* Laboratoire d’Anatomie-Pathologique,
INSERM U364, and
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
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Key Words: CNF-1 • polymorphonuclear leukocytes • cytoskeleton • Rho • Rac • Cdc42 • small GTPases • integrins
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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 View larger version (41K): [in a new window] |
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).
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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
).
  View larger version (112K): [in a new window] |
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.
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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).
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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.
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 View larger version (35K): [in a new window] |
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.
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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 ).
 View larger version (49K): [in a new window] |
Figure 7. Speculative mechanisms of the consequence of CNF1-treated PMNL during
their interaction with the epithelium. RLO , radical oxygen
intermediates; TJ, tight junction.
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Received January 12, 2000; revised May 1, 2000; accepted May 2, 2000.
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