times with DF Darmstadt, Germany). medium by centrifugation. The resulting pellets were resuspended in DF medium containing 10% FBS (PAA Lab., Linz, Austria) and incubated at o RT-PCR 37 C for 2 h under 5% CO2. Attached cells were positive for a specific mAb against macrophages (HAM56 clone; Enzo Diagnostic, Farmingdale, Total RNA was prepared from the indicated cells or tissues with an acid NY) and used as a macrophage population derived from colonic mucosa. guanidium-thiocynate-phenol chloroform mixture (14). RT-PCR was per- Collected nonadherent cells were washed with DF medium and then cul- formed using the following specific PCR primer sets: Nox1-A,5Ј-ATGG tured for 24 h in 35-mm-diameter culture dishes that had been coated with GAAACTGGGTGGTTA-3Ј and 5Ј-TAGCTGAAGTTACCATGAGAA- type IV collagen (Nitta Gelatin). The growing cells were used as primarily 3Ј; Nox1-B, 5Ј-TTCTTGGCTAAATCCCATCCA-3Ј and 5Ј-TTTCTG cultured guinea pig LIEC. These cells were completely replaced by vimen- TCCAGTCCCCTGCT-3Ј; Nox2, 5Ј-CATCATCTCTTTGTGATCTTCT-3Ј tin-positive fibroblasts after a 2-wk cultivation. Guinea pig and human PBL and 5Ј-CTTAGGTAGTTTCCACGCATC-3Ј; Nox4,5Ј-GGTCCTTTTGG were prepared, as previously described (14). T84 and Caco2 cells were AAGTCCATTTGAGG-3Ј and 5Ј-CACAGCTGATTGATTCCGCTGAG-3Ј; maintained in DMEM medium supplemented with 10% FBS, 100 U/ml p22phox,5Ј-ATGGGGCAGATCGAGTGGGCCATGT-3Ј and 5Ј-GTAGATG penicillin, and 100 ␮g/ml streptomycin. phox Ϫ CCGCTCGCAATGGCCAG-3Ј; p67 ,5Ј-TCCCGGATTTGCTTCAAC The amount of O2 release was measured by the superoxide dismutase phox Ϫ ATT-3Ј and 5Ј-TTGGCCAGCTGAGCCACTT-3Ј; p47 -A, 5Ј-ATCCG (SOD)-inhibitable reduction of cytochrome c, and O2 -producing cells TCACATCGCCCTGCT-3Ј and 5Ј-CCAACCGCTCTCGCTCTTCT-3Ј; were cytochemically visualized by detecting blue formazan precipitates of p47phox-B, 5Ј-AACAGGATCATCCCCCACCT-3Ј and 5Ј-CAGGTACAT nitroblue tetrazolium (NBT), as previously described (11). GGACGGAAAGT-3Ј; p40phox,5Ј-TGACATCGAGGAGAGAGGCT-3Ј nox Immunoblotting and 5Ј-GGAAGATCACATCTCCAGCTTTGA-3Ј; p41 ,5Ј-TTTGCCT TCTCTGTGCGCTGG-3Ј and 5Ј-TCTGGGGTGGGCAGGATCACC-3Ј; Anti-Nox1 Abs were made by immunizing rabbits with synthetic peptides p51nox,5Ј-CAAGCAGTGACTAAGGACACCTG Ϫ3Ј and 5Ј-CACAC corresponding to the 480–493 (Nox1-C1) and 544–556 (Nox1-C2) aa res- AGGACATCCACCGTGTC-3Ј; Rac1,5Ј-TGCAGGCCATCAAGTGT idues of human Nox1 (GenBank accession AF166327). The epitopes for GTGGT-3Ј and 5Ј-GCTGAGACATTTACAACAGCAGGCAT-3Ј; Rac2, the two anti-Nox1 Abs were designed not to overlap the amino acid se- 5Ј-TGCAGGCCATCAAGTGTGTGGT Ϫ3Ј and 5Ј-TAGAGGAGGCTG quences of the other Nox homologues (GenBank accession NM000397, CAGGCGCGCTT-3Ј; and GAPDH, 5Ј-TCATGACCACAGTCCATGC AF190122, NM016931, and NM024505). Nox1-C1- or Nox1-C2-immu- CATCACT-3Ј and 5Ј-GCCTGCTTCACCACCTTCTTGATGT-3Ј. PCR nized serum was further purified by affinity chromatography using the Ag products were sequenced with a DNA sequencer and confirmed to be the peptide-conjugated agarose (Amersham Pharmacia Biotech, Piscataway, corresponding cDNA fragments. NJ). The amino acid sequence of Nox1-C1 has 93% homology between human and guinea pig (GenBank, AB099629), and anti-Nox1-C1 Ab rec- Expression vectors and cDNA constructs ognized both human and guinea pig Nox1 proteins. In contrast, anti- Nox1-C2 Ab recognized only human Nox1, because guinea pig Nox1 does The cDNA encoding human p51nox was provided, as previously described not share this motif. Anti-TLR5 Ab was made by immunizing rabbits with (18), and the cDNA encoding p67phox was a gift from H. Nunoi (Miyazaki synthetic peptides corresponding to the 836–849 aa residues of human Medical School, Miyazaki, Japan). The p41nox cDNA (GenBank, TLR5. Polyclonal Abs against synthetic peptide of human p22phox (resi- AF539796) was amplified by PCR using human digestive system multiple dues 177–195), human p47phox (residues 376–390), human p40phox (resi- tissue cDNA (Clontech Laboratories, Palo Alto, CA). Full-length p67phox The Journal of Immunology 3053

ture dishes at 70–90% conﬂuence were incubated in 1 ml of serum-free DF medium for 2 h and then treated with rFliC (5 ␮g/ml) in the absence or presence of 200 U/ml SOD plus 700 U/ml catalase. After treatment for 24 h, the medium was collected and centrifuged at 1000 ϫ g for 10 min. The concentrations of IL-8 in the supernatants were measured using a human IL-8 ELISA kit (R&D Systems, Abington, U.K.), according to the manufacturer’s protocol. The amount of IL-8 was expressed as pg/ml per mg cell protein.

Results Ϫ Nox1-expressing and O2 -producing cells in guinea pig LIEC Contaminated macrophages were removed as attaching cells during an initial 2-h cultivation of isolated guinea pig colonic mucosal cells. Nonadherent cells were collected and cultured in DF medium supplemented with 10% FBS. These cells began to adhere to collagen-coated plates within 6 h and became confluent at ϳ24 h. After a 48-h culture, they started to undergo spontaneous apoptosis, mirroring their rapid turnover in vivo. At the 24-h cultivation, Ϫ 90 Ϯ 3% (mean Ϯ SD, n ϭ 8) of cultured cells possessed PAS FIGURE 1. Identification of O2 -generating cells. The majority of Downloaded from growing guinea pig LIEC at 24 h shows the PAS reaction-positive granules reaction-positive granules characteristic of surface mucous cells (A and B). Vimentin-positive fibroblasts growing at 24 h and after 2 wk are (Fig. 1, A and B). Although vimentin-positive fibroblasts were less shown in C and D, respectively. HAM56-positive macrophages do not than 5% at 24 h (Fig. 1C), they had grown to be an exclusive contaminate the final culture at 24 h (E), confirming the successful population in 2 wk (Fig. 1D). Macrophages were not detected in removal of adherent cells in the initial 2-h cultivation (F). Cultured the 24-h LIEC culture (Fig. 1E) after their removal by the adherent LIEC were counterstained with propidium iodide to visualize cell nuclei method (Fig. 1F), and the growing LIEC were used in the follow-

␮ ␮ http://www.jimmunol.org/ (right panels in B, C, and E). Scale bars indicate 100 m(A) and 20 m ing experiments. (B–F), respectively. Ϫ The cultured guinea pig LIEC spontaneously secreted O2 at 156 Ϯ 9 nmol/mg protein/h (mean Ϯ SD, n ϭ 12). This rate was cDNA and hemagglutinin-tagged p41nox and p51nox cDNAs were recom- higher than that of cultured gastric pit cells primed with H. pylori bined into pAdTrack-CMV vector (21). T84 cells were plated at a concen- LPS (112 Ϯ 5 nmol O Ϫ/mg protein/h; mean Ϯ SD, n ϭ 12). ϫ 5 2 tration of 5 10 cells/well in 24-well plates and transfected with the O Ϫ-producing cells contained granules positive for the PAS re- vectors using the FuGENE transfection reagent (Roche Biomedical Labo- 2 ratories, Burlington, NC). action (Fig. 2A) and expressed Nox1 protein (Fig. 2B). These Nox1-expressing cells (Fig. 2C) were identical with the cells cov- Measurement of IL-8

ered with precipitates of blue formazan (Fig. 2D). In the guinea pig by guest on September 27, 2021 T84 cells were transfected with mock (pAdTrack-CMV vector) or p51nox colon, surface mucous cells possessed immunoreactive materials plus p41nox vectors for 48 h. These cells growing in 35-mm-diameter cul- to anti-Nox1-C1 Ab (Fig. 2, E and F), and the synthetic Ag

FIGURE 2. Expression of Nox1 in guinea pig LIEC. Primary cultures of guinea pig LIEC were subjected to the PAS staining (A), immunocytochemistry with an anti- Nox1-C1 Ab (B and C), and NBT assay (D), as described in Materials and Methods. Cel- lular distribution of Nox1 protein in guinea pig colonic mucosa is shown in E and F. The specificity of anti-Nox1-C1 Ab was verified by a preabsorption test with a 50-fold molar excess of Ag peptide used for the Ab preparation (G). Scale bars indicate 100 ␮m(A and B), 10 ␮m(C and D), 200 ␮m(E and G), and 20 ␮m(F), respectively. Total RNA was isolated from guinea pig LIEC, Caco2 cells, human and guinea pig PBL, or guinea pig kidney, and subjected to RT-PCR, as described in Materials and Methods. Mixture with (ϩ) or without (Ϫ) reverse-transcriptase reaction was amplified using the specific primer sets for the detection of Nox1 (H), Nox2 (I), or Nox4 (J) mRNA. Data are representative of four independent experiments. 3054 Nox1 IN LIEC

Roles of p41nox and p51nox in Nox1 activity Ϫ Ͻ T84 and Caco2 cells generated only small amounts of O2 ( 2 Ϫ nmol/mg protein/h). The low output of O2 in these cells was mainly due to lower levels of Nox1 expression, compared with that in guinea pig LIEC or guinea pig gastric pit cells. It may be also due to the absence or insufﬁciency of distinct component(s) sup- portive for the Nox1 activity. The p67phox and p41nox mRNAs were absent in T84 and Caco2 cells, and the p51nox mRNA level was much lower than that in guinea pig LIEC (Fig. 4A). As shown in Fig. 4B, transduction of p67phox or overproduction of p51nox did Ϫ not change the spontaneous or PMA-stimulated release of O2 from T84 cells, but p41nox-overexpressing cells signiﬁcantly in- Ϫ creased O2 generation when stimulated by PMA (Fig. 4B). Al- though transfection of the p41nox-overexpressing cells with the p67phox vector failed to increase both spontaneous and PMA-stim- Ϫ nox nox ulated O2 productions, overproduction of p51 in the p41 - Ϫ transfected cells further increased the PMA-responsive O2 generation (Fig. 4B). P67phox is an essential activator for Nox2, and p51nox has conserved domains that possibly interact with Nox1 in Downloaded from phox a similar way as p67 does with cytochrome b558 in phagocytes (16–18, 22). However, our results suggest that p51nox is most Ϫ likely a better partner of Nox1 for achieving a high output of O2 production.

Effects of bacterial components on Nox1 activity http://www.jimmunol.org/ To elucidate physiological functions of Nox1 in LIEC, we ex- FIGURE 3. Expressions of NADPH oxidase components in guinea pig plored possible up-regulator(s) of the oxidase. Mirroring the rapid LIEC. Total RNA was isolated from human PBL or cultured guinea pig turnover of surface mucous cells in vivo, guinea pig LIEC in pri- phox phox phox phox LIEC, and the mRNAs of p22 , p67 , p47 , p40 , Rac1/2, and mary culture appeared to be already in a fully matured and acti- GAPDH were ampliﬁed by RT-PCR, as described in Materials and Meth- vated status; therefore, we investigated whether T84 cells overex- ods (A). Membranes and cytosol were fractionated from guinea pig PBL nox nox phox pressing p41 and p51 could be primed with bacterial and guinea pig LIEC. The amount of p22 in membrane fraction and of Ϫ p67phox, p47phox, p40phox, and ␤-actin in cytosol were estimated by immu- components for O2 generation. LPS from H. pylori or E. coli was nox nox demonstrated to act as a potent stimulator of Nox1 in primary

noblot analysis using the corresponding Abs (B). The p51 and p41 by guest on September 27, 2021 mRNAs were amplified by specific primer sets using mixtures with (ϩ)or cultures of guinea pig gastric mucosal cells (14). Although T84 without (Ϫ) reverse-transcriptase reaction (C). Similar results were ob- cells express the TLR4 mRNA (data not shown), they were insen- tained in three separate experiments. sitive to LPS priming: E. coli LPS up to 20 ␮g/ml did not change Ϫ the basal O2 generation (Fig. 5A). Neither peptidoglycan from S. Ϫ aureus nor CpG DNA increased the O2 production (Fig. 5A). In contrast, rFliC from S. enteritidis at 2 ␮g/ml or higher concentra- Ϫ polypeptide completely abolished this immunoreactivity (Fig. 2G). tions significantly enhanced O2 generation within 12 h (Fig. 5, B Thus, surface mucous cells of the guinea pig colon constitutively and C). Boiling did not affect the priming action of rFliC, but expressed Nox1 protein both in vitro and in vivo. We also con- treatment with trypsin completely abolished it (Fig. 5A), which are Ϫ Ͻ firmed that the prepared fibroblasts released O2 at 1 nmol/mg well-known characteristics of FliC (19). T84 cells expressed the protein/h, but these amounts were not enough to form visible blue TLR5 mRNA (Fig. 5D), and TLR5 protein was mainly present in formazan precipitates even after incubation with 0.1 mM of NBT the membrane fraction (Fig. 5E). TAK1 is a member of mitogen- for 2 h (data not shown). activated protein kinase kinase kinase. TAB1 is a specific activator RT-PCR with two different primer sets amplified the Nox1 for TAK1. Phosphorylation of TAK1 and TAB1 is a crucial event mRNA fragments in guinea pig LIEC and Caco2 (Fig. 2H), and for NF-␬B activation through the TLR and IL-1R signaling path- their nucleotide sequences were identical with the guinea pig and ways (23, 24). We confirmed that the treatment of T84 cells with human Nox1 mRNAs, respectively (data not shown). We also con- rFliC promptly phosphorylated TAK1 and TAB1 within 10 min firmed that guinea pig LIEC did not express the Nox2 nor Nox4 (Fig. 5F). mRNA (Fig. 2, I and J). ROS-dependent IL-8 release from T84 cells To address physiological roles of Nox1-derived ROS, we tested Expression of NADPH oxidase components in guinea pig LIEC Ϫ whether O2 production up-regulated IL-8 secretion from T84 We next screened the expression of Nox components. As shown in cells. Treatment of T84 cells with rFliC for 24 h increased IL-8 Fig. 3A, guinea pig LIEC expressed the p22phox, p67phox, and Rac1 release (Fig. 5G). Overproduction of p41nox and p51nox in T84 phox phox Ϫ transcripts, while the p47 , p40 , and Rac2 mRNAs were not cells enhanced O2 generation (Fig. 5, A–C), and consequently detected. Immunoblot analysis showed that they had p67phox and augmented the rFliC-stimulated IL-8 production, which was sig- p22phox proteins, but not p47phox and p40phox in line with the RT- nificantly cancelled by inclusion of SOD plus catalase (Fig. 5G). PCR results (Fig. 3B). Moreover, we have cloned the p41nox (Gen- Bank, AB105906) and p51nox (GenBank, AB105907) mRNAs ex- Induction of Nox1 protein with rFliC pressed in guinea pig LIEC (Fig. 3C). Guinea pig PBL also Finally, we studied the mechanisms by which rFliC increased expressed a small amount of p51nox mRNA, but not p41nox. Nox1 activity in T84 cells. rFliC did not change expression of the The Journal of Immunology 3055 Downloaded from http://www.jimmunol.org/

FIGURE 4. Construction of Nox1 activity in T84 cells. The levels of Nox1, p22phox, p67phox, p41nox, p51nox, Rac1, and GAPDH mRNAs in guinea pig LIEC, T84 cells, or Caco2 cells were estimated by RT-PCR analysis, as described in Materials and Methods (A). T84 cells were then transfected for 48 h

Ϫ by guest on September 27, 2021 with the indicated cDNA-containing vectors, and O2 release from the treated cells was determined in the presence or absence of 250 ng/ml PMA (B). The levels of expressed proteins were assessed by immunoblot analysis with Ab against hemagglutinin or p67phox, and results are shown in the lower panels. .p Ͻ 0.01, ANOVA and Scheffe´’s test ,ء .(Similar results were obtained in three separate experiments. Values are means Ϯ SD (n ϭ 8 p67phox, p41nox, and p51nox mRNAs (data not shown), but protein was absent in normal gastric and small intestinal mucosal RT-PCR suggested that treatment with rFliC was likely to stimu- tissues of both guinea pigs and humans (data not shown). The late the Nox1 mRNA expression (Fig. 6A). The immunoblotting expression of Nox1 in primary cultures of guinea pig gastric mu- clearly demonstrated the rFliC-primed induction of Nox1 protein cosal cells is probably due to oxidative stress during the prepara- (Fig. 6B). The induction of Nox1 was associated with the increase tion and cultivation, because the induction of Nox1 is associated Ϫ ␬ in O2 generation when primed with rFliC in the presence or ab- with activation of a redox-sensitive transcription factor NF- B sence of PMA (Fig. 6C). Recent reports have shown that TLR5- (our unpublished observation). Quiescent guinea pig gastric pit expressing cells including T84 cells activate NF-␬B in response to cells (surface mucous cells) in primary culture produced small Ϫ Ͻ bacterial flagellin (25, 26). As shown in Fig. 6, a potent inhibitor amounts of O2 ( 10 nmol/mg protein/h), but once primed with ␬ Ϫ for NF- B (PDTC) significantly blocked the rFliC-induced in- H. pylori LPS, they increased O2 generation 10-fold in associa- Ϫ ␮ crease in O2 production at concentrations over 1 M (Fig. 6D), tion with the induction of Nox1 (13). In contrast, abundant Nox1 and the induction of Nox1 protein was also concomitantly ham- protein was constitutively expressed in surface mucous cells of the pered by PDTC in a dose-dependent manner (Fig. 6E). guinea pig colon, and these cells in primary culture spontaneously Ϫ ϳ secreted O2 at a higher rate ( 160 nmol/mg protein/h), suggest- Discussion ing that Nox1 in the guinea pig colon is constitutively active. In The Nox1 mRNA is dominantly expressed in the colon (6). Intra- fact, possible activators, such as IL-1␤, TNF-␣, epidermal growth cellular regions of the Nox family are composed of highly con- factor, TGF-␤, IFN-␥, or bacterial components, did not further Ϫ served domains; therefore, specific Ab for Nox1 had not been up-regulate the O2 generation (data not shown). available. We have developed polyclonal Abs against human Nox1 In contrast to primary cultures of guinea pig LIEC, human colon useful for immunoblot and immunohistochemical analyses, and cancer cell lines (T84 and Caco2 cells) produced only small Ϫ Ͻ found that Nox1 protein was constitutively expressed in surface amounts of O2 ( 2 nmol/mg protein/h). RT-PCR analysis mucous cells of the guinea pig colon, supporting an in situ hybrid- roughly estimated that primarily cultured LIEC appeared to ex- ization study showing that the Nox1 transcript was expressed in press larger amounts of the Nox1 mRNA than the cell lines. More- epithelial cells of human colonic mucosa (27). A small amount of over, freshly isolated and cultured guinea pig LIEC constitutively Nox1 mRNA is detectable in primary cultures of guinea pig gastric expressed p67phox, its homologue p51nox, and a p47phox homo- mucosal cells even in LPS-free conditions (12, 14), while Nox1 logue p41nox, but they were absent or poorly expressed in T84 and 3056 Nox1 IN LIEC Downloaded from http://www.jimmunol.org/

nox nox by guest on September 27, 2021 FIGURE 5. Up-regulation of O2 production from T84 cells by rFliC. T84 cells cotransfected with p51 and p41 vectors were treated with native rFliC (5 ␮g/ml), boiled rFliC (5 ␮g/ml), trypsin-digested rFliC (5 ␮g/ml), LPS (5 ␮g/ml), peptidoglycan (10 ␮g/ml), or CpG DNA (50 ␮g/ml) (A). The T84 cells were incubated with different concentrations of rFliC for 24 h (B) or treated with 5 ␮g/ml rFliC for the indicated times (C) to measure spontaneous Ͻ ء Ϫ Ϯ ϭ O2 release. Values are means SD (n 6). , p 0.01, as compared with untreated cells (ANOVA and Scheffe´’s test). The TLR5 mRNA levels in T84 cells as well as human PBL were measured by RT-PCR analysis (D). Membrane (m.), cytosolic (c.), or whole cell (w.) fractions were prepared from T84 cells and subjected to immunoblot analysis with an anti-TLR5 Ab using human PBL as a control (E). After treatment of T84 cells with rFliC (5 ␮g/ml) for the indicated times, whole cell proteins were prepared and subjected to immunoblot analysis using anti-TAK1 Ab or anti-TAB1 Ab. p-TAK1, phosphorylated TAK1; p-TAB1, phosphorylated TAB1 (F). Similar results were obtained in three separate experiments. T84 cells were transfected with mock or p51nox plus p41nox vectors were primed with rFliC (5 ␮g/ml) for 24 h in the absence or presence of 200 U/ml SOD plus 700 U/ml catalase. Amounts of IL-8 production from these cells were determined in three separate experiments (G), as described in Materials and Methods. The values are expressed as pmol/ml/mg protein (means Ϯ SD, n ϭ 9). #, p Ͻ 0.01, ANOVA and Scheffe´’s test.

Caco2 cells. Mouse and human p41nox lack the regulatory domain may have other roles besides mitogenic properties. We examined corresponding to the aa 286–340 of human p47phox (16–18), whether Nox1-derived ROS exhibited bactericidal activities. For which may explain why Nox1 of guinea pig LIEC was in a self- this purpose, 2 ϫ 107 or 2 ϫ 108 CFU/ml of S. enteritidis was cocul- Ϫ activated status to generate O2 without any stimulants. When the tured with guinea pig LIEC for different incubation times (up to 8 h), nox Ϫ p41 was transfected, T84 cells augmented O2 production in and the bacterial growth was estimated by colony counts grown on response to PMA. T84 cells expressed a low level of the p51nox tryptic soy agar for12 h. Guinea pig LIEC did not affect the growth nox Ϫ mRNA, and overproduction of p51 further increased O2 -gen- rate of bacteria, and inclusion of SOD and catalase in the culture erating capability, but transfection of the p67phox was not effective medium also did not change the growth (data not shown). (Fig. 4B). P51nox lacks putative domains to interact with p40phox Surface mucous cells serve a primary protective role against (15–18), and colonic epithelial cells did not have p40phox. Based irritants by providing mucous coat. Recently, these cells have been on these findings, p51nox, rather than p67phox, may be a physio- shown to play an important role in host defense as well, producing logical partner with Nox1 and p41nox in catalyzing an electron proinflammatory mediators after the interaction with pathogenic transfer from NADPH to molecular oxygen. microbes (28). In fact, stimulation of TLR4 in guinea pig gastric At present, physiological roles of colonic Nox1 are not fully mucosal cells by H. pylori LPS activated NF-␬B within 30 min, understood. The finding that transfected Nox1 confers mitogenic followed by up-regulation of Nox1 activity within 8 h (11, 14). properties on NIH 3T3 cells bore the scenario that Nox1 may be Enhanced production of ROS further augmented NF-␬B activa- involved in the process of cell transformation (6). We demon- tion, leading to prolonged expression of the TNF-␣ and cyclo- strated that the terminally differentiated surface mucous cells in the oxygenase II mRNAs (11, 12). T84 cells specifically responded to colon constitutively expressed Nox1 protein, suggesting that Nox1 rFliC and induced Nox1, although they were insensitive to LPS, The Journal of Immunology 3057

FIGURE 6. Induction of Nox1 in T84 cells with rFliC. After T84 cells cotransfected with the p41nox and p51nox vectors were treated with rFliC (5 ␮g/ml) for the indicated times, total RNA and membrane fraction were prepared, and the Nox1 mRNA (A) and Nox1 protein (B) amounts were measured by RT-PCR using a primer set speciﬁc for Nox1-A and immunoblot analysis with anti-Nox1-C2 Ab, respectively. T84 cells were primed with or with- ␮ Ϫ out rFliC (5 g/ml) for 24 h, and their O2 production was determined in the presence or absence of 200 ng/ml PMA (C). T84 cells were pretreated with different concentrations of PDTC for 30 min and then in- Downloaded from cubated with rFliC (5 ␮g/ml) for 24 h. Ϫ PMA-stimulated O2 generation (D) and levels of Nox1 protein (E) were assessed. p Ͻ ,ء .(Values are means Ϯ SD (n ϭ 6 0.01, ANOVA and Scheffe´’s test. http://www.jimmunol.org/

CpG DNA, and peptidoglycan. Abreu et al. (29, 30) have also The present study clearly demonstrated that p41nox and p51nox, demonstrated that T84 and Caco2 cells are broadly unresponsive to novel p47phox and p67phox homologues, respectively, are essential TLR2 and TLR4 signalings. When T84 cells transfected with both components for Nox1 in achieving a potent oxidase activity. Ad- p41nox and p51nox vectors were primed with rFliC, they increased ditionally, the Nox1 may constitute early responses in epithelial by guest on September 27, 2021 Ϫ O2 -producing ability to higher than 5-fold of the vector alone cells against pathogens for the host defense. Ϫ control (Fig. 6C). This enhanced O2 production by transfection of T84 cells with p41nox and p51nox cDNAs significantly enhanced References the rFliC-stimulated IL-8 release from the cells (Fig. 5G). These 1. Kohen, R., and A. Nyska. 2002. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. results suggest that the TLR5-mediated up-regulation of Nox1 ac- Toxicol. Pathol. 30:620. tivity may contribute innate immune response by enhancing in- 2. Rey, F. E., and P. J. Pagano. 2002. The reactive adventitia: fibroblast oxidase in flammatory responses of LIEC, rather than by directly killing vascular function. Arterioscler. Thromb. Vasc. Biol. 22:1962. 3. Torres, M. 2003. Mitogen-activated protein kinase pathways in redox signaling. pathogenic bacteria. Front. Biosci. 8:D369. It has been shown that intestinal epithelial cells, including T84 4. Babior, B. M. 1999. NADPH oxidase: an update. Blood 93:1464. and Caco2 cells, express TLR5, and bacterial flagellin stimulates 5. Lambeth, J. D., G. Cheng, R. S. Arnold, and W. A. Edens. 2000. Novel homologs of gp91phox. Trends Biochem. Sci. 25:459. the TLR5 signaling, leading to the activation of proinflammatory 6. Suh, Y. A., R. S. Arnold, B. Lassegue, J. Shi, X. Xu, D. Sorescu, A. B. Chung, signals, particularly NF-␬B pathway (25, 26). T84 cells are known K. K. Griendling, and J. D. Lambeth. 1999. Cell transformation by the superoxide-generating oxidase Mox1. Nature 401:79. to show highly polarized expression of TLR5 on the basolateral 7. Banfi, B., A. Maturana, S. Jaconi, S. Arnaudeau, T. Laforge, B. Sinha, E. Ligeti, surface (26, 31). Certain flagellated bacteria are capable of trans- N. Demaurex, and K. H. Krause. 2000. A mammalian Hϩ channel generated locating flagellin and stimulating TLR5 (31). Gastric pit cells were through alternative splicing of the NADPH oxidase homolog NOH-1. Science 287:138. sensitive to LPS (11, 13, 14), while T84 cells were not. This dif- 8. Cheng, G., Z. Cao, X. Xu, E. G. van Meir, and J. D. Lambeth. 2001. Homologs ference in the sensitivity may reflect physiological environments: of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene LIEC are always exposed to Gram-negative bacteria. Thus, surface 269:131. 9. Shiose, A., J. Kuroda, K. Tsuruya, M. Hirai, H. Hirakata, S. Naito, M. Hattori, mucous cells of the stomach and colon may use different TLR Y. Sakai, and H. Sumimoto. 2001. A novel superoxide-producing NAD(P)H members to recognize respective pathogenic microbes, activate oxidase in kidney. J. Biol. Chem. 276:1417. 10. Geiszt, M., J. B. Kopp, P. Varnai, and T. L. Leto. 2000. Identification of renox, Nox1, and finally produce defensive mediators. Rapid phosphor- an NAD(P)H oxidase in kidney. Proc. Natl. Acad. Sci. USA 97:8010. ylation of TAK1 and TAB1 with rFliC confirmed that it actually 11. Teshima, S., K. Rokutan, T. Nikawa, and K. Kishi. 1998. Guinea pig gastric stimulated TLR5 signaling. Stimulation of TLR5 is suggested to mucosal cells produce abundant superoxide anion through an NADPH oxidase- like system. Gastroenterology 115:1186. activate multiple signaling pathways (25, 26, 32). Transduction of 12. Teshima, S., H. Kutsumi, T. Kawahara, K. Kishi, and K. Rokutan. 2000. Regu- dominant-negative TAK1-adenovirus vector failed to inhibit lation of growth and apoptosis of cultured guinea pig gastric mucosal cells by rFliC-primed Nox1 induction and O Ϫ increase (data not shown). mitogen oxidase 1. Am. J. Physiol. 279:G1169. 2 13. Teshima, S., S. Tsunawaki, and K. Rokutan. 1999. Helicobacter pylori lipopoly- PDTC, however, significantly blocked both responses, which sug- saccharide enhances the expression of NADPH oxidase components in cultured gest an important role of a putative NF-␬B binding site at Ϫ253 bp guinea pig gastric mucosal cells. FEBS Lett. 452:243. Ј 14. Kawahara, T., S. Teshima, A. Oka, T. Sugiyama, K. Kishi, and K. Rokutan. 2001. in the human NOX1 5 -flank (GenBank, NT010552). Further stud- Type I Helicobacter pylori lipopolysaccharide stimulates Toll-like receptor 4 and ies are necessary to settle this issue. activates mitogen oxidase 1 in gastric pit cells. Infect. Immun. 69:4382. 3058 Nox1 IN LIEC

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