Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis
Joy Carmelina Indira Singh,1
Sheena Margaret Cruickshank,1
Darren James Newton,1
Louise Wakenshaw,1
Anne Graham,2
Jinggang Lan,1
Jeremy Peter Alan Lodge,3
Peter John Felsburg,4 and
Simon Richard Carding1
1School of Biochemistry and Microbiology, The University of Leeds, Leeds; 2Department of Biomedical Sciences, University of Bradford, Bradford; 3 Departments of General Surgery, Medicine, and Anaesthesia, School of Medicine, The University of Leeds, Leeds, United Kingdom; 4Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania
Submitted 17 August 2004
; accepted in final form 10 October 2004
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ABSTRACT
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The interleukin-2-deficient (IL-2/) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2/ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2/ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-
B (p65) activation with the inhibitory p50 form of NF-
B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.
ulcerative colitis; colon
IN ADDITION TO THEIR BARRIER function, the epithelial cells that line the gastrointestinal tract play a role in sensing the external environment and communicating this information to the local immune system to effect appropriate responses (19). Disruptions or breakdowns in the signaling function of intestinal epithelial cells (IEC) are thought to be important in the development of the inflammatory bowel diseases (IBD), ulcerative colitis (UC), and Crohn's disease (20).
IEC recognition of microorganisms is based on recognition of conserved signature molecules in microorganisms called microbe (or pathogen)-associated molecular patterns (MAMP) (3, 4, 19). MAMP are shared by large groups of microorganisms and include peptidoglycans (PGN) found in most bacteria, lipopolysaccharides (LPS) of gram-negative bacteria, and lipoteichoic acids (LTA) of gram-positive bacteria. Because different microbes express distinct profiles of MAMP, recognition of the motifs informs the host about the nature of surrounding bacteria. Pattern recognition receptor (PRR) families mediate the response of immune cells to MAMP of which two main types have been identified to date. The first are the Toll-like receptor (TLR) family that share homology with the anti-fungal extracellular receptor Toll, first identified in Drosophila (4). The TLR family includes TLR4 and TLR2, which recognize LPS, PGN, and LTA, respectively. In addition to protecting the host from infection, a recent study (48) identified an additional role for TLRs in intestinal epithelial homeostasis and protection from injury. The second family of PRR consists of the cytosolic nucleotide binding site plus leucine-rich repeat proteins (29) that are related to plant disease resistance-like proteins. In mammalian cells, these include Nod1/CARD4 and Nod2/CARD15 (18). Genetic mutations in Nod2/CARD15 have been identified within groups of patients with Crohn's disease (27), and one mutation specifically deletes the terminal leucine-rich repeat proteins responsible for defective recognition of intracellular bacteria (25). Although the mechanism(s) by which mutations in NOD2/CARD15 cause Crohn's disease is not clear, NOD2/CARD15-deficient mice exhibit increased TLR2-mediated responsiveness and excessive Th1 CD4 T cell responses, consistent with NOD2/CARD15 being an anti-inflammatory and a negative regulator of TLR2 responses (65).
Intracellular signaling pathways downstream of these PRR have principally been mapped by using hematopoietic cell lines and ultimately result in NF-
B p65 activation, leading to the production of the antimicrobial factors cytokines and chemokines (3, 9, 30). Two divergent pathways have been identified (8). In the first, stimulation of PRR by MAMP leads to the recruitment of the adaptor molecule, myeloid differentiation factor 88 (MyD88), which binds to the conserved Toll/IL-1 receptor (TIR) domain (40), enabling phosphorylation of serine kinase IL1-receptor-associated kinase (IRAK), which in turn leads to recruitment and activation of TNFR-associated factor 6 (TRAF-6). The IRAK-TRAF6 complex then interacts with another complex containing TGF-
-activated kinases leading to the activation of IKK and finally NF-
B activation. Alternative or MyD88-independent pathways activate caspase-1 [IL-1
converting enzyme (ICE)], which converts the inactive form of the proinflammatory cytokine IL-18 into the secreted and active form (9, 56). Other MyD88-independent pathways involve the induction of IFN-inducible genes such as the CXC chemokine CXL10 (IP-10) (9, 30, 33).
On the basis of these observations and the close proximity of IEC with both commensal bacteria and cells of the mucosal immune system, it is possible that they have the ability to respond to different MAMPs. Alterations or disruption of this interaction might therefore contribute to the development of intestinal inflammation. To date, the majority of studies of PRR function have relied on the use of hematopoietic cells (3, 6, 30) including monocyte/macrophage cell lines and immortalized IEC lines (1, 13, 15, 41), which together have provided evidence for both positive responses as well as no response to MAMPs (10, 15, 41). We have therefore investigated the possibility that changes in or disruption of the response of IEC to microbial antigenic challenge may be a feature of chronic intestinal inflammation by comparing TLR expression and signaling in primary colonic epithelial cells (CEC) of healthy animals with CEC from animals that spontaneously develop colitis.
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MATERIALS AND METHODS
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Animals.
C57BL/6 IL2+/ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were used between 4 and 7 wk of age. IL2/ mice were obtained from heterozygote matings and were identified by PCR analysis of tail DNA samples (52). Wild-type and heterozygote littermates were used as control animals. All animals were bred and maintained in individual isolator cages under specific pathogen-free conditions at the University of Leeds.
CEC isolation and culture.
Animals were euthanized at 4 and 7 wk of age. Samples of proximal and distal colon were retained for histological determination of disease severity at time of death. Briefly, disease severity in IL2/ mice was quantitated by using a cumulative score (012) obtained by examination of the macroscopic and microscopic appearance of the spleen (maximum score of 4) and the proximal (maximum score of 4) and distal (maximum score of 4) colon. Macroscopically, spleens were examined for presence of no (0), moderate (1), or severe splenomegaly (2) and microscopically, for evidence of no (0), partial (1), or total (2) loss of normal architecture. Proximal and distal colons were examined macroscopically for evidence of no (0), moderate (1), or severe (2) inflammation and distortion/thickening and microscopically for the presence of no (0), moderate (1), or severe (2) inflammatory cell infiltrate and/or loss of goblet cells and epithelial ulceration. Normal healthy animals had a combined score of 02 with scores of 38 and 912 defining moderate and severe disease, respectively. All C57BL/6 IL2+/+, IL2+/, and IL2/ mice at 4 wk of age had a score of 02, whereas 7-wk-old IL2/ mice had a score of 38 with 9-wk-old animals having a score of 912. CEC were obtained by digesting segments of colonic mucosa with Dispase I (Roche Diagnostics, Lewes, UK) as described previously (12, 63). Before culture, RNA, or protein extraction, CEC preparations were depleted of contaminating hematopoietic cells by negative immunomagnetic selection by using a monoclonal anti-CD45-biotin antibody (BD Biosciences, Cowley, UK) and streptavidin beads (BD Biosciences). Depletion was confirmed by anti-CD45 (Caltag Laboratories, Burlingame, CA) and anti-cytokeratin antibody staining with CEC preparations being >97% cytokeratin+ and <2% CD45+. For culture, equal numbers of freshly isolated CEC were plated into tissue culture-grade plasticware coated with a 1:1 solution of Matrigel (BD Biosciences) as described previously (12). CEC were cultured for 24 h to obtain a semiconfluent monolayer of adherent cells after which nonadherent cells were removed before addition of 10 µg/ml Salmonella enteriditis LPS (Sigma, St. Louis, MO), 1 µg/ml Staphylococcus aureus PGN (Sigma), 10 µg/ml Streptococcus faecalis LTA (Sigma), or the synthetic tripalmitoglated lipopeptide Pam3 CSK4 (InVivoGen, San Diego, CA). The MAMP concentrations used were empirically determined to be optimal in a series of preliminary experiments (data not shown). Seventy-two hours later, cell counts were obtained, and supernatants were collected for cytokine analysis.
RNA isolation and cDNA synthesis.
Total cellular RNA was extracted from freshly isolated CEC by lysis in 4 M guanidinium isothiocyanate, cesium chloride density gradient centrifugation, hot (65°C) phenol extraction, and ethanol precipitation. The integrity of the RNA sample was confirmed by gel electrophoresis. Reverse transcription of RNA (10 µg) was performed with 50 U Moloney murine leukemia virus reverse transcriptase (Ambion, Huntington, UK) using 0.1 µg random hexanucleotide primers (Progema, Southampton, UK), 25 pg RNAsin (Promega), and 0.5 mM dNTPs (Amersham Biosciences, Piscataway, NJ) in buffer containing (in mM) 500 Tris·HCl, pH 8.3, 30 MgCl2, 500 KCl, and 50 DTT. Aliquots of cDNA were amplified by using primers pairs listed in Table 1.
Real-time PCR.
Primer and probe sequences for murine TLR2 were those described by Sweet et al. (59). Primer sequences for TLR4 were designed by using Primer Express software package (Applied Biosystems, Foster City, CA). All primer and probe sequences are detailed in Table 1. Primers and probe were used at 900 and 200 nM, respectively. TaqMan PCR was performed in 25 µl volumes using TaqMan Universal PCR Mastermix containing AmpliTaq Gold polymerase and run on the ABI Prism 7700 Sequence Detection System (PerkinElmer, Wellesley, MA). All samples were run in triplicate. Threshold cycle (Ct) numbers were determined with Sequence Detector Software (version 1.6; Applied Biosystems) and transformed by using the standard curve method as described by the manufacturer. Abl was used as the calibrator gene. Samples were only analyzed provided the slope of the standard curve was <10% ± 3.3, R2 > 0.97 and the SD of each triplicate Ct was < 0.4.
RT-PCR.
Expression of genes encoding components of TLR-mediated intracellular signaling pathway was examined by semiquantitative RT-PCR. Primer sequences for MyD88, Toll/IL-1R domain-containing adapter protein (TIRAP), IRAK, TRAF6, IKK
, IKK
, caspase-1, and
-actin (Table 1) were designed by using MacVector software program (Apple Computer). Samples were amplified by capillary PCR (Idaho Technology, Salt Lake City, UT) using 1 µl of sample cDNA with a PCR core mix containing 1 µl of PCR buffer, (0.5 M Tris, 5 mM MgCl2), 4 µl of ddH2O, 1 µl of 2 mM 2-deoxynucleotide-5'-triphosphates (dNTPs) (Amersham Biosciences), 12.5 pmol of forward and reverse primer and 5 units Taq polymerase (Amersham Biosciences). Each primer pair was optimized empirically using cDNA from the J774.1A macrophage cell line. Products were run on a 2% agarose gel containing 0.5 µg/ml ethidium bromide, the gel was imaged under an UV transmitting light source, and quantitative analysis of each product was determined by using Quantity One Quantitation Software, (Bio-Rad, Hercules, CA). Comparative analysis of gene expression between each littermate pair of IL-2 deficient (IL-2/) and control samples was determined by examining the amount of each product normalized to the amount of
-actin product amplified concurrently.
Western blot analysis.
Total cell lysates of freshly isolated CEC were obtained by solubilizing cells with hot (65°C) Laemmli buffer (35), and proteins were quantified spectrophotometrically using Bio-Rad DC Protein Assay kit (Bio-Rad) per manufacturer's instructions. Equivalent amounts (1030 µg) of total cellular protein were fractionated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences). Membranes were sequentially incubated with primary antibodies to TLR4 (clone MT5510) (BioCarta, Kidlington, UK), followed by goat anti-rat-biotin (Caltag), and finally, by streptavidin-horseradish peroxidase (Bio-Rad). Detection of chemiluminescence was obtained by using SuperSignal West Pico Chemluminescent Substrate (Pierce, Rockford, IL).
Flow cytometry.
To minimize any loss of cell surface TLR2, CEC were isolated by scraping the epithelial mucosa immediately after removing the colon. Cells were washed in medium, pelleted, and resuspended in PBS containing 10% FCS and 1% paraformaldehyde. Cells were stained with FITC-anti-cytokeratin and TLR2 antibody (clone 6C2; Hycult Biotechnology, Uden, The Netherlands), followed by goat anti-rat-biotin (Caltag), and finally by streptavidin-PE (Caltag). Samples were analyzed by flow cytometry. Appropriate isotype-matched control antibodies were also used.
Caspase-1 detection.
Caspase-1 activity was determined by using a Caspase-1 detection kit (BioCarta) incorporating a fluorescent-labeled caspase-1 inhibitor (FAM-YVAD-FMK), which binds irreversibly to active caspase-1, preventing further activity by caspase-1, and can be monitored by flow cytometry. The optimal concentration of FAM-YVAD-FMK was determined empirically using freshly isolated wild-type CEC. Due to the rapid loss of CEC after dissociation from the basement membrane, the FAM-caspase-1 inhibitor was incubated within the excised and intact lumen of the colon for 1 h at 37°C-5% CO2 before isolation of CEC by scraping the lumen of each colon and immediately resuspending CEC in medium before analysis. Propidium iodide staining was used to ensure that only live cells were analyzed.
ELISA.
Paired capture and detection antibodies specific for murine IL-6, IL-18, and monocyte chemoattractant protein-1 (MCP-1), (BD Biosciences) were used to develop ELISAs according to supplier's recommendations. CEC-conditioned medium was analyzed in triplicate, using recombinant cytokines to determine amounts of IL-6 and MCP-1 in the test samples. Serum samples obtained from 4-, 7-, and 9-wk-old IL-2/ and littermate age-matched controls were tested for IL-18. The sensitivity of the assay in each case was
50 pg/ml.
EMSA.
CEC pellets were lysed with buffer 1 [(in mM) 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 DTT, with 20 µl mammalian protease inhibitor cocktail, 62.5 µl of 10% vol/vol Igepal CA-630 (Sigma)] and centrifuged (800 g) for 10 min to pellet cell nuclei. Nuclear pellets were resuspended in 50 µl buffer 2 (20 mM HEPES, pH 7.9, 25% vol/vol glycerol, 0.4 M sodium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, with 20 µl mammalian protease inhibitor cocktail). Samples were then vortexed, cooled on ice for 40 min, homogenized, and centrifuged at 13,000 g for 15 min at 4°C. The supernatants were harvested and stored at 80°C. Consensus oligonucleotide probe (Promega) was labeled with [
-32P]ATP, (Amersham Biosciences) before gel shift assay was performed (according to Promega Technical Bulletin 110). Supershift gels were performed by preincubation of nuclear extracts for 20 min with 1 µl of either anti-NF-
B p50 or p65 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis.
Evaluation of parametric and nonparametric data were obtained by using the Kolmogorov-Smirnov test. Data were analyzed by using Student's t-test for parametric data and Mann Whitney's U-test for nonparametric data. Comparison of data between groups were analyzed by using ANOVA. All graphs are presented as means ± SE and significant results are determined by a P value of >0.05. Data were analyzed by using Excel and StatView statistical programs for Macintosh.
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RESULTS
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The aim of this study was to investigate the possibility that changes in or disruption of the response of CEC to microbial antigenic challenge may be a feature of chronic intestinal inflammation. Expression of TLR, TLR functionality, and components of TLR-mediated signaling by CEC in the healthy vs. inflamed colon was undertaken by using wild-type mice and IL-2/ mice that develop a spontaneous and progressive form of UC (51), which has been shown to be triggered by commensal gut bacteria (17).
Highly purified preparations of primary CEC were obtained by digesting fragments of colon with Dispase I to form an epithelial cell suspension from which contaminating hematopoietic cells were removed by immunomagnetic selection using an anti-CD45 antibody. Purity of CEC preparations, as assessed by staining with anti-cytokeratin and -CD45 antibodies (Fig. 1A) and detecting CD45mRNA expression by RT-PCR, was routinely >98% cytokeratin+ and <2% CD45+ with residual cells consisting of (vimentin+) fibroblasts. In cultures of primary CEC, it was not possible to detect CD45 mRNA using a RT-PCR assay capable of detecting <2% contaminating CD45+ cells (Fig. 1B).

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Fig. 1. Isolation and culture of primary colonic epithelial cells (CEC). A: freshly isolated CEC from 7-wk-old IL-2/ mice were analyzed for cytokeratin and CD45 expression by antibody staining and flow cytometry. The histogram shows profile of staining obtained with isotype-matched control antibodies (open profiles) and test antibodies (closed profiles) with the proportion of CD45+ or cytokeratin+ cells indicated by % values. B: level of hematopoietic cell contamination of cultured CEC was determined by RT-PCR, using primers specific for the CD45 gene. The sensitivity of the assay, as determined by adding different amounts of hematopoietic (spleen) cells to purified CEC samples, was <2%. It was not possible to detect any CD45 transcripts in any of three independent CEC cultures (13). C: growth-promoting effects of microbe-associated molecular patterns (MAMP) on primary CEC from 7-wk-old C57BL/6 mice was determined by comparing the number of cells present in cultures containing CEC alone (0) with those cultured in the presence of peptidoglycans (PGN; 10 µg/ml), lipoteichoic acid (LTA; 10 µg/ml), LPS (10 µg/ml), and the pure (synthetic) Toll-like receptor (TLR)2 ligand, Pam3Cys4 (0.5 µg/ml) after 72 h of culture. Results shown were collated from 3 independent experiments.
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Primary CEC are responsive in vitro to MAMP.
To determine whether CEC have the potential to respond to bacteria and bacterial antigens, the response of CEC from 4-wk-old C57BL/6-IL2+/+, wild-type, and IL2/ mice in vitro to MAMPs of both gram positive (PGN and LTA) and gram negative (LPS) bacteria was compared. PGN and LPS are prototypical TLR2 and TLR4 ligands, respectively. At 4 wk of age, the colons of IL2/ mice are histologically normal (disease severity score of 02; see MATERIALS AND METHODS). During a 72-h culture period, the number of CEC increased in the presence of MAMPs (Fig. 1C) and in particular, with the TLR2 ligands PGN and LTA, resulting in a more than twofold increase in cell number compared with cells cultured in complete medium alone. That this response was a result of specific TLR2-ligand interactions and not contaminants in the MAMP preparations was strongly suggested by the growth-promoting effects of the pure (synthetic) TLR2 agonist Pam3Cys4, which were comparable with that obtained with PGN and LTA (Fig. 1C). The effect of the TLR4 ligand LPS was less dramatic, resulting in only a slight (1.3- to 1.5-fold) increase in cell number (Fig. 1C). To determine the affect of MAMPs on CEC immune function, supernatants from MAMP-CEC cocultures were assessed for the presence of the cytokines IL-6 and MCP-1, which are produced by IEC both in vitro and in vivo (39). CEC from wild-type mice constitutively secreted high levels of MCP-1 and comparatively lower levels of IL-6 (Fig. 2). The levels of secretion of both cytokines were significantly increased (P < 0.002) in response to LPS. By contrast, PGN and LTA had no significant effect on MCP-1 or IL-6 production compared with cells cultured in media alone. Of note, the response of CEC from disease-free 4-wk-old IL-2/ mice to MAMPs was strikingly different to that of CEC from wild-type littermates (Fig. 2). Unlike CEC from wild-type mice, LPS did not significantly change IL-6 or MCP-1 production by IL2/ CEC, and, whereas LTA had no effect on wild-type CEC, it significantly increased (P < 0.002) both IL-6 and MCP-1 production by IL-2/ CEC. Similar to wild-type CEC, PGN did not modulate cytokine production by IL-2/ CEC. Collectively, these data suggest that primary CEC express functional TLRs and that the outcome of TLR-mediated signaling may be altered in the inflamed colon; whereas TLR2 responses of CEC from colitis-susceptible animals were augmented, their response to TLR4 ligands were reduced or absent.

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Fig. 2. MAMP-induced cytokine production by primary CEC. CEC from 4-wk-old IL-2/ or IL-2+/+ mice were cultured in complete medium alone [control (Ctrl)] or in medium containing LPS, LTA, or PGN for 72 h after which culture supernatants were assayed for monocyte chemoattractant protein-1 (MCP-1) and IL-6 by ELISA. The results shown are representative of 3 independent experiments. *P < 0.002, MCP-1 production in response to LTA by IL-2/ vs. wild-type CEC; **P < 0.002, MCP-1 production by wild-type CEC in response to LPS vs. Ctrl. * P < 0.002, IL-6 production by IL-2/ CEC in response to LTA vs. Ctrl.
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Toll receptors are expressed by CEC.
Because the ability to respond to MAMP implied the expression of functional PRR by CEC, we then investigated the profile of expression of TLR2 and TLR4 both at the mRNA and protein levels in wild-type and IL-2/ mice. Comparative analysis of gene expression using real-time PCR demonstrated that CEC from 4-wk-old IL-2/ mice, before or coincident with the onset of colitis, had 1.5- and 3.5-fold increased levels of mRNA encoding TLR2 and TLR4, respectively, compared with their healthy littermates (Fig. 3). This increase in expression of both TLR2 and TLR4 mRNA was reproduced or sustained in CEC of 7-wk-old IL2/ mice with moderate colitis (combined disease severity score 38) (Fig. 3). Similar to TLR4 mRNA analysis, examination of cellular levels of TLR4 protein by Western blot analysis demonstrated higher levels of protein expression by CEC from 7-wk-old colitic animals compared with their littermates, although this difference was not significant (Fig. 4, A and B). In contrast to the increased levels of TLR4 mRNA seen among CEC of 4-wk-old IL2/ animals (Fig. 3), there were no differences in cellular TLR4 protein levels, which were similar to that of CEC from 4-wk-old wild-type mice (Fig. 4B). This disparity in TLR4 mRNA and protein expression may reflect differences in the stability and half-life of mRNA vs. protein or sensitivity of RT-PCR analysis compared with Western blot analysis. Because TLR4 protein is normally distributed between intra- and extracellular locations, the efficiency of TLR4 extraction from these different sites in CEC by the method adopted may be different and could result in an underestimation of total cellular protein. In contrast to TLR4 expression in which changes in expression levels by IL2/ CEC were only evident once colitis was established, changes in CEC TLR2 expression were apparent before or coincident with disease onset. Flow cytometric analysis of TLR2 expression showed that CEC from 4-wk-old IL-2/ mice expressed significantly higher (P < 0.01) levels of cell surface TLR2 (Fig. 4, C and D) concordant with both mRNA analyses (Fig. 3) and with the increased sensitivity of CEC from IL2/ mice to the TLR2 ligand, LTA (Fig. 2).

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Fig. 3. Increased TLR mRNA expression in CEC of IL-2/ mice. mRNA isolated from highly purified preparations of CEC of C57BL/6 wild-type (+/+), or IL-2/ (/) mice at 4 and 7 wk of age was reverse transcribed to cDNA and TLR2 and TLR4 transcripts amplified and quantitated by real- time PCR. The Abl gene was used as a control housekeeping gene to compare relative levels of expression as described in materials and methods. Results shown were obtained from 6 or 7 paired CEC samples from 4- and 7-wk animals, respectively. The numbers in parentheses represent the average disease severity score for each group of animals (see materials and methods). Error bars indicate means ± SE.
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Fig. 4. Increased levels of cell surface TLR2 by CEC in IL-2/mice. A: lysates of highly purified CEC from two paired samples of 4- and 7-wk-old C57BL/6 wild-type (+/+) and IL-2/ (/) mice and from the J774 macrophage cell line (Ctrl) were analyzed for TLR4 expression by Western blot analysis as described in materials and methods. B: densitometric analysis of TLR4 protein levels in 5 paired samples of CEC lysates for each age group of mice. Each pair was expressed as a ratio of IL-2/ to wild-type to correct for differences between blots. Error bars are means ± SE. There was no statistical difference between the groups at either age. C: TLR2 cell surface expression was assessed by staining CEC from both 4-wk-old IL2+/+ (+/+) and IL2/ (/) mice with control and anti-TLR2 antibodies and flow cytometry. D: proportion of TLR2+ CEC in +/+ and / mice as determined from 3 paired samples of 4-wk-old / and +/+ mice. Error bars are means ± SE. *P < 0.01 comparing / with +/+ CEC samples.
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MyD88-dependant signaling pathway is not altered in mice with colitis.
Studies of TLR and their signaling pathway have been primarily restricted to macrophages and established epithelial cell lines. Very little, however, is known about these pathways in primary IEC. We therefore examined the expression levels of genes encoding molecules associated with the MyD88 signaling pathway in CEC of 4- and 7-wk-old wild-type and IL-2/ mice using semiquantitative RT-PCR. Primers used were specific for the adapter molecules MyD88, TRAF6, and TIRAP, as well as the kinases IRAK, IKK
, and IKK
. Expression of all components of MyD88-dependant signaling was detected in CEC of 4- and 7-wk-old wild-type and IL-2/ mice with higher levels of expression being evident in CEC from 4-wk-old animals (Fig. 5). At both time points, the levels of expression of MyD88 IRAK as well as IKK
and IKK
mRNA were equivalent (Fig. 5). By contrast, TRAF6 mRNA levels were significantly increased (P < 0.05) in CEC of 7-wk-old IL-2/ animals compared with CEC from wild-type animals.
Although MyD88-dependant signaling directly leads to NF-
B activation (8), it is not clear whether or what form of NF-
B is normally expressed by CEC in vivo. Studies using IEC lines (49) have demonstrated nuclear translocation of NF-
B in response to MAMPs and altered NF-
B expression in submucosal cells and within the epithelial crypts of patients with UC, the site of epithelial cell division. However, mature murine CEC have been shown to express in situ the inhibitory p50 homodimeric form of NF-
B (28), suggesting gene transcription through this pathway may not be active in mature epithelial cells. To investigate this further and determine the significance of the changes in expression of mRNAs encoding components of the MyD88-dependent signaling pathway in CEC of 7-wk-old IL2/ mice (Fig. 5), NF-
B activation was examined in IL-2/ and wild-type CEC by EMSA. Nuclear extracts from CEC were hybridized with a radiolabeled DNA consensus sequence that binds NF-
B transcription factors. Specific and nonspecific competitors were used to confirm the specificity of any resultant shifted bands. The bandshift pattern seen in Fig. 6A demonstrates the presence of low levels of NF-
B in CEC of both wild-type and IL-2/ mice at 4 and 7 wk of age. A supershift assay using antibodies to the p50 and p65 subunits of NF-
B showed that CEC from wild-type mice had minimal expression of p50 and p65 (Fig. 6B) and that p50 subunit complexes predominated in CEC from IL-2/ mice. These findings are consistent with the upregulation of the inhibitory p50 subunit, not the p65 form, of NF-
B in CEC in the inflamed intestine.
MAPKs are upregulated in CEC from animals with colitis.
Because the MAPK signaling pathway has been implicated in TLR signaling, levels of the active forms of ERK, JNK/SAPK, and p38 kinases were examined in CEC of 4- and 7-wk-old wild-type and IL-2/ mice. All CEC lysate samples were normalized for total protein content before analysis and blotted with antibodies specific for constitutive, nonphosphorylated forms of the kinases as an additional loading control. Both JNK/SAPK and ERK exist as two isoforms, p54/p46, and p44/p42, respectively. In the J77A.1A macrophage cell line, expression of the larger isoforms of JNK/SAPK (p54) and ERK (p44) were predominant (Fig. 7). By contrast, CEC displayed a different pattern of JNK/SAPK isoform expression in which both the high and low molecular weight isoforms of JNK (p54, 46) and ERK (p44, 42) were equally represented (Fig. 7). The active and phosphorylated forms of all three kinases were detected in CEC samples from 4-wk-old IL2/ mice although the levels of expression were not significantly higher than in CEC samples from wild-type animals. By contrast, analysis of MAPK expression in CEC samples from 7-wk-old animals revealed that all three members of the MAPK family had significantly (P < 0.01 for JNK/SAPK and p38 and P < 0.05 for ERK) increased levels of phosphorylation in CEC of 7-wk-old IL-2/ mice compared with their wild-type littermates (Fig. 7B). These data suggest that the activity of this family of MAPK signaling molecules is upregulated in CEC of mice with colitis.

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Fig. 7. Upregulation of MAPK signaling pathways in CEC in colitic mice. Equalized protein extracts of 2 pairs of CEC from 4- (A) and 7 (B)-wk-old C57BL/6 wild-type (+/+) and IL-2/ (/) mice were analyzed for the phosphorylated forms (P) of MAPK family members, JNK/SAPK, ERK and p38 kinases by Western blot analysis. The macrophage cell line J774 was used as a positive control. The average disease severity score of 4-wk-old IL2/ and wild-type animals was 2 and 1, respectively, and in 7-wk-old IL2/ and wild-type mice the average disease severity scores were 6 and 2, respectively. To confirm protein loading equivalency between samples, duplicate blots were probed for the nonphosphorylated forms (NP) of p38 (A) or JNK/SAPK (B). The histograms alongside each autoradiograph represent scanning densitometric analysis of a total of 5 sets of paired CEC samples from 7-wk-old C57BL/6 wild-type (+/+) and IL-2/ (/) mice analyzed for the phosphorylated forms of JNK/SAPK, ERK, and p38 kinases. Levels of expression of the different proteins in IL-2/ CEC were compared with its littermate control. Error bars represent means ± SE. Statistical analysis demonstrated significant upregulation of the phosphorylated forms of JNK/SAPK and p38 (P < 0.01) and ERK (P < 0.05) in CEC of 7-wk-old IL2/ mice (B).
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MyD88-independent signaling pathway is upregulated in CEC from mice with colitis.
Caspase-1 (ICE) is of central importance to TLR-mediated MyD88-independant signaling (7). Caspase-1 cleaves the inactive precursor of IL-18 to produce the biologically active form of the inflammatory cytokine. Caspase-1 expression was therefore examined in CEC from wild-type mice and in IL-2/ mice before and during the development of colitis. Caspase-1 gene expression was determined by semiquantitative RT-PCR, and enzyme activity was determined by using a flurogenic inhibitor and flow cytometry. Caspase-1 mRNA expression in CEC from wild-type mice revealed a significant (P = 0.01) age-related decrease between 4 and 7 wk of age (Fig. 8A). In CEC from IL-2/ mice, this age-related decline in caspase-1 mRNA levels was not seen with equivalent levels of mRNA evident at 4 and 7 wk of age (Fig. 8A). Caspase-1 activity, as determined by binding of a flurogenic inhibitor, demonstrated increased levels of active caspase-1 in CEC from IL-2/ mice compared with wild-type mice at 7 wk of age (Fig. 8B). Also, the proportion of CEC expressing caspase-1 activity was significantly (P < 0.02) higher in mice with colitis (
30%) compared with wild-type (
10-%) CEC (Fig. 8B).

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Fig. 8. Increased caspase-1 activity in CEC of IL-2/ mice. A: caspase-1 mRNA levels in CEC of 4- and 7-wk-old C57BL/6 wild-type (+/+) and IL-2/ (/) mice were determined by RT-PCR and densitometric analysis of 5 paired samples for each age group. Relative fold increase was determined by comparing levels of expression with the housekeeping gene -actin. *P < 0.01 comparing expression of caspase-1 between 4- and 7-wk-old wild-type samples and **P = 0.01 comparing 7- wk-old wild-type and IL-2/ samples. B: caspase-1 activity in CEC was determined by incubating freshly isolated CEC from 7-wk-old wild-type (+/+) and IL-2/ (/) mice with the fluorogenic caspase-1 inhibitor, FAM-YVAD-FMK, before analysis by flow cytometry. The bar graph represents the average frequency of caspase-1+ CEC in wild-type and IL-2/ mice as determined from 3 sets of paired +/+ and / samples. Live CEC were analyzed as determined by the exclusion of propidium iodide added immediately before acquisition. *P < 0.02 comparing wild-type and IL-2/ CEC.
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Increased levels of IL-18 in colitis.
IL-18 is rapidly secreted by IEC in response to various inflammatory stimuli and is bound by lymphocytes expressing IL-18R resulting in secretion of IFN-
(53). In view of the rapid secretion of this cytokine, we decided therefore to compare the levels of IL-18 in the serum of wild-type and IL-2/ mice at 4, 7, and 9 wk of age (Fig. 9). In wild-type mice, serum IL-18 levels progressively decreased with age, paralleling the downregulation of caspase-1 mRNA levels in CEC (Fig. 8A). By contrast, serum levels of IL-18 in IL-2/ mice remained high at 9 wk of age, although between 4 and 7 wk of age a decline in levels similar to that seen in wild-type mice was also evident in IL-2/ mice, although this decline did not reach statistical significance. Again, the pattern of IL-18 serum levels in IL-2/ mice was similar to that of caspase-1 mRNA levels.

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Fig. 9. Increased IL-18 levels in colitic mice. Serum samples from at least 3 paired sets of 4-, 7-, and 9-wk-old C57BL/6 wild-type (+/+) and IL-2/ littermates (/) were assayed for IL-18 by ELISA. Error bars depict the means ± SE and data were analyzed by single-factor ANOVA.
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DISCUSSION
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The findings of this study demonstrate that primary CEC express functional TLR and identify both qualitative and quantitative changes in TLR expression and signaling associated with the development of colitis in IL2/ mice. Because exposure to commensal bacteria initiates colitis in these animals (17), these findings suggest that changes in TLR responsiveness may underlie and contribute to the hypersensitivity to bacterial antigens characteristic of IBD (21, 38, 47).
One of the more striking observations made in this study is the change in TLR responsiveness during the development of colitis, characterized by augmented TLR2 responsiveness and concomitant reduction in TLR4 responsiveness. This exaggerated response by CEC from 4-wk-old IL-2/ mice to LTA most likely reflects intrinsic changes in CEC TLR responsiveness and is analogous to the increased TLR2-responsiveness and activation of NF-
B accompanied by increased TH1 CD4 T cell responses described in macrophages with NOD2/CARD15 mutations and in NOD2/CARD15-deficient mice (65). Despite differences in the downstream signaling effector molecules of TLR2 activation in macrophages (primarily MyD88- and NF-
B dependent) and CEC (MyD88- and NF-
B-independent), the findings reported by Wantanabe et al. (65) and the present study are consistent with changes in TLR responsiveness and increased TLR2-mediated signaling being central to and perhaps underlying the development of chronic inflammatory responses. The changes we have described of TLR responsiveness of CEC in IL2/ mice are unlikely to be a phenomena of the in vitro culture system, because cultured CEC retain many of the biochemical and immunological characteristics of their in vivo counterparts (60), and CEC from wild-type and IL-2/ mice were processed in parallel. It also doubtful that these findings are due solely to increased expression of TLR2 by IL-2/ CEC, because the same cells are refractory to PGN, which also binds TLR2 (40). Our studies cannot, however, rule out the possibility that CEC express receptors for LTA distinct from TLR2, or other TLRs in addition to TLR2 that are induced in response to inflammation. TLR2 is known to form receptor complexes with other TLR, and it has been shown that differential recognition of LTA is dependant on expression of both TLR2 and TLR6 (42). Unfortunately we were unable to examine CEC for expression cell-surface TLR6 due to lack of suitable reagents.
An important finding of our study is that CEC cytokine responsiveness differed between wild-type mice and animals that develop colitis. Before or coincident with disease onset, CEC from IL-2/ animals demonstrated a different pattern of cytokine production when exposed to specific MAMP. Exposure to LTA resulted in a markedly enhanced production of both IL-6 and MCP-1 with no or limited responsiveness to LPS and PGN, contrasting with the response seen in wild-type CEC. This infers that CEC from animals prone to develop intestinal inflammation have either intrinsic abnormalities predisposing them to colitis or that changes influencing functional PRR expression occurred early in the pathogenesis of intestinal inflammation. Alternatively, it may be an indirect effect and a consequence of extrinsic factors, e.g., changes in the commensal microbiota, and the emergence and overgrowth of "pathogenic" species. Another possibility explaining the changes seen in CEC may be as a result of the influence from hyperproliferative CD4 T cells that accumulate in the colonic mucosa and can transfer disease to otherwise healthy animals (11). It is difficult through this series of experiments to determine, therefore, whether our findings are a result of intrinsic differences in CEC as a direct or indirect result of IL-2 deficiency or extrinsic factors, such as altered responses to bacterial products. Examination of germ-free IL-2/ animals may help establish whether CEC express intrinsically altered functional TLR; although because these animals fail to exhibit a mature gut-associated lymphoid tissue (GALT) in the absence of bacteria, it is likely that the characteristics of CEC from these mice will be different.
Significance of MCP-1 and IL-6 production by CEC was not addressed in this study, although both cytokines are involved in the activation of the inflammatory response (16, 26, 43, 57). MCP-1 is a potent chemotactic cytokine for macrophages, dendritic cells, and antigen-primed lymphocytes (24, 50, 62), whereas IL-6 is a pleiotrophic cytokine with a wide range of biological activities in immune regulation, hematopoiesis, and inflammation (26, 46). Both are produced by a number of cell types including immune cells, endothelial cell, and smooth muscle cells, as well as epithelial cells (24, 34, 37, 46). In addition, both of these cytokines have a role in epithelial cell homeostasis. MCP-1-deficient mice display evidence of delayed wound healing with defects in reepithelialization (36); and IL-6 is an epithelial cell growth factor (23, 46, 67). The constitutive secretion of these cytokines by CEC in vitro may therefore be associated with maintenance of an intact epithelial layer, whereas an increased level of secretion after exposure to different MAMP may indicate a homeostatic response. Presumably, if sufficient stimulus occurs, it is not inconceivable that an altered response aimed at activating resident immune cells or recruiting other immune cells to the site of injury or infection would be triggered. If the stimulus persists and/or there is a failure of or breakdown in the homeostatic control of proinflammatory cytokine secretion by CEC, then the ensuing immune response would be sustained, resulting in chronic inflammation and disease.
Our findings that primary CEC in culture are functionally responsive to MAMP and that NF-
B (p65) activation is absent in primary CEC contrasts with similar studies using immortalized IEC lines (2, 41) and immature IEC (22, 45). These previous studies suggested that the absence or low levels of TLR are responsible for absent NF-
B activation and functional unresponsiveness to MAMP. Indeed, transfection of IEC lines with TLR expression vectors confers MAMP-induced NF-
B activation. Analysis of NF-
B activation in CEC in vivo, however, has shown that the transcriptionally active (p65) form of NF-
B is restricted to proliferating cells within the crypts, and that similar to our own findings, mature enterocytes express the p50 inhibitory form of NF-
B (28). The conflicting results obtained by using primary CEC vs. those using established CEC lines may therefore reflect differences in their maturation states rather than specific nonresponsiveness of IEC to MAMP. Indeed, Cario et al. (14) have shown that the response of T84 CEC to MAMP is related to their maturation state. Whereas less differentiated cells failed to respond to LPS and PGN, fully differentiated cells were capable of responding. In the same study, surface expression of TLR2 and TLR4 by T84 cells was shown to be restricted to differentiated cells. These data support the argument that the level of differentiation of IEC influences the expression and functional response of TLR. Finally, the functionality of PRR expressed by CEC may be influenced or determined by the presence of negative regulators of TLR signaling, i.e., the single immunoglobulin-IL-1 receptor-related molecule (64) or cofactors such as MD-2 and CD14, which have been implicated in LPS binding to TLR4 (5, 58).
Although the MyD88-dependant-NF
B signaling pathway is the most well-characterized TLR signaling pathway, the results obtained in this study raise questions as to the requirement for MyD88-NF-
B in the response of CEC to MAMP. Activation of MAPK family members after stimulation of CEC with MAMP suggests the existence of divergent intracellular signaling pathways that ultimately converge at the level of activation of genes encoding proteins involved in the inflammatory cascade. The identification of MAMP-induced intracellular signaling pathways resulting in activation of NF-
B and MAPKs in MyD88 (31)- and TRAF6 (44)-deficient cells is consistent with the existence of distinct and divergent TLR-mediated intracellular signaling pathways. Although additional studies are required to fully dissect the signaling pathways involved in TLR signaling in CEC, our study identifies the utilization of MyD88-independent signaling pathways characterized by the sustained expression and lack of downregulation of caspase-1 activity between 4 and 7 wk of age in IL-2/ mice. The failure or breakdown in the regulation of caspase-1 activity in CEC may therefore play a role in the development or maintenance of intestinal inflammation. The observation that caspase-1/ animals are resistant to chemically induced colitis (55) and that anti-IL-18 antibodies ameliorate colitis in a number of murine models of IBD (32, 54, 61, 66) are consistent with this interpretation. With regard to human IBD, upregulation of IL-18 expression has been demonstrated in the inflamed lesions of the intestine of IBD patients localized primarily to macrophages and epithelial cells, and caspase-1 has been identified as a potential therapeutic target for IBD (reviewed in Ref. 53).
In conclusion, this study supports the role of CEC as microbial sensors with the ability to respond to different classes of bacterial antigens and that CEC from animals that develop colitis have altered TLR-mediated responses to MAMP and may use nonconventional TLR signaling pathways, which may contribute to the intestinal inflammation seen in IBD.
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GRANTS
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This work was supported, in part, by grants from the National Institutes of Health (to S. R. Carding and P. J. Felsburg). J. C. I. Singh was supported by the Ann Gloag Fellowship of the Royal College of Surgeons of Edinburgh.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. R. Carding, School of Biochemistry and Microbiology, The Univ. of Leeds, Leeds, LS2 9JT, United Kingdom (E-mail: S.R.Carding{at}Leeds.ac.uk)
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.
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REFERENCES
|
---|
- Abreu MT, Arnold ET, Thomas LS, Gonsky R, Zhou Y, Hu B, and Arditi M. TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells. J Biol Chem 277: 2043120437, 2002.[Abstract/Free Full Text]
- Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, and Arditi M. Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol 167: 16091616, 2001.[Abstract/Free Full Text]
- Aderem A. Role of Toll-like receptors in inflammatory response in macrophages. Crit Care Med 29: S16S18, 2001.[CrossRef][ISI][Medline]
- Aderem A and Ulevitch RJ. Toll-like receptors in the induction of innate immunity. Nature 406: 728787, 2000.[CrossRef]
- Akashi S, Ogata H, Kirikae F, Kirikae T, Kawasaki K, Nishijima M, Shimazu R, Nagai Y, Fukudome K, Kimoto M, and Miyake K. Regulatory roles for CD14 and phosphotidylinositol in the signaling via Toll-like receptor 4-MD-2. Biochem Biophys Res Commun 268: 172177, 2000.[CrossRef][ISI][Medline]
- Akira S. Toll-like receptors: lessons from knockout mice. Biochem Soc Trans 28: 551556, 2000.[ISI][Medline]
- Akira S. Toll-like receptors and innate immunity. Adv Immunol 78: 156, 2001.[ISI][Medline]
- Akira S. Toll-like receptor signaling. J Biol Chem 278: 3810538108, 2003.[Free Full Text]
- Akira S, Takeda K, and Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immun 2: 675680, 2001.[CrossRef][ISI]
- Andoh A, Kinoshita K, Rosenberg I, and Podolsky DK. Intestinal trefoil factor induces decay-accelerating factor expression and enhances the protective activities against complement activation in intestinal epithelial cells. J Immunol 167: 38873893, 2001.[Abstract/Free Full Text]
- Ashcroft A, Cruickshank SM, Croucher PI, Perry MJ, Rollinson S, Lippitt JM, Child JA, Dunstan C, Felsburg PJ, Morgan GJ, and Carding SR. Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin. Immunity 19: 849861, 2003.[CrossRef][ISI][Medline]
- Baumgart DC, Olivier WA, Reya T, Peritt D, Rombeau JL, and Carding SR. Mechanisms of intestinal epithelial cell injury and colitis in interleukin 2 (IL-2)-deficient mice. Cell Immunol 187: 5266, 1998.[CrossRef][ISI][Medline]
- Bocker U, Yezerskyy O, Feick P, Manigold T, Panja A, Kalina U, Herweck F, Rossol S, and Singer MV. Responsiveness of intestinal epithelial cell lines to lipopolysaccharide is correlated with Toll-like receptor 4 but not Toll-like receptor 2 or CD14 expression. Int J Colorectal Dis 18: 2532, 2003.[CrossRef][ISI][Medline]
- Cario E, Brown D, McKee M, Lynch-Devaney K, Gerken G, and Podolsky DK. Commensal-associated molecular patterns induce selective Toll-like receptor-trafficking from apical membrane to cytoplasmic compartments in polarized intestinal epithelium. Am J Pathol 160: 165173, 2002.[Abstract/Free Full Text]
- Cario E, Rosenberg IM, Brandwein SL, Beck PL, Reinecker HC, and Podolsky DK. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J Immunol 164: 966972, 2000.[Abstract/Free Full Text]
- Conti P and DiGioacchino M. MCP-1 and RANTES are mediators of acute and chronic inflammation. Allergy Asthma Proc 22: 133137, 2001.[ISI][Medline]
- Contractor NV, Bassiri H, Reya T, Park AV, Baumgart DC, Wasik M, Emerson SG, and Carding SR. Lymphoid hyperplasia, autoimmunity and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic interleukin 2-deficient mice. J Immunol 160: 385394, 1998.[Abstract/Free Full Text]
- Dangl JL and Jones JDG. Plant pathogens and integrated defence responses to infection. Nature 411: 826833, 2001.[CrossRef][ISI][Medline]
- Didierlaurent A, Sirard JC, Kraehenbuhl JP, and Neutra MR. How the gut senses its content. Cell Microbiol 4: 6172, 2002.[CrossRef][ISI][Medline]
- Duchmann R, Kaiser I, Hermann E, Mayet W, Ewe K, and Meyer Zum Buschenfelde K-H. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin Exp Immunol 102: 448455, 1995.[ISI][Medline]
- Duchmann R, May E, Heike M, Knolle P, Neurath M, and Meyer zum Buschenfelde K-H. T cell specificity and cross reactivity towards enterobacteria, Bacteriodes, Bifidobacterium, and antigens from resident intestinal flora. Gut 44: 812818, 1999.[Abstract/Free Full Text]
- Fusunyan RD, Nanthakumar NN, Baldeon ME, and Walker WA. Evidence for an innate immune response in the immature human intestine: toll-like receptors on fetal enterocytes. Pediatr Res 49: 589593, 2001.[Abstract/Free Full Text]
- Grossmann RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT, Kupper TS, Seghal BL, and Gottlieb AB. Interleukin-6 is expressed in high levels in psoriasis skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 86: 63676371, 1989.[Abstract]
- Gu L, Rutledge B, Fiorillo J, Ernst C, Grewal I, Flavell R, Gladue R, and Rollins B. In vivo properties of monocyte chemoattractant protein-1. J Leukoc Biol 62: 577580, 1997.[Abstract]
- Hampe J, Cuthbert A, Croucher PJP, Mirza MM, Mascheretti S, Fisher S, Frenzel H, King K, Hasselmeyer A, MacPherson AJS, Bridger S, van Deventer S, Forbes A, Nikolaus S, Lennard-Jones JE, Foelsch UR, Krawczak M, Lewis C, Schreiber S, and Mathew CG. Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet 357: 19251928, 2001.[CrossRef][ISI][Medline]
- Hibi M, Nakajima K, and Hirano T. IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med 74: 112, 1996.[CrossRef][ISI][Medline]
- Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, Almer S, Tysk C, O'Morain CA, Gassull M, Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M, and Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411: 599603, 2001.[CrossRef][ISI][Medline]
- Inan MS, Tolmacheva V, Wang QS, Rosenberg D, and Giardina C. Transcription factor NF-
B participates in regulation of epithelial cell turnover in the colon. Am J Physiol Gastrointest Liver Physiol 279: G1282G1291, 2000.[Abstract/Free Full Text]
- Inohara N, Ogura Y, and Nunez G. Nods: a family of cytosolic proteins that regulates the host responses to pathogens. Curr Opin Microbiol 5: 7680, 2002.[CrossRef][ISI][Medline]
- Irie T, Muta T, and Takeshige K. TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-
B in lipopolysaccharide-stimulated macrophages. FEBS Lett 467: 160164, 2000.[CrossRef][ISI][Medline]
- Kaisho T and Akira S. Dendritic-cell function in Toll-like receptor and MyD88 knockout mice. Trends Immunol 22: 7883, 2001.[CrossRef][ISI][Medline]
- Kanai T, Watanabe M, Okazawa A, Sato T, and Hibi T. Interleukin-18 and Crohn's disease. Digestion 63: 3742, 2001.[CrossRef][ISI][Medline]
- Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, and Akira S. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 167: 58875894, 2001.[Abstract/Free Full Text]
- Kishimoto T, Akira S, Narazaki M, and Taga T. Interleukin-6 family of cytokines and gp130. Blood 86: 12431254, 1995.[Free Full Text]
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 from random aggregation. Nature 47: 6985, 1970.
- Low QE, Drugea IA, Duffner LA, Quinn DG, Cook DN, Rollins BJ, Kovacs EJ, and DiPietro LA. Wound healing in MIP-1
(/) and MCP-1(/) mice. Am J Pathol 159: 457463, 2001.[Abstract/Free Full Text]
- Malik AA, Radhakrishnan N, Reddy K, Smith AD, and Singhal PC. Tubular cell-Escherichia coli interaction products modulate migration of monocytes through generation of transforming growth factor-
and macrophage-monocyte chemoattractant protein-1. J Endourol 16: 599603, 2002.[CrossRef][ISI][Medline]
- Matsuda H, Fujiyama Y, Andoh A, Ushijima T, Kajinami T, and Bamba T. Characterization of antibody responses against rectal mucosa-associated bacterial flora in patients with ulcerative colitis. J Gastroenterol Hepatol 15: 6168, 2000.[CrossRef][ISI][Medline]
- McGee D. Inflammation and mucosal cytokine production. In: Mucosal Immunology, edited by Ogra P, Mestecky J, Lamm ME, Strober W, Bienenstock J, and McGhee JR. San Diego, CA: Academic, 1999, p. 559573.
- Means TK, Golenbock DT, and Fenton MJ. The biology of Toll-like receptors. Cytokines Growth Factor Rev 11: 219232, 2000.[CrossRef][ISI][Medline]
- Melmed G, Thomas LS, Lee N, Tesfay SY, Lukasek K, Michelsen KS, Zhou Y, Hu B, Arditi M, and Abreu MT. Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host-microbial interactions in the gut. J Immunol 170: 14061415, 2003.[Abstract/Free Full Text]
- Morr M, Takeuchi O, Akira S, Simon MM, and Muhlradt PF. Differential recognition of structural details of bacterial lipopeptides by toll-like receptors. Eur J Immunol 32: 33373347, 2002.[CrossRef][ISI][Medline]
- Mukaida N, Harada A, and Matsushima K. Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine Growth Factor Rev 9: 923, 1998.[CrossRef][ISI][Medline]
- Muzio M, Natoli G, and Saccani S. The human toll signaling pathway: divergence of nuclear factor
B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J Exp Med 187: 20972101, 1998.[Abstract/Free Full Text]
- Naik S, Kelly EJ, Meijer L, Pettersson T, and Sanderson IR. Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium. J Pediatr Gastroenterol Nutr 32: 449453, 2001.[CrossRef][ISI][Medline]
- Naka T, Nishimoto N, and Kishimoto T. The paradigm of IL-6: from basic science to medicine. Arthritis Res 4: S233S242, 2002.[CrossRef][Medline]
- Prindiville T, Sheikh R, and Cohen S. Bacteriodes fragilis enterotoxin gene sequences in patients with inflammatory bowel diseases. Emerg Infect Dis 6: 171174, 2000.[ISI][Medline]
- Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, and Medzhitov R. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118: 229241, 2004.[CrossRef][ISI][Medline]
- Rogler G, Brand K, Vogl S, Page R, Hofmeister T, Andus T, Knuechel R, Baeuerle PA, Scholmerich J, and Gross V. Nuclear factor-
B is activated in macrophages and epithelial cells of inflammed mucosa. Gastroenterology 115: 357369, 1998.[ISI][Medline]
- Rollins BJ. Monocyte chemoattractant protein 1: a potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today 2: 198204, 1996.[CrossRef][ISI][Medline]
- Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, and Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75: 253261, 1993.[CrossRef][ISI][Medline]
- Schorle H, Holtschke T, Hunig T, Schimpl A, and Horak I. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352: 621624, 1991.[CrossRef][ISI][Medline]
- Seigmund B. Interleukin-1
converting enzyme (caspase-1) in intestinal inflammation. Biochem Pharmacol 64: 18, 2002.[CrossRef][ISI][Medline]
- Seigmund B, Fantuzzi G, Rieder F, Gamboni-Robertson F, Lehr HA, Hartmann G, Dinarello CA, Endres S, and Eigler A. Neutralisation of interleukin-18 reduces severity in murine colitis and intestinal IFN-
and TNF-
production. Am J Physiol Regul Integr Comp Physiol 281: R1264R1273, 2001.[Abstract/Free Full Text]
- Siegmund B, Lehr HA, Fantuzzi G, and Dinarello CA. IL-1
converting enzyme (caspase-1) in intestinal inflammation. Proc Natl Acad Sci USA 98: 1324913254, 2001.[Abstract/Free Full Text]
- Seki E, Tsutsui H, Nakano H, Tsuji NM, Hoshino K, Adachi O, Adachi K, Futatsugi S, Kuida K, Takeuchi O, Okamura H, Fujimoto J, Akira S, and Nakanishi K. Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1
. J Immunol 166: 26512657, 2001.[Abstract/Free Full Text]
- Shephard RJ. Cytokine responses to physical activity, with particular reference to IL-6: sources, actions, and clinical implications. Crit Rev Immunol 22: 165182, 2002.[ISI][Medline]
- Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, and Kimoto M. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189: 1777, 1999.[Abstract/Free Full Text]
- Sweet MJ, Campell CC, Sester DP, Xu D, McDonald RC, Stacey KJ, Hume DA, and Liew FY. Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of Toll-like receptor but enhances responses to lipopolysacharide in murine macrophages. J Immunol 168: 392399, 2002.[Abstract/Free Full Text]
- Telega G, Baumgart DB, and Carding SR. Uptake and presentation of antigen to T cells by primary colonic epithelial cells in normal and diseased states. Gastroenterology 119: 15481559, 2000.[ISI][Medline]
- Ten Hove T, Corbaz A, Amitai H, Aloni S, Belzer I, Graber P, Drillenburg P, Van Deventer SJH, Chvatchko Y, and Te Velde AA. Blockade of endogenous IL-18 ameliorates TNBS-induced colitis by decreasing local TNF-
production in mice. Gastroenterology 121: 13721379, 2001.[ISI][Medline]
- Traynor TR, Herring AC, Dorf ME, Kuziel WA, Toews GB, and Huffnagle GB. Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity. J Immunol 168: 46594666, 2002.[Abstract/Free Full Text]
- Vidrich A, Ravindranath R, Farsi K, and Targan S. A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures. In Vitro Cell Dev Biol 24: 188194, 1988.[ISI][Medline]
- Wald D, Qin J, Zhao Z, Qian Y, Naramura M, Tian L, Towne J, Sims JE, Stark GR, and Li X. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat Immun 4: 920927, 2003.[CrossRef][ISI]
- Wantanabe T, Kitani A, Murray PJ, and Strober W. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat Immun 5: 800808, 2004.[CrossRef][ISI]
- Wirtz S, Becker C, Blumberg R, Galle PR, and Neurath MF. Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA. J Immunol 168: 411420, 2002.[Abstract/Free Full Text]
- Yoshizaki K, Nishimoto N, Matsumoto K, Tagoh H, Taga T, Deguchi Y, Kuritani T, Hirano T, and Kishimoto T. Interleukin-6 and its receptor expression on the epidermal keratinocytes. Cytokine 2: 381387, 1990.[CrossRef][Medline]