Alteration of gene expression by intestinal epithelial cells precedes colitis in interleukin-2-deficient mice

Maarten A. C. Meijssen, Steven L. Brandwein, Hans-Christian Reinecker, Atul K. Bhan, and Daniel K. Podolsky

Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114-2696

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intestinal epithelial cells may be actively involved in the immunoregulatory pathways leading to intestinal inflammation. The aim of this study was to assess expression by intestinal epithelial cells of cytokines with potential involvement in the development of intestinal inflammation in interleukin (IL)-2-deficient [(-/-)] mice. Wild-type mice, mice heterozygous for the disrupted IL-2 gene, and IL-2(-/-) mice were studied at 6, 16, and 24 wk of age. The mRNA levels of transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha ), IL-1beta , IL-6, IL-15, KC, JE, and CD14 in colonic and small intestinal epithelial cells were assessed by Northern blot analysis. CD14 was also measured by Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). TGF-beta 1 mRNA was constitutively expressed in both colonic and small intestinal epithelial cells with increased expression in the colonic epithelium of colitic mice. CD14 was detected only in colonic epithelial cells, and mRNA levels increased severalfold in IL-2(-/-) mice with colitis. Northern analysis demonstrated increased levels of TGF-beta 1 and CD14 mRNA in colonic epithelial cells of IL-2(-/-) mice before the development of signs of colitis. CD14 mRNA and protein expression in the epithelial cells of colitic mice were confirmed by RT-PCR and Western blot analysis of isolated cells. In addition, IL-2(-/-) mice also expressed increased levels of IL-15 mRNA in small intestinal and colonic epithelial cells compared with heterozygous control mice. TNF-alpha , IL-1beta , IL-6, KC, and JE mRNAs were only detectable in colonic epithelial cells of mice after the onset of colitis. Enhanced expression of TGF-beta 1, IL-15, and CD14 by colonic epithelial cells may play a role in the subsequent development of colitis in IL-2(-/-) mice.

lipopolysaccharide; CD14; interleukin-15; transforming growth factor-beta

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MICE DEFICIENT [(-/-)] in interleukin (IL)-2 develop normally during the first 3-4 wk of age but subsequently develop anemia leading to ~50% mortality between 4 and 9 wk after birth. Surviving mice develop an inflammatory bowel disease with histological alterations resembling human ulcerative colitis (26). The absence of intestinal inflammation in germ-free IL-2(-/-) mice suggests that luminal bacteria are involved in the development of inflammation (26). Lipopolysaccharide (LPS), a component of the outer membrane of all gram-negative bacteria, is present in large amounts in the lumen of the colon. This common bacterial product is a potent stimulus for the production of a variety of inflammatory mediators, such as IL-1 (5), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha ; see Ref. 15), as well as lipid mediators derived from arachidonic acid, and toxic oxygen radicals (19). CD14, the membrane receptor for complexes of LPS and LPS-binding protein on myeloid cells (33), has recently been shown to be expressed by nonintestinal epithelial cells (10). LPS is able to induce CD14 mRNA in most tissues, including epithelial cells in the respiratory, gastrointestinal, urinary, and reproductive tracts and the liver (10). Increased numbers of macrophages expressing CD14 have been found in inflammatory bowel disease mucosa, which may give rise to increased production of proinflammatory mediators (12).

Recent studies have shown the importance of transforming growth factor-beta 1 (TGF-beta 1) in maintaining homeostasis in intestinal epithelial cell populations. TGF-beta 1 inhibits intestinal epithelial cell proliferation, stimulates cell differentiation, and promotes epithelial restitution after mucosal injury (7). Although TGF-beta 1 increases in association with inflammatory activity, evidence for a protective role for TGF-beta 1 in intestinal inflammation has also been obtained (29).

Development of colitis in IL-2(-/-) mice suggests that IL-2 plays a role in mucosal immunoregulatory pathways. These mice exhibit normal thymic development and thymocyte and peripheral T cell subset composition, suggesting that other cytokines may compensate for the absence of IL-2. IL-15 is a recently identified cytokine that is produced by nonlymphoid cells, including intestinal epithelial cells (11, 23), and utilizes the IL-2 receptor beta  and common gamma c subunits for signal transduction. As a result, IL-15 exerts functional effects on T and B cells similar to those of IL-2 (11).

In addition to their long-recognized role in absorptive function, recent evidence suggests that intestinal epithelial cells are closely integrated into mucosal immune responses. Intestinal epithelial cells are able to present antigen through major histocompatibility complex class II expression (16) and secrete proinflammatory cytokines such as TNF-alpha , IL-1beta , IL-8 (8), IL-6 (28), and monocyte-chemoattractant protein-1 (MCP-1; see Ref. 22).

The present study demonstrates that altered gene expression in intestinal epithelial cells may contribute to the development of intestinal inflammation in IL-2(-/-) mice. Markedly increased levels of CD14 mRNA and TGF-beta mRNA preceded epithelial expression of mRNA of the proinflammatory cytokines TNF-alpha , IL-1beta , IL-6, KC, and JE during the development of colitis in IL-2(-/-) mice. Most importantly, the expression of CD14 and TGF-beta 1 mRNAs increased before histological signs of mucosal inflammation. In addition, IL-15 gene expression by small intestinal and colonic epithelial cells of IL-2(-/-) mice was constitutively increased. In the small and large intestine, IL-15 may compensate in part for the loss of IL-2 in IL-2(-/-) mice.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

IL-2(-/-) mice from a mixed C57BL/6 × 129 Ola background (27) were provided by the Genetic Animal Model Core of the Center for the Study of Inflammatory Bowel Disease. Breeding of heterozygous parents resulted in homozygous [IL-2(-/-)], heterozygous [IL-2(+/-)], and wild-type mice [IL-2(+/+)]. Animals were identified by polymerase chain reaction (PCR) products of genomic DNA on a 2% agarose gel, as described previously (27). Mice were kept in a conventional specific pathogen-free environment and fed with normal mouse chow and water ad libitum.

Peritoneal macrophages. Activated mouse peritoneal macrophages were used as positive controls for the expression of CD14 (20). These were obtained by injecting 6-wk-old C57BL/6 mice intraperitoneally with 3 ml of 3% fluid thioglycollate. Four days later, peritoneal exudate macrophage cells were extracted for use.

Intestinal Epithelial Cells

Animals were killed by cervical dislocation, and the intestines were removed. The total small intestine and colon were separated and flushed with 4°C calcium- and magnesium-free phosphate-buffered saline (CMF). Intestinal epithelial cells were obtained using a modification of previously described methods (3). Briefly, the small intestine or colon was everted over an animal feeding needle (Popper, New Hyde Park, NY) and filled with 4°C CMF, and both ends were closed with surgical silk. The everted intestinal segments were incubated in 50 ml freshly made 3 mM EDTA in CMF, pH 7.4, at 37°C and gently shaken every 5 min. After 20 min, the epithelial cells were centrifuged, washed in CMF, and centrifuged again. Cytospin preparations of epithelial cell populations showed <5% of contaminating nonepithelial cells, using CD45 as leukocyte common antigen marker, CD3 (rat anti-mouse and hamster anti-mouse) as T cell marker, CD45R/B220 as B cell marker, and IM290 as intraepithelial lymphocyte marker. Viability of epithelial cells was >95%, as assessed by 0.1% trypan blue exclusion.

Northern Blot Analysis

Immediately after isolation, epithelial cells were lysed and homogenized in 4 M guanidium isothiocyanate and stored at -80°C until RNA extraction. Frozen homogenized epithelial cells were thawed, and total cellular RNA was isolated by a modification of the method of Chirgwin et al. (4). mRNA (2-4 µg/lane) obtained by oligo(dT)-cellulose column chromatography (Bio-Rad, Hercules, CA) was electrophoresed in a 1% agarose-formaldehyde gel and blotted onto nylon transfer membranes (MSI, Westboro, MA) by standard methods. Isolated cDNA were radiolabeled using a DNA labeling kit (Pharmacia Biotech, Piscataway, NJ) and 50 µCi [I-32P]dCTP (NEN, Boston, MA). The TGF-beta 1 probe was a 592-bp EcoR I/Xma I insert of a human TGF-beta 1 cDNA clone designated phTGF-beta 1; the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a 0.78-kb Pst I/Xba I insert of a human GAPDH plasmid designated phcGAP; the IL-1beta probe was a 1.047-kb Pst I insert of a human IL-1beta clone named IL-1 X-14; the IL-6 probe was a 750-bp Sst I/Hinc II/Xba I fragment of a murine IL-6 phagemid called OMRF 2189; and the TNF-alpha probe was a 1.101-kb EcoR I insert of a murine TNF-alpha clone named pMuTNF, all obtained from American Type Culture Collection (Rockville, MD). The CD14 probe was a 1.022-kb Bgl II/Hind III insert of the murine CD14 cDNA and was a generous gift of Dr. Richard J. Ulevitch. The KC probe was a 800-bp Pst I fragment of the murine KC cDNA, and the JE probe was a 800-bp EcoR I insert of the murine JE cDNA, both generously provided by Dr. Andrew Luster. The IL-15 probe was a 335-bp Dpn I fragment representing part of the coding region of rat IL-15 (23). Hybridization was performed using Rapid-hyb buffer (Amersham International, Arlington Heights, IL) at 65°C. Blots were successively washed at room temperature in 2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) and at 65°C in 1.0-0.1× SSC and 0.1% SDS. Blots were exposed to X-ray film for 16-72 h using an intensifying screen at -80°C. Blots were stripped in 0.1× SSC and 0.1% SDS at 100°C and then were rehybridized with a GAPDH probe to standardize mRNA loading, as described previously (30).

Selection of single primary epithelial cells. A previously described method was used to isolate pure primary murine intestinal epithelial cells (24). After EDTA preparation, the extracted cells were resuspended in sodium tetraphenylborate. Briefly, the crypts were dissociated into single epithelial cells to allow selection of individual cells with a micropipette under microscopic inspection. Fifty cells were resuspended in RPMI media and frozen at -80°C in Trizol reagent (GIBCO-BRL, Gaithersburg, MD). RNA was isolated after addition of 50 µg of carrier rRNA from Escherichia coli W (Sigma).

Reverse transcription and PCR amplification. Total cellular RNA was isolated from Trizol according to the manufacturer's suggested protocol (GIBCO-BRL). Reverse transcription was performed by using 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL), 20 units of RNasin (Promega, Madison, WI), 1 µm dGTP, 1 µm dATP, 1 µm dTTP, 1 µm dCTP, and 1 µg of hexanucleotide random primer (Boehringer Mannheim, Indianapolis, IN) in 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol for 1 h at 37°C.

Oligonucleotide primers were designed by using OLIGO 4.0 software and synthesized on a PCR-mate DNA synthesizer (Applied Biosystems). Primers were intron spanning to allow for differentiation of mRNA from genomic DNA (Table 1). PCR was performed using 1× PCR buffer, 2 mM MgCl2, all four dNTPs (each at 50 µM), each 5' and 3' primer at 1 µM, and 2.5 units AmpliTaq DNA polymerase LD (Perkin-Elmer) in a total volume of 100 µl. Five microliters of the reverse-transcribed RNA from the single cell isolations was run in each reaction in the thermal reactor (Perkin-Elmer). Forty-five cycles were run for 95°C for 1 min, 62°C for 1 min, and 72°C for 1.5 min. PCR products from peritoneal macrophages were cloned into pCR2.1 vector (Invitrogen, San Diego, CA) and sequenced by using the Sequenase 2.0 kit (United States Biochemical, Cleveland, OH). In addition, negative control PCRs were performed with water to rule out amplification contamination products.

                              
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Table 1.   Oligonucleotides used for detection of mRNA by RT-PCR

Immunoblot analysis. Intestinal epithelial cell isolates and peritoneal macrophages were suspended in lysis buffer (1% Nonidet P-40, 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). After 30 min on ice, cell lysates were cleared by centrifugation at 12,000 g for 20 min. The protein concentration in each sample was quantified by the Bradford method (Bio-Rad).

Protein preparations were denatured for 5 min at 95°C in Laemmli buffer and separated by 10% SDS-gel electrophoresis. After transfer onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), the blots were saturated in 5% milk proteins in CMF-0.05% Tween 20. Subsequent washings were in CMF-0.05% Tween 20. The primary antibody was rat anti-mouse CD14 monoclonal antibody (Pharmingen, San Diego, CA) diluted 1:250 in CMF-1% bovine serum albumin. The bound antibodies were detected by peroxidase-labeled sheep anti-rat immunoglobulin (Ig; Amersham International, Buckinghamshire, UK). After incubation with Renaissance chemiluminescence reagents (NEN), the immunoblots were exposed to autoradiography film (Kodak, Rochester, NY).

Histology

At 6, 16, and 24 wk of age, mouse tissues were fixed in 4% paraformaldehyde, treated with graded alcohols and xylenes, and embedded in paraffin, and sections were cut at 4 mm. Light microscopy examination of tissues from IL-2(-/-) mice revealed the same histological pattern as described previously (26). Briefly, after 12 wk of age, all IL-2(-/-) mice developed a diffuse inflammatory bowel disease of the colon with increasing intensity of inflammation from the cecum to the rectum, resulting in pronounced thickening of the bowel wall. The histological features were characterized by marked elongation of the crypts with epithelial cell proliferation, crypt branching, goblet cell depletion, occasional crypt abscesses, and presence of inflammatory cells in the mucosa.

Immunohistochemical Detection of TGF-beta 1

To localize TGF-beta 1 protein, a rabbit polyclonal antibody to a synthetic peptide corresponding to the NH2-terminal 30 amino acids of TGF-beta 1, anti-LC-(1---30), was used, which was generously provided by Dr. Kathy C. Flanders. This antibody was recently reported to localize an active form of TGF-beta 1 (2). After deparaffinization and blocking of endogenous peroxidase with 3% hydrogen peroxide in Tris-buffered saline (TBS), the sections were blocked with 5% normal goat serum-1% bovine serum albumin in TBS. Tissue sections were incubated overnight at 40°C with the primary antibody at an IgG concentration of 5 mg/ml, washed extensively, and then incubated with biotinylated goat anti-rabbit IgG and avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA). Subsequently, sections were incubated with 3,3'-diaminobenzidine and hydrogen peroxide (Sigma Chemical, St. Louis, MO) and counterstained with hematoxylin. In control sections, normal rabbit serum IgG (5 mg/ml) replaced the primary antibody.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CD14 Gene Expression by Intestinal Epithelial Cells

Using Northern blot analysis, we found CD14 mRNA to be constitutively expressed by colonic epithelial cells in all mice. As demonstrated in Fig. 1, colonic epithelial cells of colitic IL-2(-/-) mice at 16 and 24 wk of age expressed severalfold increased levels of CD14 mRNA compared with colonic epithelial cells of wild-type control mice. Most importantly, increased expression of CD14 mRNA in IL-2(-/-) mice was present by 6 wk of age, before the onset of colitis. In contrast, no expression of CD14 mRNA was found in small intestinal epithelial cells from control or IL-2(-/-) mice, before or after the onset of colitis in the latter (Fig. 1).


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Fig. 1.   CD14 mRNA expression by intestinal epithelial cells of interleukin (IL)-2-deficient [(-/-)] mice by Northern analysis. Northern blot of mRNA (4 µg/lane) from colonic and small intestinal epithelial cells; mRNA was prepared, electrophoresed, and hybridized with a probe for CD14 and the constitutively expressed transcript glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described in MATERIALS AND METHODS. C, colonic epithelial cells; SI, small intestinal epithelial cells. Lanes 1 and 5, wild-type control mouse; lanes 2 and 6, 6-wk-old noncolitic IL-2(-/-) mouse; lanes 3 and 7, 16-wk-old colitic IL-2(-/-) mouse; lanes 4 and 8, 24-wk-old colitic IL-2(-/-) mouse.

The epithelial derivation of CD14 was confirmed using isolated primary epithelial cells. As shown in Fig. 2, the absence of leukocyte contamination in preparations of isolated primary colonic intestinal epithelial cells was demonstrated by the absence of CD45 (leukocyte common antigen) transcript. The ability to detect even minimal contamination by this technique was confirmed by the demonstration of transcripts for CD45 in a preparation of isolated epithelial cells to which two leukocytes were added (Fig. 2, lane 7). After confirmation of the purity of isolated primary intestinal epithelial cell preparations, epithelial cells were tested for the presence of CD14 and GAPDH. As shown in Fig. 2, primary epithelial cells from IL-2(-/-) mice contained transcripts for CD14. In contrast, the epithelial cell isolates from control mice did not demonstrate CD14 mRNA.


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Fig. 2.   Determination of CD14 mRNA expression from isolated intestinal epithelial cells by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR). Fifty primary colonic intestinal epithelial cells were isolated through a technique described in MATERIALS AND METHODS. After RNA isolation, RT-PCR was performed for CD14, CD45, and GAPDH. Lanes 1-3, isolations from three 16-wk-old wild-type IL-2(+/+) mice; lanes 4-6, isolations from three 16-wk-old colitic IL-2(-/-) mice; lane 7*, RT-PCRs carried out with RNA from intestinal epithelial cells intentionally contaminated with 2 lymphocytes to test the sensitivity of the PCR for CD45. MØ, murine peritoneal macrophages.

The presence of CD14 protein in the colonic epithelial cells of colitic mice was demonstrated by Western analysis. As shown in Fig. 3, colonic epithelial cells from IL-2(-/-) mice expressed CD14 protein, whereas CD14 was undetectable in epithelial cells from IL-2(+/+) mice. CD14 could not be detected by Western blotting in small intestinal epithelial cell preparations from either wild-type or IL-2(-/-) mice (data not shown).


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Fig. 3.   Protein expression of CD14 by intestinal epithelial cells from IL-2(-/-) mice by Western blotting. Cell lysates (50 µg/lane) from colonic epithelial cells were separated electrophoretically on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After blocking with milk, blots were incubated with rat anti-mouse CD14 monoclonal antibody. Bound antibodies were detected by peroxidase-labeled sheep anti-rat immunoglobulin followed by a chemiluminescence-based detection system. Lanes 1-3, three 24-wk-old wild-type control mice; lanes 4-6, three 24-wk-old colitic IL-2(-/-) mice. Molecular weight markers expressed as kDa.

TGF-beta 1 Gene and Protein Expression by Intestinal Epithelial Cells

TGF-beta 1 mRNA expression was measured by Northern blot analysis. All mice constitutively expressed TGF-beta 1 mRNA in their colonic epithelial cells, as demonstrated in Fig. 4. Steady-state levels of TGF-beta 1 mRNA expression were higher in IL-2(-/-) mice before the onset of colitis than in wild-type mice. Levels of TGF-beta 1 mRNA were further increased in mice after development of colitis. In addition to the 2.5-kb TGF-beta 1 transcript, a smaller transcript, ~2.0 kb in size, was observed (Fig. 4). In contrast to CD14, small intestinal epithelial cells constitutively expressed TGF-beta 1 mRNA, as shown in Fig. 5. In the small intestine, the level of expression was highest in epithelial cells of noncolitic IL-2(-/-) mice. Progressively lower levels of gene expression were found in older, colitic mice.


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Fig. 4.   Transforming growth factor-beta 1 (TGF-beta 1) mRNA expression by colonic epithelial cells. Northern blot of mRNA (2 µg/lane) from colonic epithelial cells hybridized sequentially with probes for TGF-beta 1 and GAPDH. Lanes 1 and 2, wild-type control mice; lane 3, heterozygous control mouse; lanes 4 and 5, 6-wk-old noncolitic IL-2(-/-) mice; lane 6, 16-wk-old colitic IL-2(-/-) mouse; lane 7, 24-wk-old colitic IL-2(-/-) mouse.


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Fig. 5.   TGF-beta 1 mRNA expression by small intestinal epithelial cells. Northern blot of mRNA (2 µg/lane) from small intestinal epithelial cells hybridized sequentially with probes for TGF-beta 1 and GAPDH. Lane 1, wild-type control mouse; lane 2, heterozygous control mouse; lanes 3 and 4, 6-wk-old noncolitic IL-2(-/-) mice; lane 5, 16-wk-old colitic IL-2(-/-) mouse; lane 6, 24-wk-old colitic IL-2(-/-) mouse.

Using immunohistochemical analysis, we localized active TGF-beta 1 in the supranuclear cytoplasm of both colonic (Fig. 6) and small intestinal epithelial cells (data not shown). In the colon, TGF-beta 1 protein was detected in colonocytes along the crypts. Expression of active TGF-beta 1 in intestinal epithelial cells was increased in IL-2(-/-) mice before the onset of inflammation (Fig. 6B) and during active colitis (Fig. 6C) compared with in wild-type mice (Fig. 6A). Staining for active TGF-beta 1 was specific, as demonstrated by the absence of staining when the primary antibody was replaced with normal rabbit serum (Fig. 6D). In the terminal ileum, immunoreactivity for TGF-beta 1 was present in the enterocytes of the middle and upper crypts and along the villi. The expression of TGF-beta 1 in small intestinal epithelial cells did not change during the development of colitis in IL-2(-/-) mice (data not shown).


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Fig. 6.   Immunohistochemical staining of TGF-beta 1 in colonic intestinal epithelial cells. Expression of active TGF-beta 1 in intestinal epithelial cells was increased in IL-2(-/-) mice before onset of inflammation (B) and during active colitis (C) compared with wild-type mice (A). Staining for active TGF-beta 1 was specific, as demonstrated by lack of staining when primary antibody was replaced with normal rabbit serum (D).

IL-15 Gene Expression by Intestinal Epithelial Cells

IL-15 mRNA expression from colonic and small intestinal epithelial cells from IL-2(-/-) and heterozygous control mice was analyzed. The IL-15 mRNA expression by colonic (Fig. 7, lane 3) and small intestinal epithelial cells (Fig. 7, lane 7) of IL-2(-/-) mice was found to be greatly increased compared with the expression of IL-15 mRNA by colonic epithelial cells and small intestinal epithelial cells of wild-type (Fig. 7, lanes 1 and 5) and heterozygous (Fig. 7, lanes 2 and 6) control mice. The IL-15 mRNA expression was not further increased in inflamed colonic tissue (Fig. 7, lane 4).


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Fig. 7.   Northern blot analysis of IL-15 mRNA in isolated intestinal epithelial cells from large (lanes 1-4) and small (lanes 5-7) intestine. Intestinal epithelial cells isolated from IL-2(-/-) mice expressed increased amounts of IL-15 mRNA in the large (lane 3) and small (lane 7) intestine compared with intestinal epithelial cells from wild-type mouse (lanes 1 and 5) and heterozygous control mice (lanes 2 and 6). IL-15 mRNA expression by colonic intestinal epithelial cells in IL-2(-/-) mice did not increase further during inflammation (lane 4). mRNA (2 µg/lane) were analyzed by sequential hybridization with probes specific for IL-15 and GAPDH.

Expression of Proinflammatory Cytokines and Chemokines by Mouse Intestinal Epithelial Cells

Finally, the expression of the proinflammatory cytokines TNF-alpha , IL-1beta , and IL-6 and the mRNA levels of the chemokines KC (the mouse homologue of human GRO-alpha and functionally related to IL-8) and JE (the mouse homologue of the human MCP-1) were assessed (Fig. 6). These cytokines and chemokines are known to attract and activate inflammatory cells and thus are important in the initiation and amplification of the mucosal inflammatory activity (33). In addition, induction of CD14 mRNA results in increased production of proinflammatory mediators (16). Only colonic epithelial cells from 24-wk-old colitic mice expressed detectable mRNA for TNF-alpha , IL-1beta , IL-6, KC, and JE. Colonic epithelial cells from 16-wk-old colitic mice expressed TNF-alpha and JE mRNAs. Colonic and small intestinal epithelial cells from wild-type control and noncolitic IL-2(-/-) mice did not express detectable mRNAs of these cytokines and chemokines. Interestingly, even colitic mice did not express gene transcripts of these cytokines and chemokines in small intestinal epithelial cells, consistent with the inference that expression was related to the inflammatory process (Fig. 8).


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Fig. 8.   Cytokine mRNA expression by intestinal epithelial cells. Northern blot analysis of mRNA (4 µg/lane) from colonic and small intestinal epithelial cells sequentially hybridized with probes for tumor necrosis factor-alpha (TNF-alpha ), IL-1beta , IL-6, KC, JE, and GAPDH. Lanes 1 and 5, wild-type control mouse; lanes 2 and 6, 6-wk-old noncolitic IL-2(-/-) mouse; lanes 3 and 7, 16-wk-old colitic IL-2(-/-) mouse; lanes 4 and 8, 24-wk-old colitic IL-2(-/-) mouse.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal models of colitis offer the opportunity to gain new insight into the mucosal immune system in inflammatory bowel disease. Several new rodent models support the importance of the bacterial flora in causing intestinal inflammation (9). IL-2(-/-) mice that do not succumb to an early anemia develop progressive colonic inflammation by 6-15 wk of age, resulting in death by 10-35 wk of age (26). A role for the enteric bacterial flora in intestinal inflammation is supported by the absence of disease in IL-2(-/-) mice bred in a germ-free environment (26). Another model of colitis, the HLA-B27/beta 2m transgenic rat, also fails to develop intestinal inflammation when raised in germ-free conditions (31). IL-10(-/-) mice develop enteritis when reared under conventional conditions but have attenuated disease when raised in a specific pathogen-free environment (14). Antibiotic treatment of CD45RBhi CD4+ T cell reconstituted severe combined immunodeficient mice diminishes intestinal inflammation (18). Collectively, these animal models show that, in addition to an immunologic defect, nonpathogenic bacteria are required for the development of inflammatory bowel disease.

At present, the exact mechanism by which bacterial components contribute to the pathogenesis of inflammatory bowel disease is unknown. Binding of LPS in the presence of LPS binding protein to the LPS receptor CD14 results in protein tyrosine phosphorylation and the initiation of multiple cellular events, resulting in the production of proinflammatory cytokines (5, 15). The results of Northern studies demonstrated that mouse colonic epithelial cells constitutively express CD14 mRNA. However, wild-type control mice demonstrated low levels of CD14 mRNA compared with IL-2(-/-) mice. This could be caused by downregulation of CD14 mRNA expression in wild-type mice, upregulation of CD14 gene transcription in IL-2(-/-) mice, or contamination by leukocytes.

To fully determine if the CD14 mRNA detected was from colonic intestinal epithelial cells, individual cells were isolated to allow for a pure population of epithelial cells. These were found to be free of mRNA for the leukocyte common antigen CD45. This excluded possible contamination by any cells belonging to leukocyte lineage, including macrophages, 90% of which express CD45 (13). Using single cell reverse transcriptase-PCR, we found colonic epithelial cells from colitic IL-2(-/-) mice to express CD14 mRNA, whereas age-matched wild-type mice did not.

To assess whether CD14 protein was produced, immunoblotting was performed. Colonic epithelial cells from IL-2(-/-) mice expressed CD14, whereas IL-2(+/+) mice lacked CD14. Thus the CD14 mRNA found in the colonic epithelial cells of colitic IL-2(-/-) mice is translated to protein.

CD14 expression has been shown to be upregulated in other systems in response to LPS (10). Tolerance to LPS seen in wild-type mice might be lost in IL-2(-/-) mice, resulting in the activation of a proinflammatory cascade. Of note, no expression of CD14 mRNA or protein was found in the small intestinal epithelial cells in any of the studied mice populations. This is consistent with the inference that CD14 expression by colonic intestinal epithelial cells may be driven directly or indirectly by the higher concentrations of bacteria in the large intestine. This may influence the development of colitis in IL-2(-/-) mice, since they express the receptor for LPS on their epithelial cells and do not develop disease when raised germ free.

In inflammatory bowel disease, TGF-beta 1 can play dual roles. TGF-beta 1 has proinflammatory activities by modulation of isotype switching among B lymphocytes, enhanced cytokine expression by T lymphocytes and macrophages (32), and chemotactic activity for neutrophils and monocytes (21). TGF-beta also has anti-inflammatory properties, including suppression of lymphocyte proliferation, induction of IL-1 receptor antagonist (32), and enhancement of the process of restitution after mucosal injury (7). In the present study, we show that colonic and small intestinal epithelial cells in mice constitutively express TGF-beta 1 mRNA. In noncolitic IL-2(-/-) mice, the levels of TGF-beta 1 mRNA in colonic and small intestinal epithelial cells are higher than wild-type and heterozygous control mice, which could result in proinflammatory activity in IL-2(-/-) mice. In colitic mice, TGF-beta 1 mRNA, minimal in small intestinal epithelial cells, is expressed in high amounts by colonic epithelial cells, suggesting that it could play a role in migration of the colonic epithelial cells into a wounded surface. The increased levels of TGF-beta 1 mRNA correlated with an increased amount of TGF-beta 1 protein in colonic epithelial cells in IL-2(-/-) mice. These observations are in accordance with recent studies in which increased contents of TGF-beta 1 mRNA were found in the colonic mucosa of patients with inflammatory bowel disease (1, 17).

IL-15 appears to be abundantly and constitutively expressed in a variety of tissues and shares many biological properties with IL-2. The present data suggest a regulatory pathway between IL-2 and IL-15 expressions. Increased expression of IL-15 mRNA by epithelial cells may compensate for the lack of IL-2 in IL-2(-/-) mice. Further studies are needed to determine whether increased IL-15 mRNA expression contributes directly to the initiation of colitis in IL-2(-/-) mice.

The expression of proinflammatory cytokines TNF-alpha , IL-1beta , IL-6, KC, and JE by intestinal epithelial cells was only upregulated after the onset of inflammation. Previously, the production of these proinflammatory cytokines has been largely attributed to secretion by leukocytes. This study demonstrates that a similar repertoire of proinflammatory cytokines and chemokines can be expressed by colonic epithelial cells of colitic IL-2(-/-) mice. The cytokine pattern during colitis in the IL-2(-/-) mice resembled the expression of their human homologs in the intestinal mucosa of patients with ulcerative colitis and Crohn's disease (8, 22, 25). Increased expression of TNF-alpha , IL-1beta , IL-6, and the chemokine macrophage inhibitory protein-1 in mouse colon tissue has been demonstrated in a mouse model after induction of colitis by acetic acid (6). In this model, the expression of proinflammatory cytokines was closely correlated with the histological signs of inflammation. These cytokines may therefore be a marker for "nonspecific" inflammation and tissue repair. In contrast, increased gene expression of CD14, TGF-beta 1, and IL-15 in IL-2(-/-) mice is present before signs of colitis and therefore may play a role in the initiation of colitis in these mice. These findings implicate intestinal epithelial cells in the initiation and subsequent development of colitis in IL-2(-/-) mice.

    ACKNOWLEDGEMENTS

We thank Dr. Stephen Simpson, Center for the Study of Inflammatory Bowel Disease (CSIBD) Genetic Animal Model Core for providing interleukin-2-deficient mice and Dr. Emiko Mizoguchi, CSIBD Morphology Core, for performing analysis of cytospin preparations.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41557, DK-07191, and DK-43351 (to D. K. Podolsky) and DK-51003, a research grant from the Crohn's and Colitis Foundation of America (to H.-C. Reinecker), the Dutch Organization for Scientific Research, a Fulbright Scholarship, and Tramedico (M. A. C. Meijssen).

Address for reprint requests: D. K. Podolsky, Gastrointestinal Unit, GRJ-719, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696.

Received 3 September 1996; accepted in final form 19 November 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 274(3):G472-G479
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