Epithelial: Lamina Propria Lymphocyte Interactions Promote Epithelial Cell Differentiation
2007; Elsevier BV; Volume: 134; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.10.022
ISSN1528-0012
AutoresStéphanie Dahan, Giulia Roda, David Pinn, Franziska Roth‐Walter, Okebugwu Kamalu, Andrea P. Martin, Lloyd Mayer,
Tópico(s)Genetic factors in colorectal cancer
ResumoBackground & Aims: Intestinal lymphoepithelial interactions occur in the epithelium and the subepithelial space. We asked whether normal, Crohn's disease (CD), or ulcerative colitis (UC) lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) growth and differentiation. Methods: T84 cells were cocultured with isolated LPL. IECs were then lysed and subjected to measurement of intestinal alkaline phosphatase (IAP) activity; Western blot analysis for MAPK and Akt activation; and real-time polymerase chain reaction to assess caudal-related homeoprotein 2 (CDX2) messenger RNA (mRNA) levels. Tissue sections were immunostained for evidence of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) activation, CDX2, and IAP; and CDX2 mRNA expression was assessed in human colonic biopsy specimens. Results: IAP activity was increased in T84 cells cocultured for 8 days with normal LPL (P < .05) and even greater with CD LPL (P < .001). Crypt IECs in active CD mucosa expressed IAP ex vivo. Phospho-MAPK (extracellular signal-regulated kinase 1/2, p38, and c-Jun-N-terminal kinase) and phospho-Akt were seen as early as 30 minutes after coculture. MAPK activation was greatest in T84 cells cocultured with CD LPL. There was a specific increase in Phospho-p38 MAPK and Phospho-Akt staining in the nuclei of crypt IECs in active vs inactive CD, normal mucosa, and UC mucosa. CDX2 mRNA expression was increased in CD LPL cocultured T84 cells, which did not correlate with CDX2 protein localization ex vivo. Conclusions: There is cross talk between LPL and IECs, which leads to IEC differentiation. The differentiation is accelerated in CD mucosa. Background & Aims: Intestinal lymphoepithelial interactions occur in the epithelium and the subepithelial space. We asked whether normal, Crohn's disease (CD), or ulcerative colitis (UC) lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) growth and differentiation. Methods: T84 cells were cocultured with isolated LPL. IECs were then lysed and subjected to measurement of intestinal alkaline phosphatase (IAP) activity; Western blot analysis for MAPK and Akt activation; and real-time polymerase chain reaction to assess caudal-related homeoprotein 2 (CDX2) messenger RNA (mRNA) levels. Tissue sections were immunostained for evidence of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) activation, CDX2, and IAP; and CDX2 mRNA expression was assessed in human colonic biopsy specimens. Results: IAP activity was increased in T84 cells cocultured for 8 days with normal LPL (P < .05) and even greater with CD LPL (P < .001). Crypt IECs in active CD mucosa expressed IAP ex vivo. Phospho-MAPK (extracellular signal-regulated kinase 1/2, p38, and c-Jun-N-terminal kinase) and phospho-Akt were seen as early as 30 minutes after coculture. MAPK activation was greatest in T84 cells cocultured with CD LPL. There was a specific increase in Phospho-p38 MAPK and Phospho-Akt staining in the nuclei of crypt IECs in active vs inactive CD, normal mucosa, and UC mucosa. CDX2 mRNA expression was increased in CD LPL cocultured T84 cells, which did not correlate with CDX2 protein localization ex vivo. Conclusions: There is cross talk between LPL and IECs, which leads to IEC differentiation. The differentiation is accelerated in CD mucosa. Intestinal epithelial cells (IECs) provide the first line of defense for the host by preventing the entry of potentially dangerous microorganisms into underlying lymphoid tissues. In the intestine, 2 major lymphocyte populations exist: the intraepithelial lymphocytes (IEL), which remain associated with the basolateral membrane of the IECs, and the lamina propria lymphocytes (LPL), which localize to the subepithelial lamina propria and are in contact with IECs via basolateral projections through the semiporous basement membrane.1Hershberg R.M. Mayer L.F. Antigen processing and presentation by intestinal epithelial cells—polarity and complexity.Immunol Today. 2000; 21: 123-128Google Scholar, 2Dahan S. Roth-Walter F. Arnaboldi P. et al.Epithelia: lymphocyte interactions in the gut.Immunol Rev. 2007; 215: 243-253Google Scholar Previous studies from our laboratory suggested that IECs function as antigen-presenting cells and, as such, can promote regulatory T-cell responses in the mucosa.3Allez M. Brimnes J. Dotan I. et al.Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells.Gastroenterology. 2002; 123: 1516-1526Google Scholar, 4Allez M. Brimnes J. Shao L. et al.Activation of a unique population of CD8(+) T cells by intestinal epithelial cells.Ann N Y Acad Sci. 2004; 1029: 22-35Google Scholar, 5Allez M. Mayer L. Regulatory T cells: peacekeepers in the gut.Inflamm Bowel Dis. 2004; 10: 666-676Google Scholar, 6Brimnes J. Allez M. Dotan I. et al.Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease.J Immunol. 2005; 174: 5814-5822Google Scholar, 7Nakazawa A. Dotan I. Brimnes J. et al.The expression and function of costimulatory molecules B7H and B7-H1 on colonic epithelial cells.Gastroenterology. 2004; 126: 1347-1357Google Scholar, 8Perera L. Shao L. Patel A. et al.Expression of nonclassical class I molecules by intestinal epithelial cells.Inflamm Bowel Dis. 2007; 13: 298-307Google Scholar In a reverse interaction, studies by Chen et al suggested that IEL could induce IEC differentiation via the production of keratinocyte growth factor (KGF).9Chen Y. Chou K. Fuchs E. et al.Protection of the intestinal mucosa by intraepithelial δγ T cells.Proc Natl Acad Sci U S A. 2002; 99: 14338-14343Google Scholar Thus, lymphoepithelial interactions have the potential to promote barrier function as well as regulate mucosal immune responses. The control of immune responses in the gut is critical for normal immune homeostasis in the host. Failure to control such responses has been proposed as one mechanism in the development of inflammatory bowel disease (IBD).1Hershberg R.M. Mayer L.F. Antigen processing and presentation by intestinal epithelial cells—polarity and complexity.Immunol Today. 2000; 21: 123-128Google Scholar In one murine model of IBD, the interleukin-10 (IL-10)-deficient mouse,10Kuhn R. Lohler J. Rennick D. et al.Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Google Scholar lymphocyte development and antibody responses are normal, but most animals are growth retarded and anemic. Profound alterations are present in the intestine of these animals, such as a chronic enterocolitis that can involve the entire intestinal tract and is associated with either hyperregenerative or degenerative lesions of the intestinal epithelia. The typical architecture of the mucosa is disturbed by the formation of abnormal crypt and villus structures consisting of branched and fused villi, enlarged and branched crypts, and labyrinthine sheets of enterocytes on the surface.10Kuhn R. Lohler J. Rennick D. et al.Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Google Scholar These findings suggest that IL-10 and probably other factors involved in regulating inflammation may have a role in controlling IEC homeostasis: proliferation vs differentiation. Cell proliferation, lineage-specific differentiation, migration, and finally apoptosis and/or cell shedding are tightly regulated processes that are spatially and temporally regulated along the crypt/surface axis in the colon. The epithelium is characterized by its rapid and constant renewal. This process involves cell generation and migration from the stem cell populations located at the bottom of the crypt to the extrusion of terminally differentiated cells at the tip of the villus.11Grossmann J. Mohr S. Lapentina E.G. et al.Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells.Am J Physiol. 1998; 274: G1117-G1124Google Scholar, 12Mariadason J.M. Nicholas C. L'Italien K.E. et al.Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis.Gastroenterology. 2005; 128: 1081-1088Abstract Full Text Full Text PDF Scopus (163) Google Scholar Thus, the crypt is mainly composed of proliferative and poorly differentiated cells, whereas the villus is lined with functional absorptive, goblet, and endocrine cells.13Crosnier C. Stamataki D. Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.Nat Rev Genet. 2006; 7: 349-359Google Scholar The molecular and cellular mechanisms responsible for the fine coordination between proliferation, migration, and differentiation along the crypt-villus axis are still largely unknown. Several studies suggest that the intestine-specific, caudal-related cdx1 and cdx2 homeobox genes encode nuclear transcription factors that play a critical role in IEC proliferation and differentiation. In contrast to caudal-related homeoprotein (CDX) 1, which is mainly expressed in the crypt compartment (although not restricted to proliferative cells),14Escaffit F. Pare F. Gauthier R. et al.Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells.Biochem Biophys Res Commun. 2006; 342: 66-72Google Scholar the CDX2 homeoproteins are mainly expressed in differentiating enterocytes,15Alkhoury F. Malo M.S. Mozumder M. et al.Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G285-G290Google Scholar triggering growth retardation and cell differentiation by overexpression in several intestinal lines in vitro.16Houde M. Laprise P. Jean D. et al.Intestinal epithelial cell differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor CDX2.J Biol Chem. 2001; 276: 21885-21894Google Scholar Furthermore, genes regulated by either CDX1 or CDX2 generally define a functional differentiated phenotype, such as sucrase-isomaltase,16Houde M. Laprise P. Jean D. et al.Intestinal epithelial cell differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor CDX2.J Biol Chem. 2001; 276: 21885-21894Google Scholar dipeptidyl peptidase IV,14Escaffit F. Pare F. Gauthier R. et al.Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells.Biochem Biophys Res Commun. 2006; 342: 66-72Google Scholar or intestinal alkaline phosphatase (IAP).15Alkhoury F. Malo M.S. Mozumder M. et al.Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G285-G290Google Scholar The mitogen-activated protein kinase (MAPK) family, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, has been shown to play various roles in regulating gene expression via transcription factor phosphorylation. This signaling pathway has been implicated in IEC differentiation. Elevated ERK1/2 activities stimulate cell cycle progression of IECs, whereas low-level activities are correlated with G1 arrest and differentiation.17Boucher M.J. Rivard N. Regulation and role of brush border-associated ERK1/2 in intestinal epithelial cells.Biochem Biophys Res Commun. 2003; 311: 121-128Google Scholar Moreover, p38 MAPK is rapidly activated in IECs induced to differentiate, and this activation enhances CDX2 transcriptional activity.16Houde M. Laprise P. Jean D. et al.Intestinal epithelial cell differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor CDX2.J Biol Chem. 2001; 276: 21885-21894Google Scholar Another signaling pathway, phosphatidylinositol 3-kinase/Akt (PI3K/Akt), has been implicated in cellular differentiation with conflicting reports regarding its ability to promote or inhibit IEC differentiation.18Kim S. Domon-Dell C. Wang Q. et al.PTEN and TNF-α regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-κB-dependent pathway.Gastroenterology. 2002; 123: 1163-1178Google Scholar, 19Laprise P. Chailler P. Houde M. et al.Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.J Biol Chem. 2002; 277: 8226-8234Google Scholar, 20Sheng H. Shao J. Townsend Jr, C.M. et al.Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.Gut. 2003; 52: 1472-1478Google Scholar, 21Wang Q. Wang X. Hernandez A. et al.Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation.Gastroenterology. 2001; 120: 1381-1392Google Scholar Laprise et al19Laprise P. Chailler P. Houde M. et al.Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.J Biol Chem. 2002; 277: 8226-8234Google Scholar described that PI3K is necessary for functional and morphologic differentiation of IECs. The recruitment of PI3K appears to be essential for the integrity of the adherens junctions via Akt and p38 MAPK activation.19Laprise P. Chailler P. Houde M. et al.Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.J Biol Chem. 2002; 277: 8226-8234Google Scholar In contrast, Evers et al reported that the PI3K/Akt pathway mediates proliferative signals in IECs,18Kim S. Domon-Dell C. Wang Q. et al.PTEN and TNF-α regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-κB-dependent pathway.Gastroenterology. 2002; 123: 1163-1178Google Scholar, 20Sheng H. Shao J. Townsend Jr, C.M. et al.Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.Gut. 2003; 52: 1472-1478Google Scholar, 21Wang Q. Wang X. Hernandez A. et al.Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation.Gastroenterology. 2001; 120: 1381-1392Google Scholar and, by the antagonistic effect of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) on the PI3K/Akt pathway, regulates the intestine-specific cdx2 homeobox gene.18Kim S. Domon-Dell C. Wang Q. et al.PTEN and TNF-α regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-κB-dependent pathway.Gastroenterology. 2002; 123: 1163-1178Google Scholar The purpose of this study was to understand better the dysregulation of IECs that occurs in the mucosa of IBD patients. To do so, the role of LPL in IEC differentiation was investigated using a new experimental model set up in our laboratory, ie, freshly isolated LPL derived from the mucosa of normal or Crohn's disease (CD) patients were cocultured with IEC lines, such as T84. T84 cells cocultured with CD LPL displayed a greater increase of IAP activity than with normal LPL. Both the Akt- and the MAPK-signaling pathways were activated in T84 cells cocultured with normal and CD LPL, but the level of CDX2 messenger RNA (mRNA) was significantly higher in T84 cells cocultured with CD LPL. These findings were validated ex vivo using tissue sections of normal, inactive and active CD, and ulcerative colitis (UC) colonic mucosa. Our findings suggest that there is cross talk between LPL and IECs, leading to IEC differentiation, and in CD mucosa this differentiation is accelerated. T84, a human carcinoma cell line derived from a colon carcinoma, and Caco-2, a human colon carcinoma cell line with enterocytic properties, were obtained from the American Type Culture Collection (Manassas, VA). T84 cells were cultured in Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen, Carlsbad, CA)/5% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT) and Caco-2 cells in DMEM (Invitrogen)/10% heat-inactivated FCS and 1% nonessential amino acids (Invitrogen). HT29 Clone 16E, a goblet cell line and derivative of the HT29 human colonic cancer cell line was a gift of Pr. C. L. Laboisse.22Augeron C. Laboisse C.L. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate.Cancer Res. 1984; 44: 3961-3969Google Scholar This cell line was cultured in DMEM/10% heat-inactivated FCS. Surgical specimens from patients undergoing colon resection for IBD or cancer or biopsy specimens from patients undergoing colonoscopy at the Mount Sinai Medical Center were used as a source of IEC and LPL. The surgical specimens obtained from IBD patients were all from noninflamed areas. Isolation of IECs and LPLs was performed as previously described in our laboratory using Dispase II (Roche Diagnostics, Alameda, CA) and collagenase treatment.6Brimnes J. Allez M. Dotan I. et al.Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease.J Immunol. 2005; 174: 5814-5822Google Scholar The biopsy specimens were incubated in RPMI containing 3 mg/mL Dispase II for 15–30 minutes, with vortexing every 5 minutes, in a 37°C water bath. During this treatment, IECs were released from the tissues. The cell suspension resulting from the dispase treatment was washed twice, pelleted, and processed for Trizol extraction (Invitrogen) or further analysis. The IEC lines were seeded in 6- or 12-well plates the night before the experiment in a nonconfluent state (40%–50% confluency) or on regular and inverted Transwells with a diameter of 6.5 mm and a pore size of 3 μm (Corning Inc, Corning, NY) in a confluent state. During the LPL isolation, the cells were serum deprived. The cocultures were set up with a ratio of 1 IEC/1 LPL. For the Transwell studies, the LPL were incubated on the basolateral side of the filter. In some experiments, the IEC lines were preincubated with 100 nmol/L wortmanin (Calbiochem, San Diego, CA), a PI-3K inhibitor, for 90 minutes prior to coculture. The inhibitor was removed before the coculture. The IEC line and LPL alone served as negative controls for all studies. After 1 hour or 1, 2, 4, or 8 days, T84 cells were washed with cold phosphate-buffered saline (PBS) containing Ca2+ and Mg2+ to remove the LPL then frozen at −20°C. The cells were lysed in 350 μL 0.5% Triton X-100, 10 mmol/L Tris-HCl (pH 8), and 150 mmol/L NaCl. Fifty microliters of each sample were mixed with 150 μL of a p-nitrophenyl phosphate solution (Sigma-Aldrich, St. Louis, MO). After a 30-minute incubation at room temperature, the absorbance at 405 nm was measured. The protein content of each sample was quantified using the Bio-Rad DC kit (Bio-Rad, Hercules, CA). Caco-2 cells exhibit a constitutively high level of IAP activity,23Jumarie C. Malo C. Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro.J Cell Physiol. 1991; 149: 24-33Google Scholar, 24Matsumoto H. Erickson R.H. Gum J.R. et al.Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2.Gastroenterology. 1990; 98: 1199-1207Abstract Full Text PDF Scopus (156) Google Scholar so we used this cell line as a positive control in our experiments. After 4 days, the T84 cells were washed with PBS to remove the LPL and then processed for Trizol extraction (Invitrogen). We used a previously described protocol.25Yuen T. Zhang W. Ebersole B.J. et al.Monitoring G-protein-coupled receptor signaling with DNA microarrays and real-time polymerase chain reaction.Methods Enzymol. 2002; 345: 556-569Google Scholar Briefly, 5 μg total RNA was converted into complementary DNA (cDNA) and used for a 40 cycle, 3-step polymerase chain reaction (PCR) using an ABI Prism 7900 (PE Applied Biosystems, Foster City, CA) in 20 mmol/L Tris, pH 8.4, 50 mmol/L KCl, 5 mmol/L MgCl2, 200 μmol/L deoxynucleoside triphosphates, 0.5X SYBR Green I (Molecular Probes, Inc, Eugene, OR), 200 nmol/L each primer, and 0.5 U Platinum Taq (Invitrogen). The number of target copies in each sample was extrapolated from its detection threshold cycle (CT) value using a plasmid or purified PCR product standard curve included on each plate. Three different housekeeping genes were used as controls. Each transcript in each sample was assayed 3 times, and the median CT values were used to calculate the fold increase in gene expression as 2−ΔΔCT, where the ΔΔCT corresponds to (mean CTTARGET NORMAL− CTTARGET UC/CD) − (mean CTCONTROL NORMAL−CTCONTROL UC/CD). By definition, ΔΔCT in the control group equals 0, and 20 equals 1. With this method, we normalized our results to the reference genes and to the control group. The primers used in this study are presented in the Supplementary Data (see Supplementary Table 1 online at www.gastrojournal.org). The same experimental procedure was used for real-time PCR analysis using RNA from freshly isolated IECs from biopsy specimens.Table 1Primers Used for Real-Time PCRCDX2 SenseGCCTGTCACCAGAGCTTCTC AntisenseAGACCAACAACCCAAACAGCβ-actin SenseACTGGAACGGTGAAGGTGAC AntisenseGTGGACTTGGGAGAGGACTGRps 11 SenseGCCGAGACTATCTGCACTAC AntisenseATGTCCAGCCTCAGAACTTCα-tubulin SenseGCCTGGACCACAAGTTTGAC AntisenseTGAAATTCTGGGAGCATGAC Open table in a new tab After 30 minutes to 3 hours of coculture, the T84 cells were washed with cold PBS to remove the LPL and frozen at −80°C. The cells were scraped from the plastic at 4°C into 150 μL lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1% NP-40, 2 mmol/L Na3VO4, 1 mmol/L 4-(2-aminoethyl)-benzene sulfonyl fluoride, 10 mmol/L NaF, 5 mmol/L NaPPi, 10 mmol/L glycerophosphate). The lysate was then sonicated and solubilized for 30 minutes at 4°C and centrifuged at 14,000g for 20 minutes at 4°C. The protein concentration of the supernatant was determined as described above. Equal protein concentrations of whole-cell lysates were resolved by 10% SDS-PAGE. The proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA) and incubated overnight at 4°C with either antiphospho-Akt, antiphospho-ERK1/2, antiphospho-p38, antiphospho-JNK, anti-Akt (Cell Signaling, Danvers, MA) or anti-ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Cell Signaling). The binding of antibodies was revealed with an enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Pittsburgh, PA). Biopsy specimens from patients undergoing endoscopic examination (normal and inactive and active CD and UC) and colonic tissues from C57BL/6 (wild-type [WT]) and RAG1−/− mice, obtained from The Jackson Laboratory, Bar Harbor, ME, were paraffin embedded. All mice were housed under specific pathogen-free conditions in individually ventilated cages at the Mount Sinai School of Medicine Animal Facility. All experiments were performed following institutional guidelines. Tissue sections were dewaxed and rehydrated. IAP was stained according to the manufacturer's protocol using the BCIP/NBT Alkaline Phosphatase Substrate Kit IV (Vector Laboratories, Burlingame, CA). The slides were then mounted with a coverslip using Vectashield Hard Set (Vector Laboratories). The slides were examined with a Zeiss Axioskop light microscope at ×20 magnification. For immunohistochemistry, tissue sections were dewaxed and rehydrated, and endogenous peroxidase activity was quenched with 1.5% H2O2 in methanol (15 minutes room temperature). Antigen retrieval was performed by heating for 10 minutes at 100°C in 0.01 mol/L sodium citrate. The stainings were done according to the manufacturer's protocol using a rabbit or mouse Histostain-Plus kit (Invitrogen). The tissues were incubated overnight with either antiphospho-Akt, antiphospho-PTEN, anticyclin D1, antiphospho-ERK1/2, antiphospho-p38, antiphospho-JNK (dilution 1/50, Cell Signaling), or anti-CDX2 (Biogenex) antibodies in PBS containing 0.1% Triton X-100 or Ready-to-Use anti-CDX2 (Biogenex, San Ramon, CA). The slides were counterstained with Mayer's Hematoxylin Solution (Sigma-Aldrich). The slides were then mounted with a coverslip using Vectashield Hard Set (Vector Laboratories). The slides were examined with a Zeiss Axioskop light microscope at ×20 and ×100 magnification. Results are presented as the mean ± standard deviation. Because of the variability of human samples, statistical significance was determined by 1-way ANOVA followed by posttest Newman–Keuls, t test followed by Mann–Whitney U test, or 1-way ANOVA followed by Kruskal–Wallis test. P < .05 was considered significant. The changes in band intensity were quantified by densitometric analysis using the Scion Image program for PC (Scion Corp, Frederick, MD). All experiments were repeated at least 3 times, and a representative result is shown for each experiment. IAP is exclusively expressed in villus enterocytes in normal mucosa and hence serves as an excellent marker of crypt-villus differentiation.23Jumarie C. Malo C. Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro.J Cell Physiol. 1991; 149: 24-33Google Scholar, 24Matsumoto H. Erickson R.H. Gum J.R. et al.Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2.Gastroenterology. 1990; 98: 1199-1207Abstract Full Text PDF Scopus (156) Google Scholar The putative effect of normal LPL on epithelial differentiation was investigated using T84, a crypt-like intestinal epithelial cell line, cocultured with freshly isolated normal LPL for 1 hour, 4 and 8 days (Figure 1A). These different time points were based on the kinetics of T84 cell differentiation. We followed differentiation by quantifying IAP activity. Normal LPL induced a slight increase in IAP activity after 4 days of coculture with nonpolarized T84 cells (179.3 ± 60.9 vs 67.2 ± 41.6 nmol pNP/mg prot, respectively), and this reached significance after 8 days (287.6 ± 151.8 vs 110.9 ± 67.7 nmol pNP/mg prot, respectively, P < .05), suggesting that normal LPL have an effect on IEC differentiation. The colonic mucosa of IL-0 knockout mice has been reported to display abnormal biclonal crypts10Kuhn R. Lohler J. Rennick D. et al.Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Google Scholar before the presence of active inflammation. We were interested to determine whether IBD LPL played a role in this process. T84 cells were cocultured with freshly isolated CD or UC LPL for 1 hour and 4 and 8 days. CD LPL induced a highly significant increase in IAP activity as rapidly as after 4 days of coculture with T84 cells (Figure 1A) (355.7 ± 217.8 vs 67.2 ± 41.6 nmol pNP/mg prot, respectively, P < .001); this increase was even greater after 8 days (576.4 ± 51.2 vs 110.9 ± 67.7 nmol pNP/mg prot, respectively, P < .001). We performed this assay using Caco-2 and HT29 Cl16E cells (data not shown), and each cell line showed a different pattern of differentiation in the presence of LPL, particularly with CD LPL. The T84 cell line was the most responsive in terms of an increase in IAP activity. This cell line mimics an intestinal crypt cell, which can potentially explain its susceptibility to differentiate. Nonpolarized Caco-2 cells are known to be highly differentiated (baseline IAP activity was already quite high), so further differentiation was difficult to detect. The HT29 Cl16E cell line is a goblet-like cell line, and IAP activity is not an accurate marker of differentiation in this cell type. Freshly isolated LPL from UC patients had a cytotoxic effect on the IEC lines, so these results are not shown. We are currently trying to better understand this phenomenon. Taken together, these findings are consistent with enhanced differentiation of intestinal crypt cells induced by CD LPL. To determine whether IEC differentiation in the presence of CD LPL was mediated by soluble factors or required cell contact, T84 cells were seeded on conventional or inverted Transwells the night before the experiment. The inverted Transwells were flipped the morning of the LPL isolation. LPL were added to the basolateral side of the IECs allowing contacts between T84 cells and LPL while assessing the involvement of soluble factors in conventional Transwells. IAP activity was assessed after 1 or 2 days of coculture (Figure 1B). In T84 cells cocultured with either normal or CD LPL in conventional Transwells, the IAP activity was comparable with the control (data not shown). In T84 cells cocultured with. When T84 cells were cocultured with CD LPL in inverted Transwells, IAP activity was increased after 1 day (781.3 ± 550 vs 377 ± 85.7 nmol pNP/mg prot, respectively), and this induction was significant after 2 days of coculture (858 ± 753.9 vs 377 ± 69.9 nmol pNP/mg prot, respectively, P < .05). In contrast, with normal LPL, the IAP activity was comparable with controls. These findings strongly suggest that the enhancement of IEC differentiation by CD LPL is contact dependent. The PI3K/Akt pathway has been shown to affect proliferation, differentiation, and apoptosis in a number of cells,26Garcia Z. Kumar A. Marques M. et al.Phosphoinositide 3-kinase controls early and late events in mammalian cell division.EMBO J. 2006; 25: 655-661Google Scholar including IECs. PI3K was shown to modulate enterocyte-like differentiation in HT29 and Caco-2 cells, as well as goblet cell differentiation.18Kim S. Domon-Dell C. Wang Q. et al.PTEN and TNF-α regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-κB-dependent pathway.Gastroenterology. 2002; 123: 1163-1178Google Scholar, 20Sheng H. Shao J. Townsend Jr, C.M. et al.Phosphatidylinositol 3-kinase mediates proliferative s
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