Hepatocyte Growth Factor Alters Renal Epithelial Cell Susceptibility to Uropathogenic Escherichia coli

MDCK Culture and HGF Treatment

Low-passage type II MDCK cells were obtained from K. Mostov (University of California San Francisco, San Francisco, CA) and used between passages 3 to 10 as previously described (8,16). Cells were cultured in modified Eagle’s minimum essential medium (MEM) containing Earl’s balanced salt solution and glutamine supplemented with 5% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 μg/ml amphotericin B. Ca²⁺-free medium was used to open the intercellular junctions and alter MDCK cell polarity as described previously (17). For all our experiments, MDCK cells were seeded at confluency on Transwell filter units (Costar, Cambridge, MA). Pore size on all filters was 0.4 μm, except for bacterial translocation experiments for which filters with 3-μm pores were used. No bacteria were recovered from the basolateral compartment after apical application of bacteria when blank 0.4-μm pore filters were used. Cell monolayers were used for experiments after 3 to 5 d of culture with daily changes in medium. In some experiments, HGF at 100 ng/ml was added to the basolateral compartment of MDCK cell monolayers for a 72-h pretreatment. This dose was shown to dedifferentiate MDCK cell monolayers (8). Recombinant human HGF was generously provided by R. Schwall (Genentech, San Francisco, CA).

Bacteria Strains, Culture, and Concentration

Escherichia coli uropathogenic strain J96 was obtained from American Type Culture Collection (Manassas, VA) and expresses type 1 and P pili, hemolysin, and colicin (18). XL1-Blue (Stratagene, La Jolla, CA) is a laboratory strain that is used for plasmid transformation and is known to be nonpathogenic. Bacteria stocks were stored at −20°C in 50% glycerol and broth. All bacteria were cultured in broth overnight at 37°C. Broth consisted of 32 g of tryptone, 20 g of yeast extract, 5 g of NaCl, and 5 ml of 1 N NaOH per liter. Bacterial concentrations were determined by OD 600 nm with each 0.1 OD equal to 10⁸ bacteria/ml. The actual number of colony counts was confirmed by growing bacteria on liquid broth plates overnight at 37°C.

Determination of Bacterial Translocation and Transepithelial Resistance

Monolayers of MDCK cells were grown on 12-mm-diameter Costar transwell filters with 3-μm pores for bacterial translocation experiments. The final volumes of antibiotic free medium with or without bacteria in the apical and basolateral compartments were 600 and 1200 μl, respectively. Freshly cultured bacteria (J96 or XL1-Blue) were added to the apical compartments (200 μl of bacteria suspension at 1 × 10¹⁰/ml of antibiotic-free medium to the apical or 400 μl to the basolateral compartment). Aliquots (20 μl from basolateral compartment) were removed at 0, 2, 4, and 8 h. Dilutions from each aliquot were made and plated on LB agar plates and incubated for 12 to 16 h at 37°C, and colonies were counted. For transepithelial resistance (TER) experiments, MDCK monolayers were grown on 12-mm-diameter, 0.4-μm pore size, filters, and electrical resistance was measured with the EVOM electrical resistance system (World Precision Instruments, New Haven, CT) TER was measured at 0, 2, 4, 6, and 8 h after exposure of either the apical or basolateral surface to bacteria. TER across MDCK cells on filters with 3-μm pores was similar to that seen using filters with 0.4-μm pores (189 ± 4 ohms/cm²).

Immunofluorescence and Propidium Iodide Staining

MDCK II cells were grown on 6.5-mm-diameter Costar transwell filters with 0.4-μm pores. Bacteria (XL1-Blue or J96) or medium alone (control) were added to the apical compartment of each filter. HGF (100 ng/ml) was added to the basolateral compartment of half of the filters for 72 h before bacterial exposure. In addition, some filters received either Ca⁺²-free media with J96 or Ca⁺² media alone (control). Wells with bacteria were incubated for 0, 2, and 4 h at 37°C. After incubation, wells were washed with PBS⁺ (PBS containing Ca⁺² and Mg⁺²) and then fixed with 4% paraformaldehyde for 20 min at 4°C. After fixation, wells were washed three times with PBS⁺ and quenched with 75 mM NH₄CL and 20 mM glycine for 20 min at room temperature and washed again with PBS⁺. Permeabilization of cells was performed by incubating wells at 37°C for 15 min in PBS⁺ with 0.7% fish skin gelatin–0.025% saponin (PFS). During permeabilization, RNase at 25 μg/ml was added to each filter. Rat mAb R40.76 hybridoma against ZO-1, a peripheral membrane protein associated with the cytoplasmic aspect of tight junctions (19), was obtained from B. Stevenson (University of Alberta, Alberta, Canada). R40.76-conditioned medium was used at 1:2 dilution in PFS for 1 h. Before adding the secondary antibody, each well was washed with PFS 4 times for 5 min each. The secondary antibody donkey anti-rat IgG FITC (1:100 dilution) and propidium iodide (2 μg/ml) were added to the wells and incubated for 1 h. The wells were washed with PFS (4×, 5 min) and then with PBS⁺ with 0.1% TX-100 (2×, 3 min) and postfixed with 4% paraformaldehyde for 20 min each. Filters were then cut from wells and mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed with laser scanning confocal microscopy (Leica LSCM, Heidelberg, Germany). Surface fluorescence labeling of apically accessible E-cadherin in MDCK cells after HGF treatment was performed as described previously (8).

Cell Lysate Preparation and Immunoprecipitation

MDCK II cells grown on 24-mm-diameter Costar filters with 0.4-μm pores were used for preparation of cell lysates for Western blot analysis. Adherent cells on filters were exposed to 0.2 ml of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% TX-100, 1% deoxycholic acid, and 5 mM ethylenediaminetetraacetic acid (radioimmunoprecipitation assay [RIPA] buffer) containing inhibitors of proteases (2 mM phenylmethylsulfonyl fluoride, 50 μg/ml pepstatin, 50 μg/ml chymostatin, and 10 μg/ml antipain) for 15 min on ice. Nonadherent cells in the apical compartment medium were collected by microfuge centrifugation at 3000 rpm and added to the RIPA buffer on the respective filters. Adherent cells were scraped from the filter with a rubber policeman to generate total cell lysates. Total cell lysates were sedimented in a 4°C microfuge at 14,000 rpm. The protein concentration of each cell lysate was determined by using the bicinchoninic acid determination assay (Pierce Chemical Co., Rockford, IL). For the immunoprecipitation of E-cadherin, MDCK cells were cultured in 10-cm-diameter tissue culture petri dishes until confluent. Confluent monolayers were rinsed once in ice-cold PBS⁺ and lysed in RIPA buffer. Total cell lysates were sedimented in a 4°C microfuge at 14,000 rpm. Soluble lysates were rotated with E-cadherin mAb (Transduction Laboratories, Lexington, KY) for 2 h at 4°C. Immunocomplexes were collected with affinity-purified, rabbit anti-mouse IgG (Jackson Immuno Research Labs, West Grove, PA) coupled to protein A-Sepharose beads. Immunoprecipitate beads were washed three times with PBS⁺ and resuspended in 200 μl of the same, and 20 μl of immunoprecipitated E-cadherin were exposed to 5 μl of the bacterial suspension (1 × 10¹⁰/ml) or PBS⁺ (control) for various times. The reactions were terminated by the addition of 8 μl of 4X Laemmli buffer containing 100 mM dithiothreitol and boiling for 5 min.

Electrophoresis and Western Blot Analysis

Western blot analysis was performed as described previously (8). Briefly, equal amounts of protein in lysates of MDCK cells incubated with bacteria or immunoprecipitated E-cadherin incubated with bacteria were run on 7.5% acrylamide gels at 160 v for 45 min. Proteins were transferred to Immobilon P membranes (Milipore Corp., Bedford, MA). Membranes were blocked for 30 min with PBS⁻ (PBS without Ca⁺² and Mg⁺²) 5% milk, 0.1% Tween 20 (block solution). Filters were probed with E-cadherin (1:500) (Transduction Laboratories) for 1 h and then washed with PBS⁻ 0.1% Tween 20 (4×, 5 min). Filters were then probed with either horseradish peroxidase-labeled goat anti-mouse at 1:25,000 dilution in block solution for 1 h. Filters were again washed (4×, 5 min) with PBS⁻ 0.1% Tween 20. Filters were developed by using the enhanced chemiluminescence kit (ECL; Amersham Corp., Piscataway, NJ) and visualized on Kodak X-OMAT film (Eastman Kodak, Rockester, NY).

Gentamicin Protection Assay/Invasion Assay

To quantitate intracellular bacterial invasion, gentamicin protection assay was performed as described previously (13). MDCK cells grown on transwell filters were exposed to varying concentrations of J96 or XL1-Blue. After incubation with bacteria for 0, 2, 4, or 8 h, filters were washed with PBS⁺ to remove nonadherent bacteria. Each filter was then incubated with MEM medium containing 200 μg/ml gentamicin for 45 min to kill external bacteria. The filters were washed with PBS⁺, and the cells were lysed osmotically with sterile H₂O. Lysates were then plated on LB agar plates and incubated at 37°C overnight, and colonies were counted the next day.

Cytotoxicity Assay

Cytotoxicity was determined by using the LIVE/DEAD viability/cytotoxicity assay (Molecular Probes, Inc., Eugene, OR). Differentiation of living and dead cells was determined by cell uptake of either green or red dyes, respectively. MDCK monolayers grown on 6.5-mm-diameter Costar transwell filters were incubated with LIVE/DEAD reagents after exposure to bacteria (J96 or XL1-Blue) at 3.33 × 10⁹ bacteria/ml or media without bacteria for 0, 2, 4, and 8 h. The filters were then cut from the wells and mounted on glass slides. Conventional immunofluorescence images of live and dead cells were obtained with a Leica fluorescence microscope (Leica, Wetzlar, Germany) that was equipped with a Hamamatsu C5810 digital camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). The generated photomicrographs were labeled by using Photoshop (Adobe, Mountain View, CA).

Basolateral Exposure of Polarized MDCK Cells to J96 Decreases TER

Basolateral exposure of MDCK cells to 3.33 × 10⁹ bacteria/ml of J96 resulted in a drop in TER such that the TER approached that of blank filters by 2 to 4 h after exposure (Figure 1A). There appeared to be a threshold effect in which the TER of filters exposed to lower concentrations of J96 (3.33 × 10⁷ or 10⁵ bacteria/ml) did not differ from controls at up to 8 h (Figure 1A). In contrast, basolateral exposure to the XL1-Blue strain had no effect on barrier function as measured by TER (Figure 1A). The apical surface was resistant to an effect of J96 on the integrity of the epithelial cells because the TER across the monolayer did not drop below that of controls by 8 h, even with exposure to J96 (3.33 × 10⁹ bacteria/ml) (Figure 1A). Apical application of XL1-Blue also had no effect on the TER (Figure 1A). These data show that the apical surface of MDCK cells is resistant to disruption of TER by both J96 and XL1-Blue and that the basolateral surface is susceptible to disruption of barrier function by uropathogenic Escherichia coli (J96), but not XL1-Blue.

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Figure 1. The effect of bacteria on TER and bacterial translocation. (A) The surface of polarized monolayers of Madin-Darby canine kidney (MDCK) cells grown on filter supports were exposed to increasing concentrations of basolateral J96, apical J96, basolateral XL1-Blue, or apical XL1-Blue strains of Escherichia coli. The bacteria were diluted in antibiotic-free medium to the indicated concentration (bacteria/ml) before addition to wells. The TER was measured at the indicated time points after addition of bacteria. (B) Hepatocyte growth factor (HGF) pretreatment renders MDCK monolayers susceptible to apical disruption of TER by J96. Control and HGF-treated MDCK cells were exposed to apical or basolateral J96 (3.33 × 10⁹ bacteria/ml). HGF-pretreated cells were exposed to HGF (100 ng/ml) for 72 h. TER was then measured at the indicated time points after addition of bacteria to indicated cultures. (C) Quantitation of apical to basolateral translocation of J96 across control and HGF-pretreated MDCK monolayers. Escherichia coli (3.33 × 10⁹ bacteria/ml) was added to the apical compartment of control and HGF-pretreated (72 h) MDCK cell monolayers grown on 3.0-μm Transwell filter units (bacteria were unable to traverse filters with 0.4-μm pores). Aliquots were removed from the basolateral (20 μl) compartment at 2-h intervals up to 8 h and plated on LB plates. All values represent the mean of three determinations ± SD. The error bars are not visible when smaller than the size of the symbol.

HGF Pretreatment Increases Apical Susceptibility of MDCK Cell Layers to J96

HGF disrupts epithelial polarity by causing epithelial cells to dedifferentiate and proliferate (8–10,20). Apically applied antibody against an extracellular epitope of E-cadherin failed to label E-cadherin in control MDCK cell monolayers grown on filters (Figure 2; control). In contrast, cells pretreated for extended periods of time with HGF showed an increasing population of cells expressing apically accessible E-cadherin (Figure 2; 24, 48, and 72 h). These observations confirm that HGF treatment of polarized MDCK cells grown on filters decreases cell polarity as previously reported in detail (8). After pretreatment with HGF, membrane components that were once restricted to the basolateral surface only become accessible to the apical surface and vice versa. When MDCK cell monolayers were pretreated with HGF, apically applied J96 dropped TER to that equivalent to empty filters after 2 to 4 h (Figure 1B). This drop in TER was similar to what was seen with basolateral treatment of control MDCK cells with J96 (Figure 1, A and B). Treatment of MDCK cell monolayers with lower concentrations of HGF (1 and 10 ng/ml) also increased the apical susceptibility to J96. However, at these HGF concentrations, the drop in TER was observed after 10 and 6 h after exposure to J96 (data not shown). The drop in TER of MDCK cells treated with HGF and exposed to basolateral J96 was not as rapid as seen in control MDCK cells exposed to basolateral J96 (Figure 1, A and B). This effect is most obvious at the 2-h time point. In the absence of HGF, the MDCK cell monolayers were resistant to disruption by apically applied J96 at 3.33 × 10⁹ bacteria/ml (Figure 1, A and B). The threshold concentration of J96 required to disrupt the TER in HGF-pretreated MDCK cell layers from both the apical and basolateral surface was 3.33 × 10⁹ bacteria/ml. Exposure of HGF-pretreated MDCK cell layers to lower concentrations of J96 (3.33 × 10⁷ and 10⁵ bacteria/ml) had no effect on TER. MDCK cell monolayers maintained TER when exposed to either apical or basolateral XL1-Blue with and without HGF pretreatment (Figure 1A and data not shown). Interestingly, there was an increase in TER of filters pretreated with HGF and not exposed to bacteria.

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Figure 2. Time course of HGF-induced apically accessible E-cadherin. MDCK cell monolayers were treated for 0 (control), 24, 48, or 72 h with HGF and subjected to apical immunolabeling for E-cadherin as described in Materials and Methods. White line equals 10 μM.

HGF Pretreatment Increases Apical to Basolateral Translocation of J96 across MDCK Cell Layers

When the J96 bacteria were added to the apical compartment of control and HGF-pretreated MDCK cell layers cultured on filters with 3.0-μm pores, the number of bacteria recovered from the basolateral medium increased over time in only the HGF-pretreated MDCK cells (Figure 1C). In control MDCK cells, no J96 bacteria was detected in the basolateral medium after incubation periods of up to 8 h. HGF pretreatment of MDCK cells increased the ability of J96 to cross the epithelial monolayer. No XL1-Blue was recovered from the basolateral medium, even after 8 h of incubation with or without HGF pretreatment (data not shown).

Effect of J96 and XL1-Blue on MDCK Cell Viability

A possible mechanism by which J96 disrupts TER and enhances apical to basolateral translocation across the monolayer is by J96-induced MDCK cell cytotoxicity. Piliated hemolytic strains of uropathogenic Escherichia coli have been shown to be cytotoxic to nonpolarized human renal epithelial cells (21). We used the LIVE/DEAD cytotoxicity assay to determine the effect of MDCK cell exposure to J96 and XL1-Blue on cell viability. Control MDCK cells were exposed to either J96 or XL1-Blue in the apical or basolateral compartment at a concentration of 3.33 × 10⁹ for 4 h (Figure 3). Apical (Figure 3B) and basolateral (Figure 3D) XL1-Blue and apical J96 (Figure 3C) had no appreciable effect on cell death compared with cells not exposed to bacteria (Figure 3A). However, significant cytotoxicity was observed after basolateral exposure to J96 (Figure 3E). On the filters that were exposed to basolateral J96, many of the cells had fallen off the filter, and the few remaining cells either stained red or were rounded and detached from the filter, suggesting cell necrosis. The appearance of a large number of dead cells after 4 h incubation with basolateral J96 coincides with the decrease in TER seen between 2 to 4 h after exposure to basolateral J96 (Figure 1, A and B).

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Figure 3. LIVE/DEAD stain (Molecular Probes, Inc., Eugene, OR) of MDCK cells grown on filters after exposure to bacteria. LIVE/DEAD stain was performed on MDCK cells after 4-h incubation with (A) control (no bacteria), (B) apical XL1-Blue, (C) apical J96, (D) basolateral XL1-Blue, and (E) basolateral J96. LIVE/DEAD stain was also performed on MDCK cells that were pretreated with HGF for 72 h and incubated for 4 h with (F) control (no bacteria), (G) apical J96, (H) basolateral J96. LIVE/DEAD stains of filters that were exposed to either apical or basolateral XL1-Blue bacteria after pretreatment with HGF looked similar to controls (data not shown). The LIVE/DEAD stain differentiates between viable and dead cells by staining with green and red fluorescent dyes, respectively.

We next assessed the effects of HGF pretreatment on cytotoxicity after apical and basolateral exposure to J96 (Figure 3). Apical exposure to J96 resulted in marked cell death (and many cells falling off the filter) in the HGF-pretreated (Figure 3G) filters compared with non-HGF–treated (Figure 3C) and HGF-pretreated filters not exposed to bacteria (Figure 3F). Basolateral exposure of HGF-pretreated cell monolayers to J96 resulted in an impressive increase in the number of dead cells (Figure 3H). However, the cell monolayer was still intact. This was in contrast with the effect of basolateral exposure of control MDCK cell monolayers to J96, which resulted in significant detachment of the cells from the filter. These data suggest that. although HGF pretreatment renders the apical surface more sensitive to the cytotoxic effects of J96, the basolateral surface becomes more resistant to the cytotoxic effects of J96. These data in conjunction with the TER data support the conclusion that HGF-induced loss of cell polarity has reciprocal effects on the relative sensitivities of the epithelial cell surface domains to the effects of J96. Apical and basolateral exposure of HGF-pretreated MDCK cell monolayers to XL1-Blue did not result in cell cytotoxicity (data not shown).

Effect of Escherichia coli on Cell-Cell Junctional Protein E-Cadherin

Another possible mechanism by which J96 could disrupt TER and facilitate apical to basolateral translocation is by hydrolysis of cell-cell junctional proteins. This mechanism would also cause MDCK cells to fall off the filter as seen in Figures 3E and 3G. We have previously provided evidence that this mechanism is potentially important for epithelial tissue invasion by the periodontal disease pathogen Porphyromonas gingivalis (13). Western blot analysis of lysates prepared from control and HGF-pretreated MDCK cell layers apically or basolaterally exposed to J96 was done to determine if the disruption of TER and bacterial translocation was partially due to hydrolysis of the cell-cell junctional protein, E-cadherin. Incubation of control MDCK cell layers with basolateral exposure J96 resulted in a decrease in E-cadherin first seen by 2 h after exposure to J96 (Figure 4A). However, the amount of E-cadherin remained constant in control MDCK cell layers that were apically exposed to J96 for up to 4 h of incubation. In contrast to control MDCK cell layers, pretreatment with HGF resulted in a time-dependent decrease in E-cadherin after apical exposure to J96 (3.33 × 10⁹ bacteria/ml) that was similar to the decrease seen with basolateral exposure of HGF-pretreated cells. Although there was evidence of hydrolysis of E-cadherin in both control and HGF-pretreated MDCK cells that were basolaterally exposed to J96, this effect was not seen until after 4-h incubation in HGF-pretreated cells compared with decreased E-cadherin seen after 2 h in control cells that were similarly exposed. The reciprocal effects on the relative sensitivities of the epithelial domains to reductions in TER and to cell cytotoxicity after pretreatment with HGF corresponds to the effect HGF pretreatment has on the sensitivity of E-cadherin to hydrolysis in the presence of J96. In basolaterally exposed cells, pretreatment with HGF appears to impart a slight resistance to hydrolysis of E-cadherin while there is increased hydrolysis in apically exposed MDCK cells pretreated with HGF compared with controls. Apical and basolateral exposure to XL1-Blue had no effect on the amount of E-cadherin detected in lysates from both HGF-pretreated and control MDCK cell layers (data not shown).

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Figure 4. Effect of bacteria on MDCK cell E-cadherin and immunoprecipitated E-cadherin. (A) Western blot analysis of equal amounts of protein from control and HGF-pretreated MDCK cell lysates apically or basolaterally exposed to J96 (3.33 × 10⁹ bacteria/ml) for 0, 2, and 4 h for E-cadherin. Western blot analysis of these lysates for α-actin confirmed the presence of equal amounts of protein in each lane (data not shown). (B) Effect of J96, XL1-Blue, or medium alone on immunoprecipitated E-cadherin after 2, 4, and 8 h. The Western blot analysis was performed as described in Materials and Methods.

J96 Does Not Directly Hydrolyze Immunoprecipitated E-Cadherin Molecules

To test whether the observed degradation of junctional proteins was directly due to a protease produced by J96 or by a mechanism in which epithelial cell protease(s) are activated by exposure to J96, we next examined the proteolytic effect of J96 on immunoprecipitated E-cadherin molecules (Figure 4B). Immunoprecipitated E-cadherin molecules were incubated with either J96 or XL1-Blue (3.33 × 10⁹ bacteria/ml) or medium alone (control) for 0, 2, 4, and 8 h. After incubation for up to 8 h, there was no evidence of hydrolysis of immunoprecipitated E-cadherin by J96 or XL-1 Blue. This is in striking contrast to the effect seen with Porphyromonas gingivalis, where significant hydrolysis occurs (13). These studies indicate that J96 and XL1-Blue have no proteolytic activity against the junctional protein E-cadherin and that the degradation of E-cadherin after basolateral exposure of control MDCK cell layers and apical exposure of HGF-pretreated MDCK cell layers is due to activation of epithelial cell proteolysis.

Adherence of J96 from the Apical Compartment to Control and HGF-Pretreated MDCK Cell Layers

To gain some insight into whether or not J96 is able to invade epithelial cells from the apical compartment, we apically exposed control and HGF-pretreated MDCK cell layers to J96 (3.33 × 10⁹ bacteria/ml) for 2 h. A 2-h exposure time was chosen because the HGF-pretreated MDCK cell layer remains largely attached to the filter at this time point but becomes mostly detached after 4 h of exposure (Figure 3G). After exposure, the cell layers were gently washed to remove nonadherent bacteria, fixed, and then the DNA of both the MDCK cells and bacteria were stained with the fluorescent dye, propidium iodide. The MDCK cells were also FITC-labeled with a rat mAb against ZO-1 to mark the tight junctions. Scanning laser confocal microscopy was used to analyze the three-dimensional architecture of the bacteria-exposed MDCK cell layers by using X-Y (a section parallel to the plane of the filter) and X-Z (a section perpendicular to the plane of the filter) views. Figure 5A shows only occasional J96 bacterium attached to the apical surface of control MDCK cell layers by X-Y view at the level of ZO-1 (tight junction). The X-Z view confirmed that the rare bacteria were on the apical surface and most likely not internalized (Figure 5B). However, after HGF pretreatment, J96 attachment to the MDCK cells from the apical compartment was dramatically increased as demonstrated by numerous bacteria seen on both X-Y (Figure 5C) and X-Z (Figure 5D) sectional views. Even with HGF pretreatment and enhanced adherence to the apical surface, no definitely internalized bacteria could be seen on multiple X-Z views. Figure 5D also demonstrates that HGF treatment induces morphologic changes, i.e., increases thickness of the monolayer and more tortuous interrelationships between adjacent cells as described previously (8).

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Figure 5. Immunofluorescence analysis of control and HGF-treated MDCK cells after exposure to apical J96 viewed with confocal microscopy. HGF treatment of MDCK cells for 72 h led to increased adherence of bacteria to the surface of MDCK cells. (A and B) are X-Y and X-Z sections of control cells that were exposed to J96 compared with X-Y and X-Z sections of HGF-treated cells (C and D). MDCK cells treated with HGF become stratified (D) with numerous adherent bacteria. White line equals 10 μm.

Disruption of MDCK Cell Polarity with Ca⁺²-Free Medium Mimics the Effects of HGF

To test the hypothesis that the effects of HGF on MDCK cell susceptibility to HGF are at least partially the result of reduction of cell polarity, we examined the effects of Ca⁺²-free medium on adherence of apically applied J96 to MDCK cells. Ca⁺²-free medium disrupts MDCK cell polarity by opening the adherens and tight junctions (17). MDCK cells that were incubated in Ca⁺²-free medium and MDCK cells that were pretreated with HGF exhibited similar morphologic alterations. Both pretreatment with HGF and incubation in Ca⁺²-free medium induced mislocalization of the occludens junction and stratification of the MDCK cell layer (Figures 5D and 6B). Similar to what was seen with HGF pretreatment, there was markedly increased adherence of J96 bacteria to MDCK cell layers that were incubated in Ca⁺²-free medium (Figures 6D and 6E) compared with MDCK layers that were grown in Ca⁺²-containing medium. In addition, Ca⁺²-free medium also renders MDCK cell monolayers susceptible to apically applied J96-induced cytotoxicity (Figure 6F). Ca⁺²-free medium alone disrupted the normal chicken-wire distribution of ZO-1 (green) (Figure 6A) but did not affect cell viability (Figure 6C).

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Figure 6. The effect of Ca⁺²-free medium on bacterial adherence and cytotoxicity in MDCK cells apically exposed to J96. Exposure of MDCK cells to Ca⁺²-free medium led to increased adherence of bacteria to the surface of MDCK cells, using propidium iodide stain (red) and ZO-1 labeling (green). (A and B) X-Y and X-Z sections of MDCK cells that were exposed to Ca⁺²-free medium alone (2 h) compared with X-Y and X-Z sections of MDCK cells exposed to Ca⁺²-free medium and apical J96 (2 h) (D and E). Viability was examined by using the LIVE/DEAD stain. Exposure of MDCK cells to Ca⁺²-free medium (4 h) increased MDCK cell susceptibility to J96-induced cytotoxicity (F). Ca⁺²-free medium alone (4 h) did not alter MDCK cell viability (C). White line equals 10 μm in (A, B, D, and E). White line equals 50 μm in (C and F).

MDCK Cell Layer Invasion Assays

The possibility of invasion was further investigated by using the gentamicin protection assay that is more sensitive than propidium iodide staining for determining intracellular invasion. Gentamicin at high enough concentrations kills external bacteria and leaves any internalized bacteria alive. Cytosolic extracts prepared by osmotic lysis of control and HGF-pretreated MDCK cells after apical exposure to J96 or XL1-Blue grew no bacteria after washing the external surface of cells with gentamicin-containing medium, which suggests that there was no internalization of J96 into MDCK cell monolayers (data not shown). However, this assay does not rule out the possibility that J96 is able to invade and kill the MDCK cells in a process that also allows the passive movement of gentamicin into the dead MDCK cells and inhibition of bacterial colony growth on LB plates.