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Am J Physiol Gastrointest Liver Physiol 294: G808-G818, 2008. First published January 17, 2008; doi:10.1152/ajpgi.00362.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Flagellin is required for salmonella-induced expression of heat shock protein Hsp25 in intestinal epithelium

Elaine O. Petrof,1 Mark W. Musch,2 Mae Ciancio,2 Jun Sun,3 Michael E. Hobert,3 Erika C. Claud,4 Andrew Gewirtz,5 and Eugene B. Chang2

1Section of Infectious Disease and 2Martin Boyer Laboratories and IBD Research Center, Department of Medicine, 3Department of Pathology, and 4Section of Neonatology, Department of Pediatrics, The University of Chicago, Chicago, Illinois; and 5Department of Pathology, Emory University, Atlanta, Georgia

Submitted 9 August 2007 ; accepted in final form 16 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.

intestinal epithelial cells; oxidant stress; innate immunity; Toll-like receptors

FLAGELLIN, a 55-kDa bacterial protein found in flagellated and motile bacteria such as Salmonella, is one of many bacterial ligands recognized by the Toll-like receptors (TLRs) of the innate immune system, being recognized by TLR5. Flagellin is also thought to be a prominent antigen that incites a chronic inflammatory response in some patients with Crohn's disease (12).

In the intestine, TLR5 is expressed on the basolateral membranes of intestinal epithelial cells (4), most likely accounting for the relative insensitivity of these cells to the continuous and large exposure to flagellin on the luminal side. If, however, the basolateral membrane comes in contact with flagellin, an intense inflammatory response mediated by TLR5 is initiated (20). This may occur during situations when the intestinal cell or mucosal barrier is breached, either by translocation of flagellin across the epithelial cell monolayer as occurs with the pathogen Salmonella enterica Serovar Typhimurium (5) or through mucosal injury that exposes the intestinal basolateral membrane to the hostile environment of the gut luminal contents. The resulting inflammatory response forms a major line of innate immune defense against bacterial infection.

In the very hostile environment of the intestine, the induction of cytoprotective heat shock proteins can be important for protection of host cells against further injury (17). Presence of bacteria appears to upregulate expression of inducible heat shock proteins (1), and both TLR signaling and the induction of heat shock proteins by luminal bacteria are thought to play important roles in intestinal homeostasis (19). There are vast numbers of flagellated bacteria in the normal enteric flora of the intestine, and hence there are large quantities of flagellin circulating in the gut luminal contents, and yet the relative contribution and role of flagellin in intestinal homeostasis remains largely unknown. The purpose of this study was to investigate the relative contribution and role of flagellin in heat shock protein induction by measuring the ability of flagellin to induce a known cytoprotective heat shock protein, Hsp25.

The present study demonstrates that exposure of intestinal epithelial cells to flagellin induces Hsp25 expression in vitro in a time-dependent manner, an effect mediated by activation of p38 MAPK and Hsp25 gene transcription, and results in enhanced protection against oxidant-induced cellular injury. In vivo, using a mouse model of Salmonella colitis, flagellin also induces Hsp25 expression. Data from experiments utilizing infection of intestinal epithelial cells with live wild-type Salmonella, an aflagellate mutant strain, and a Salmonella mutant that produces flagellin but is nonmotile indicate that flagellin is necessary for the induction of Hsp25. The aflagellate strain lacking flagellin failed to elicit induction of Hsp25 both in vitro and in vivo. Together, these data suggest that the induction of Hsp25 expression by bacterial flagellin may play an important role in intestinal homeostasis and in protecting the gut against oxidant injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bacterial strains and growth conditions. Bacterial strains included wild-type S. enterica Serovar Typhimurium (American Type Culture Collection no. 14028s), S. fliC–/fljB–, a strain that lacks flagellin, and fliD, a strain that synthesizes flagellin but is nonmotile (7, 8). Nonagitated microaerophilic bacterial cultures were prepared by inoculation of 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture, followed by overnight incubation (~18 h) at 37°C, as previously described (15).

Purification of flagellin. Flagellin was purified from wild-type S. enterica Serovar Typhimurium as previously described (4). Briefly, Salmonella supernatants were filtered through 0.2-µm filters to remove all bacteria, then were concentrated with Amicon 30-kDa filters (Millipore, Billerica, MA) and boiled for 20 min. The pH of the flagellin concentrate was adjusted to pH 6.0, and the concentrate was mixed for 1 h at room temperature with 0.5 ml High S cation exchange support (pH 6.0; Bio-Rad Laboratories, Hercules, CA) and then polymyxin-B agarose (Sigma, St. Louis, MO) to remove LPS. Resins were then removed by centrifugation, the flagellin solution was diluted fivefold in 20 mM Tris, pH 8.0, and the diluted solution was applied to a 2-ml High Q anion-exchange column (Bio-Rad). The column was washed with 10 ml of 20 mM Tris, pH 8.0, at a flow rate of 1.5 ml/min. A linear gradient of NaCl (0.0–1.0 M, flow rate 1.5 ml/min) was applied to the column, and fractions were collected and analyzed for flagellin by Western blot analysis [performed with MAb 15D8 (IGEN International, Gaithersburg, MD) to Escherichia coli flagellin that cross-reacts with Salmonella typhimurium flagellin]. Fractions containing flagellin were pooled and assayed for bioactivity and concentration.

Tissue culture. Young adult mouse colon (YAMC) cells, a gift from Dr. R. Whitehead (Vanderbilt University, Nashville, TN) (27), were maintained under permissive conditions (33°C) in RPMI 1640 medium with 5% (vol/vol) fetal bovine serum, 5 U/ml murine IFN-{gamma} (GIBCO-BRL, Grand Island, NY), 50 µg/ml streptomycin, and 50 U/ml penicillin, supplemented with ITS+ Premix (BD Biosciences, Bedford, MA), and plated at confluence on fibrillar collagen-coated polyethylene Transwells (BD Falcon) or on 60-mm culture dishes. After 24 h of growth at 33°C to allow for cell attachment, the medium was replaced with IFN-{gamma}-free medium, and cells were moved to 37°C (nonpermissive conditions) for 24 h to allow the development of the differentiated intestinal epithelial cell phenotype for all experiments. Cells were treated with 100 ng/ml flagellin and were left for 16 h or for the times indicated before harvest.

The rat small intestinal cell line IEC-18 (ATCC no. CRL-1589) was grown in DMEM (high glucose, 4.5 g/l) containing 5% (vol/vol) fetal bovine serum, 0.1 U/ml insulin, 50 µg/ml streptomycin, and 50 U/ml penicillin. IEC-18 cells were used at or near confluence between passages 20 and 35. Cells were grown until confluence for use and were fed the day before each experiment. Heat-shock-positive controls were exposed to 42°C for 23 min and were allowed to recover at 37°C for 2 h before harvest.

For bacterial coculture experiments, IEC-18 cells were cocultured for 3 h (multiplicity of infection 300:1, i.e., 2.4 x 108 bacteria colony-forming units per well of cells) with either wild-type S. typhimurium, a Salmonella strain lacking flagellin that still possesses the ability to invade (fliC–/fljB–), a flagellated strain that is nonmotile (fliD), or E. coli (strain F18). Cells were then washed three times in PBS, media were replaced with fresh media containing 300 µg/ml gentamicin, and cells harvested for Western blot analysis.

Western blot analysis. Intestinal epithelial cells grown in culture were washed and then harvested by gentle scraping with a rubber policeman into ice-cold PBS, pelleted (14,000 g for 20 s), and then resuspended in lysis buffer [10 mM Tris, pH 7.4, 5 mM MgCl2, 50 U/ml DNAse and RNAse, plus complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN)] to disrupt the cells as previously described (25). Protein concentrations were determined from an aliquot by using the bicinchoninic acid procedure (23). After protein determination, 3x Laemmli stop buffer was added to the remainder and samples were heated to 75°C for 5 min, then stored at –80°C until use. Cells harvested from mouse tissue for Western blot analysis were processed as described under In vivo experiments and Salmonella treatment.

Samples were electrophoresed on a 12.5% SDS-PAGE gel (20 µg protein/lane) and then immediately transferred in 1x Towbin buffer onto PVDF membranes (Perkin-Elmer NEN, Boston, MA) as previously described (9) and were analyzed immediately. Membranes were blocked in 5% (wt/vol) nonfat milk in Tris-buffered saline (TBS)-Tween [150 mM NaCl, 5 mM KCl, 10 mM Tris, pH 7.4 with 0.01% (vol/vol) Tween 20] for 1 h at room temperature. Primary antibody was added to TBS-Tween, and blots were incubated overnight at 4°C. Primary antibodies used were either a polyclonal anti-Hsp25 anti-rabbit antibody (SPA801; Stressgen, Victoria, BC, Canada), a phospho-p65 antibody (Cell Signaling Technology, Beverly, MA); an I{kappa}B antibody (Santa Cruz Biotechnology), a TLR5 antibody (Axxora, San Diego, CA), a monoclonal anti-heat shock cognate (Hsc) 70 antibody (SPA 815, Stressgen), or a β-actin antibody (Sigma). Either Hsc70 or β-actin was used as a loading control for all blots. Membranes were washed in TBS-Tween and then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Labs, Fort Washington, PA) for 1 h at room temperature followed by washing in TBS-Tween and development with an enhanced chemiluminescence system reagent (Supersignal; Pierce, Rockford, IL) as per the manufacturer's instructions.

Treatment with small interfering RNA. To silence Hsp25 expression, IEC-18 cells were treated with a silencing oligonucleotide. A 25-mer Stealth oligonucleotide (Invitrogen, Carlsbad, CA) comprised of bases 366–390 of the rat Hsp25 mRNA sequence (Genbank M86389 [GenBank] ) or bases 388–412 of the human Hsp27 mRNA sequence (Genbank NM_001540 [GenBank] ) was complexed with Silentfect reagent (Bio-Rad). (Note: Hsp27 is the human homolog of rat Hsp25). The sequence of the human oligonucleotide for Hsp27 is not present in the rat Hsp25 sequence and was used as a negative control. Cells were treated with a final concentration of 20 nM oligonucleotide and 0.6 µl of Silentfect reagent per square centimeter. The oligonucleotides and Silentfect were individually diluted into Optimem medium (Invitrogen) and then mixed and allowed to complex for 15 min at room temperature. Medium was removed from the cells and was replaced with Optimem. The complexed double-stranded RNA oligoncucleotides/Silentfect reagent were added, and after 30 min, complete medium was added and cells were returned to the incubator overnight. A second application of oligonucleotide was applied at 24 h, and cells were used for experiments 24 h after the second introduction of oligonucleotide. Silencing of Hsp25 was monitored in all experiments by examining Hsp25 expression by Western blotting.

Treatment with actinomycin D. To determine whether transcription is required for flagellin induction of Hsp25, IEC-18 cells were treated for 2 h with the RNA polymerase II inhibitor actinomycin D (10 µg/ml), which was added directly to the culture medium. Flagellin was then added (100 ng/ml), both agents were removed after 2 h, and cells were kept in the 37°C-5% CO2 incubator for an additional 22 h. Cells were then harvested, and levels of Hsp25 protein were determined by Western blotting.

Luciferase assays. IEC-18 cells were plated in 60-mm culture dishes, and, when 50% confluent, cells were transfected for luciferase assays in four groups: 1) a promoterless firefly luciferase plasmid (pGL3, Promega, Madison, WI); 2 and 3) five copies of either the NF-{kappa}B response element or heat shock element attached to luciferase in the Mercury Reporter System (Clontech, Palo Alto, CA); and 4) a section of the mouse Hsp25 promoter (GenBank L07577 [GenBank] ) of bases –72 to 728 from the initiation ATG site cloned into pGL3. All cells were also transfected with a Renilla reporter gene under control of the thymidine kinase promoter to control for transfection efficiency. IEC-18 cells were transiently transfected with 3 µg of the respective firefly luciferase plasmid, along with 100 ng of the Renilla reporter plasmid using 30 µl of LT-1 transfection reagent (Mirus, Madison, WI) as per the manufacturer's directions. Twenty-four hours after transfection, monolayers were treated with flagellin (100 ng/ml) or heat shocked (42°C for 23 min), and cells were harvested 24 h later. With the use of the protocol provided with the dual-luciferase assay system (Promega), cells were harvested in lysis buffer provided, and firefly and Renilla luciferase activities were measured in a Berthold Lumat luminometer (Berthold, Oak Ridge, TN). Data are presented as relative light units of firefly luciferase (n = 4 for each group). No treatments altered the expression of Renilla luciferase (data not shown).

Real-time PCR. Real-time PCR was performed in an iCycler (Bio-Rad) using iQSYBR Green PCR supermix (Bio-Rad). Total RNA was extracted from IEC-18 cells treated with flagellin (100 ng/ml) for varying times or immediately after heat shock at 42°C for 23 min. RNA was extracted by using Trizol reagent (Invitrogen) as per the manufacturer's instructions and then extracted with acid phenol:chloroform once to remove residual DNA. Two micrograms of RNA were reverse transcribed with the Superscript II cDNA synthesis kit (Invitrogen) according to the manufacturer's directions. Primers used for PCR were designed by using MacVector software (Accelrys, San Diego, CA), and for rat Hsp25 this corresponded to bases 405–540 of the coding sequence (GenBank accession no. M86389) or bases 93–187 of the coding region of rat GAPDH (GenBank XM_216453). In all cases, Hsp expression levels were normalized to the GAPDH levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCR reactions were performed in triplicate, triplicates were averaged for both Hsp25 and GAPDH, and data were presented as {Delta}{Delta}Ct, where Ct is cycle threshold.

Cell-viability assay. IEC-18 cells were treated with either flagellin, MAPK inhibitors, or silencing RNA as described (see also figure legends), then loaded with 51Cr (50 µCi/ml; Sigma) for 60 min, washed, and incubated in media with differing concentrations of the oxidant monochloramine to induce epithelial cell damage. Medium was harvested after 60 min, and the 51Cr remaining in the cells was extracted with 1 N HNO3 for 4 h. 51Cr in the released and cellular fractions was counted by liquid scintillation spectroscopy. 51Cr released was calculated as released divided by released plus cellular remainder.

In vivo experiments and Salmonella treatment. In vivo experiments of Salmonella infection were performed by using a previously described streptomycin-pretreated mouse model (2). The protocol was approved by the University of Chicago Animal Care (IACUC) committee. Mice used were 6- to 7-wk-old specific-pathogen-free female C57BL/6 mice (Jackson Laboratories). Water and food were withdrawn 4 h before oral gavage, with 7.5 mg/mouse of streptomycin given as 75 µl of sterile solution or 75 µl of sterile water (control). Animals were then supplied with water and food ad libitum. As previously described (2), at 20 h after streptomycin treatment, water and food were withdrawn again for 4 h and the mice were gavaged with either 1 x 107 colony-forming units of wild-type S. enterica Serovar Typhimurium (50-µl suspension in HBSS), the flagellin-deficient mutant strain fliC–/fljB–, or with sterile HBSS. Six hours after gavage, mice were killed and intestinal tissue was harvested for analysis. Epithelial cells were collected by gently scraping the jejunum with a glass slide. Cells were sonicated in lysis buffer [1% Triton X-100 with (in mM) 150 NaCl, 10 Tris, pH 7.4, 1 EDTA, 1 EGTA, pH 8.0, and 0.2 sodium orthovanadate, plus protease inhibitor cocktail (Roche Diagnostics)], and the protein concentration was determined. Samples were then analyzed by Western blot analysis.

Immunohistochemistry. Experiments using the streptomycin-Salmonella model of colitis were performed as above with the wild-type S. typhimurium or the flagellin-deficient mutant strain fliC–/fljB–. Six hours after gavage, mice were killed and the cecum was harvested for analysis and fixed in formalin. Tissue was paraffin embedded and then cut into 4-µm-thick sections by microtome (Leica RM2235). The sections were deparaffinized at 56°C overnight three times with xylene for 20, 10, and 5 min each time and were then immersed in ethanol (two times with 100% and 95% ethanol and one time with 75% ethanol) for 5 min each. For antigen unmasking, slides were heated to 96°C in 10 mM sodium citrate buffer (pH 6.0) for 15 min. Slides were pretreated with 0.3% hydrogen peroxide for 30 min and then blocked with 5% BSA in PBS for 30 min at room temperature followed by overnight incubation with rabbit anti-Hsp25 polyclonal antibody (Stressgen no. SPA-801; 1:300 in 1% BSA ) at 4°C. Slides were washed, then incubated with polymer-horseradish peroxidase anti-rabbit (Dako no. K4003) for 30 min at room temperature. Positive staining was visualized with diaminobenzidine chromogen (Dako no. K3467), nuclei counterstain was performed in hematoxylin (Sigma no. HHS32), and slides were photographed (Zeiss Axiocam, model 412-312).

MAPK assays and MAPK inhibitors. For MAPK assays, IEC-18 cells were treated with 100 ng/ml of flagellin for varying times and then replaced with fresh media. Cells were then harvested immediately after treatment with flagellin for Western blot analysis (MAPK phosphorylation). MAPK assays were performed as previously described elsewhere (25). Antibodies used were specific for p38 MAPK (no. 9212, Cell Signaling), phospho-p38 MAPK (no. 9211S, Cell Signaling), p44/42 ERK MAPK (no. 9102, Cell Signaling), phospho-p44/42 ERK MAPK (no. 9101S), SAPK/JNK (no. 9252, Cell Signaling), and phospho-SAPK/JNK (no. 9251S, Cell Signaling). The phosphorylated form of the kinase corresponds to the activated form. As positive controls, 37.7 µM anisomycin (Alexis, San Diego, CA) was used for p38 and SAPK/JNK, and 100 nM phorbol 12-myristate 13-acetate, (Sigma) was used for ERK1/2. For the MAPK-inhibitor experiments, MAPK inhibitors were used to pretreat the intestinal epithelial cells for 2 h, then cells were treated with flagellin for 2 h, the medium was changed, and cells were harvested the next day for Hsp25 analysis. MAPK inhibitors (Alexis Biochemicals, Carlsbad, CA) used were the p38 inhibitor SB-203580 (20 µM), the ERK inhibitor PD-98059 (50 µM), and the JNK inhibitor SP-600125 (20 µM).

Statistical analysis. Densitometry values were determined by using NIH ImageJ software and are shown along with standard error (*P < 0.05, +P < 0.01, and ++P < 0.01 by ANOVA with Bonferroni correction).

For chromium-release viability assays, data are presented as means ± SE and, where appropriate, are compared with Student's t-test or ANOVA with Bonferroni correction. A value of P < 0.05 was accepted as a level of statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Flagellin induces Hsp25 in intestinal epithelial cells in a time-dependent and concentration-dependent manner. A number of bacterial cell components and metabolites, such as LPS and butyrate, have been shown to induce Hsp expression (10, 21), which is postulated to play an important role in intestinal homeostasis (19). However, one of the most abundant bacterial components, flagellin, has yet to be investigated for its ability to induce Hsp expression in intestinal epithelial cells. To determine whether purified bacterial flagellin would induce Hsp expression, a cell line derived from rat small intestine epithelial cells (IEC-18) was treated with flagellin for various time periods. This non-transformed, diploid cell line has the advantage of possessing low levels of Hsp expression during the basal state, making it ideal for studying induction of heat shock proteins. There was an increase in Hsp25 expression over time that began around 6 h in the IEC-18 cells in response to flagellin treatment (Fig. 1A). Expression of the constitutively expressed heat shock protein Hsc70, which serves as a loading control, remained relatively constant and is also shown. The induction of Hsp25 by flagellin treatment was concentration dependent, with a maximal effect observed at concentrations of 100 ng/ml as determined by densitometry of Hsp25 bands on Western blot (Fig. 1B).


Figure 1
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  		Fig. 1. Heat shock protein (Hsp) 25 expression is induced by purified Salmonella flagellin in intestinal epithelial cells. A: Western blot analysis of levels of Hsp25 in IEC-18 cells exposed to 100 ng/ml of purified flagellin and then harvested at times (h) indicated, demonstrating a time-dependent increase in Hsp25 expression. HS, heat shocked cells (positive control). Heat shock cognate 70 (Hsc70) serves as a loading control. Densitometry is shown at bottom. Results are representative of 3 separate experiments (*P < 0.05, +P < 0.01, and ++P < 0.001). B: Western blot analysis of levels of Hsp25 in IEC-18 cells following exposure to flagellin purified from Salmonella typhimurium at concentrations indicated. A concentration-dependent increase in Hsp25 expression can be seen. Hsc70 serves as a loading control. Densitometry is also shown. Results are representative of 3 separate experiments (*P < 0.05, +P < 0.01, and ++P < 0.001). C: Western blot analysis before (lane 1) and after treatment with 100 ng/ml flagellin (lanes 3 and 4), demonstrating that IEC-18 cells respond appropriately to flagellin by degrading I{kappa}B (inhibitor of NF-{kappa}B) and by activating NF-{kappa}B (p-p65, phosphorylated and activated subunit of NF-{kappa}B), in response to flagellin. Treatment with 50 ng/ml TNF-{alpha}, which serves as a positive control for activation of NF-{kappa}B pathway, is also shown (lane 2). IEC-18 cells also express Toll-like receptor 5 (TLR5), and this, along with actin (bottom), served as a loading control. For each panel, results shown are representative of 3 separate experiments.

 
The expression of TLR5 was confirmed by Western blot analysis (Fig. 1C). In addition, flagellin treatment resulted in increased expression of the activated, phosphorylated form of the p65 subunit of NF-{kappa}B and subsequent I{kappa}B-{alpha} degradation followed by resynthesis at 60 min after exposure, indicating the presence of functional TLR5 receptors.

Flagellin induces Hsp25 in intestinal epithelial cells in a polarized manner. One limitation of IEC-18 cells is that they do not form well-polarized monolayers. Because the TLR5 receptor is expressed on the basolateral side of intestinal epithelial cells (4), YAMC cells were grown on collagen-coated, semipermeable Transwells to form tight, polarized monolayers. Flagellin was then administered either from the apical side, the basolateral side, or both. After timed incubations, cells were harvested and analyzed for Hsp25 expression by Western blot analysis to determine whether the heat shock protein-inducing abilities of flagellin would exhibit a polarized response. Hsp25 induction was seen only in those samples where flagellin was used to treat the basolateral side (or both sides together), indicating that the induction requires administration to the basolateral side of the intestinal epithelial cell (Fig. 2A). This is consistent with the basolateral location of the TLR5 receptor. A time course of flagellin exposure to YAMC cells shows that the colonic YAMC cell line exhibits a time-dependent increase in Hsp25 expression on exposure to flagellin (Fig. 2B).


Figure 2
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  		Fig. 2. Induction of Hsp25 expression in polarized intestinal epithelial cell monolayers requires basolateral administration of flagellin, consistent with location of TLR5 receptor. A: young adult mouse colon (YAMC) cells were grown on collagen-coated Transwells to form polarized monolayers and then were either left untreated (Con) or were treated with 100 ng/ml of purified flagellin administered to either apical side, basolateral side, or both, as indicated. Heat shock protein expression was determined by Western blotting. Hsc70 serves as a loading control. Densitometry (means ± SE) is shown under representative Western blot. ANOVA was performed by using a Bonferroni correction comparing all groups to control (C; +P < 0.01). Results shown are representative of 3 separate experiments. B: Western blot analysis of levels of Hsp25 in YAMC cells following exposure to 100 ng/ml of purified flagellin from S. typhimurium for times (h) indicated, demonstrating a time-dependent increase in Hsp25 expression. HS cells were positive control. Hsc70 serves as a loading control. Densitometry values are presented in graph form (means ± SE). Results shown are representative of 3 separate experiments.

 
Flagellin induces transcription of Hsp25 RNA. To determine whether flagellin-associated Hsp25 induction was due to an effect on transcription, several approaches were used. First, the effect of flagellin treatment on RNA levels of Hsp25 was examined. IEC-18 cells were treated with flagellin and then harvested for RNA isolation. Using real-time PCR to examine induction of Hsp25 mRNA (Fig. 3A), it was determined that exposure to flagellin increased Hsp25 mRNA levels. Second, this effect was blocked by treatment with the RNA II polymerase inhibitor actinomycin D, also indicating that exposure to flagellin resulted in transcriptional upregulation of Hsp25 expression (Fig. 3B).


Figure 3
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  		Fig. 3. Flagellin induces Hsp25 mRNA expression, and this process is inhibited by actinomycin D, suggesting a transcriptional mechanism of action. A: real-time PCR of Hsp25 mRNA after flagellin treatment is shown. IEC-18 cells were either untreated (time 0), treated with 100 ng/ml of flagellin for times indicated, or heat shocked (HS), as described in MATERIALS AND METHODS. RNA was harvested and analyzed for Hsp25 as well as GAPDH by real-time PCR as described in MATERIALS AND METHODS. Data are means ± SE for 3 separate experiments. ANOVA with Bonferroni correction was used to compare all groups to 0 h with flagellin (*P < 0.05, ++P < 0.001). Induction of Hsp25 mRNA was normalized to GAPDH signal of each sample. B: Western blot analysis of control cells (C), or cells treated with flagellin (+F), either in absence or presence of RNA II polymerase inhibitor actinomycin D as described in MATERIALS AND METHODS. A treatment timeline is shown at bottom. Treatment with actinomycin D abolishes expression of Hsp25 normally induced by flagellin (100 ng/ml), suggesting a transcriptional mechanism is involved. Densitometry values are graphed along with standard error below representative Western blot (n = 3). All groups were compared with C without actinomycin by using ANOVA with Bonferroni correction (*P < 0.05). Hsc70 serves as a loading control. C: IEC-18 cells were transiently transfected with 3 µg of firefly luciferase plasmid, along with 100 ng of Renilla reporter plasmid to control for transfection efficiency, using LT-1 transfection agent, in 4 groups as follows: 1) a promoterless firefly luciferase plasmid (pGL3; Promega, Madison, WI), 2 and 3) 5 copies of either NF-{kappa}B response element or heat shock element on luciferase plasmid in Mercury Reporter System (Clontech, Palo Alto, CA), and 4) a section of mouse Hsp25 core promoter (GenBank L07577) of bases –72 to 728 from initiation ATG cloned into pGL3 luciferase plasmid. Twenty-four hours after transfection, monolayers were treated with flagellin (Flag, 100 ng/ml, black bars) or heat shocked (42°C for 23 min, gray bars) and then were harvested 24 h later. Luciferase and Renilla activity were determined by using dual-luciferase assay system (Promega) as per manufacturer's instructions. Data are presented as relative light units of firefly luciferase as means ± SE with n = 4 for each group (*P < 0.05, ++P < 0.001 by ANOVA using method of Bonferroni). No treatments altered expression Renilla luciferase (data not shown).

 
Third, since there are no NF-{kappa}B binding sites in the core promoter of the Hsp25 gene, luciferase reporter assays were used to further explore the role of flagellin in the transcriptional regulation of Hsp25 and to determine whether flagellin activates the core promoter of Hsp25. IEC-18 cells were transiently transfected with luciferase reporter plasmids, using the following: 1) a promoterless pGL3 firefly luciferase plasmid; 2 and 3) luciferase plasmid containing five copies of either the NF-{kappa}B response element or heat shock element, respectively; and 4) a luciferase plasmid containing the base core promoter of the Hsp25 gene cloned into pGL3. Cells were treated with 100 ng/ml flagellin or were heat shocked and then tested for luciferase activity as described in MATERIALS AND METHODS. Results are shown in Fig. 3C. Heat shock treatment resulted in greatly increased expression of the Hsp25 luciferase reporter gene, with a smaller but statistically significant fold increase in luciferase activity when treated with flagellin, indicating that flagellin treatment is able to activate the core promoter of Hsp25.

Flagellin induces intestinal epithelial cell Hsp25 expression through a p38 MAPK-mediated pathway. Flagellin has previously been shown to activate MAP kinases (28). In addition, MAP kinase activation by some nonflagellated bacteria results in induction of heat shock proteins in intestinal epithelial cells (25). Therefore, to further elucidate the mechanism behind Hsp25 induction by flagellin, the role of flagellin-mediated MAPK activation in the induction of Hsp25 expression was explored. It was first determined that flagellin treatment activated MAPK in IEC-18 intestinal epithelial cells (Fig. 4A), consistent with previous reports (28). In Fig. 4A, cells were collected after 15 min of flagellin treatment to examine the effects on MAPK activations (i.e., MAPK phosphorylation), which is an early and rapid event. Flagellin activated p38 and ERK1/2 (Fig. 4A) but had no effect on SAP/JNK. Next, IEC-18 cells were pretreated with MAPK inhibitors and then treated with flagellin. The effect of treatment with different MAPK inhibitors on the expression of Hsp25 was then examined by using Western blot analysis. Cells were harvested after 48 h of recovery for Hsp25 induction (Fig. 4B) because on the basis of time-course data this is how long it takes to see optimal induction of Hsp25 protein by flagellin (see Fig. 1 time course). A time line at the bottom of Fig. 4B is included to better illustrate the experimental design. Treatment with the inhibitors to SAP/JNK and ERK1/2 had no inhibitory effect on flagellin-mediated Hsp25 induction. In contrast, the p38 MAPK inhibitor SB-203580 blocked the induction of Hsp25 expression by flagellin, indicating that Hsp25 induction by flagellin requires activation of the MAPK p38 (Fig. 4B).


Figure 4
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  		Fig. 4. Flagellin activates MAPK, and Hsp25 induction requires activation of MAPK p38. A: IEC-18 cells were treated with 100 ng/ml S. typhimurium flagellin for times as shown, and then MAPK activation was determined by Western blotting with antibodies against total and activated ERK1/2, p38, or SAPK/JNK. MAPK assays were performed by Western blot analysis as previously described elsewhere (25). Phosphorylated form of kinase corresponds to activated form. As positive controls, 37.7 µM anisomycin (Aniso) was used for p38 and SAPK/JNK, and 100 nM phorbol 12-myristate 13-acetate (PMA) was used for ERK1/2 activation. Image shown is representative of 3 separate experiments. B: MAPK inhibitors were used to pretreat IEC-18 cells for 2 h, then cells were treated with 100 ng/ml flagellin for 2 h, media were changed, and cells were allowed to recover and were harvested next day for analysis of Hsp25 expression by Western blot as described in MATERIALS AND METHODS. For clarity, a treatment timeline is shown. MAPK inhibitors used were p38 inhibitor SB-203580 (20 µM), ERK inhibitor PD-98059 (50 µM), and JNK inhibitor SP-600125 (20 µM). Induction of Hsp25 in IEC-18 cells by flagellin requires activation of MAPK p38. Hsc70 serves as a loading control. Image shown is representative of 3 separate experiments. Densitometry is as shown comparing control to flagellin (1; +P < 0.01) and comparing last 4 columns to each other by ANOVA (2); only p38 inhibitor SB-203580 had an effect on flagellin-mediated Hsp25 expression (*P < 0.05).

 
Flagellin treatment of intestinal epithelial cells provides protection against oxidant stress, mediated through heat shock protein induction, and is dependent on p38 MAPK activation. Given that one of the well-described functions of inducible heat shock proteins in intestinal epithelial cells is to protect against oxidant stress and to preserve cytoskeleton integrity, the effect of flagellin-mediated Hsp25 induction on the effects of oxidant injury was investigated. Cells were pretreated with flagellin, and then a well-established model of oxidant injury that uses monochloramine treatment followed by a chromium-release viability assay was used to determine whether the induction of Hsp25 by flagellin would provide protection against oxidant injury.

Flagellin conferred a statistically significant protective effect over one log range of monochloramine concentrations (from 0.03 to 0.3 mM, Fig. 5A). To determine whether this protective effect was specific to Hsp25, small interfering RNAs (siRNAs) against Hsp25 were used to selectively inhibit expression of Hsp25 in IEC-18 cells, and then cells were exposed to oxidant injury and tested for viability by using chromium-release assays as described above. An siRNA sequence against the human isoform (Hsp27), which does not recognize the rat Hsp25 isoform, was used as a negative control. Those cells treated with the human Hsp27 siRNA exhibited no loss of viability, whereas the protective effect of flagellin treatment was decreased in those cells treated with the Hsp25 siRNA, indicating that flagellin-mediated protection against oxidant stress is at least partially mediated through flagellin-induced Hsp25 upregulation (Fig. 5B). Inhibition of Hsp25 expression by siRNA treatment was confirmed by Western blot analysis (Fig. 5B).


Figure 5
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  		Fig. 5. Flagellin protects intestinal epithelial cells against oxidant (NH2Cl)-induced injury. A: IEC-18 cells were untreated (open bars) or treated with 100 ng/ml purified flagellin (black bars) followed by removal and recovery overnight. Cells were labeled with 51Cr for 60 min and were treated with increasing concentrations of NH2Cl, as shown. NH2Cl-induced release of Cr was then measured as described in MATERIALS AND METHODS. Data are means ± SE for 3 separate experiments, and each point was determined in duplicate (*P < 0.05 as determined by Student's t-test, flagellin vs. untreated at each concentration). B: small interfering RNA (siRNA) was used to knock down Hsp25 expression as described in MATERIALS AND METHODS by using rat sequence rHsp25 (middle columns) and human oligonucleotide hHsp27 for Hsp27 (right columns), which is not present in rat Hsp25 sequence and serves as a negative siRNA control. IEC-18 cells were then treated with 100 ng/ml flagellin a day before chromium loading and injury with 0.6 mM NH2Cl as above. Data are means ± SE for 3 separate experiments; in each experiment, each point was determined in duplicate (*P < 0.05, ++P < 0.001 by ANOVA using method of Bonferroni). Western blot analysis was performed to confirm silencing of Hsp25 expression, and this is also shown. C: MAPK inhibitors [ERK inhibitor PD-98059 (50 µM), p38 inhibitor SB-203580 (20 µM), JNK inhibitor SP-600125 (20 µM)] were used to pretreat cells, which were then treated with 100 ng/ml flagellin, then media were changed and cells were left to recover overnight. Cells were labeled with 51Cr for 60 min and were treated with 0.6 mM NH2Cl for 60 min to induce oxidant injury. Data are means ± SE for 3 separate experiments; in each experiment, each point was determined in duplicate (*P < 0.05, ++P < 0.001 by ANOVA using method of Bonferroni).

 
The use of MAPK inhibitors had suggested that the induction of Hsp25 depended on p38 activation, and so one would predict that blockade of p38 function should result in loss of protection against oxidant stress. To test this hypothesis, cells were treated with MAPK inhibitors plus flagellin before monochloramine insult, to determine whether the protective effect against monochloramine treatment required the activation of p38 MAPK (Fig. 5C). Consistent with this hypothesis, treatment with the inhibitor against p38 MAPK, but not the ERK1/2 or SAP/JNK inhibitor, resulted in loss of protection against oxidant injury.

Flagellin is a major heat shock protein-inducing effector and is required for heat shock protein induction by live Salmonella both in vitro and in vivo. To determine whether the same phenomenon of flagellin-mediated heat shock protein induction would hold true in the physiological setting of live bacterial exposure, intestinal epithelial cells were cocultured with either a wild-type S. typhimurium strain or with fliC–/fljB–, a flagellin-deficient mutant strain that is still able to invade. Aflagellate mutants are nonmotile, so differences in Hsp25 induction by the flagellin-deficient strain in this experiment could potentially be due to impaired motility and consequent decreased contact between the bacteria and the intestinal epithelial cells. To control for this possibility, the nonmotile strain fliD, which still expresses flagellin, was included in the experimental design. The fliC–/fljB– flagellin-deficient mutant strain induced much less Hsp25 than the wild-type strain (Fig. 6). Comparison of the fliC–/fljB– flagellin-deficient mutant strain with the non-motile fliD strain by densitometric analysis indicated that lack of motility alone was not enough to explain the decrease in Hsp25 induction observed with the fliC–/fljB– flagellin-deficient strain (Fig. 6). E. coli is also shown.


Figure 6
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  		Fig. 6. Flagellin is major Hsp25-inducing effector during Salmonella infection in vitro. IEC-18 cells were cocultured for 3 h (multiplicity of infection 300:1, i.e., 2.4 x 108 bacteria colony-forming units per well of cells) with either wild-type S. typhimurium (wt), a Salmonella strain lacking flagellin that still possesses ability to invade (fliC–/fliB–; C-B-), a flagellated strain that is nonmotile (fliD), or E. coli (strain F18). Cells were then washed 3 times in PBS, media were replaced with fresh media containing 300 µg/ml gentamicin, and cells were harvested for Western blot analysis as previously described (9). Results shown are representative of 3 separate experiments (+P < 0.01 compared with no stimulation).

 
The effects of live Salmonella on Hsp25 expression in intestinal epithelium were then investigated in a live-animal model. To determine the contribution of flagellin to the induction of intestinal epithelial Hsp25 in vivo, the streptomycin animal model of Salmonella infection was used (2). Mice are intrinsically resistant to developing enterocolitis from Salmonella infection, but this model allows development of a murine intestinal salmonellosis that closely resembles the enterocolitis caused by S. typhimurium in humans. Mice were infected with either a wild-type S. typhimurium strain or with fliC–/fljB–, the flagellin-deficient mutant strain that is still able to invade. Tissue was then harvested after 6 h and was examined for changes in Hsp25 expression by Western blot analysis (Fig. 7A) or by immunohistochemistry staining for Hsp25 expression (Fig. 7B). The wild-type strain of Salmonella induced expression of Hsp25, whereas the aflagellate strain fliC–/fljB– did not cause an increase in Hsp25 expression. These data indicate that, in this animal model of Salmonella colitis, the bacterial protein flagellin is responsible for the majority of intestinal Hsp25 induction in vivo.


Figure 7
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  		Fig. 7. Flagellin is a major Hsp25-inducing effector during Salmonella infection in vivo. A: normal C57BL/6 mice were pretreated with streptomycin as described in MATERIALS AND METHODS and then gavaged with either wild-type (WT) S. typhimurium or fliC–/fljB– (C-B-), a Salmonella strain lacking flagellin that still possesses LPS and ability to invade. Mice were killed after 6 h as shown. Small intestine (jejunum) epithelial cells were harvested, and Western blot analysis for Hsp25 was performed. fliC–/fljB– strain of Salmonella was unable to induce Hsp25 in mouse small intestine. Hsc70 served as loading control. Densitometry is as shown and was normalized to Hsc70 (++P < 0.001 compared with no stimulation). Results shown are representative of 3 separate experiments. B: mice were pretreated with streptomycin and then infected with Salmonella WT or fliC–/fljB– as described. Tissues were harvested at 6 h, and cecum was stained for Hsp25 expression. Hsp25 expression was increased in cecum of WT-treated animals (c), compared with fliC–/fljB– (b) or untreated controls (a). Results shown are representative of 3 separate animals for each treatment group.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 
Cross-talk between the intestinal mucosa and the resident microbial flora is a critical and fascinating aspect of intestinal biology. Studies have shown that bacteria are crucial for the maintenance of intestinal homeostasis (11, 19). This study demonstrates that the bacterial product flagellin may also contribute to intestinal homeostasis: flagellin induces expression of the cytoprotective protein Hsp25, which helps protect intestinal epithelial cells against oxidant damage.

Flagellin, a highly conserved molecule expressed by all motile bacteria, induces cytoprotective heat shock proteins in intestinal epithelial cells over time in a concentration-dependent fashion. This effect was observed in different intestinal epithelial cell lines derived from both small (IEC-18) and large (YAMC) intestinal epithelial cells, demonstrating that the effect is not species or cell-line specific. Hsp25 induction by flagellin was markedly more prominent when administered to the basolateral side of YAMC cells (grown on collagen-coated transwells to form polarized monolayers), as opposed to the apical side. This is consistent with previous reports describing the location of the TLR5 receptor on the basolateral side of intestinal epithelial cells, as well as reports describing TLR5-mediated activation of p38 MAPK (28), suggesting that the effect may be mediated through TLR5. Interestingly, disruption of TLR signaling (as demonstrated with TLR2, TLR4, and MyD88 knockout mice) results in increased susceptibility of the intestine to injury, partially through the loss of expression of important cytoprotective factors such as heat shock proteins (19). The current study further shows that the mechanism of Hsp25 induction by flagellin involves transcriptional activation of Hsp25 expression and activation of the MAPK p38. The luciferase-reporter experiments indicate that flagellin is able to activate the core promoter of the Hsp25 gene, but it should be recognized that other factors likely play a role in the regulation of Hsp25. For example, although there are no NF-{kappa}B consensus binding sites in this core promoter region, it is possible that NF-{kappa}B is able to bind outside of this core promoter region or that effects of NF-{kappa}B on other proteins may play a contributing role in the regulation of Hsp25. These possibilities remain to be explored.

One of the well-established functions of inducible heat shock proteins in intestinal epithelial cells is to protect against oxidant injury, and so it was hypothesized that one of the functions of flagellin-mediated heat shock protein induction may be to preserve cytoskeleton integrity and protect against oxidant damage. Pretreatment of epithelial cells with flagellin resulted in protection against oxidant injury, as demonstrated by improved cellular viability. In addition, it was again determined that p38 MAPK played an important role, this time in the mechanism of flagellin-mediated protection against oxidant stress. Although these in vitro studies using MAPK inhibitors point to p38 as being a key player in cytoprotection, it should be noted that the activation of NF-{kappa}B via TLR signaling plays a critical role in maintaining mucosal integrity in the intestinal tract, and therefore it is possible that, in vivo, some of the cytoprotective effect of flagellin may also be contributed by this signaling pathway.

To determine whether these in vitro observations made using purified flagellin in epithelial cell lines could be extended to live bacteria, an in vitro coculture model and an in vivo mouse model of S. typhimurium infection were used (2). In the cell-culture model, IEC-18 intestinal epithelial cells were cocultured with either wild-type Salmonella or the Salmonella strain fliC–/fljB–, which still possesses the necessary cellular machinery to invade but lacks flagellin expression (29). A previous report had suggested that decreased motility, rather than a lack of TLR5 activation, was the basis for why intestinal epithelial cells displayed less response to the fliC–/fljB– aflagellate strain compared with a wild-type strain of Salmonella (24). Therefore, the fliD Salmonella strain (which possesses flagellin but is nonmotile) was included as an additional control. By Western blot analysis, Hsp25 expression in IEC-18 cells infected with live Salmonella was determined to be due primarily to flagellin, as the fliC–/fljB– strain lacking flagellin did not induce as much Hsp25 as the wild-type strain, and the fliD control (which has flagellin but is nonmotile) was still able to induce Hsp25 expression. Interestingly, E. coli F18, which possesses flagellin, did not induce appreciable Hsp25 expression. This observation suggests that flagellin was not delivered effectively to the basolateral membrane to activate Hsp25 expression, which may be related to previous reports indicating that IEC-18 cells do possess some degree of polarity (3, 13). This would also be consistent with another report that apical E. coli flagellin from noninvasive commensal bacteria is unable to activate epithelial gene expression when added apically to intestinal epithelial cells (4).

To further explore the in vitro findings in an in vivo model of bacterial infection, mice were pretreated with streptomycin and then gavaged with either wild-type Salmonella or the aflagellate Salmonella strain (fliC–/fljB–). Again, in contrast to what was observed with the fliC–/fljB– strain lacking flagellin, increased Hsp25 expression was observed in animals infected with live wild-type Salmonella, both by Western blot analysis and by immunohistochemistry.

In small intestine, there is normally very low basal expression of inducible heat shock protein, and blind-loop studies have shown that heat shock protein expression increases in the presence of bacteria (1). When blind loops crafted from small intestine are surgically attached to the colon so that they self-empty with peristalsis into the colon, they do not become colonized with bacteria and heat shock protein expression remains low. If, however, the intestinal loops are surgically attached so that they "self-fill" with peristalsis and become colonized with bacteria, then expression of inducible Hsp25 increases (1). Flagellin is not the only bacterial product to induce heat shock protein production. Probiotic bacteria derived from normal commensal gut flora also induce cytoprotective heat shock proteins that protect intestinal epithelial cells against oxidant injury and stress (18, 25). Because Lactobacillus probiotic bacteria are nonmotile and do not contain flagellin, yet they are strong inducers of Hsp25 expression in intestinal epithelial cells, other factors must be present in these bacteria that are able to induce Hsp25 expression. Another strong inducer of Hsp25 is SEB, the enterotoxin B from the pathogen Staphylococcus aureus, which has been shown to induce heat shock protein expression in intestinal epithelial cells both in vitro and in vivo. This heat shock protein induction provides protection against oxidant stress and F-actin depolymerization (16). These observations, in conjunction with the observation that loss of inducible heat shock protein expression has an adverse effect on the ability of intestinal epithelial cells to survive under hostile conditions and in stressful environments (22, 26), provide further evidence that bacteria-induced expression of inducible heat shock proteins may serve a beneficial role in the gut. It should be acknowledged that direct evidence for the in vivo physiological role of Hsp25 in the intestinal tract still remains to be confirmed. The development of an Hsp25 transgenic mouse is currently underway to address the issue of the true physiological role of Hsp25 in the gut in vivo.

It is tempting to speculate that induction of Hsp25 is a highly conserved response of the intestinal epithelium that occurs in reaction to exposure to bacterial signals in the lumen, particularly in reaction to exposure to bacterial signals on the basolateral side of enterocytes, and which may be protective against oxidant injury. For example, while beneficial in killing bacterial pathogens, the physiological oxidant monochloramine, which is normally produced when hypochlorous acid released from innate immune cells reacts with ammonia, has potent direct antimicrobial activity but also affects epithelial cells by causing cytoskeletal disruption and eventual cell death through oxidative-mediated injury (6). The results of this study show that intestinal epithelial cells respond to flagellin treatment by the production of inducible heat shock proteins that provide a degree of cytoprotection against oxidative stress from monochloramine. Exposure to flagellin may send signals simultaneously to the cell to increase heat shock protein expression in anticipation, to protect itself from oxidative damage while at the same time allowing massive production of oxidants, with the intention of killing off the invading pathogen, to proceed.

In vivo, Salmonella uses its type-three secretory system needle complex to inject bacterial effector proteins into the host cytoplasm. These bacterial effector proteins cause loss of apical microvilli and membrane ruffling through the local disruption and reorganization of the actin cytoskeleton immediately beneath the invading Salmonella. Heat shock protein induction, particularly Hsp25, which binds to actin filaments, may help block invasion through stabilization of the actin cytoskeleton. In this situation, upregulation of inducible heat shock proteins likely serves as a protective mechanism for the epithelial cell. Thermal stress and butyrate, both known to be strong inducers of inducible heat shock proteins, reduce IL-8 release in intestinal epithelial cells and decrease the inflammatory response to Salmonella (14). Salmonella invasion, or even breach of the epithelial barrier by flagellin-possessing enteric flora, may be interpreted by the cell as a hostile condition. In this situation, upregulation of inducible heat shock proteins likely serves as a protective mechanism for the epithelial cell. Perhaps flagellin-induced expression of Hsp25 may actually protect cells from being overwhelmed by invading Salmonella and provide the host sufficient time to mount an effective response.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Digestive Disease Research Core Center Grant DK-42086. E. O. Petrof is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant K08-DK-64840, and E. C. Claud is supported by National Institute of Child Health and Human Development Grant K08-HD-43839. E. B. Chang is supported by the Crohn's and Colitis Foundation of America and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47722.


   ACKNOWLEDGMENTS
 
We thank Dr. R. H. Whitehead for his generous gift of YAMC cells used in this study. We also thank K. Drabik, V. Vulakaite, T. Waypa, T. Abramova, and Y. Guo for technical assistance.

Present address of J. Sun: Department of Gastroenterology and Hepatology, University of Rochester, Rochester, NY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. B. Chang, Dept. of Medicine, The Univ. of Chicago, 5841 S. Maryland Ave., MC6084, Chicago, IL 60637 (e-mail: echang{at}medicine.bsd.uchicago.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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