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(Journal of Leukocyte Biology. 2000;68:522-528.)
© 2000 by Society for Leukocyte Biology

Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) increases the adherence to epithelia and the oxidative burst of human polymorphonuclear leukocytes but decreases bacteria phagocytosis

Paul Hofman*,{dagger}, Gaëlle Le Negrate{dagger}, Baharia Mograbi{dagger}, Véronique Hofman*, Patrick Brest{dagger}, Annie Alliana-Schmid{dagger}, Gilles Flatau{ddagger}, Patrice Boquet{ddagger} and Bernard Rossi{dagger}

* Laboratoire d’Anatomie-Pathologique,
{dagger} INSERM U364, and
{ddagger} INSERM U452, IFR 50, Faculté de Médecine, Nice, France

Correspondence: Paul Hofman, M.D., Ph.D., INSERM unité 364, IFR 50, Faculté de Médecine, avenue de Valombrose, 06107 Nice, Cédex 02, France. E-mail: hofman{at}unice.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recruitment of polymorphonuclear leukocytes (PMNL) is a hallmark of both urinary and digestive infections caused by Escherichia coli. Cytotoxic necrotizing factor 1 (CNF-1) is a toxin produced by uropathogenic E. coli strains that mediates its effects via the activation of small GTP-binding proteins. However, the role and the consequences of CNF-1 on PMNL physiology remain largely unknown. In this study, we provide evidence that CNF-1 dramatically affects the PMNL cytoskeleton architecture by inducing an increased content of F-actin. Furthermore, we demonstrate that CNF-1 increases functional features of PMNL, such as superoxide generation and adherence on epithelial T84 monolayers, but significantly decreases their phagocytic function. Our results suggest that CNF-1 may behave as a virulence factor in urinary or digestive infection by stimulating PMNL cytotoxicity as a result of its enhancing effect on their adherence to epithelial cells as well as the production of radical oxygen products. Moreover, the decreased phagocytosis of PMNL induced by CNF-1 likely facilitates growth of bacteria. In these conditions, CNF-1 would intervene in the initiation and in the perpetuation of the inflammatory process.

Key Words: CNF-1 • polymorphonuclear leukocytes • cytoskeleton • Rho • Rac • Cdc42 • small GTPases • integrins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli belongs to normal microflora of the gastrointestinal tract, but certain E. coli strains are associated with urogenital diseases, pleuro-pneumonitis, gastroenteritis, and septicemia. Cytotoxic necrotizing factor 1 (CNF-1), discovered by Caprioli et al. [1 ] as a cell-associated product of E. coli strains isolated from young children with diarrhea, causes necrosis of rabbit skin and multinucleation of different types of tissue culture cells. CNF-1 is produced by 40% of pathogenic E. coli strains involved in urinary tract infections [2 ] and by 5%–30% of the E. coli strains isolated from diarrhea [3 ]. CNF-1 acts on epithelial [4 ] or endothelial cells [5 , 6 ], or monocyte-macrophages [7 ], via activation of the Rho, small, guanosine 5'-triphosphate (GTP)-binding proteins. The effects of the small GTP-binding proteins on cell shape are mediated by regulation of the actin-myosin cytoskeleton, and each of the proteins has been shown to have different specific effects on actin. RhoA stimulates formation of stress fibers and impacts on focal adhesion formation; Rac promotes cell ruffles and lamellipodia; and Cdc42 controls the extension of filopodia [8 ]. CNF-1 modifies RhoA by deamidation of Gln63, thereby producing a chemical transformation of this residue into a glutamic acid [9 ].

In addition, RhoA, Cdc42, and Rac can also be substrates for the glutamine deaminase activity of CNF-1 in epithelial cells [10 ]. In epithelial cell systems, the toxin has been shown to induce the accumulation of thick stress fibers and to rescue cells from apoptosis [11 , 12 ]. Recently, we have shown that CNF-1 decreases transepithelial migration of polymorphonuclear leukocytes (PMNL) and modifies microvillus structure of epithelial cells [13 ]. In human endothelial cells monolayers, CNF-1 induces prominent stress fiber formation without significant modifications of the peripheral localization of VE-cadherin [6 ]. Finally, in hematopoietic lineages, the effect of CNF-1 has been studied on monocyte-macrophages, showing an increased content in filamentous actin associated with a decreased phagocytic function [7 ].

The presence of PMNL in urinary or alimentary tracts is frequently associated to bacterial infection by pathogenic E. coli strains [14 , 15 ]. In these diseases, contact of PMNL to epithelial linings has been shown to be responsible for important damages toward epithelial cells.

The effects of CNF-1 on human PMNL have not been explored yet. We provide evidence in the present study that CNF-1 induces a remodeling of the PMNL actin cytoskeleton and a stimulation of their oxygen radical production in accord with its activating effect toward Rho and Rac, respectively [16 , 17 ]. Furthermore, we show that CNF-1 provokes an increased adherence of PMNL on epithelial cells and a decreased bacteria phagocytosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The source of CNF-1 was highly purified CNF-1, used throughout this work and prepared as described previously [18 ]. The bacteria strain was E. coli (HMS174; DE3) and was provided by Novagen (Abingdon, UK).

PMNL preparation and treatment
Human PMNL were isolated from whole blood using a gelatin-sedimentation technique [19 ]. Briefly, whole blood anticoagulated with citrate/dextrose was centrifuged at 300 g for 20 min (20°C). The plasma and buffy coat were removed, and the gelatin/cell mixture was incubated at 37°C for 30 min to remove contaminating red blood cells (RBC). Residual RBCs were then lysed with isotonic ammonium choride. After washing in Hanks’ balanced saline solution (HBSS) without Ca2+ or Mg2+, the cells were counted and resuspended at 5 x 107 PMNL/ml. PMNL (95% pure) with 98% viability by trypan blue exclusion were used within 1 h after isolation. PMNL were incubated with CNF-1 (10-9 M) for 16 h using low-attachment Costar (Cambridge, MA) in RPMI 1640 supplemented with 10% FBS plates.

Morphological analysis
F-actin fluorescence staining of control and CNF-1-treated PMNL was processed as follows. Cells were fixed with 3.7% paraformaldehyde [in phosphate-buffered saline (PBS), pH 7.4] for 30 min at room temperature and rinsed in buffer containing 0.2% gelatin and 0.01% Triton X-100 [7 ]. Cells were then incubated for 45 min in the dark with 500 nM rhodamine-phalloidin (Molecular Probes, Junction City, OR), diluted in PBS, and washed in HBSS; slides were mounted in a phenylenediamine-glycerol-PBS medium.

For CD11b fluorescence staining, cells were fixed as described above and incubated for 1 h with anti-CD11b antibody (Bear1; Immunotech, Luminy, France; diluted 1/200). The cells were washed twice in PBS and then exposed to fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Ig; Sigma, Paris, France) for 45 min at room temperature in the dark, washed in PBS, and then mounted. The slides were observed and photographed with a laser-scanning fluorescence microscope (Leica, DMIRBE, Lyon, France) equipped for epifluorescence.

For electron microscopy studies, control and CNF-1-treated PMNL were fixed with 2% formaldehyde, in 0.1 M Na cacodylate, pH 7.4, for 1 h at 4°C. Pellets were rinsed in cacodylate buffer, post-fixed in 1% OsO4 for 1 h, dehydrated through graded alcohols, and embedded in epoxy resin. Oriented 1-mm sections were obtained with diamond knives, and multiple areas were thin-sectioned, mounted on copper mesh grids, and stained with uranyl acetate and lead citrate. Ultrathin sections were examined on a Jeol 1200 XII electron microscope.

Resting or CNF-1-stimulated PMNL pellets were fixed in 3.7% paraformaldehyde and embedded at low temperature into LR White resin (Hard LR White, London, UK) for immunoelectron microscopy [20 ]. Ultrathin sections were put on 300 mesh nickel grids, washed with PBS, and then incubated for 60 min at room temperature with CD11b antibody (Bear1; Immunotech; diluted: 1/100). After washing with PBS, the grids were incubated for 60 min with 10 nm colloidal gold-conjugated rabbit anti-mouse secondary antibody (British Biocell International, Cardiff, UK). The grids were washed with PBS and then with distilled water and stained with uranyl acetate.

Viability of control PMNL after 16 h and CNF-1-treated PMNL (10-9 M for 16 h) was assessed by trypan blue exclusion. The morphological changes of apoptosis were examined by light and electron microscopy. Briefly, control PMNL after 16 h and CNF-1 treated PMNL (10-9 M for 16 h) were fixed with methanol and stained with Wright-Giemsa, and the slides were examined by light microscopy. At least 400 cells of each preparation in various fields were counted. For electron microscopy, PMNL were processed as described above. Apoptotic cells were easily distinguishable on the basis of their reduced volume, chromatin condensation, and nuclear fragmentation.

Flow cytometry analysis
To determine F-actin, control and CNF-1-treated PMNL were fixed with 3.7% formaldehyde and incubated for 45 min with PBS containing 500 nM rhodamine-phalloidin. After being washed in PBS, the cellular content in F-actin was determined. Analysis was performed on a FACScan (Becton Dickinson, Rutherford, NJ) with the channel (log scale) representing the mean fluorescence intensity for 10,000 cells.

Control PMNL, CNF-1-treated PMNL, and PMNL stimulated by N-formyl-L-methionyl-leucyl-L-phenylalanine peptide (fMLP; 10-7 M for 30 min in HBSS) were fixed in 1% formalin for 30 min at room temperature for expression of integrins. The cells were then washed once in HBSS and incubated with polyclonal goat Ig for 20 min. PMNL were washed again in HBSS and incubated with OKM1 (anti-CD11b; American Type Culture Collection, Rockville, MD; diluted: 1/1000), K20 (anti-CD29; INSERM 343, Nice, France), or isotype-matched control antibodies for 20 min at room temperature and then washed twice. Cells were then exposed to FITC-conjugated goat anti-mouse Ig (Sigma) for 20 min at room temperature in the dark, and then washed and resuspended in 500 µl HBSS. Analysis was performed on a FACScan (Becton Dickinson) with the channel number (log scale) representing the mean fluorescence intensity of 10,000 cells.

PMNL adherence to epithelial colonic monolayers (T84) and transmigration assay
The physiologically (basolateral-to-apical) directed PMNL transepithelial migration was then performed as previously described [21 ]. PMNL transmigration experiments were performed at 37°C on 0.33-cm2 inverts. The PMNL were suspended in modified HBSS (without Ca2+ and Mg2+) with 10 mM HEPES (pH 7.4; Sigma) at a concentration of 5 x 107/ml. PMNL were incubated for 16 h before transmigration without CNF-1 or with 10-9 M CNF-1. We added 0.5 x 106 PMNL to the inverts. Transmigration of PMNL was initiated in the presence or absence of 10-7 M fMLP to the lower reservoir and incubated for 15 min to allow a transepithelial chemotactic gradient to form before the addition of PMNL. The number of adherent PMNL to the epithelial cells and the number of PMNL that transmigrated into the lower reservoirs were assayed by quantification of the azurophil granule marker myeloperoxidase (MPO), as described previously [19 ]. Briefly, after 2 h of transmigration, T84 monolayers were rapidly cooled to 4°C, washed with HBSS, and solubilized in 1% Triton X-100 containing HBSS. The pH was adjusted to 4.2 with a 1:10 dilution of 1.0 M Na citrate, pH 4.2, and peroxydase activity was assayed by the addition of an equal volume of 1 mM 2,2'-azino-di-(3-ethyl) dithiazoline sulfonic acid and 10 mM H2O2 in 100 mM citrate, pH 4.2. To quantitate PMNL, which migrated through the monolayer into the lower reservoir, 10% Triton X-100 was added to the reservoir and assayed as described above. Color development was quantitated on a microtiter plate reader at 405 nm. Data are pooled from 6–12 individual monolayers for each condition, and results are means ± SE of five experiments.

In some experiments, the morphological consequences of PMNL adherence to epithelial colonic monolayers were assessed by electron microscopy. Three different T84 monolayers were examined for each experimental condition after 24 h of contact with PMNL, treated or not treated with CNF-1 (16 h, 10-9 M). After removal from the inverts, the monolayers were fixed with 2% freshly prepared formaldehyde in 0.1 M Na cacodylate, pH 7.4, for 1 h at 4°C. Tissues were rinsed in cacodylate buffer and then processed as describe above.

Superoxide assay
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-catalyzed superoxide generation was assayed by luminol-dependent chemiluminescence [22 ]. CNF-1-treated (for 2 h and 16 h) and control PMNL were resuspended at 2.5 x 105 cells/ml in 96-well microtiter plates (Dynatech Laboratories, Guyancourt, France). Opsonized zymosan (OPZ, 0.05 ml, of 108 particles/ml) was used to trigger radical oxygen intermediate (ROI) production before the addition of 80 mM luminol (Sigma) in the dark. The chemiluminescence resulting from the reaction of luminol with radicals was measured at 37°C using a luminometer (ML 3000 Microtiter Plate Luminometer, Dynatech Laboratories).

Phagocytosis determination
Phagocytosis capacity of the control and CNF-1-treated (for 2 h and 16 h) PMNL was monitored by measuring the engulfment of the E. coli (HMS174; DE3) strain (Novagen), expressing the green fluorescent protein (GFP). The GFP was amplified by polymerase chain reaction from the pEGFP-1 vector (Clontech, Palo Alto, CA), ligated in the plasmid pET28a (+) (Novagen) and thus inducible by isopropylthiogalactoside (IPTG; ICN, Irvine, CA). IPTG (0.5 mM) was added for expression of GFP. The number of bacteria incubated with PMNL was determined by optical density (OD), considering that OD600 = 1 corresponds to 109 bacteria. PMNL were incubated for 120 min with bacteria (ratio PMNL/E. coli: 1/25 ) in HBSS. The number of phagocytosed E. coli in PMNL was estimated by fluorescent microscopy or quantified by flow cytometry using a FACScan (Becton Dickinson).

Data analysis
Myeloperoxidase and flow cytometric assays were compared by Student’s t-test. Values are expressed as the mean and SEM of n number of experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CNF-1 induces morphologic changes of PMNL and F-actin reorganization
The morphology of control PMNL (16 h) and CNF-1-treated PMNL was assessed by electron microscopy. Changes in the shape of CNF-1-stimulated PMNL were detectable as soon as 6 h, with the greatest effect observed after 16 h of treatment. Cells often covered a much larger area than control PMNL and exhibited cytoplasmic projections ressembling pseudopodia or filopodia (Fig. 1A and B ).



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  		Figure 1. Morphologic modifications and F-actin reorganization induced by CNF-1 (10-9 M for 16 h) on PMNL. Transmission electron microscopic photographs of control (A) and CNF-1-treated PMNL (B ; original bar size, 10 µm). F-actin distribution in control (C) and CNF-1-treated (D) PMNL were observed with confocal microscopy. Determination of PMNL F-actin by flow cytometry. PMNL incubated with CNF-1 (F) presented a shift in the fluoresence peak, indicating a polymerization of actin in comparison with control cells (E).

 
Considering the fact that CNF-1 induces a prominent F-actin reorganization in various models, we tested the effect of this toxin (10-9 M for 16 h) on the PMNL actin cytoskeleton. F-actin distribution was investigated by conventional fluorescence and confocal microscopy. In control cells, an evenly distributed subcortical actin band was observed (Fig. 1C) . In contrast, CNF-1 treatment caused a concentration of subcortical F-actin in broad membrane extension. A fraction of treated cells (60±5%) remained spherical but exhibited numerous peripheral foci of F-actin organized as microspikes (Fig. 1D) . The other fraction of PMNL displayed a polarized morphology with a large membrane expansion rich in F-actin (unpublished results). The CNF-1-treated PMNL displayed an increased cell size.

The content of F-actin assessed by flow cytometry was significantly higher in CNF-1-treated PMNL (10-9 M for 16 h; Fig. 1F ) than in control cells (Fig. 1E) (p<0.02). F-actin content of PMNL treated with CNF-1 increased to reach a plateau between 10 h and 24 h of treatment. A similar amount of F-actin was obtained by incubating PMNL with 10-7 M fMLP for 30 min (unpublished results).

By the trypan blue exclusion method, no significant difference was noted between control PMNL and CNF-1-treated PMNL (72% vs. 74% of viability for control PMNL vs. CNF-1-treated PMNL, respectively). By light microscopy, control PMNL showed 24% ± 8% apoptotic cells, whereas this proportion was 26% ± 7% in CNF-1-treated PMNL (the difference is not significant; unpublished results). Observation by transmission electron microscopy demonstrated similar results (unpublished results).

CNF-1 does not modify the expression but induces the clustering of ß2 integrin (CD11b) on human PMNL
We quantitatively evaluated by flow cytometry whether CNF-1 treatment could modulate the expression of CD11b and CD29, which have been shown to play a crucial role in PMNL adhesion. Exposure of PMNL to CNF-1 for 16 h was without effect on CD11b (Fig. 2B ) or CD29 expression (Fig. 2D) in comparison with control PMNL (16 h) (Fig. 2A and 2C) . CD11b, as revealed by confocal microscopy, adopted a punctuated distribution in small peripheral patches in CNF-1-treated PMNL (Fig. 2F) , whereas this integrin formed an homogeneous ring at the periphery of control cells (Fig. 2E) . Similar results were obtained for CD29 (unpublished results). Electron microscopy confirmed that CD11b was preferentially clustered into filopodia in CNF-1-treated cells (Fig. 2H) , although it was regularly distributed along the plasma membrane in control cells (Fig. 3G ).




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  		Figure 2. Effect of CNF-1 on the CD11b membrane receptor. Exposure to CNF-1 did not cause any increase in CD11b (B) or in CD29 expression (D) determined by flow cytometry, in comparison with control PMNL (A, C). In CNF-1-treated PMNL, CD11b observed by confocal microscopy was detected concentrated in small peripheral patches (F), whereas CD11b in control cells was detected at the periphery as a fluorescent ring (E). CD11b staining observed by electron microscopy showed numerous beads noted preferentially and regrouped on filopodia in CNF-1-treated cells (H) or regularly distributed along the plasma membrane in control cells (G).

 


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  		Figure 3. Myeloperoxydase assay of PMNL in the monolayers and reservoirs after 2 h of transmigration (basolateral-to-apical direction) induced with or without fMLP (10-7 M). PMNL were incubated for 16 h before transmigration without CNF-1 or with 10-9 M CNF-1. Data are pooled from 6–12 individual monolayers for each condition, and results are means ± SE of five experiments.

 
CNF-1 enhances the adherence of PMNL to T84 cells
Because CNF-1 treatment induced the clustering of ß2 integrin on PMNL, we then investigated whether CNF-1 treatment of PMNL could affect their adherence on epithelial cells. Incubation of PMNL with 10-9 M CNF-1 for 16 h in the absence of fMLP increased by five-fold the number of adherent PMNL associated with the colonic epithelial cells, whereas control PMNL did not adhere to T84 cells (5±1.21 vs. 0.8±0.9x104 cell equivalent for treated PMNL and control cells, respectively; p<0.05; Fig. 3 ). Conversely, a pretreatment of PMNL with CNF-1 (10-9 M for 16 h) decreased the PMNL migration in response to fMLP (10-7 M), as assessed by measuring the myeloperoxydase activity in the lower reservoirs (4±1.9 vs. 10±1.2x104 cell equivalent for treated PMNL vs. control cells, respectively; p<0.05; Fig. 3 ). Concomitantly, incubation of PMNL with CNF-1 increased the number of PMNL associated with the monolayers after PMNL migration induced by fMLP (10-7 M; 9±1.1 vs. 2±1.5x104 cell equivalent for treated PMNL vs. control cells, respectively; p<0.05; Fig. 3 ). To verify that the observed effects were not a result of a direct action on epithelial cells of residual CNF-1 molecules associated with CNF-1-treated PMNL after the washing step, experiments were conducted with T84 cells incubated for 2 h with 10-9 M CNF-1 before being tested for PMNL transmigration. In these conditions, no difference in transmigration was observed after CNF-1 treatment, demonstrating that CNF-1 acted on PMNL and not on epithelial cells (unpublished results).

T84 epithelial cells are altered by prolonged contact with CNF-1-treated PMNL
Electron microscopic analysis of epithelial cells after contact (24 h) with CNF-1-treated PMNL demonstrated necrosis features of T84 cells with disorganization of the monolayer and loss of microvilli (Fig. 4A ). Many PMNL exibited a spontaneous adherence to the monolayers (Fig. 4A) . By contrast, electron microscopic analysis of the epithelial cells after contact with control PMNL did not show severe modification of the monolayers (Fig. 4B) . PMNL associated with the T84 cells were not observed (Fig. 4B) .



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  		Figure 4. Electron micrographs illustrating PMNL-induced epithelial damages in T84 monolayers after contact (24 h) with CNF1-treated PMNL. (A) CNF1-treated PMNL caused epithelial necrosis. PMNL (arrows) in contact with the epithelial cells (E). (B) The morphological features of T84 cells are not altered after contact with control PMNL. Mv, microvilli; Ap, apical side; Bl, basolateral side (original bar size=0.2 µm).

 
CNF-1 enhances superoxide generation by OPZ-stimulated PMNL
ROI production by control and CNF-1-treated PMNL was triggered by addition of OPZ and measured by photon emission using luminol. The extent of ROI production was similar in PMNL treated for 2 h with 10-9 M CNF-1 or in control cells, in the presence or absence of OPZ stimulation (Fig. 5 ). In contrast, when CNF-1 treatment was extended to 16 h with 10-9 M toxin, PMNL exhibited a significant increase in ROI production when challenged with OPZ, in comparison with control cells (61±4 vs. 30±3 for treated vs. control PMNL, respectively; p<0.01). No difference in ROI production could be detected between CNF-1 treated for 16 h or nontreated cells in the absence of OPZ stimulation (5±1.5 vs. 6±2 for treated PMNL vs. control cells, respectively; the difference is nonsignificant), suggesting that CNF-1 treatment affected the NADPH oxydase machinery specifically.



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  		Figure 5. ROI production by control and CNF-1-treated PMNL. The kinetics of ROI production in PMNL with (hatched bars) and without (solid bars) OPZ were measured by luminol-dependent chemiluminescence assay after 2 h and 16 h of incubation in RPMI SVF 10% with or without CNF-1 at 10-9 M.

 
CNF-1 decreases bacteria phagocytosis in PMNL
We next investigated the capacity of CNF-1-treated PMNL to engulf E. coli. CNF-1-treated PMNL exhibited a decreased level of fluorescence intensity in comparison with control PMNL when their phagocytic function was estimated by their capacity to engulf GFP-labeled E. coli. This was visible when PMNL were treated for 16 h (24±7 vs. 82±8 arbitrary units (A.U.) for treated vs. control PMNL, respectively; p<0.001; Fig. 6 ). In contrast, no significant differences were noted in the level of fluorescence intensity between CNF-1-treated PMNL for 2 h and control PMNL (80±6 vs. 82±8 A.U. for treated vs. control PMNL, respectively), indicating that CNF-1 required the same delay to exert its inhibitory effect on PMNL phagocytosis and ROI production. To better assess the number of E. coli really phagocytosed by PMNL, electron-microscopy experiments were performed. The number of E. coli per cell observed in a thin section decreased from 4 ± 2 in control PMNL to 1 ± 2 E. coli per cell in CNF-1-treated PMNL (unpublished results).



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  		Figure 6. Phagocytosis of GFP expressing E. coli by control and CNF-1-treated PMNL. The number of phagocytosed bacteria after 2 h and 16 h of incubation in RPMI FBS 10% with or without CNF-1 at 10-9 M was quantified by flow cytometry.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Uropathogenic E. coli strains that express CNF-1 evoke colibacillosis with a dramatic recruitment of PMNL. Although the mode of action of CNF-1 has been extensively studied in epithelial, endothelial, and monocytic cells [5 , 7 , 11 , 12 , 17 ], no information is available to date on possible effects of CNF-1 on PMNL.

To gain information, we first verified that, in agreement to its effect on other cell types [1 , 4 , 5 ], CNF-1 produced in PMNL morphological changes associated with a profound actin cytoskeleton remodeling. We found that CNF-1 elicited in PMNL an increase in F-actin content after 16 h of treatment compatible with the necessity for CNF-1 to reach the cytosolic compartment for exerting its effect on small GTPases [16 , 17 ]. Actin polymerization produced cell projections rich in F-actin and intracellular formations evoking actin cables. These morphological changes were associated to an accentuated cellular spreading that likely involves the activation of GTPase RhoA [16 , 17 ], because similar cell flattening and actin polymerization have been shown to result from the activation of RhoA by CNF-1 in macrophages [7 ].

Previous studies have shown that ß2 integrin (CD11b/CD18) plays a crucial role in the passage of PMNL across the epithelium [23 ]. We observed in our model that the level of CD11b expression at the cell surface of PMNL was not increased after 16 h of incubation with 10-9 M CNF-1. However, spontaneously or after fMLP stimulation, CNF-1-treated PMNL displayed an increased adherence to the basolateral side of colonic epithelial cells (T84) grown on filters. Interestingly, increased PMNL adhesiveness to epithelial cells paralleled a clustering of the C11b/CD18 integrin into filopodia upon CNF-1 treatment, suggesting a causal relationship between these two events. Previous study has shown a similar clustering of CD11b in CNF-1-treated monocytes [7 ].

It is well established that adhesion and production of ROI are linked event, because adhesion is a prerequiste for a large oxidative burst in response to cytokines [24 ]. At the functional level, we have shown that treatment of PMNL with CNF-1 induced an enhancement of ROI generation triggered by OPZ. Considering that 1) activation of the GTPase Rac is required for building up the NADPH oxidase complex in PMNL [25 ], and 2) CNF-1 has been shown to activate Rac [10 ], one can hypothesize that CNF-1 mediated its effect on PMNL oxidative burst via Rac activation.

Bacterial-epithelial cell interaction usually triggers recruitment of PMNL and epithelium necrosis. In our study, we have observed that prolonged contact of CNF-1-treated PMNL by itself may also induced profound alteration of epithelial cells’ ultrastructure. This is consistent with the necrosis features of epithelium observed during pyelonephritis, cystitis, or enterocolitis because of certain E. coli strains.

Phagocytosis requires reorganization of the actin cytoskeleton and is mainly mediated by the complement receptor type III (CR3 or CD11b/CD18) or by the Fc receptors [26 , 27 ]. We observed that CNF-1 decreased bacteria phagocytosis by PMNL in accord with a previous study obtained on monocytes [7 ]. We suggest that the toxin likely mediates its inhibitory effect by blocking Rho GTPase.

Taken together, our data support a model where excessive adherence of PMNL at the basolateral pole of epithelia in response to CNF-1 may impair their transepithelial migration and might induce an exagerated adhesion-mediated oxidative burst resulting in possible epithelial cells damage (Fig. 7 ).



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  		Figure 7. Speculative mechanisms of the consequence of CNF1-treated PMNL during their interaction with the epithelium. RLO , radical oxygen intermediates; TJ, tight junction.

 
In conclusion, CNF-1 promotes actin cytoskeleton reorganization in human PMNL. This toxin acts on PMNL by increasing their adherence on epithelial cells and by activation of the oxidative burst. Moreover, these effects are associated with a decrease of the phagocytosis function. We hypothesize that CNF-1 enhances the virulence of certain E. coli infections by decreasing the number of phagocytosed bacteria and by promoting an increased release of ROI that results in dramatic epithelial damages.


   ACKNOWLEDGEMENTS
 
The authors thank Ms. Mireille Mari, Dominique Sadoulet, and Anne Doye for their technical assistance.

Received January 12, 2000; revised May 1, 2000; accepted May 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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