Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines

GASTROENTEROLOGY 1996;111:8–18

ALIMENTARY TRACT Immunologic, Functional, and Morphological Characterization of Three New Human Small Intestinal Epithelial Cell Lines GERALD PANG,* ANDRE BURET,‡ EDWARD O’LOUGHLIN,§ ARABELLA SMITH,x ROBERT BATEY,§ and ROBERT CLANCY* *Faculty of Medicine and Australian Institute of Mucosal Immunology, University of Newcastle, Newcastle, New South Wales, Australia; ‡ Biological Science and Gastrointestinal Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; § Gastroenterology Unit and Department of Paediatrics, John Hunter Hospital, Hunter Area Health, Newcastle, New South Wales, Australia; and xDepartment of Cytogenetics, Royal Alexandria Children’s Hospital, Camperdown, New South Wales, Australia

Background & Aims: Epithelial cell cultures can be used for the study of epithelial cell biology, although human small intestinal cultures have not been available to date. The aim of this study was to characterize three cell lines derived from normal human duodenum. Methods: Cells were cultured from tissue fragments obtained from endoscopic biopsy specimens and characterized with respect to morphology and cytokine gene expression and for the presence of vectorial transport. Results: All cell lines grew as polarized continuous monolayers and were mostly cuboidal in shape but were not immortalized. Cells showed junctional complexes and sparse microvilli. All cell lines showed cytokeratins and mucin antigen but not chromagranin and messenger RNA for epidermal growth factor, interleukin 6, and vascular cell adhesion molecule 1. Disaccharidase activities were low and correlated with the low proportion of cells (1%–10%), showing positive immunocytochemistry for sucrase. Monolayer resistance varied from 30 to 200 V. One monolayer (BN) consistently showed secretion in response to forskolin (10 mmol/L), which could be inhibited by chloride-free buffer and apical addition of the chloride channel blocker diphenylamine decarboxylate. No monolayer had evidence of glucose transport. Conclusions: These three nonimmortalized lines show morphological, phenotypic, and transport characteristics of crypt-like intestinal epithelial cells. The pattern of messenger RNA expression suggests a growth-promoting and immunomodulatory role.

T

he study of function and structure in human intestinal epithelia has been facilitated by the establishment of cultured colonic epithelial cell monolayers. Because of the unavailability of cultured human small intestinal epithelial cell lines, earlier studies have used Madine–Darby canine kidney monolayers1,2 or rat small intestinal epithelial cells (IEC-6)3 as alternative model systems. Therefore, investigations using human intestinal epithelial cell lines in vitro have been restricted to / 5E0F$$0011

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transformed enterocytes derived from colon adenocarcinoma.4 – 6 In general, these cell lines cultured as monolayers on porous supports show several features characteristic of intestinal epithelium, including brush borders, junctional complexes, and defined lateral spaces. This in vitro model has been used to study cellular differentiation,4,5 regulation of epithelial transport processes,7 – 9 polymeric immunoglobulin (Ig) A receptors (secretory component),10 and HLA molecules.11,12 In addition, recent studies have reported that colonic epithelial cells were capable of presenting antigen to primed T cells13 and expressed cytokine genes,14 indicating that intestinal epithelial cells are active participants in inflammatory responses in mucosal defense. The establishment in vitro of epithelial cell monolayers from human small intestine would represent a major breakthrough and provide researchers with a powerful system for novel investigation of development and function in the human small intestine. The aim of this study was to culture in vitro nontransformed epithelial cell lines obtained from histologically normal duodenal biopsy tissues of patients undergoing investigation for diarrhea. Monolayers were established and characterized on the basis of their morphology, phenotype, ion transport properties, and cytokine messenger RNA (mRNA) gene expression.

Materials and Methods Duodenal Biopsy Three patients had biopsy specimens taken from the duodenum in the course of upper gastrointestinal endoscopy Abbreviations used in this paper: DMEM, Dulbecco’s modified Eagle medium; GM-CSF, granulocyte-macrophage colony–stimulating factor; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MMLV-RT, Moloney murine leukemia virus–reverse transcriptase; VCAM-1, vascular cell adhesion molecule 1. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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for diarrhea (50-year-old man and 78-year-old woman) and iron deficiency (49-year-old woman). In each patient, no macroscopic abnormality was noted, and histology was normal with no evidence of mucosal inflammation or neoplasia. The study was approved by the University of Newcastle Human Ethics Committee and the Hunter Area Research Ethics Committee.

Tissue Culture Biopsy tissue (5–10 mg) was placed in a Petri dish containing Dulbecco’s modified Eagle medium (DMEM) (Commonwealth Serum Laboratory, Melbourne, Victoria, Australia) with streptomycin, penicillin, and gentamicin (Trace Biosciences, Sydney, New South Wales, Australia) and 5% fetal calf serum (Trace Biosciences) and finely minced with a scalpel. Tissue fragments were resuspended in 2 mL Iscove’s DMEM containing 5% pooled human AB serum from 4 normal patients and 5% fetal calf serum, 2.5 mmol/L L-glutamine, 2-mercaptoethanol (1 1 1005 mol/L), and antibiotics; seeded in 200-mL aliquots into wells of a 96-well round-bottomed microtiter plate; and incubated at 37⬚C and 5% CO2 in air. The cultures were fed every 2–3 days by removing half of the medium and replacing it with fresh warm medium. This procedure was repeated until outgrowth of fibroblasts and epithelial cells occurred, i.e., 2–3 weeks from the time of initiation of culture. At this point, culture supernatants were removed, and 100 mL of Ca2//Mg2/-free trypsin/versene solution containing 0.05% trypsin and 0.50 mmol/L ethylenediaminetetraacetic acid (EDTA) (Commonwealth Serum Laboratory) was added to each well to detach adherent cells, which were transferred to a 24-well microtiter plate with each well containing 2 mL of Iscove’s DMEM. The cells were cultured for 3 days, at which time epithelial cells were the dominant population with few fibroblasts. After treatment with trypsin/ versene solution, the cells were pooled and then transferred into two 25-cm2 flasks and cultured in DMEM containing 10% fetal calf serum, L-glutamine, and antibiotics. The slowgrowing fibroblasts remaining were eliminated by limiting dilution so that cultures containing epithelial cells only were selected for further expansion. After 1–2 passages, pure epithelial cells, as verified by cytokeratin staining, were obtained. Three cell lines designated BN, LG, and WT were established from 3 patients. All three cell lines tested negative for mycoplasma contamination (data not shown).

Immunocytochemistry Adherent cells were grown in an eight-well Lab-Tek glass slide (Nunc, Roskilde, Denmark) until 60%–70% confluency, washed in phosphate-buffered saline (PBS), fixed in cold acetone, air-dried, and stained using an indirect immunoperoxidase method (Dako–Patt ABC kit; Dako, Carpinteria, CA). Monoclonal antibody (1:40 dilution) was added to each well and incubated for 30 minutes at room temperature in a humid chamber. After washing, biotinylated rabbit anti-mouse IgG at 1:500 dilution was added, and the slides were incubated

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for another 30 minutes. The slides were then washed in PBS, and peroxidase-labeled avidin-streptavidin complex at 1:1000 dilution was added. After a final washing in PBS, the slides were developed using 3,3ⴕ-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Chemical, Sydney, New South Wales, Australia) as a substrate, were counterstained with hematoxylin, and then were mounted. Monoclonal antibodies to brush border–associated and mucin antigens were kindly provided by Dr. R. Whitehead15 (Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia). Antibodies to brush border enzymes, dipeptidyl peptidase, and sucrase isomaltase (HBB 2/19/20) were obtained previously from Dr. H.-P. Hauri16 (Basel University, Basel, Switzerland). HBB antibody (1/23) specific for a brush border–associated protein of 180 kilodaltons was obtained from Dr. A. Quaroni (Cornell University, Ithaca, NY). Antibody to chromagranin granules was obtained from Hybritech Inc. (San Diego, CA). Small intestinal mucus antigen and large intestinal mucus antigen antibodies were obtained from Dr. P. Hertzog (Monash University, Melbourne, Victoria, Australia). Antibodies raised against the MUC1 core peptide repeat sequence (BC3) and against the MUC2 core peptide repeat sequence (3A2) were obtained from Dr. I. F. C. McKenzie (Austin Research Institute, Heidelberg, Victoria, Australia). Monoclonal antibodies to cytokeratin 8 and 18 were purchased from Boehringer Mannheim (Mannheim, Germany) and used at 1:100 dilution. Monoclonal antibody to human fibroblast (Dako-Patt 5B5; Dako) was used as described above. Tissue sections from a normal duodenal biopsy specimen were used as positive controls.

Immunoblotting For detection of cytokeratins, epithelial cells grown in 25-cm2 culture flask were rinsed four times with cold PBS and then scraped into a sterile centrifuge tube containing 10 mL cold PBS. Human foreskin fibroblasts were used as negative controls. After centrifugation, the cells were resuspended in 0.1 mL of sodium dodecyl sulfate sample buffer (10 mmol/L Tris-HCl, pH 8.0; 1 mmol/L EDTA; 2.5% sodium dodecyl sulfate; and 5% 2-mercaptoethanol) and transferred to a microfuge tube. The sample was boiled for 10 minutes and then centrifuged at 12,000 rpm for 20 minutes in a microfuge. The total protein content of the supernatant was determined using a protein assay reagent kit (Pierce Chemical Co., Rockford, IL). One-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed according to Laemmli.17 Equal amounts of protein (20 mg) were loaded onto the gels. Electrophoretic transfer of separated proteins on a 12% polyacrylamide slab gel was performed in a solution consisting of 48 mmol/L Tris-HCl, 38 mmol/L glycine, 20% methanol, and 0.05% sodium dodecyl sulfate, pH 8.3. To visualize the protein bands, the blots were stained with Ponceau S solution (Sigma Chemical Co., St. Louis, MO) for 5 minutes with constant agitation. The blots were destained by several washes with distilled water and then incubated with a mixture of primary monoclonal antibodies to cytokeratins (8 and 18) or

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an isotype-matched mouse IgG1 at 1:1000 dilution in blocking buffer (Tris-buffered saline, pH 8, and 5% bovine skim milk) for 60 minutes. After washing twice in 0.05% Tween 20/Tris-buffered saline, the blots were incubated with horseradish peroxidase–conjugated rabbit anti-mouse IgG (1:5000 dilution; Amersham Corp., Little Chalfont, England) in Tris-buffered saline containing 5% skim milk and 0.05% Tween 20. Finally, the blots were developed using an enhanced chemiluminescence kit (Amersham Corp.) and exposed for 5 minutes on a blue light–sensitive Hyperfilm (Amersham Corp.).

Disaccharidase Assay Sucrase- and maltose-hydrolyzing activity was performed on the cell homogenates according to the method described by Dahlqvist.18 Briefly, cells grown to confluency in 75 cm2 culture flasks were scraped off and homogenized in a homogenizer with cold 2.5 mmol/L EDTA solution. Freshly isolated mucosa from rat intestine was used as positive control. The homogenates were centrifuged to remove debris at 400g at 4⬚C. Ten microliters of homogenates was added in duplicate to wells of a 96-well round-bottomed plate. Ten microliters of sucrose or maltose was added to each well. After incubation at 37⬚C for 60 minutes, the enzymatic reaction was stopped by placing the plates in a 120⬚C oven for 2 minutes. Blanks were prepared by heating the enzyme and substrate mixture immediately after mixing. For determination of glucose or maltose, 300 mL of Tris–glucose oxidase solution was added to each well. After incubation at 37⬚C for 1 hour for development of color, the plates were read at 450 nm, and the amount of sugar was determined against a known standard. Results were expressed as milliunits per milligram of protein. One unit is defined as the activity that hydrolyzes 1 mmol of substrate per minute at 37⬚C. Proteins were assayed by the method of Lowry using a Bio-Rad kit (Bio-Rad Laboratories, Richmond, CA).

Ultrastructural Studies Epithelial cells were cultured in a 25 cm2 flask until confluent. The cells were removed by treatment with trypsin/ versene solution, centrifuged, resuspended in 5 mL DMEM, and cultured on collagen-coated Transwell chamber polycarbonate filters (6.5 mm; Costar, Cambridge, MA) for 24 hours. The chambers were removed and fixed overnight at 4⬚C in Karnovsky fixative (2.5% gluteraldehyde and 2% formaldehyde), postfixed for 2 hours in 1% osmium tetroxide, dehydrated in ethanol, and infiltrated in Spurr’s medium (FSE, Sydney, New South Wales, Australia). Membrane sheets were then excised and embedded in Spurr’s medium. Sections (80 nm) were stained with saturated uranyl acetate in 50% ethanol and 0.04% lead citrate. Micrographs were obtained with a Phillips CM 10 transmission electron microscope at 80 kV.

In Vitro Transport Confluent epithelial cells were grown on collagencoated Costar Transwell filters (0.4 mmol/L and 24.5 mm;

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Costar) as described above. After 24 hours, confluency of the monolayers was verified under an inverted microscope, and monolayers were mounted in Ussing chambers. Each chamber exposed 1.12 cm2 of the basal or apical side of the monolayer to 10 mL of Krebs’ buffer (37⬚C, pH 7.4; 140 mmol/L Na/; 127.5 mmol/L Cl0; 25 mmol/L HCO30; 10 mmol/L K/; 1.25 mmol/L Ca2/; 1.1 mmol/L Mg2/; 2 mmol/L H2PO4 ; and 300 mOsm Krebs’ buffer) with glucose (10 mmol/L) on the serosal side and mannitol (10 mmol/L) on the mucosal side. Buffers were oxygenated with 95% oxygen and 5% CO2 . Spontaneous transepithelial potential difference was determined, and the monolayer was clamped at zero voltage by continuously introducing an appropriate short-circuit current with an automatic voltage clamp (DVC 1000; World Precision Instruments, New Haven, CT), except for 3–5 seconds at the times when opencircuit current was measured. Measurement of tissue conductance was performed by voltage clamping the monolayer and recording the resultant short-circuit current. Tissue conductance was then calculated from change in short-circuit current and change in potential difference according to Ohm’s law.19 Monolayers were discarded when tissue conductance was ú30 millisiemens/cm2 because this indicated the presence of an incomplete confluency not detected under light microscopy (data not shown). Readings were obtained after 5 minutes of equilibration and then every 5 minutes for 20 minutes, and full tracings of short-circuit current were recorded continuously on a pen chart recorder. Electrical parameters were measured under basal conditions followed by a second period when the secretagogue 10 mmol/ L forskolin was added to the serosal side of the tissue. Experiments were repeated in the presence of chloride-free buffer. In a third experiment, 1 mmol/L of the Cl0 channel blocker diphenylamine decarboxylate was added to the mucosal buffer after stimulation with forskolin as described above. To assess glucose-coupled sodium transport, 30 mmol/L glucose was added to both sides of the monolayer after the basal period in a separate experiment. The change in short-circuit current was determined from the peak increase after secretagogue addition. In addition, the percentage decrease in short-circuit current was determined after adding diphenylamine decarboxylate when the short-circuit current had reached the steady state.

Reverse Transcription and Polymerase Chain Reaction Confluent monolayers of epithelial cells from cultures maintained for 3 days were treated with trypsin/versene, and the cells were recovered by centrifugation. After washing in PBS, the cells were counted in a hemocytometer chamber and adjusted to 0.5–1.0 1 106 cells/mL PBS. One milliliter of the cell suspension in an Eppendorf tube was centrifuged at 6000 rpm for 15 seconds. After removing the supernatant, the cell pellet was used for mRNA extraction. mRNA gene expression of cytokines and adhesion molecules was determined by reverse-transcription polymerase chain reaction as described previously.20

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Briefly, complementary DNA (cDNA) synthesis was performed at 42⬚C for 1 hour in 30 mL final volume containing the following: 3 mL of Moloney murine leukemia virus–reverse transcriptase (MMLV-RT) (200 U/mL); 1.0 mL of ribonuclease inhibitor (40 U/mL); 6.0 mL of 51 MMLV-RT buffer (250 mmol/L Tris-HCl, 375 mmol/L KCl, and 15 mmol/L MgCl2 ); 3.0 mL of dithiothreitol (0.1 mol/L); 3.0 mL of oligo(dT)15 (0.5 ng/mL); and 3.0 mL of acetylated bovine serum albumin (1 mg/ mL) and 1.5 mL of deoxynucleoside triphosphate (10 mmol/L each). Five microliters of first-strand cDNA was added to the polymerase chain reaction mix containing the following: 0.2 mL Taq DNA polymerase (50 U/mL); 1.2 mL of 25 mmol/L MgCl2 ; 2 mL 101 polymerase chain reaction buffer; 1 mL of 4 mmol/L mix; 8.6 mL sterile distilled water; and 1 mL of 20 mmol/L antisense primer and 1 mL of 20 mmol/L sense primer. The mixture was subjected to amplification using a thermal cycler (Pharmacia LKB, Uppsala, Sweden) set at 94⬚C for 1 minute, 57⬚C for 2 minutes, and 72⬚C for 3 minutes for 25– 35 cycles. Polymerase chain reaction fragments were separated on ethidium bromide–stained 2% agarose gel and then blotted onto Hybond nylon membrane (Amersham Corp.) for Southern blot analysis with oligoprobes. Hybridization was performed using enhanced chemiluminescence and detection kits (Amersham Corp.). Blots were exposed on a blue light–sensitive Hyperfilm MP (Amersham Corp.) to detect light output. Primers and oligoprobes for interleukin (IL) 1, IL-6, IL-8, epidermal growth factor, and granulocyte-macrophage colony–stimulating factor (GM-CSF) were purchased from Clontech (Palo Alto, CA). The following primers and oligoprobe for vascular cell adhesion molecule 1 (VCAM-1) were synthesized according to published DNA sequences21,22 by Bresatec (Adelaide, South Australia, Australia): intercellular adhesion molecule 1 (ICAM1) primers, 5ⴕ AGA ACC TTA CCC TAC GCT GCⴕ 3 (443– 462) and 5ⴕ CAG TAT TAC TGC ACA CGT CAG 3ⴕ (939– 918); ICAM-1 probe, 5ⴕ GCT GGC AGG ACA AAG GTC TGG AGC TGG TAG (704–675); VCAM-1 primers, 5ⴕ GGA AGT GGA ATT AAT TAT CCA A 3ⴕ (1599–1622) and 5ⴕ CTA CAC TTT TGA TTT CTG TG 3ⴕ (2040–2021); and VCAM-1 probe, 5ⴕ GAT TCA CAT TCA TAT ACT CCC GCA TCC TTC 3ⴕ (1839–1810).

Tumorigenicity Test The cell lines were tested for tumorigenic potential in the nude mouse.23 Mice were injected with 106 cells subcutaneously and evaluated for tumor growth from 7 to 48 days.

Analysis of Data Electrical parameters are depicted as mean { SE. Effect of forskolin on short-circuit current was assessed by paired Student’s t test.

Results

Tumorigenicity Test No tumor formed in all mice inoculated with BN, LG, or WT cells, indicating that the cell lines were not tumorigenic. Immunochemistry The proportion of cells expressing brush border– associated antigens is shown in Table 1. The proportions of cells in each cell line stained with brush border peptidase and disaccharidase antibodies were low, ranging from 10% to 1%. All cell lines stained with anti-small intestinal mucus antigen antibody but not anti-large intestinal mucus antigen antibody. In addition, a large proportion of the cells in all three cell lines were stained with antibodies to mucin core peptide repeat sequences (MUC1 and MUC2). None of the cell lines was stained with antibody to chromogranin. Continued passage in culture did not significantly alter the staining pattern in the cell lines. Finally, all three cell lines were stained with antibody to cytokeratin (Figure 3) but did not stain for fibroblast-specific antibody (data not shown). The cytokeratin antibody showed a distinct filamentous staining that is characteristic of epithelial cells. The presence of keratin polypeptides was shown by immunoblotting, showing the expression of a strong 52.6-kilodalton and a weak 45-kilodalton band (Figure 4), which correspond to K8 and K18 keratins, respectively.24 Disaccharidase Activity Maltase and maltose-hydrolyzing activity caused by either sucrase, maltase, or isomaltase was low compared with fresh rat mucosa. Of all the cell lines, BN showed the highest level of activity (Table 2). Transport In Vitro

Characterization of the Cell Lines by Light and Electron Microscopy All three cell lines (BN, LG, and WT) grew as confluent monolayers with doubling times of 32 hours / 5E0F$$0011

at serial passage 18 (BN) or 24 hours (LG and WT) at serial passages 9 and 11, respectively (Figure 1). The ultrastructure of the cell lines is shown in Figure 2. Cells formed characteristic continuous epithelial monolayers, were mostly cuboidal in shape, and showed a typical, although not well-differentiated, apical microvilli. The adluminal ends of cells were joined by junctional complexes with well-developed desmosomes and zonulae occludens and/or adherens. Cells showed characteristics of generic epithelial cells and of cells containing uncharacterized granules, possibly secretory in nature (Figure 2).

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Data from the transport studies are shown in Table 3 and Figures 5 and 6. The figures show traces from confluent monolayers derived from subject BN. Initial experiments indicated monolayer viability of more than WBS-Gastro

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60 minutes after mounting in the Ussing chambers. Baseline electrical data are shown in Table 2. Monolayers showed a wide variability in electrical parameters, particularly conductance, which varied from 1.3 to 27 millisiemens/cm2 and short-circuit current varied from 0 to 13 mA/cm2. Stimulation of monolayers from BN with 10 mmol/L forskolin resulted in an increase in short-circuit current (3.0 { 0.4 mA/cm2; n Å 13) (Figure 4), although the response in monolayers from LG and WT was inconsistent. This increase seemed to be caused by Cl0 secretion because it was significantly reduced (P õ 0.025) in the presence of Cl0-free buffer (1.5 { 0.1 mA/cm2; n Å 7). In addition, 1006 mol/L diphenylamine decarboxylate, a chloride channel blocker, caused a significant reduction in short-circuit current (3.9 { 0.1 mA/cm2 to 1.0 { 0.3 mA/cm2; P õ 0.0001) in monolayers from BN. Two different patterns of inhibition were observed and are shown in Figure 6A and B. Diphenylamine decarboxylate resulted in an immediate reduction in short-circuit current to baseline. In the second pattern, diphenylamine decarboxylate caused a rapid reduction, followed by a transient increase and then persistent decrease to baseline levels in 50% of monolayers. The ionic basis of this second pattern has not been examined. Addition of 30 mmol/L glucose to the mucosal side of the monolayer did not induce a change in short-circuit current in any preparation (n Å 10). The negative results were consistent with the lack of mRNA gene expression for Na/ glucose cotransporter isoform SLGT1, as determined by reverse-transcription polymerase chain reaction (data not shown). mRNA Gene Expression Cell lines were examined for the expression of a range of proinflammatory cytokine mRNA genes, including IL-1, IL-6, IL-8, and GM-CSF, and adhesion molecules, such as ICAM-1 and VCAM-1. As shown in Figure 7, mRNA gene expression was detected only for IL-6, VCAM-1, and epidermal growth factor but not IL-1b, IL-8, or GM-CSF (data not shown).

Discussion

Figure 1. Monolayer cultures of BN, LG, and WT small intestinal epithelial cells. Formation of domes (arrows) occurred in all preparations (original magnification 1251).

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In this report, we characterize the first human small intestinal epithelial cell lines growing as monolayers in vitro. The cells were cultured from histologically normal human duodenal mucosa and did not form tumors when inoculated into nude mice. The cell lines form confluent polarized monolayers and show a number of feature characteristics of intestinal epithelium, including microvilli, brush border enzymes, junctional complexes, defined apical and basolateral surfaces, and apical Cl0

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Figure 2. Transmission electron micrograph of human small intestinal epithelial cell monolayers (BN) cultured in vitro on synthetic membranous supports. (A ) Microvilli (arrow) protrude from the apical surface of the monolayer and some of the cells contain cytoplasmic granules (arrowhead), possibly secretory in nature (bar Å 5 mm). (B ) Adjacent cells are joined by typical electron-dense junctional complexes with zonulae occluders/ adherens (z) and desmosomes (d) (bar Å 1 mm).

secretion. In addition, they constitutively express mRNA for IL-6, VCAM-1, and epidermal growth factor. The most prominent morphological features of epithelial enterocytes are the well-defined apical microvilli and well-developed junctional complexes.5,6 Ultrastructural studies showed that the cells described in this report showed features characteristic of small bowel epithelium, including apical microvilli and junctional complexes. The cells appeared morphologically heterogeneous with respect to cell types because some contained well-defined cytoplasmic granules, possibly secretory in nature, whereas others did not. In the light of the fact that all cell lines stained strongly with antibodies to mucin epitopes, it remains to be established whether these granules belong to morphologically undifferentiated goblet cells. When inoculated into nude mice, all three cell lines did not form tumors, suggesting that the cells were nonmalignant. However, all three cell lines showed abnormal karyotype with modal chromosomal numbers 78,

76, and 75 for LG, BN, and WT, respectively (results not shown). All the cell lines studied grew rapidly as adherent polarized monolayers with varying degree of pleomorphism. Although all the cell lines stained strongly with antibodies to mucin epitopes specific for the small intestine, only a small percentage of cells (1%–10%) in each line showed staining with antibodies to brush border enzymes. The results obtained when the cells were measured for disaccharidase activities were consistent with the low proportions of cells expressing brush border enzymes. None of the cell lines stained with antibody to chromagranin, a marker for enteroendocrine cells. The cells characteristically expressed cytokeratin proteins that were similar in pattern in the first and serially passaged cultures, suggesting that the cells continued to express cytokeratins. In all three cell lines, the relatively short and sparse microvilli observed under transmission electron microscopy are indicative of immature enterocytes from small intestinal crypts.

Table 1. Proportion of Epithelial Cells Expressing Brush Border–Associated Antigens Cell line BN LG WT

Dipeptidyl peptidase IV (%)a

Sucrase isomaltase (%)

BC3 (%)

3A2 (%)

SIMA (%)

LIMA

Chromagranin A

Cytokeratin (%)

10 5 5

5 1 1

90 100 90

80 90 100

100 100 100

— — —

— — —

100 100 100

NOTE. BC3, MUC1 core peptide repeat sequence. 3A2, MUC2 core peptide repeat sequence. Chromagranin A, endocrine cell granules. LIMA, large intestinal mucus antigen; SIMA, small intestinal mucus antigen. a Percentage of cells stained with antibody.

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Figure 4. Western blot analysis of cytokeratin in the first (top) and 25th (bottom) passage cultures of BN, LG, and WT cells.

In the mammalian small intestine, enterocyte differentiation from the crypt to the villus epithelium is accompanied by the appearance of glucose-coupled sodium absorption, whereas Cl0 secretion occurs in both immature and mature cells.25,26 Cell monolayer showed basal electrical parameters to T84 colon cancer cells27 and human airway epithelial cells.28 However, conductance of our small intestinal cell lines was substantially lower than values observed in T84 cells,27 although similar to what we have reported previously for intact human small intestine.29 Stimulation with forskolin, a known stimulator of intracellular adenosine 3ⴕ, 5ⴕ-cyclic monophosphate, induced a small increase in short-circuit current that was inhibited in the presence of Cl0-free buffer and diphenylamine decarboxylate, indicative of Cl0 secretion. The degree of secretion elicited in the enterocyte monolayer was similar to that observed in the airway epithelia but less than maximal stimulation observed in T84 cells.30 Basal potential difference and short-circuit current in LG and WT monolayers were lower than observed in BN monolayers, and the response to forskolin was not as Table 2. Disaccharidase Activities in Cultured Small Intestinal Epithelial Cell Lines (BN, LG, and WT), Rat Mucosa, and Cultured Foreskin Fibroblasts Specific enzyme activity (U/mg protein)

Figure 3. Indirect immunoperoxidase staining of anticytokeratin antibodies in the 25th passage culture of BN, LG, and WT cells (original magnification 4001).

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Cell lines

Sucrase

Maltose-hydrolyzing activitya

BN LG WT Rat mucosa Foreskin fibroblast

2.08 0.68 0.51 12.79 0

1.05 0.39 0.26 80.20 0

NOTE. Enzyme activities were measured on cell homogenates after 1 week in culture. Results obtained from the cell lines are the mean of two different passages. a Either sucrase, isomaltase, or maltase.

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Table 3. Basal Electrical Parameters From Human Small Intestinal Monolayers

Cell line

Potential difference (mV )

Short-circuit current (mA/cm2)

Conductance (millisiemens/cm2)

BN (n Å 20) LG (n Å 9) WT (n Å 13)

00.311 { 0.08 00.06 { 0.05 00.03 { 0.06

2.42 { 0.70 0.56 { 0.66 0.23 { 1.1

10 { 2 10 { 2 12 { 2

consistent. One possible explanation for the differences in transport characteristics in our three lines may be related to the rapidity of cellular division and, thus, the degree of cellular differentiation. Both LG and WT were rapidly dividing cells with doubling time of 18 hours, whereas BN cells required 32 hours. Others have shown that cellular differentiation is required for the expression of adenosine 3ⴕ,5ⴕ-cyclic monophosphate–dependent Cl0 channels in colonic epithelial cells.31 Our cell lines did not show evidence of glucose-coupled sodium absorption, a finding consistent with our failure to show mRNA gene expression for SGLT1. However, the presence of Cl0 secretion, especially in BN, and the apparent lack of a functional glucose-coupled sodium transporter are consistent with the characteristics of immature cryptlike enterocytes. The observation that all three lines are of an immature nature is further supported by transmission electron microscopy, which showed a population of enterocytes that were cuboidal in shape and lined with a well-defined but poorly differentiated brush border. Intestinal epithelial cells express mRNA and/or secrete

Figure 5. Effect of forskolin (10 mmol/L) on short-circuit current in (A ) normal Krebs’ bicarbonate buffer and (B ) chloride-free buffer. Monolayers were derived from subject BN.

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Figure 6. Effect of 1 mmol/L diphenylamine decarboxylate (DPC ) on forskolin-stimulated short-circuit current. (A ) Rapid inhibition (2 of 8 preparations). (B ) A biphasic response characterized by transient inhibition followed by a brief increase in short-circuit current (6 of 8 preparations). In both patterns, the final short-circuit current decreased to the baseline. Monolayers were derived from subject BN.

IL-1a, IL-1b, IL-6, IL-8, transforming growth factor b, and tumor necrosis factor a.14,32 IL-8 and transforming growth factor b mRNA genes are constitutively expressed in human colonic cell lines T84, Caco-2, SW620, and HT29, whereas mRNA for IL-1a, IL-1b, IL-10, and tumor necrosis factor a are expressed differentially in some cell lines.14 The rat small intestine epithelial cell line IEC-6 has been shown to produce IL-6, which was enhanced by exposure to IL-1b or transforming growth factor b.32 In the present study, all three cell lines constitutively expressed mRNA for IL-6, epidermal growth factor, and VCAM-1. However, cytokine expression for IL-1b or IL-8, as reported for colon epithelial cells, was not detected by polymerase chain reaction. IL-6, a pleiotropic cytokine, may have an important role in the regulation of inflammation and immune responses in the small intestine. This inflammatory cytokine is not expressed in colonic epithelial cells,14 suggesting that there may be a regional difference in the expression of cytokines along the gut. Alternatively, variation in cytokine message expression in colon cancer epithelial cell lines may have been the result of the transformation process. In murine small intestine, transforming growth factor b is prominent in enterocytes located on the villus and not in those in the crypt.33 Because transforming growth factor b message was undetectable in all cell lines reported in this study, it further supports the evidence that WBS-Gastro

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Figure 7. Constitutive expression of IL-6, VCAM-1, and epidermal growth factor mRNA genes in BN, LG, and WT small intestinal epithelial cells. mRNA was extracted from confluent monolayers and the cDNA products obtained by reverse-transcription polymerase chain reaction were detected by Southern blot analysis using IL-1b, IL-8, IL6, VCAM-1, and epidermal growth factor oligoprobes. Equivalent loading of each sample was verified by the glyceraldehyde 3-phosphate dehydrogenase message shown below. C, positive controls for each message derived from cDNA of cell lines known to express the specific cytokines.

epidermal growth factor is also an autocrine growth factor for small intestinal epithelial cells is not clear. Adhesion molecules influence inflammatory and immune responses through interactions between leukocytes and tissue structural cells.36,37 The ICAM-1 mediates interactions in immunity and inflammation by binding to the leukocyte adhesion receptor molecule lymphocyte function–associated antigen (CD11a/CD18).38 Likewise, the VCAM-1 mediates the selective migration of lymphocytes into an inflammatory site by binding to the b1 integrin receptor very late activation 4 on lymphocytes.39 In addition, VCAM-1 and/or very late activation 4 selectively promotes the recruitment of eosinophils and basophils in allergic reactions.40 In this study, it was shown that the VCAM-1 mRNA gene was expressed by all three epithelial cell lines, indicating that epithelial cells in the small intestine may play a central role in the initiation and regulation of inflammation and immune responses. Recent studies have shown that colonic epithelial cell lines HT29 and Caco-2 express mRNA for ICAM-1, and the protein product41 is expressed on the cell surface. Whether VCAM-1 is expressed in these colonic cells is not known. However, ICAM-1 mRNA expression was undetectable in the cell lines described in this report. Regional specialization in the mucosal immune system in the gut has been reported for colonic and small bowel intraepithelial lymphocytes, which differ in terms of phenotype and function.42 Thus, epithelial cells may play a role in determining the preferential recruitment of intraepithelial lymphocytes. In summary, this study reports the first characterization of small intestinal epithelial cell monolayers cultured from histologically normal human duodenal mucosa. All the cell lines showed morphological and functional characteristics typical of small intestinal crypt cells. They secrete Cl0 and express mRNA genes for IL-6, VCAM1, and epidermal growth factor. Importantly, the findings in all three normal cell lines are similar and they are nonmalignant, which contrasts with the considerable variation in colon cancer–derived epithelial cell lines. Therefore, these cell lines represent a valuable in vitro model system for the study of differentiation, function, and immunity in the human small intestine.

References they are immature crypt enterocytes. All the cell lines constitutively express the mRNA gene for epidermal growth factor that has been shown to play a role in regulating intestinal transport.34 In particular, epidermal growth factor stimulates the absorption of intestinal nutrients and electrolytes, an effect caused by an increase in the brush border surface area.35 However, whether / 5E0F$$0011

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phils and basophils but not neutrophils to endothelium. J Immunol 1992;148:1066–1092. 41. Kvale D, Krajci P, Brandtzaeg P. Expression and regulation of adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) in human intestinal epithelial cell lines. Scand J Immunol 1992;35:669– 676. 42. Camerini V, Panwala C, Kronenberg M. Regional specialization of the mucosal immune system. Intraepithelial lymphocytes of

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Received October 31, 1994. Accepted February 21, 1996. Address requests for reprints: Gerald Pang, M.D., Faculty of Medicine, Royal Newcastle Hospital, Newcastle, New South Wales 2300, Australia. Fax: (61) 49-292-591.

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