ABSTRACT
The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb3/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb3/CD77+ cells were activated CD3+ CD6+ CD8α+ T cells, whereas only some CD4+ T cells and B cells expressed Gb3/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb3/CD77+ cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host.
Persistently infected ruminants—especially cattle—are the main natural reservoir of Shiga toxin (Stx)-producing Escherichia coli (STEC) (14). STEC can be detected in as many as 60% of bovine fecal samples from several countries all over the world (5). The pathogenicity of STEC for cattle is low and limited to sporadic cases of diarrhea in young calves. For adult animals, no disease condition has been linked to STEC infections to date (35). Nevertheless, STEC represents an emerging group of zoonotic enteric pathogens (39): when transmitted to humans, some STEC strains cause epidemics and sporadic cases of diarrhea, and in most cases intestinal inflammation then progresses to hemorrhagic colitis (HC). Patients with HC may develop life-threatening sequelae such as the hemolytic-uremic syndrome (HUS) (32). Once HUS is clinically established, there is no curative therapy (44). Most human STEC infections for which a source has been identified have been traced to direct or indirect contact with bovine feces (13). Strategies aimed at reducing the incidence of human STEC infections by lowering the prevalence of STEC in cattle must be based on an understanding of how STEC is able to persist in the intestine of the bovine host.
Because there are no overt signs of intestinal inflammation in STEC-infected cattle, it has been hypothesized that STEC has adapted to a purely “commensal” lifestyle in cattle (35). However, even under normal conditions, the intestinal mucosa displays a state of “physiological inflammation,” manifested by the presence of abundant leukocytes in the intraepithelial and subepithelial compartments (7). Most STEC strains have an array of virulence determinants, including the induction of a characteristic histopathological feature known as the “attaching and effacing” lesion (2, 6, 10, 46), that allows them to interact with intestinal cells in multiple ways. It is tempting to assume that at least one of these determinants represents a danger signal that generates a response beyond physiological inflammation and terminates STEC colonization, even in the bovine host (34, 40). The frequent detection of anti-Stx antibodies in the sera and colostrum of cows (17, 31) provides additional evidence that the persistence of bovine STEC infections does not result from a general inability of cattle to respond to STEC or its products. STEC must have elaborated a mechanism that actively limits intestinal inflammation, maintains intestinal homeostasis, and finally allows persistent colonization.
The main virulence factors of STEC are the Stxs, potent cytotoxins of the ribosome-inactivating type comprising five B subunits that mediate Stx binding to a eukaryotic cell surface receptor (globotriaosylceramide [Gb3]/CD77) and one A subunit that contains the enzymatic activity (33). Stxs may also play a role in facilitating intestinal persistence of STEC. Experimentally infected adult sheep shed STEC strains longer than they shed strains lacking the stx gene (4). Our hypothesis that Stxs are able to modulate intestinal inflammation in the bovine mucosa was based on observations that activated bovine blood-derived lymphocytes express Gb3/CD77 molecules, a subset of which represents functional Stx1 receptors (23, 37). Stx1 binds directly to bovine blood lymphocytes and blocks activation and proliferation of these cells in vitro (21, 24). However, Stx1 does not affect all subpopulations equally but mainly blocks proliferation of CD21+ B cells and CD8+ T cells (24).
Approximately 10 to 15% of the cells in the bovine intestinal epithelium are intraepithelial lymphocytes (IEL), and the majority of those belong to the CD8+ subset (C. Menge, unpublished data). IEL are located between the epithelial cells adjacent to the basement membrane and are the only lymphocyte population situated so close to Stxs in the entire body (27). Given that bovine IEL resemble blood-derived lymphocytes with regard to Stx receptor expression and susceptibility to Stx1, we hypothesized that Stx1 modulates intestinal inflammation by acting directly on Gb3/CD77+ IEL. In a first attempt to prove this hypothesis, we obtained IEL from the intestines of adult cattle and characterized them phenotypically ex vivo and functionally after incubation in the presence and absence of purified Stx1 in vitro.
MATERIALS AND METHODS
Protein purification.Stx1 was produced from bovine STEC1 strain 2403 (rough, H− [47]) and purified by a procedure described previously (24). At the end of the purification process, toxin preparations were passed through Detoxi-Gel columns (Pierce, Old-Beijerland, The Netherlands) to reduce contamination with endotoxin. Cytotoxic activities of toxin preparations were determined on Vero cells (ATCC CRL 1587) by the method of Gentry and Dalrymple (9) with minor modifications (24). Cellular metabolic activity was assessed by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay as described previously (22, 24). The 50% cytotoxic dose (CD50) was calculated from dose-response curves geometrically as the reciprocal of the toxin dilution causing a 50% reduction in cellular metabolic activity. The Stx1 preparation contained 50,000 CD50s of Stx1 and 0.17 ng of endotoxin per ml, as determined by the Limulus amoebocyte lysate assay. Recombinant StxB1 (rStxB1) was purified from E. coli DH5α(pSU108) (42) by the method of Niebuhr (29) with slight modifications (37). The rStxB1 preparation used in the present study contained 411 μg of rStxB1/ml.
Preparation and stimulation of bovine ileal IEL (iIEL).Gut specimens (distal ileum) were obtained from healthy adult (>24-month-old) cows (German black pied) slaughtered at the local slaughterhouse. Digesta were removed by washing with tap water. After incubation with phosphate-buffered saline (PBS) supplemented with 1 mM dithiothreitol at 37°C and 100 rpm for 25 min, the specimens were cut into strips (width, 0.5 to 1 cm). The strips were then transferred to 50-ml centrifugation tubes filled with 35 ml of PBS-EDTA-AB solution (PBS supplemented with 2 mM EDTA, 100 IU of penicillin/ml, 100 μg of streptomycin/ml, and 50 μg of gentamicin/ml) and incubated (at 37°C and 100 rpm for 20 min). After vortexing (for 2 min at maximum speed), the liquid contents of the tubes were passed through nylon wool. Cells were collected by centrifugation (at 250 × g for 8 min), resuspended in 25 ml of Percoll solution (density adjusted to 1.0500 g/ml with PBS-EDTA [PBS supplemented with 5.4 mM EDTA]; Biochrom, Berlin, Germany), and layered onto 10 ml of Percoll solution (density adjusted to 1.0816 g/ml) in a 50-ml centrifugation tube. The gradient was covered with 15 ml of Leibowitz L15 medium. After centrifugation (at 652 × g for 20 min), cells were recovered from the Percoll-Percoll interface, washed twice with PBS-EDTA solution (at 202 × g for 7 min), and resuspended at 5 × 106 cells/ml in modified cell culture medium (IEL medium, containing 89% RPMI 1640 [Biochrom], 10% fetal calf serum [Life Technologies GmbH, Karlsruhe, Germany], 100 IU of penicillin/ml-100 μg of streptomycin/ml [Life Technologies GmbH], and 3 μM mercaptoethanol [Fluka, Taufkirchen, Germany]).
For stimulation assays, 2.5 × 105 IEL/150 μl of IEL medium were plated in triplicate onto 96-well flat-bottom microtiter plates (Nunc, Wiesbaden, Germany). The medium was additionally supplemented with recombinant human interleukin-2 (rhuIL-2) (200 IU/ml; kindly provided by H. Jomaa, Institute for Biochemistry, Academic Hospital Centre, Giessen, Germany) with or without purified Stx1 (24) at 200 CD50s/ml, and for neutralization studies the latter was preincubated (for 90 min at room temperature) with purified anti-StxB1 (41) (monoclonal antibody 13C4; 1.5 μg of immunoglobulin per ml). Previous experiments had shown that this concentration of antibody is sufficient to completely neutralize the biological activity of at least 200 CD50s/ml in the Vero cell assay (21, 24). For IEL stimulation, the medium was supplemented with nine different combinations (see Fig. 3) of commonly used mitogens with different modes of action (all purchased from Sigma, Taufkirchen, Germany): concanavalin A (final concentration, 2.5 μg/ml), phytohemagglutinin P (PHA-P; 2.5 μg/ml), pokeweed mitogen (2.5 μg/ml), phorbol-12-myristate-13-acetate (PMA; 5 and 20 ng/ml), and ionomycin (500 ng/ml). The plates were incubated for 72 h at 37°C under 5% CO2 at 95% humidity. Blast cell transformation of lymphocytes was quantified by flow cytometry according to the cells' light scatter characteristics by acquiring 5,000 events as described previously (22).
Immunophenotyping studies.Immediately after preparation or at the end of the cultivation period, IEL were thoroughly resuspended and transferred to V-shaped microtiter plates (Greiner, Frickenhausen, Germany) for immunolabeling as described previously (22, 25). Briefly, the cells were centrifuged (at 137 × g and 4°C for 7 min) and resuspended in 50 μl of cell culture medium as a negative control or with supernatants or diluted ascites fluid of hybridoma cell lines. Antibodies kindly provided by J. Naessens (International Livestock Research Institute [ILRI], Nairobi, Kenya) were as follows: IL-A11 (specific for bovine CD4), IL-A57 (CD6), IL-A105 (CD8α), IL-A65 (CD21), IL-A111 (CD25), IL-A118 (CD44), IL-A77 (CD71), IL-A30 (surface immunoglobulin M [IgM]), J11 (major histocompatibility complex class II), IL-A29 (WC1), and IL-A96 (WC9). Antibodies purchased from VMRD (Pullman, Wash.) were MM1A (specific for CD3), BAT82A (CD8β), CACT61A (TcR1-N12), CACTB6A (TcR1-N6), CACTB81A (TcR1-N7), and CACT26A (ACT-2). Alternatively, the cells were resuspended with 25 μl of rat IgM (1 mg/ml; dilution, 1:50 in PBS; Camon, Wiesbaden, Germany) as a negative control or with an anti-human CD77 antibody (clone 38.13; dilution, 1:10 in PBS; Beckman-Coulter, Krefeld, Germany). Cells were incubated on ice for 20 min, washed once with PBS supplemented with 1% bovine serum albumin and 0.01% sodium azide (BSA-azide), resuspended with either 50 μl of an anti-mouse γ-chain antibody conjugated with fluorescein isothiocyanate (dilution, 1:100 in PBS; Medac, Hamburg, Germany) or an anti-rat μ-chain antibody conjugated with R-phycoerythrin (R-PE) (diluted 1:200 in PBS containing 2 μg of propidium iodide [PI; Sigma]/ml; Beckman-Coulter), and kept on ice for 20 min. In double-staining experiments, cells were incubated with anti-CD77 and an anti-rat μ-chain antibody conjugated with PE (Beckman-Coulter) after the staining procedure described above. Finally, the cells were washed twice and analyzed with an EPICS ELITE Analyser (Beckman-Coulter). A total of 5,000 events were acquired from each sample. Data analysis was performed with the ELITE (version 4.01) software provided by the manufacturer. Electronic gates were set according to the negative control included in each test series, defining less than 2% of the cells as positive. When freshly prepared iIEL were analyzed, a region was set around a dense population of events with indistinguishable light scatter characteristics. Similarly, when cultured iIEL were investigated, two populations of non-blast cells and enlarged blast cells were defined according to their light scatter characteristics as described previously (22, 23) and were analyzed separately.
rStxB1 binding studies.For binding studies, cells were sequentially incubated with 50 μl of rStxB1 (30 μg/ml) for 30 min on ice (37), washed once with BSA-azide, resuspended in 50 μl of anti-StxB1 (45 μg/ml), incubated for 30 min on ice, washed, resuspended in 50 μl of an anti-mouse γ-chain antibody conjugated with fluorescein isothiocyanate (Dianova, Hamburg, Germany), incubated for 30 min on ice, washed twice, and analyzed by flow cytometry as described above. In some instances, cells were additionally immunolabeled as described above, with the primary antibodies from ILRI used in biotinylated form and detection carried out with PE-conjugated streptavidin (Dianova). A supernatant of a hybridoma cell line producing an irrelevant antibody was used between the staining steps to block nonspecific binding.
Quantitation of natural killer cell activity of bovine iIEL.Cells from a homologous B lymphoma cell line (BL-3; ECACC 86962401) were used as target cells. Cells were maintained in a medium (BL-3 medium) consisting of 56% RPMI 1640 (Biochrom), 23% Leibowitz L15 medium, 20% fetal calf serum, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (all purchased from Life Technologies), and 3 μM 2-mercaptoethanol (Fluka). To differentiate between target and effector cells, 106 BL-3 cells were stained with 25 μg of 3,3′-dioctadecyloxacarbocyaninperchlorate (DiO; Molecular Probes, Leiden, The Netherlands) in 2 ml of the medium at 37°C (for 4 h at 100 rpm). The cells were washed twice with PBS and incubated in the medium at 37°C under 5% CO2 and 95% humidity overnight. Prior to addition of the cells to the test mixture, the washing step was repeated twice and cells were resuspended in 2 ml of BL-3 medium.
Freshly prepared effector cells (iIEL) were incubated in the presence or absence of Stx1 (200 CD50s/ml) with or without anti-StxB1 (1.5 μg/ml) at a density of 2.5 × 105/150 μl of IEL medium in 6-well multiwell plates (5 to 10 ml per well) for 24 h at 37°C under 5% CO2 and 95% humidity. Anti-StxB1 (final concentration, 1.5 μg/ml) was then added to wells supplemented only with Stx1. After an additional incubation period of 2 h, IEL were then harvested and plated onto 96-well flat bottom microtiter plates in triplicate in threefold dilution series starting with 106 cells/well in 200 μl of IEL medium. A total of 104 BL-3 cells (stained with DiO as mentioned above) were added to each well, resulting in effector/target ratios of 100:1, 33:1, 11:1, 3.6:1, and 1.2:1. Target cell controls without effector cells were included. After incubation for 18 h (at 37°C under 5% CO2), cells were analyzed by flow cytometry, as described above. Samples were measured in PBS containing 2 μg of PI/ml. The ratio of dead (PI-positive) to viable (PI-negative) target cells was calculated after gating on 2,500 target cells according to their FL1 (DiO) signal. Natural killer cell activity was determined as the difference between the mean percentages of lysed target cells in test and control samples.
Preparation of mRNAs from bovine iIEL.A total of 3.75 × 107 freshly prepared IEL in 15 ml of IEL medium (with the fetal calf serum concentration raised to 20%) were plated onto 10-cm-diameter petri dishes. The medium was supplemented with 2.5 μg of PHA-P/ml. Cells were incubated for 4.5 h at 37°C under 5% CO2 in the presence or absence of purified Stx1 (200 CD50s/ml). Cells were then harvested and counted by trypan blue exclusion. A total of 107 viable cells from each treatment were washed twice with ice-cold PBS and finally resuspended in 2 ml of RNAzol (Wak-Chemie, Steinbach, Germany). Samples were stored at −80°C for as long as 3 days until isolation of RNA.
After thawing, samples were intensively mixed with 200 μl of chloroform, incubated on ice for 5 min, and centrifuged (at 12,000 × g and 4°C for 15 min). The upper clear phase was recovered, mixed with the same volume of isopropanol, and incubated on ice for 15 min. After centrifugation (at 12,000 × g and 4°C for 15 min), pellets were washed twice in 75% ethanol, air dried, and resuspended in diethyl pyrocarbonate-treated distilled water. The nucleic acid content was estimated spectrophotometrically. The quality of isolated RNA was checked by gel electrophoresis.
Reverse transcription.cDNA was obtained from RNA by reverse transcription of 1 μg of RNA per sample by using 200 U of Moloney murine leukemia virus reverse transcriptase (H−) (Promega, Mannheim, Germany) and 0.1 nmol of oligo(dT)16 primers (Applied Biosystems, Darmstadt, Germany) in a 40-μl reaction volume according to the manufacturer's instructions. After annealing at 70°C for 5 min, the reaction was performed at 37°C for 60 min, followed by 94°C for 2 min. Negative controls were performed without Moloney murine leukemia virus reverse transcriptase (H−).
Quantitation of cytokine-specific mRNA from bovine iIEL.Cytokine-specific PCR was performed in a 20-μl reaction volume including 1 U of AmpliTaq polymerase (Applied Biosystems), 1 μM concentrations of each primer, and 1 μl of cDNA template. Previously published cytokine primers (11, 26) were used with minor modifications: for IL-2, 5′-TCT TGC ATT GCA CTA ACT CT-3′ (sense) and 5′-GCT TTG ACA AAA GGT AAT CC-3′ (antisense); for IL-4, 5′-GCC ACT TCG TCC ATG GAC AC-3′ (sense) and 5′-TCC CAA GAG GTC TCT CAG CG-3′ (antisense); for IL-8, 5′-GCA GTT CTG TCA AGA ATG AG-3′ (sense) and 5′-GGA TCT TGC TTC TCA GCT C-3′ (antisense); for IL-10, 5′-TGT TGC CTG GTC TTC CTG-3′ (sense) and 5′-TCT CTT CAC CTG CTC CAC-3′ (antisense); for gamma interferon (IFN-γ), 5′-GCT TTA CTG CTC TGT GTG CT-3′ (sense) and 5′-GAC TTC TCT TCC GCT TTC TG-3′ (antisense); and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-ATC ACT GCC ACC CAG-3′ (sense) and 5′-CAT GCC AGT GAG CTT-3′ (antisense). GAPDH was used as control for constitutive gene expression. The amplification reaction was carried out for a total of 35 cycles as follows: 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s, with a precycle of 94°C for 15 s and postextension at 72°C for 5 min. PCR products were separated by electrophoresis on a 1.75% agarose gel and stained with ethidium bromide. Densitometry values for cytokine signals were evaluated with EasyWin 32 software (Herolab, Wiesloch, Germany) and normalized to those for GAPDH.
Quantitation of the migratory activity of bovine granulocytes.Neutrophil migration was assayed by the method of Galligan and Coomber (8) with some modifications. Transwell inserts with 3-μm pores (diameter, 12 mm; Corning Costar, Badhoevedorp, The Netherlands) were used in 12-well multiwell plates (Corning Costar), thus producing a two-chambered system. Neutrophils were separated from whole blood by density gradient centrifugation using Ficoll-Hypaque (Amersham Biosciences, Freiburg, Germany). After that, cells were resuspended in 2 ml of IEL medium. The purity of each fraction was assessed by flow cytometry. Only fractions with a purity higher than 90% were pooled. Purified neutrophils were diluted to 5 × 105 cells 0.5 ml−1 in IEL medium and were added to the top well. At the same time, 1.5 ml of an agonist was added to the bottom well. Agonists were supernatants of bovine iIEL preparations, which were incubated in either IEL medium alone, IEL medium plus Stx1 (200 CD50s/ml), or IEL medium plus Stx1 (200 CD50s/ml) and anti-StxB1 (1.5 μg/ml). Filter plates were incubated at 37°C under 5% CO2 and 95% humidity for 2 h. Fluids of top and bottom wells were resuspended. A suspension of counting beads (Fluoresbrite Calibration Grade 3.0 Micron YG Microspheres; Polysciences, Eppelheim, Germany) was added, and neutrophils were then quantitated by flow cytometry.
Statistical analysis.After log10 transformation, data were analyzed statistically by one-way repeated-measures analysis of variance followed by the Student-Newman-Keuls test using SigmaStat (version 2.03) software (SPSS Inc., Chicago, Ill.). Results were evaluated as highly significant (P ≤ 0.001), significant (P ≤ 0.01), weakly significant (P ≤ 0.05), or not significant (P > 0.05).
RESULTS
Stx receptor expression by bovine iIEL.When analyzed by flow cytometry after preparation from intestinal specimens, 14.9 ± 1.96% of iIEL (mean ± standard error of the mean [SEM] from 12 determinations with 6 iIEL preparations) stained with anti-CD77, but the intensity of anti-CD77 staining differed among different iIEL subpopulations ex vivo (Fig. 1 and Table 1). The majority of cells expressing high levels of the Gb3/CD77 antigen (estimated from the mean fluorescence intensity of the cells) were activated mature T cells expressing CD3, CD6, ACT-2, and CD8α. A high level of Gb3/CD77 expression was also found on CD8β+ cells and subpopulations of γδ T cells (WC1, TcR1-N7). Although mean fluorescence intensities for Gb3/CD77 expression were different for iIEL prepared from different intestinal specimens (i.e., different donor animals), CD4+ T cells and B cells (CD21+; surface IgM+) consistently expressed only low numbers of Gb3/CD77 molecules on their surfaces. The notion of a differential Gb3/CD77 expression was supported by the calculation of the percentage of Gb3/CD77+ cells within a given subpopulation of iIEL (Table 1). While more than 40% of iIEL belonging to γδ T-cell subpopulations (TcR1-N7+, WC1+) expressed Gb3/CD77 ex vivo, only 20.0% of CD4+ iIEL were Gb3/CD77+. The calculation also revealed further differences within certain subpopulations, since 30.2% of CD21+ B cells coexpressed Gb3/CD77 ex vivo but only 16.9% of cells positive for surface IgM were Gb3/CD77 positive. Moreover, 38.7% of CD8α+ T cells coexpressed Gb3/CD77 as opposed to 18.2% of the CD8β+ T cells. Gb3/CD77 was only partially coexpressed with the activation markers CD25 and ACT-2.
Flow cytometer dot plots illustrating Gb3/CD77 expression by bovine iIEL subpopulations ex vivo. IEL were prepared from the ileum of an adult animal at slaughter and were subsequently double labeled with anti-CD77 and subpopulation-identifying monoclonal antibodies. Dot plots depict cells of the lymphocyte region in the forward- versus side-scatter plot from a single representative of six experiments. Percentages of cells positive for one or both of the antigens are given in the upper right corners of the dot plots.
Gb3/CD77 expression by bovine iIEL subpopulations ex vivo
Binding studies with rStxB1 (the subunit responsible for receptor binding of the holotoxin) showed that 7.9% (mean from 10 determinations with 5 iIEL preparations; range, 5.7 to 11.5%) of the iIEL ex vivo bound the protein. In particular, rStxB1 bound to CD8α+ iIEL and to iIEL coexpressing TcR1-N12, a pan-γδ T-cell marker in cattle (Fig. 2). Double-staining experiments with rStxB1 and anti-CD77 at limiting concentrations that avoided complete masking of Gb3/CD77 by one of the ligands (37) showed that the populations of iIEL that bound these two ligands overlapped but were not identical. Although double-positive cells were detected (Fig. 2, right), a significant number of cells bound only one of the ligands.
Abilities of bovine iIEL subpopulations to bind rStxB1 ex vivo. IEL were prepared from the ileum of an adult animal at slaughter and subsequently double labeled with rStxB1 and subpopulation-identifying monoclonal antibodies. Each flow cytometer dot plot shows results for lymphocytes from a single experiment representative of five experiments. Percentages of cells positive for one or both of the antigens are given in the upper right corners of the dot plots.
Effect of Stx1 on lymphoblast transformation and natural killer cell activity in bovine iIEL cultures.Although iIEL generally respond poorly to mitogenic stimulation in vitro (27), detectable levels of blast cell transformation were induced in bovine iIEL cultures by applying a number of different stimuli alone or in combination. The percentage of blast cells in iIEL cultures incubated with Stx1 for 72 h was reduced (40 to 70% depending on the stimulus) compared to that with the respective mitogen control (Fig. 3). The presence of the anti-StxB1 monoclonal antibody 13C4 partially neutralized the effect of Stx1 on the average proliferative response of five iIEL preparations under seven out of nine conditions tested. Stx1 reduced the percentage of blast cells within all subpopulations identified to similar extents (Fig. 4). Individual differences between the iIEL preparations from different donors occurred, but Stx1 significantly reduced the percentage of Gb3/CD77-expressing blast cells, and this effect was completely neutralized by preincubation with anti-StxB1. Although Gb3/CD77 was expressed mainly by cells with an activated morphology, i.e., blast cells, Stx1 also affected the percentage of Gb3/CD77+ cells within the non-blast cell population. The cellular distribution of Gb3/CD77 on the iIEL further stimulated in vitro was strikingly different from that observed ex vivo (Table 2). Following incubation, comparable portions of CD8α+ and CD8β+ iIEL coexpressed Gb3/CD77. Furthermore, the highest percentage of cells coexpressing Gb3/CD77 could now be detected within the CD4+ and CD21+ subpopulation. Pretreatment with Stx1 did not affect the ability of iIEL to exert natural killer cell activity against a homologous lymphoma cell line (Fig. 5).
Effect of purified Stx1 on transformation and proliferation of bovine iIEL subpopulations in vitro. Cells were incubated with purified Stx1 (200 CD50s/ml; quantified on Vero cells as described in Materials and Methods) at 37°C for 72 h either in the absence (solid bars) or in the presence (striped bars) of 1.5 μg of the anti-StxB1 monoclonal antibody 13C4/ml. Culture medium was supplemented with rhuIL-2 (200 U/ml) and different stimuli as indicated. Lymphocytes that were transformed to blast cells were identified by flow cytometry according to their light scatter characteristics and quantified by acquiring 5,000 events in total. Data analysis was performed by calculating the percentage of viable (PI-negative) cells belonging to the blast cell population in relation to a mitogen control (i.e., cells that were incubated in the absence of Stx1 and anti-StxB1). Data are arithmetic means + SEMs from 15 determinations with 5 IEL preparations. PWM, pokeweed mitogen; ConA, concanavalin A.
Effect of purified Stx1 on transformation and proliferation of bovine iIEL subpopulations in vitro. Cells were incubated with purified Stx1 (200 CD50s/ml; quantified on Vero cells as described in Materials and Methods) at 37°C for 72 h either in the absence (solid bars) or in the presence (striped bars) of 1.5 μg of the anti-StxB1 13C4 monoclonal antibody/ml. Cells that were incubated in the absence of Stx1 and anti-StxB1 were included as controls (open bars). The culture medium was supplemented with rhuIL-2 (200 U/ml) and PHA-P (2.5 μg/ml). Lymphocyte subpopulations were identified by immunophenotyping and quantified by flow cytometry acquiring 5,000 events in total. Data analysis was performed by using the software provided with the instrument to calculate the percentages of viable (PI-negative) and antigen-positive blast cells among all cells in culture. Data are geometric means and dispersion factors from 6 to 8 determinations with 4 IEL preparations (and from 56 determinations with 4 IEL preparations in the case of CD77). Statistical significance was obtained after log10 transformation by one-way repeated-measures analysis of variance followed by the Student-Newman-Keuls test. Horizontal brackets indicate bars that are different (*, P ≤ 0.05; **, P ≤ 0.01).
Effect of purified Stx1 on natural killer cell activity of bovine iIEL in vitro. iIEL were incubated with purified Stx1 (200 CD50s/ml; quantified on Vero cells as described in Materials and Methods) at 37°C either in the absence (solid bars) or in the presence (striped bars) of 1.5 μg of the anti-StxB1 monoclonal antibody 13C4/ml. Cells that were incubated in the absence of Stx1 and anti-StxB1 were included as controls (open bars). The culture medium was supplemented with rhuIL-2 (200 U/ml). Upon 24 h of incubation, homologous dye-labeled lymphoma cells (BL-3) were added to the cultures for an additional 18 h. The percentage of target cell lysis was quantified by flow cytometry. By live-gating, 2,500 events identified as target cells by their FL-1 fluorescence were acquired and analyzed with regard to their membrane integrity (PI uptake). Data are arithmetic means + SEMs from 15 determinations with 5 IEL preparations.
In vitro effect of Stx1 on the percentage of bovine iIEL subpopulations coexpressing Gb3/CD77a
Effect of Stx1 on cytokine expression in bovine iIEL cultures.iIEL preparations obtained from different donors showed different levels of cytokine gene expression and different effects of Stx1 (Fig. 6). Stx1 reduced the IL-2 and IFN-γ signals in some preparations, but the expression of these TH1-type cytokines was unaffected or even slightly enhanced in other preparations. IL-10-specific mRNA was undetectable in four preparations, and Stx1 did not consistently affect its expression in the remaining two preparations. In contrast, Stx1 enhanced the expression of IL-4 in all of the four iIEL preparations in which IL-4-specific mRNA was detectable (four of six preparations). In addition, treatment with Stx1 for 4.5 h reduced the amount of IL-8-specific mRNA in all of the iIEL preparations investigated. The migration of bovine granulocytes through a filter membrane toward the supernatants obtained from two Stx1-treated iIEL cultures did not differ from that of supernatants of controls treated with Stx1 plus anti-StxB1 (data not shown).
Effects of purified Stx1 on cytokine gene expression by bovine iIEL in vitro. iIEL were incubated in the absence and presence of purified Stx1 (200 CD50s/ml; quantified on Vero cells as described in Materials and Methods) at 37°C. The culture medium was supplemented with PHA-P (2.5 μg/ml). Upon 4.5 h of incubation, mRNA was harvested from the cells and subjected to semiquantitative reverse transcription-PCR. Each value is the band intensity of the specific PCR product expressed as a percentage of the GAPDH signal obtained from the same sample. Each symbol represents results for a particular IEL preparation.
DISCUSSION
Some STEC strains are able to induce intestinal inflammation in infected humans. Neutrophils attracted and activated by Stxs may contribute to the pathogenesis of HC (18), and it has been further suggested that intestinal T cells are activated in the course of human STEC infections (15). In contrast, intestinal STEC infections in bovines do not induce inflammatory signs. These differences might be partially explained by the absence of functional Stx receptors on the surfaces of bovine granulocytes (C. Menge, T. Eisenberg, I. Stamm, and G. Baljer, Abstr. 5th Int. Symp. Shiga Toxin [Verocytotoxin]-Producing Escherichia coli Infect. 2003, abstr. O-11, p. 22, 2003). Another possible explanation is that Stx receptors are expressed on bovine T cells but not on human T cells (37). The observation that Stx1 suppresses bovine peripheral blood mononuclear cell (PBMC) functions (24) led us to hypothesize that by secreting Stx, STEC is able to limit the activation of intraepithelial T cells in cattle. This could explain both the persistence of bovine STEC infections and the absence of inflammation. Consistent with this hypothesis, the present study showed that bovine iIEL from adult animals express the Stx receptor ex vivo and are sensitive to purified Stx1 in vitro. The terminal rectum had recently been reported as the principal site of STEC O157:H7 colonization in the bovine host (28), but colonization of the ileum, cecum, and colon was shown to occur in calves infected with E. coli O157:H7 (5, 12), and non-O157 enterohemorrhagic E. coli apparently does not share a tropism for the terminal rectum (28, 39). The high similarity among the patterns of expression of cell surface molecules, including Gb3/CD77, by IEL obtained from ileal, colonic, or cecal sites (Menge, unpublished) suggests that sensitivity to Stx1 is widely distributed among IEL of the intestinal tract in cattle.
CD77 expression was detected on the surfaces of all iIEL subpopulations, including αβ and γδ T cells, that were analyzed directly ex vivo. It was recently confirmed by biochemical analysis that the antigen on bovine lymphocytes recognized by an anti-human CD77 antibody belongs to the Gb3 family (23). Gb3 molecules share the carbohydrate moiety that serves as the Stx binding site in human Stx receptors (19). However, double-labeling studies with anti-CD77 and rStxB1 identified three distinct population of cells. One population clearly stained with both of the ligands; the other two populations bound only rStxB1 or anti-CD77. Although corresponding experiments utilizing the Stx1 holotoxin have not been performed yet, bovine iIEL resemble their PBMC counterparts in expressing different isoforms of Gb3/CD77 with different affinities for the two ligands (37). Resting PBMCs predominantly express Gb3 isoforms with high affinity for rStxB1, but upon activation these cells synthesize Gb3 molecules with a preference for anti-CD77. This shift in affinity comes along with an increase in the percentage of Gb3 molecules that have incorporated long-chain fatty acids (23), a feature known to lower the affinity of Stx receptors for the holotoxin (30). Expression of anti-CD77 binding Gb3 isoforms by bovine iIEL suggests, therefore, that the cells display an activated state. Waters et al. (45) defined an activated phenotype of bovine IEL by the expression of the null cell and CD8+ T-cell activation marker ACT-2. ACT-2 was expressed by 42.8% (mean from 12 determinations with 6 iIEL preparations; range, 28.4 to 61.2%) of the iIEL in the present study. However, Gb3/CD77 expression did not correlate closely with the expression of activation markers (Table 1). Gb3/CD77 was found mainly on the TcR1-N7+ and WC1+ subsets of γδ T cells as well as on CD8α+ cells. Detailed analysis of the IEL subsets further revealed that CD8α is coexpressed by 31.8% (mean from nine determinations with five iIEL preparations; range, 22.5 to 48.1%) of TcR1-N12+ cells (pan-γδ T cells), while only 18.3% (range, 7.3 to 38.9%) of TcR1-N12+ cells coexpress CD8β. Hence, CD8αα homodimer-positive γδ T cells—as identified by Wyatt et al. (48)—and probably other CD8α+ IEL represent the iIEL subpopulation with the highest proportion of Gb3/CD77+ cells in cattle. In mice and humans, CD8αβ+ αβ T IEL are considered to lyse infected cells, while γδ T IEL (and perhaps CD8αα+ αβ T IEL) may stimulate renewal of the epithelial layer. Although the information available on the function of bovine IEL is very limited, it is tempting to speculate that, by affecting this type of cell, STEC is able to interfere with the local immune response as well as with epithelial-cell turnover. In fact, both aspects of intestinal barrier homeostasis have previously been linked to the persistent character of STEC infections in ruminants (20; M. Hoffman, T. Casey, and B. Bosworth, Abstr. 3rd Int. Symp. Workshop Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infect. 1997, abstr. V67/VIII, p. 117, 1997).
Comparable to what is seen with PBMCs (24), the addition of Stx1 to iIEL cultures from several donors reduced the average proliferative response to several mitogenic stimuli. This effect of Stx1 was partially prevented by preincubation of the toxin with anti-StxB1. However, the addition of anti-StxB1 seemed to have had an adverse effect when the cells were stimulated with PHA-P plus PMA or with pokeweed mitogen plus PMA. One possible explanation, based on previous findings (37), is that anti-StxB1 at low antigen-to-antibody ratios ameliorates rather than prevents the binding of rStxB1 to bovine lymphocytes. In contrast to the differential expression pattern of Gb3/CD77 by iIEL subpopulations ex vivo, Stx1 affected all subpopulations of cultured iIEL similarly and, in particular, reduced the percentage of Gb3/CD77+ cells. Calculation of the percentages of Gb3/CD77+ cells within the different subpopulations after 72 h of culture showed that high percentages of CD4+ and CD21+ cells now coexpressed Gb3/CD77, suggesting that each type of mucosal lymphocyte is capable of expressing functional Stx1 receptors in principle.
When we quantified the mRNAs specific for TH1 or TH2 prototype cytokines in iIEL after 4.5 h of treatment, Stx1 was found to induce a prominent increase in IL-4-specific mRNA levels in four out of six iIEL preparations examined. Previous investigations of peripheral lymphocytes from cattle showed that IL-4 is solely produced by CD4+ cells with a CD45RO+ memory phenotype (36), while activated γδ T cells primarily secrete IFN-γ (1). The proliferation experiments in this study showed that Stx1 is able to affect CD4+ cells at least upon stimulation in vitro, despite the low level of Gb3/CD77 expression on the surfaces of these cells ex vivo. Recently, several apoptosis-inducing toxins that, like Stx1, belong to the ribosome-inactivating proteins were found to stimulate intracellular IL-4 expression in human PBMCs (38). Although the proliferation-inhibiting effect of Stx1 on bovine iIEL is not accompanied by a significant increase in the number of apoptotic cells (data not shown), we cannot rule out the possibility that induction of IL-4 in bovine iIEL cultures by Stx1 results from the onset of apoptosis in one or several lymphocyte subpopulations. IL-4 represents a highly attractive target for future investigations, because it is able to retard granulocyte migration into and across human intestinal epithelial cell monolayers (3). IEL-regulated granulocyte migration at mucosal sites is part of the local immune response against colonizing bacteria (43), and Stx1-induced IL-4 from IEL might thus prevent STEC microcolonies from being attacked by granulocytes in situ. Strikingly, Stx1 also acts directly on Gb3/CD77+ bovine colonic epithelial cells and causes a reduction in the release of chemoattractive signals by the epithelial cells (Menge et al., Abstr. 5th Int. Symp. Shiga Toxin-Producing E. coli Infect.). Stx1-treated iIEL examined in the present study also showed reduced levels of IL-8-specific mRNA synthesis. However, IL-8 is only one of numerous chemokines discovered thus far. The observation that supernatants of Stx1-treated iIEL cultures did not consistently contain reduced chemotactic activities led us to hypothesize that Stx1 differentially influences inflammatory signals in the intestinal barrier rather than generally downregulating them.
The intestinal system comprises a rich network of reciprocal and finely orchestrated interactions between immune, epithelial, endothelial, mesenchymal, and nerve cells and the extracellular matrix (7). Dysfunction of any component of this highly integrated mucosal system may lead to a disruption in communication which influences the course of intestinal infections. The proof that bovine IEL represent target cells for Stx1, together with recent reports on the expression of functional Stx receptors on bovine intestinal epithelial cells (16; Menge et al., unpublished data), is thus highly relevant for our understanding of the interplay of STEC with its bovine reservoir host and for the development of strategies aimed to limit these infections.
ACKNOWLEDGMENTS
We thank J. Naessens of ILRI, Nairobi, Kenya for generously supplying hybridoma cell lines producing antibodies to bovine leukocyte antigens. Klaus Failing, biomathematics workgroup, Faculty of Veterinary Medicine of the Justus-Liebig University, is acknowledged for statistical analysis.
This work was supported by grants from the Deutsche Forschungsgemeinschaft to C.M. (Sonderforschungsbereich 535) and to M.B. and T.E. (Graduiertenkolleg 455).
FOOTNOTES
- Received 25 July 2003.
- Returned for modification 9 October 2003.
- Accepted 8 January 2004.
- Copyright © 2004 American Society for Microbiology
