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Am J Physiol Gastrointest Liver Physiol 273: G1014-G1022, 1997;
0193-1857/97 $5.00
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Vol. 273, Issue 5, G1014-G1022, November 1997

Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa

Martin Riegler1, Roland Sedivy2, Tacettin Sogukoglu1, Ignazio Castagliuolo3, Charalabos Pothoulakis3, Enrico Cosentini1, Georg Bischof1, Gerhard Hamilton1, Bela Teleky1, Wolfgang Feil4, J. Thomas Lamont3, and E. Wenzl1

1 University Clinic of Surgery and 2 Institute of Clinical Pathology, University of Vienna, A-1090 Vienna; 4 Department of Surgery, Danube Hospital, A-1220 Vienna, Austria; and 3 Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Epidermal growth factor (EGF) exhibits a cytoprotective effect on gastrointestinal epithelia via a receptor-mediated mechanism. We investigated the effect of EGF on Clostridium difficile toxin A (TxA)- and toxin B (TxB)-induced damage of human colon. Ussing-chambered colonic mucosa was exposed serosally to EGF before and during luminal exposure to TxA and TxB. Resistance was calculated from potential difference and short-circuit current. Epithelial damage was assessed by light microscopy and alteration of F-actin by fluoresceinated phalloidin. Luminal exposure of colonic strips to TxA and TxB caused a time- and dose-dependent decrease in electrical resistance, necrosis and dehiscence of colonocytes, and disruption and condensation of enterocyte F-actin. These effects were inhibited by prior, but not simultaneous, serosal application of EGF (20 nM). Administration of the tyrosine kinase inhibitor genistein (10-6 M) inhibited the protective effects of EGF. We conclude that EGF protects against TxA and TxB probably by stabilizing the cytoskeleton, the main target of these toxins.

enterotoxins; genistein; human colonic epithelium

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

EPIDERMAL GROWTH FACTOR (EGF), originally isolated from mouse submaxillary gland (9), is mainly involved in the regulation of gastrointestinal epithelial barrier function. In addition to modulating epithelial maturation, proliferation, and differentiation (18, 30), EGF also exhibits acute and chronic cytoprotective effects on gastrointestinal epithelia (28, 35, 36, 38, 46). For example, EGF stimulates healing of chronic gastric and duodenal ulcers in rats (28, 36) and inhibits gastric acid secretion in rats when applied subcutaneously (35). Furthermore, luminal application of EGF increases mucosal blood flow in ethanol-exposed rat gastric mucosa (23), and EGF promotes epithelial restitution of intestinal cell monolayers (6, 7, 11) and of rabbit duodenum in vitro (49). Salivary glands, duodenum, and kidney are the major sources of EGF in humans (27). Because EGF is cleaved to less active forms in gastric acid, duodenal and pancreatic EGF are suggested to be essential for protection of small and large bowel epithelium (38). EGF mediates its biological effects via binding to its receptor on the basolateral membrane of epithelial cells (50, 54), thus activating an intracellular signal transduction pathway (54) that modulates cell growth and cell metabolism (52) and may also regulate the cytoskeleton (56). Recently, it was shown that systemic EGF attenuated epithelial damage in an experimental model of acute colitis in rats (46).

Clostridium difficile, the causative agent of antibiotic-associated enterocolitis in animals (1, 5) and in humans (3, 4), produces two high-molecular-weight exotoxins: toxin A and toxin B (40, 51). In addition to causing fluid secretion and intestinal inflammation through activation of inflammatory cells (26, 31) and nerves (8, 41) and release of mediators of inflammation (16, 42, 53), both toxins have direct effects on intestinal epithelial cells (20, 21, 32, 45, 48). For example, toxin A damages villus tip epithelium of guinea pig ileum in vitro (32), and both toxins A and B impair epithelial barrier function of human colonic cancer T84 cell monolayers in vitro (20, 21). We recently demonstrated that both toxins severely damage human colonic epithelium in vitro (48). Electrophysiological studies show that toxin-induced damage is paralleled by a decline of transepithelial resistance, indicating impaired epithelial barrier function (20, 21, 32, 48). Interestingly, epithelial cell damage by toxins is confined to the surface epithelium, whereas the epithelium of the crypts remains intact (32, 48). This distribution of damage is probably related to the expression of toxin receptors on villus cell brush borders with no receptors on crypt cells (44).

Both toxins induce disruption of epithelial cell cytoskeletal F-actin, thus impairing paracellular permeability (20, 21, 32, 48). Both toxins are suggested to elicit their action on target cells via a receptor-mediated mechanism involving release of intracellular calcium (17, 44). The cellular target of C. difficile toxins is the small GTP-binding protein rho (12, 24, 25), which regulates assembly of F-actin in fibroblasts in vitro (10, 22, 29, 33, 34, 37, 47). In contrast, recent studies indicated that EGF modulates F-actin assembly via activation of small GTP-binding proteins in fibroblasts (34, 47) and that the EGF receptor is associated with F-actin in vitro (56). According to the above considerations, the aim of the present study was to investigate the effects of EGF on toxin A- and toxin B-induced epithelial damage of human colonic mucosa in vitro. Epithelial integrity was assessed by light microscopy, morphometry, electrophysiology (15, 48), and immunofluorescent microscopy of enterocyte F-actin as previously described (48).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Toxin preparation. Toxins A and B were purified to homogeneity as previously described by us (40, 44). Enterotoxic activity of toxin A was assayed in rat ileal loops (8, 43, 53), and cytotoxic activity of both toxins was tested against IMR-90 fibroblasts (40, 44). Purity of toxins A and B was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (40, 44). Concentrations of toxins A and B were expressed in moles based on molecular masses of 308 kDa for toxin A (14) and 279 kDa for toxin B (2).

Experimental design. Human colonic mucosal sheets were first equilibrated in Ussing chamber with buffer alone for 30 min. Thereafter, tissues were paired and exposed to buffer alone or serosal buffer containing 20 nM EGF (no. E 1257, Sigma Chemie, Deisenhofen, Germany) for 60 min. Luminal solution was then replaced with buffer containing 32 nM toxin A or 3.0 nM toxin B for 3 h, while tissues remained incubated serosally with buffer or buffer containing 20 nM EGF (n = 6, paired). In separate experiments, tissues were incubated with serosal buffer or serosal buffer containing 10 nM EGF for 60 min before and during 3 h of luminal exposure to 32 nM toxin A or 3.0 nM toxin B (n = 3 per group). In some experiments, after 60 min of baseline incubation, tissues were incubated with luminal buffer containing 32 nM toxin A or 3.0 nM toxin B and serosal buffer containing 20 nM EGF for 3 h (n = 4 per group). Control tissues were incubated for 4.5 h with buffer or serosal buffer containing 20 nM EGF (n = 6, paired).

In an additional series of experiments, four human colonic explants from a single individual were mounted in parallel and incubated with or without serosal buffer containing EGF (20 nM) or EGF (20 nM)/genistein (10-6 M; no. G 6776, Sigma Chemie) 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure; one tissue was incubated with buffer alone for 4.5 h and served as control (n = 6, quadruplicate). Similar doses of EGF and genistein were previously used to test the effects of these compounds in vitro (6, 7, 11, 49, 55).

In another series of experiments, two human colonic explants from a single individual were mounted in parallel and incubated with serosal buffer with or without 10-6 M genistein for 4.5 h (n = 5, paired).

Dose-response studies on the effect of C. difficile toxins A and B on human colonic mucosa were described previously (48). Doses of C. difficile toxins A and B used in this study caused comparable electrophysiological and morphological changes in human colonic mucosal strips in vitro (48).

Ussing chamber measurements. In this study, a total of 62 individual specimens of histological normal sigmoid colon was used. After removal of the seromuscular layer by blunt dissection, one to five mucosal sheets from each specimen measuring 4-10 cm2 were mounted vertically in Ussing chambers (19) (1 cm2 surface area; Precision Instruments, Lake Tahoe, CA) as previously described (48, 49). Luminal and serosal sides were bathed at 37°C with a nutrient buffer containing (in mM) 122.0 NaCl, 2.0 CaCl2, 1.3 MgSO4, 5.0 KCl, 20.0 glucose, and 25.0 NaHCO3 (pH 7.51 when gassed with 95% O2-5% CO2; temperature 37°C). The top level of fluids in both luminal and serosal reservoirs was identical. Potential difference (PD) and short-circuit current (Isc) were continuously recorded every 10 min. Luminal and serosal solutions were connected via Ag-AgCl electrodes and Ringer-agar bridges to a voltmeter (voltage-current clamp model VCC600, Physiologic Instruments). Resistance (R) was calculated using Ohm's law from the open-circuit PD and the Isc. PD values were given in millivolts, Isc in microamperes per square centimeter, and R in ohms times square centimeter. PD values were corrected for the junction potentials (<0.1 mV) between luminal and serosal solutions.

Morphometry. After Ussing chamber experiments were performed, tissues were processed for light microscopy. Mucosal damage was assessed by a histopathologist (R. Sedivy), who performed morphometry on coded, paraffin-embedded, hematoxylin and eosin-stained slides as previously described (15, 48, 49). The histopathologist was not informed about the experimental conditions to which the tissues had been exposed. The surface area of each tissue mounted into chambers was 1 × 1 cm. Morphometry was performed on nine vertical sections from different locations, each of which represented the total height and length (1 cm) of the mucosal preparation. Thus a total length of 8-9 cm of mucosal surface was examined for each tissue. We used a Leitz Diaplan research microscope (objective magnification, ×4, ×6.3, ×16, ×25, ×40; ocular magnification, ×12.5; Wild Leitz, Heerbrugg, Switzerland), a Panasonic color charge-coupled device video camera (model WV-CD 130/G, Matshushita), an analog-to-digital monitor screen (model PVM-1271Q, superfine pitch, Sony), a personal computer (IBM-PS/2, model PS2; IBM, Armonk, NY), extended by insertion of a frame grabber (ITI PCVision plus board; Imaging Technology, Woburn, MA), and a graphic tablet (Summasketch plus 12" × 12" MM 1201; Summagraphics, Fairfield, CT). Downloading MIPSY (the Micro-based image processing system) real-time morphometry was performed, and the extent of epithelial damage was measured in micrometers and expressed as percent of total mucosal surface.

Histological criteria for epithelial damage were as follows: reduced staining intensity of epithelial cells, kariopyknosis, kariolysis and kariorrhexis, cell disruption, formation of subepithelial blebs, and lifting off of cells from the basal lamina (15, 48, 49).

Fluorescent microscopy. Fluorescent staining of F-actin was performed using fluorescein isothiocyanate-labeled phalloidin (Molecular Probes) on fresh-frozen tissue. Sections (4 µm) were fixed in 4% paraformaldehyde (pH 8.0), washed in phosphate-buffered saline solution (PBS; pH 8.0), and incubated for 45 min. Subsequently, slides were mounted with glycerol-PBS (1:9) and examined and photographed using a Zeiss Axiophot fluorescence microscope (48).

Statistics. All data were expressed as means ± SE. Statistical analyses were performed by the Student's t-test for paired and unpaired observations. Probabilities were regarded as significant if they reached a 95% level of confidence (P < 0.05).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Effect of EGF on toxin-induced electrophysiological changes. The effects of EGF on toxin-induced changes on colonic electrophysiology were assessed by comparing PD, Isc, and electrical resistance (R). As expected (48), luminal exposure of tissues to 32 nM toxin A or 3.0 nM toxin B for 3 h caused a time-dependent decrease of PD, Isc, and R as compared with buffer alone (P < 0.05, Table 1). Exposure of tissues to EGF (20 nM) 60 min before and during 3 h of luminal toxin A exposure completely prevented all colonic electrophysiological changes (P < 0.05; Table 1, Fig. 1). Under the same experimental conditions, EGF (20 nM) partially, but significantly, reduced toxin B-mediated PD and R decline (P < 0.05), whereas Isc remained the same (Table 1). However, 10 nM EGF failed to inhibit any of the toxin-mediated electrophysiological responses (Table 1). These results indicate that EGF is more potent in preventing toxin A-induced impairment of epithelial barrier function when compared with toxin B (Table 1, Fig. 1).

                              
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  		Table 1.   Effect of epidermal growth factor and Clostridium difficile toxins A and B on electrophysiology of human colonic mucosa


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  		Fig. 1.   Time course of 20 nM epidermal growth factor (EGF) on 32 nM toxin A (A)- and 3.0 nM toxin B-induced (B) decline of colonic resistance in vitro. Human colonic mucosal sheets were incubated with or without serosal buffer containing 20 nM EGF 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure. Resistance was calculated from potential difference and short-circuit current. Values represent means ± SE of 6 paired experiments. * P < 0.05, dagger  P < 0.01 vs. toxin.

The time course effect of 20 nM serosal EGF on toxin A (32 nM)- or toxin B (3.0 nM)-induced decline of transepithelial resistance is shown in Fig. 1. When compared with tissues treated with EGF before and during toxin A or toxin B exposure, decline of resistance of buffer-treated, toxin-exposed tissues became statistically significant after 60 and 120 min for 32 nM toxin A and 3.0 nM toxin B, respectively (P < 0.05, n = 6, paired) (Fig. 1).

Coadministration of serosal EGF (20 nM) with luminal toxin A or toxin B exposure did not have an effect in toxin-induced decline of electrophysiological parameters (Table 2).

                              
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  		Table 2.   Effect of simultaneous epidermal growth factor and Clostridium difficile toxins A and B on electrophysiology of human colonic mucosa

Effect of genistein on EGF-mediated protective effects. The EGF receptor is a tyrosine kinase that is autophosphorylated upon ligand binding (54). Thus we next investigated the effect of the tyrosine kinase inhibitor genistein on the protective effects of EGF on toxin A- and toxin B-induced electrophysiological changes. As shown in Fig. 2, coadministration of serosal 20 nM EGF with 10-6 M genistein (EGF/genistein) 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure caused an ~40% R decline when compared with buffer-treated controls (P < 0.01, n = 6). However, this R decline was statistically indistinguishable from the R decline seen in EGF/genistein-untreated, toxin-exposed tissues (Fig. 2). In contrast, serosal 20 nM EGF before and during luminal toxin A or toxin B exposure inhibited the R decrease seen after toxin A and attenuated toxin B-induced R decline (Fig. 2).


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  		Fig. 2.   Effect of EGF on C. difficile toxin A- and toxin B-induced changes of colonic resistance in vitro. Human colonic mucosal sheets were incubated with or without serosal buffer containing 20 nM EGF (EGF + toxin) or EGF (20 nM)/genistein (Gen; 10-6 M) 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure. Controls were incubated with buffer alone for 4.5 h. Resistance was calculated from potential difference and short-circuit current. Values for resistance were obtained after 3 h of luminal toxin A or toxin B exposure. Bars represent means ± SE of 6 experiments performed in quadruplicate. * P < 0.01, dagger  P < 0.05 vs. controls.

The time course effect of serosal EGF/genistein on colonic resistance is shown in Fig. 3. Coadministration of serosal EGF/genistein induced a R decrease that became statistically significant after 60 and 120 min of luminal exposure to toxins A (32 nM) and B (3.0 nM), respectively, when compared with EGF-preincubated, toxin-exposed tissues (P < 0.05, n = 6).


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  		Fig. 3.   Time course of EGF and EGF/genistein on 32 nM toxin A- (A) and 3.0 nM toxin B-induced (B) decline of colonic resistance in vitro. Human colonic mucosal sheets were incubated with or without serosal buffer containing 20 nM EGF or EGF (20 nM)/genistein (10-6 M) 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure. Resistance was calculated from potential difference and short-circuit current. Values represent means ± SE of 6 paired experiments. * P < 0.05, dagger  P < 0.01 vs. EGF + toxin.

Incubation of tissues with either buffer alone or serosal buffer containing 20 nM EGF for 4.5 h did not alter basal PD, Isc, and R (Table 1). Incubation of tissues with serosal genistein (10-6 M) for 4.5 h caused a statistically significant decrease of Isc, whereas R remained unchanged. (Isc baseline vs. 4.5 h for controls: 80 ± 12 vs. 82 ± 11 µA/cm2; 10-6 M genistein: 87 ± 9.2 vs. 58 ± 8.6 µA/cm2; P < 0.01; n = 5, paired).

Histology. No tissue strip used in these experiments showed histological criteria of malignancy. Tissues incubated with buffer (Fig. 4A), serosal buffer containing 20 nM EGF or 10-6 M genistein (data not shown) showed normal colonic architecture (n = 6, paired). As shown by morphometry (Fig. 5), incubation of tissues with buffer alone or serosal buffer containing 20 nM EGF or 10-6 M genistein (data not shown) for 4.5 h did not impair epithelial integrity.


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  		Fig. 4.   Morphological effect of EGF on toxin A- and toxin B-induced epithelial damage on human colonic mucosa in vitro. Human colonic mucosal sheets were incubated with luminal buffer alone for 4.5 h (A). Tissues were incubated with luminal buffer containing 32 nM toxin A (B), 3.0 nM toxin B for 3 h (C), or incubated with serosal 20 nM EGF 60 min before and during toxin A (D) and toxin B (E) exposure. At end of experiments, tissues were fixed, stained (hematoxylin and eosin), and processed for light microscopy. Toxins A and B caused disruptions of superficial epithelium (B and C); in contrast, only minor epithelial defects are detected in EGF-treated tissues (D and E). Magnification, ×200.


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  		Fig. 5.   Morphometric analysis of colonic epithelial cell damage. Human colonic mucosal sheets were incubated with or without serosal buffer containing 10 or 20 nM EGF 60 min before and during 3 h of luminal toxin A (32 nM) or toxin B (3.0 nM) exposure. Toxin-unexposed tissues were incubated with buffer alone or serosal buffer containing 20 nM EGF for 4.5 h. Mucosal sheets were fixed in Formalin and processed for light microscopy, and morphometric analysis was performed as described in METHODS. Results are expressed as means ± SE; n, number of experiments. dagger  P < 0.01, * P < 0.05 vs. toxin alone, paired experiments. ** P < 0.05 toxin A vs. toxin B EGF treated. · P < 0.05 toxin unexposed vs. EGF treated and toxin exposed.

The effect of serosal EGF (20 nM) on toxin A (32 nM)- or toxin B (3.0 nM)-induced mucosal damage is shown in Fig. 4, B-E. Both toxins caused damage of the superficial epithelium while the epithelium of the crypts remained intact (Fig. 4, B and C). Moreover, epithelial cell damage had a patchy distribution as intact epithelium was adjacent to damaged areas (Fig. 4, B and C). In contrast, tissues incubated with 20 nM EGF 60 min before and during 3 h of luminal exposure to toxin A (32 nM) (Fig. 4D) or toxin B (3.0 nM) (Fig. 4E) retained an almost confluent epithelium, with only few areas showing damage and detachment of individual epithelial cells. The amount of epithelial discontinuities was less in toxin A-exposed (Fig. 4D) than in toxin B-exposed tissues (Fig. 4E) after pretreatment with EGF.

Serosal exposure of tissues to EGF (20 nM)/genistein (10-6 M) 60 min before and during 3 h of luminal toxin A or B exposure blocked the protective effect of EGF on toxin A- or toxin B-induced epithelial damage and caused morphological changes that did not differ from untreated, toxin-exposed tissues (data not shown).

The dose-dependent effect of EGF on toxin A- or toxin B-induced morphological damage expressed as the percent of damaged mucosa is shown in Fig. 5. Incubation of tissues with 20 nM of serosal EGF 60 min before and during 3 h of luminal toxin A or B exposure significantly reduced the extent of damaged surface (P < 0.05). Furthermore, EGF was more potent in reducing toxin A-induced damage when compared with toxin B (P < 0.05 toxin A vs. toxin B, n = 6 per group). Serosal application of EGF (20 nM) at the same time with luminal exposure to toxins A or B did not alter toxin-mediated epithelial cell damage (n = 4). Furthermore, 10 nM serosal EGF did not influence toxin A- or toxin B-mediated epithelial cell damage (Fig. 5).

Serosal exposure of tissues to EGF (20 nM)/genistein (10-6 M) 60 min before and during 3 h of luminal toxin A or toxin B exposure caused morphological damage that was statistically indistinguishable from the damage seen in EGF/genistein-untreated, toxin A- or toxin B-exposed tissues (EGF/genistein-toxin A vs. toxin A alone: 27 ± 4 vs. 24 ± 5%; EGF/genistein-toxin B vs. toxin B alone: 28 ± 6 vs. 26 ± 4%; n = 6, paired).

Distribution of F-actin. Recent studies demonstrated that both toxins caused a marked decrease in fluorescent staining for F-actin in monolayers of the T84 colonic adenocarcinoma cell line (20, 21), guinea pig ileal enterocytes (32), and human colonic epithelium in vitro (48). We therefore assessed the effect of EGF on toxin A- and toxin B-induced changes of F-actin distribution. In keeping with our recent study (48), control cells had a polygonal shape with F-actin distributed in the peripheral actinomyosin ring associated with the cell membrane (Fig. 6A). Tissues treated with 32 nM toxin A (Fig. 6B) or 3.0 nM toxin B (Fig. 6C) showed disorganization, disruption, and condensation of intracellular F-actin, confirming our previous observations (48). In contrast, F-actin distribution of tissues incubated with serosal 20 nM EGF 60 min before and during 3 h of luminal toxin A (32 nM) exposure (Fig. 6D) or toxin B (3.0 nM) exposure (Fig. 6E) was similar to buffer-exposed controls, and in the majority of cells F-actin ring retained a polygonal shape. Furthermore, the F-actin ring showed dotted condensations of increased staining activity, and no condensations of F-actin were detected in the cytoplasm of epithelial cells (Fig. 6, D and E). Very few epithelial cells showed interruptions and disorganization of intracellular F-actin ring. Incubation of tissues with serosal buffer containing 20 nM EGF alone did not alter F-actin distribution (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The main finding of this study was that EGF attenuated C. difficile toxin A- and toxin B-induced electrophysiological changes and epithelial damage of human colon in vitro. Furthermore, administration of the tyrosine kinase inhibitor genistein blocked the effect of EGF.

In keeping with our recent in vitro studies (20, 21, 32, 48), toxins A and B induced a time-dependent decline of transepithelial resistance, indicating toxin-induced impairment of epithelial barrier integrity (Fig. 1). In contrast, incubation of tissues with serosal EGF before and during luminal toxin exposure inhibited toxin A-induced decline of resistance and attenuated toxin B-induced decrease in tissue resistance (Table 1, Figs. 1 and 2). As also shown in Table 1, EGF prevented toxin A- and toxin B-induced decline of Isc. These data indicate that EGF-treated, toxin-exposed tissues retained the ability for active transepithelial electrogenic transport, whereas decline in PD and R appears to reflect a slight increase in paracellular conductance (20, 21).

Our results show that EGF was more potent in reducing toxin A- than toxin B-induced epithelial cell damage, although at the doses used both toxins caused similar electrophysiological (Fig. 2) and morphological changes (Fig. 5). The reason(s) for the different potency of EGF in the prevention of toxin A's vs. toxin B's intestinal effects is unclear, although they may reflect differences in the molecular pathways used by these toxins in the human colonocyte.

Coadministration of EGF together with the tyrosine kinase inhibitor genistein blocked the preventive effect of EGF on toxin A- and toxin B-induced electrophysiological (Figs. 2 and 3) and morphological changes. It is well accepted that upon binding to EGF, the EGF receptor undergoes autophosphorylation, which in turn activates a tyrosine kinase-dependent signal transduction pathway (54). Studies in fibroblasts in vitro also indicate that tyrosine kinases are involved in EGF-mediated modulation of the F-actin cytoskeleton (10, 33, 47). Taken together, our data indicate that the mechanism of the preventive effects of EGF in toxin-induced damage involves binding of EGF to its receptor and a tyrosine kinase-dependent signal transduction pathway.

An important finding of this study was that EGF prevented toxin A- and toxin B-induced disruption of cytoskeletal F-actin (Fig. 6). It is well documented that both toxins act on F-actin of the cytoskeleton, thus causing cell rounding and detachment of cells (20, 21, 32, 48). Although toxin B disrupts F-actin of nonepithelial cells (12, 13, 40) and toxin A affects F-actin of guinea pig ileal epithelial cells (32) in vitro, both toxins cause disorganization of F-actin of human colonic epithelial cells in vitro (20, 21, 48). According to the results of recent biochemical and cell culture studies, the mechanism of C. difficile toxins involves inactivation of the small GTP-binding protein rho (12, 24, 25). Several in vitro studies also showed that rho is involved in F-actin assembly and organization in fibroblasts in vitro (10, 22, 29, 33, 34, 37, 47). These results indicate that rho may be involved in interactions between the cytoskeleton and the extracellular matrix proteins required for cell adhesion (10, 22, 29, 33, 34, 37, 47). Although the exact mechanism is not completely understood, rho was demonstrated to be involved in the activation of focal adhesion kinase 125 (37, 47), which in turn promotes binding of F-actin to the intracellular domain of integrin receptors via the cytoskeletal glycoproteins talin, tensin, vinculin, and alpha -actinin (22, 33). These results indicate that rho stimulates cellular adhesion of cultured fibroblasts to an underlying matrix. Microinjection of rho into toxin A- or toxin B-exposed fibroblasts inhibited cell rounding and F-actin disorganization (12). In contrast, microinjection of C. difficile toxin B-ribosylated rho into toxin-unexposed fibroblasts caused disorganization of the cytoskeleton and cell rounding (25). Thus C. difficile toxin-induced inactivation of rho may represent the molecular switch that initiates disruption, disorganization, and clumping of F-actin (12, 24, 25). A growing body of evidence indicates that EGF is capable of modulating actin assembly (10, 34, 47). EGF was demonstrated to stimulate F-actin polymerization during stress fiber formation and focal adhesion assembly when added to fibroblasts in vitro (47). This effect could be blocked by Clostridium botulinum exoenzyme C3-induced ADP-ribosylation of rho, which is known to inactivate rho, thus indicating that EGF mediated its effect via activation of rho (47). Furthermore, the EGF receptor was demonstrated to directly bind the actin cytoskeleton (56). Because there exist no data on the role of rho in epithelial cells, the action of EGF in our system remains speculative. Several intracellular targets are suggested to enable EGF to inhibit toxin-induced disorganization of the cytoskeleton: EGF could activate rho, prevent toxin-induced inactivation of rho, or stimulate actin polymerization via rho or via direct binding of the activated EGF receptor to F-actin, thus resulting in stabilization of the cytoskeleton (10, 22, 29, 33, 34, 37, 47, 56). Net activation of rho would in turn promote the assembly of integrin adhesion complexes and stabilize the cytoskeleton (22). Furthermore, EGF was shown to induce expression of integrin receptors that in turn could mediate increased adhesion of epithelial cells to the basal lamina (7). Concerning time dependency of rho and EGF-induced cytoskeletal alterations, Ridley and Hall (47) found that actin polymerization could be observed by 30 min after microinjection of rho into cultured fibroblasts, whereas EGF-induced actin polymerization required up to 1 h (47). In keeping with these observations, we report here that EGF treatment had to be initiated 1 h before toxin exposure to prevent C. difficile toxin-induced disruption of enterocyte F-actin. Taken together, EGF probably stabilizes the actin cytoskeleton by enabling epithelial cells to withstand the cytotoxic action of C. difficile toxins A and B. 


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  		Fig. 6.   Effect of EGF on toxin A- and toxin B-induced disruption of cellular F-actin. Fluorescent photomicrograph of human colonic mucosal sheet incubated with buffer (A), 32 nM toxin A (B), and 3.0 nM toxin B (C) or with serosal 20 nM EGF 60 min before and during toxin A (D) and toxin B (E) exposure. Samples were fixed, fresh frozen, and stained for F-actin as described in METHODS. Note disruption of F-actin in toxin-exposed tissues (B and C), as opposed to ringlike distribution of F-actin seen in controls (A) and EGF-treated toxin-exposed tissues (D and E). Magnification, ×300.

Because we used mucosal preparations containing epithelial and nonepithelial cells, we cannot exclude the possibility that the protective effects seen are also mediated through EGF-induced activation of lamina propria cells. It is also possible that EGF attenuated toxin-induced biological effects via direct binding to the toxin or via inhibition of toxin binding to its receptor. However, binding studies showed that EGF does not bind to radiolabeled toxin A, nor does it inhibit toxin A binding to intestinal membranes (C. Pothoulakis, unpublished observations).

Taken together, our results show that EGF attenuated C. difficile toxin A- and B-induced damage of human colonic epithelium in vitro. Although the exact mechanism remains unclear, EGF probably elicits its effect by stabilizing intracellular F-actin, the main intracellular target of C. difficile toxins. Further studies are required to elicit the molecular events by which EGF maintains intestinal epithelial barrier function.

    ACKNOWLEDGEMENTS

We thank Ingrid Hammer for expert technical support.

    FOOTNOTES

This study was supported by grants from the Jubiläumsfonds der Österreichischen Nationalbank, the Anton Dreher-Gedächtnisschenkung des Medizinischen Dekanats der Universität Wien, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34583 and DK-47343, and the Crohn's and Colitis Foundation of America.

Address for reprint requests: C. Pothoulakis, Beth Israel Deaconess Medical Center, Div. of Gastroenterology, 330 Brookline Ave., Boston, MA 02215.

Received 26 August 1996; accepted in final form 15 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(5):G1014-G1022
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