Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1

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University of Nebraska - Lincoln of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty USDA Agricultural Research Service --Lincoln, Nebraska Epithelial and
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University of Nebraska - Lincoln of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty USDA Agricultural Research Service --Lincoln, Nebraska Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1 Ivonne Stamm Institut für Hygiene und Infektionskrankheiten der Tiere Melanie Mohr Institut für Hygiene und Infektionskrankheiten der Tiere Philip S. Bridger Institut für Hygiene und Infektionskrankheiten der Tiere Elmar Schröpfer Institut für Hygiene und Infektionskrankheiten der Tiere Matthias König Institut für Virologie See next page for additional authors Follow this and additional works at: Part of the Agricultural Science Commons Stamm, Ivonne; Mohr, Melanie; Bridger, Philip S.; Schröpfer, Elmar; König, Matthias; Stoffregen, William C.; Dean-Nystrom, Evelyn A.; Baljer, Georg; and Menge, Christian, Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1 (2008). Publications from USDA-ARS / UNL Faculty. Paper This Article is brought to you for free and open access by the USDA Agricultural Research Service --Lincoln, Nebraska at of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of of Nebraska - Lincoln. Authors Ivonne Stamm, Melanie Mohr, Philip S. Bridger, Elmar Schröpfer, Matthias König, William C. Stoffregen, Evelyn A. Dean-Nystrom, Georg Baljer, and Christian Menge This article is available at of Nebraska - Lincoln: INFECTION AND IMMUNITY, Nov. 2008, p Vol. 76, No /08/$ doi: /iai Copyright 2008, American Society for Microbiology. All Rights Reserved. Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1 Ivonne Stamm, 1 Melanie Mohr, 1 Philip S. Bridger, 1 Elmar Schröpfer, 1 Matthias König, 2 William C. Stoffregen, 3 Evelyn A. Dean-Nystrom, 3 Georg Baljer, 1 and Christian Menge 1 * Institut für Hygiene und Infektionskrankheiten der Tiere, 1 and Institut für Virologie, 2 Justus-Liebig-Universität, D Giessen, Germany, and Pre-Harvest Food Safety and Enteric Diseases Research, National Animal Disease Center, USDA, Agriculture Research Service, Ames, Iowa Received 6 May 2008/Returned for modification 19 June 2008/Accepted 26 August 2008 Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77 cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-, MCP-1, and RANTES mrna. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mrna. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa. * Corresponding author. Mailing address: Institut für Hygiene und Infektionskrankheiten der Tiere, Frankfurter Strasse 85-89, D Giessen, Germany. Phone: Fax: Published ahead of print on 2 September Some Shiga toxin-producing Escherichia coli (STEC) strains, enterohemorrhagic E. coli strains, are food-borne pathogens which evoke life-threatening diseases in humans (26). Cattle and other ruminants can shed STEC for long periods and are a major reservoir for zoonotic STEC (6, 61, 70, 72). Emerging human STEC infections make the reduction of STEC shedding by reservoir species a current challenge in veterinary public health. Several lines of evidence indicate that STEC adherence to bovine intestinal epithelial cells is essential for long-term STEC colonization of ruminants. Within hours after oral infection, STEC O157:H7 can be detected throughout the gastrointestinal tract, including the rumen, of cattle (6, 18). As early as 4 days after inoculation, STEC strains colonize epithelial cells in the ileum, cecum, colon, rectum, and gall bladder in weaned calves (8, 59, 62). STEC O157:H7 strains principally colonize the rectoanal junction of weaned calves and older cattle (10, 33, 44), but O157:H7 colonization also can occur at other sites of the bovine intestinal tract (8, 23, 55). The ability of the majority of bovine STEC isolates to intimately attach to cells and rearrange the actin cytoskeleton (attachingand-effacing [AE] lesions) (71) may facilitate adherence to the intestinal epithelium (5, 9, 44). Signature-tagged mutagenesis studies showed that factors not involved in AE lesion formation further support STEC colonization of the bovine intestinal epithelium (11, 68). The duration of STEC shedding correlates with epithelial cell turnover in the bovine intestine (35). Vaccination strategies directed against proteins involved in STEC adherence to the bovine intestinal mucosa have been successful in reducing STEC O157:H7 infection in cattle (49, 50, 52, 67). Other STEC factors also may influence the duration of colonization. Recent studies suggest that STEC suppresses the bovine host s immune response, limits mucosal inflammation, and maintains intestinal homeostasis. Lymphostatin (2) and Shiga toxin 1 (Stx1) (39) block the proliferation of bovine lymphocytes in vitro. Stx1 alters the cytokine response of bovine intraepithelial lymphocytes (42), cells that are scattered within the epithelial layer and are affected in vivo by Stx1 from STEC strains that do not colonize next to organized lymphoid tissues (38). Some STEC O157:H7 strains exhibit a tropism for the follicle-associated epithelium of Peyer s patches in the bovine intestine (51) and may release modulating factors adjacent to induction sites of the immune response. Development of a cellular immune response against STEC antigens is significantly delayed in calves inoculated with Stx2-producing E. coli O157:H7 compared to that of calves inoculated with a nontoxigenic O157:H7 strain (22). Immune-modulating STEC factors are potential targets for future strategies aimed at reducing STEC shedding in cattle, but their mode of action in the bovine intestine is only partially understood. Stx proteins are potent 1A:5B-structured cytotoxins with RNA N-glycosidase activity that inhibit protein synthesis of sensitive cells (13). Independent of cytolethal effects, Stx-in- 5381 5382 STAMM ET AL. INFECT. IMMUN. duced expression of chemokines in human intestinal epithelial cells (66, 74) may be a key event in the pathogenesis of human STEC-related diseases (47). There is conflicting evidence about whether Stx are involved in STEC-epithelial cell interactions in cattle. Pruimboom-Brees et al. (54) did not detect Stx receptors in the bovine intestinal epithelium, but other researchers described the presence of Gb 3 /CD77 (a glycosphingolipid Stx receptor found on sensitive cells) and Stx binding sites in the bovine intestinal mucosa (20, 21, 57). Stx receptor expression was ascribed to proliferating epithelial cells at the bases of the crypts (21). However, Stx1 binding to these receptors in vitro is not cytolethal but leads to a rapid degradation of the toxin in the cellular lysosomal compartment (21). We hypothesized that bovine colonic epithelial cells are resistant to the cytotoxic, but not the modulatory, effects of Stx1. We used flow cytometry, fluorescence microscopy, and mrna quantitation by real-time PCR to analyze Stx receptor distribution and the effects of Stx1 in a colonic crypt cell culture system. MATERIALS AND METHODS Collection of bovine tissues. Intestinal specimens for the isolation of bovine colonic crypts were obtained from freshly slaughtered cattle (18 to 24 months old) of different breeds from a local abattoir (Giessen, Germany). Frozen bovine distal colon tissues were available from earlier infection studies at the National Animal Disease Center in Ames, IA, for the detection of CD14 and CD77 by immunofluorescence. These last tissue specimens had been collected from 4-month-old weaned calves at necropsy, snap-frozen in isopentane, and stored at 80 C. Because STEC pathogenicity for cattle is restricted to the immediate neonatal phase (7, 8, 10, 62), we considered both 4-month-old and older cattle representative of cattle prone to asymptomatic STEC colonization. Isolation of bovine colonic crypts. Crypts were isolated according to Föllmann et al. (15), with some modifications. Tissue samples, consisting of approximately 30-cm-long sections from the ascending colon of each animal, were extensively washed with isotonic NaCl solution (4 C) and cut open longitudinally. Mucus was removed by slightly scraping tissue with a glass slide. Specimens were transported to the laboratory in phosphate-buffered saline (PBS) supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2.5 g/ml amphotericin, 4 mm L-glutamine (PAA Laboratories GmbH, Pasching, Austria), 2.5 g/ml gentamicin (Biochrom AG, Berlin, Germany), and 0.2% glucose (Merck, Darmstadt, Germany). Mucosal tissue was separated from the lamina propria by scraping with a sterile glass slide, homogenized by a razor blade in Hank s buffered saline solution (HBSS; 4 C) and transferred to 50-ml tubes (Greiner, Frickenhausen, Germany). After centrifugation (5 min, 130 g, 4 C) the mucus-containing layer was withdrawn together with the supernatant and discarded. The remaining pellet was washed twice and enzymatically digested in 60 ml of Dulbecco s modified Eagle s medium (DMEM; Invitrogen, Karlsruhe, Germany) plus 60 ml of HBSS, 150 U/ml collagenase I (Biochrom), 2 mm L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 2.5 g/ml gentamicin, and 2.5 g/ml amphotericin. After incubation (45 min, 37 C, 8% CO 2 ) with stirring (100 rpm), the solution was passed twice through 0.6-by-25-mm needles to further disintegrate cell clots. After a second digestion step (10 min) and subsequent centrifugation (7 min, 202 g, 4 C), the remaining pellet was resuspended in a 2% D-sorbitol solution (in HBSS) and centrifuged (5 min, 50 g, 4 C). The pellet was resuspended in 2% D-sorbitol. The procedure was repeated several times until the supernatant was clear. The isolated crypts (the pellet) were washed with HBSS (3 min, 65 g, 4 C) and resuspended in DMEM. Primary bovine colonic crypt cell cultures. Crypts were seeded at a density of 350 to 400 crypts/cm 2 (microscopic counts; 200 to 300 cells per crypt) in 25-cm 2 culture flasks (Costar, Bethesda, United Kingdom) coated with rat tail collagen (2.8 l/cm 2 [CollagenR; Serva, Heidelberg, Germany], 1:10 in distilled water) and cultivated (37 C, 8% CO 2 ). Crypt culture medium consisted of DMEM supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2.5 g/ml gentamicin, 2.5 g/ml amphotericin, 5 g/ml bovine transferrin (Invitrogen), 10 g/ml bovine insulin (Biochrom), 0.15 mm nonessential amino acids (Biochrom), 1 g/ml hydrocortisone (Sigma-Aldrich, Steinheim, Germany), 30 ng/ml epidermal growth factor (AF Schützdeller GmbH, Germany), 4 mm L-glutamine, and 2.7 mg/ml glucose (Sigma-Aldrich). For the first 24 h, 10% fetal calf serum (FCS; Invitrogen) was added. Further cultivation was performed in medium containing the supplements named above with FCS at 2% only and an addition of 0.5% bovine pituitary extract (C.C.Pro GmbH, Neustadt, Germany). In some experiments, cells were cultured for 48 to 72 h in medium additionally supplemented with lipopolysaccharide (LPS) from E. coli O111:B4 (25 g/ml; Sigma- Aldrich) or sodium butyrate (2 mm or 4 mm; Sigma-Aldrich). Flow cytometry analysis of cells harvested from representative cultures (n 16) 96 h after seeding of the crypts showed that the cultures consisted of 93.82% 2.61% (mean standard deviation) cytokeratin-positive (i.e., epithelial) and 6.07% 4.16% vimentin-positive (mesenchymal) cells. These cultures are referred to as primary colonic crypt cell cultures. Generation and cultivation of bovine colonic crypt mesenchymal cell lines. Primary bovine colonic crypt cells grown for 96 h in collagen-coated petri dishes (diameter, 3.5 cm; Falcon), were lipofected with the psv3neo plasmid carrying the SV40 large-t antigen (48). Four micrograms of plasmid and 20 l of Superfect transfection reagent (Qiagen, Hilden, Germany) were mixed in 100 l of DMEM, incubated for 10 min at 25 C and added to 1 ml of DMEM in each dish. After incubation (3 h, 37 C), supernatants were withdrawn, and cells were covered with 5 ml of fresh medium. After 48 h at 37 C, the selection of transformed cells was started by adding 40 l of Geneticin solution (50 mg/ml; Sigma- Aldrich). Medium was changed every 48 to 72 h. When clonal proliferation became visible, cells were detached by trypsin (0.25% in HEPES buffer supplemented with 0.2% EDTA; PAA), seeded into fresh cell culture flasks (Falcon), and further propagated with RPMI 1640 medium containing 2 mm stabilized L-glutamine and 2.0 mg/ml NaHCO 3 (PAN Biotech GmbH, Aidenbach, Germany), 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS. Cytotoxicity assays. The effect of Stx1 on the cellular metabolic activity was quantified by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide, Sigma) reduction assay as described previously (39). Cells ( per well) were seeded into microtiter plates and incubated (72 h, 37 C) with purified Stx1 ( to 2,000 CD 50 /ml; CD 50 [50% cytotoxic dose] as determined on Vero cells, where 1 CD 50 was calculated to be equivalent to 0.4 to 0.8 pg/ml of purified Stx1 [46]) without or after preincubation with purified mouse anti-stxb1 (immunoglobulin G1 [IgG1], clone 13C4; 1.5 g/ml [63]). Methods for Stx1 and anti-stx1 subunit B (StxB1) purification were previously published (39). Epithelial cell-specific apoptosis was quantified using an M30 CytoDEATH fluorescein isothiocyanate (FITC) test kit (Roche, Mannheim, Germany) and flow cytometry analysis. Flow cytometry. There is no indication that trypsin cleaves the Stx receptor Gb 3 /CD77, a glycosphingolipid, or that trypsin cleavage of proteinaceous Gb 3 / CD77 ligands on the surface of bovine cells unmasks Stx receptors and makes them more accessible for toxin binding. The only physiological Gb 3 /CD77 ligand detected so far in cattle is the type I interferon receptor protein (IFNAR) (17). However, the affinity of Stx1 for Gb 3 /CD77 is greater than the affinity of IFNAR for Gb 3 /CD77 (27). Thus, the use of trypsin was considered appropriate to generate cell suspensions for flow cytometry analysis of StxB1 binding to bovine colonic cells. Cells were detached by trypsinization, transferred to V-shaped microtiter plates (Greiner), and pelletized by centrifugation (150 g, 7 min, 4 C). Detection of cell surface antigens and surface binding sites for the recombinant StxB1 (rstxb1) was performed with native cells as described previously (37, 60). Leukocyte antigens were detected using antibodies listed in Table 1. Binding of rstxb1 was detected by subsequent incubation with purified rstxb1 (30 g/ml PBS [60]), anti-stxb1 clone 13C4 (45 g/ml PBS), and anti-mouse IgG( ) FITC (Medac, Hamburg, Germany). For detection of intracellular antigens, cells were fixed in paraformaldehyde (2% in PBS; 30 min, 25 C), washed with PBS, and permeabilized with digitonin (0.005% in PBS, 10 min, 25 C [Sigma-Aldrich]). Subsequently, cells were incubated with FITC-conjugated antipan cytokeratin antibody (clone C-11, 1:100 in PBS [Sigma-Aldrich]) or antivimentin antibody (clone 3B4, 1:13 in PBS [Dako, Hamburg, Germany]) and phycoerythrin-conjugated anti-mouse IgG antibody (1:50 in PBS [Sigma-Aldrich]). Finally, cells were washed twice and analyzed on an Epics Elite analyzer (Beckman-Coulter, Krefeld, Germany), acquiring 5,000 events per sample. Data were analyzed with FCS Express version 2 software (DeNovo software, Thornhill, Canada). Gates were defined according to the negative control (PBS) and secondary antibody control included in each test series, defining less than 2% of the cells as positive. Immunofluorescence staining for microscopy. Cells grown in 12-well culture plates (Costar) were fixed and permeabilized as described for flow cytometry but omitting trypsinization. While being washed in PBS, the floors of the wells were punched out using a heated copper cutter and placed in PBS-prefilled 12-well plates for further processing. Labeling was carried out as described above, and samples were mounted on glass slides and dried overnight at 4 C. Immunofluorescence microscopy was performed using a Leica DMRB Laborlux 12 micro- VOL. 76, 2008 STX1-SUSCEPTIBLE CELLS IN BOVINE COLONIC MUCOSA 5383 Target antigen TABLE 1. Antibodies used in this study Antibody Host Isotype Label Clone Source a Working dilution CD11a Mouse IgG2a IL-A99 DW Nondiluted CD11b Mouse IgG1 IL-A15 JN Nondiluted CD11c Mouse IgG1 IL-A16 JN Nondiluted CD14 Mouse IgG1 CC-G33 DW Nondiluted CD21 Mouse IgG2a IL-A65 JN Nondiluted CD25 Mouse IgG1 IL-A111 JN Nondiluted CD71 Mouse IgM IL-A77 JN Nondiluted CD77 Rat IgM Beckman-Coulter 1:10 in PBS CD80 Mouse IgG1 IL-A190 DW Nondiluted CD86 Mouse IgG1 IL-A158 DW Nondiluted CD172a Mouse IgG1 IL-A24 JN Nondiluted MHC-I Mouse IgG2a IL-A88 JN Nondiluted MHC-II DQ Mouse IgG2a CC158 DW Nondiluted MHC-II DR Mouse IgG1 CC108 DW Nondiluted ACT-2 Mouse IgG1 CACT26A VMRD Inc. 1:400 in PBS Vimentin Mouse IgG1 3B4 Dako A/S 1:13 in PBS Pan-cytokeratin Mouse IgG1 FITC C-11 Sigma-Aldrich 1:100 in PBS Mouse IgG( ) FITC Medac 1:200 in PBS Mouse IgG Phycoerythrin Sigma-Aldrich 1:50 in PBS Rat IgM( ) Phycoerythrin Beckman-Coulter 1:200 in PBS Rat IgM FITC Dianova 1:100 in PBS a DW, Dirk Werling, Royal Veterinary College London, United Kingdom; JN, Jan Naessens, International Livestock Research Institute, Nairobi, Kenya. scope with an analog camera (for conventional fluorescence microscopy) or a Leica DM IRBE microscope (for confocal fluorescence microscopy). For immunofluorescence detection of CD14- and CD77-positive cells in situ, 5- to 6- m sections of isopentane-flash-frozen bovine colon tissues were cut, fixed in cold ethanol ( 20 C) for 10 s, and stored at 80 C until used. Slides were removed from 80 C storage and set directly 6 inches under a fluorescent light source for 18 to 20 h to quench nonspecific fluorescence, rehydrated (0.023 M PBS [ph 7.4], 30 min), and incubated in diaminobenzidine solution (Histo- Mark kit; Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 10 min to block naturally fluorescent eosinophils (28). Sections were incubated for 30 min at room temperature with Image it FX signal enhancer (Molecular Probes, Eugene, OR), washed with PBS with 0.125% Tween 20 (PBS-T), incubated for 18 h at 4 C with combined rat anti-human CD77 IgM (clone 38.13, 1:20 in PBS-T [Serotec, Raleigh, NC]) and mouse anti-m-m9 (CD14) IgG1 (clone CAM36A, 1:200 [VMRD, Pullman, WA]), washed in PBS-T, and incubated for 30 min (25 C) with Alexa Fluor 594-labeled goat anti-rat IgM and Alexa Fluor 488- labeled goat
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