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*
Unité des Rickettsies, Université de la Méditerranée, Centre National de la Recherche Scientifique, UPRESA 6020, Faculté de Médecine, and
Laboratoire d’Hématologie, Hôpital de la Conception, Marseille; and
Institut National de la Santé et de la Recherche Médicale, Unité 452, Faculté de Médecine, Nice, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RII (CD32) but
selectively impaired the colocalization of CR3 with F-actin. It is
likely that CNF1-induced reorganization of actin cytoskeleton
down-modulates integrin activation-dependent phagocytosis by preventing
the codistribution of CR3 with F-actin. CNF1 may control some features
of integrin-dependent phagocytosis in myeloid cells through its action
on Rho GTP binding proteins and cytoskeletal
organization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 integrin to VCAM-1 (9).
IL-2-driven lymphocyte proliferation requires the integrity of actin
cytoskeleton and is under the control of Rho proteins (10). Mice
lacking thymic function of the GTPase Rho exhibit severe defects in
fetal and adult thymopoiesis, establishing the critical role of Rho in
the development of early thymic progenitors (11).
In phagocytes, the role of Rho GTPases has been studied for few
cell functions. Rac1 and Rac2 appear to be essential for the activation
of NADPH oxidase complex in neutrophils and macrophages (12, 13). C3
exoenzyme-mediated inactivation of Rho reduces neutrophil chemotaxis
(9). The abnormality of an effector of Cdc42 is related to the
deficiency of neutrophil chemotaxis observed in Wiskott-Aldrich
syndrome (14). The spreading of monocyte-derived macrophages correlates
with the expression of Cdc42 (15) and is enhanced by the ADP
ribosylation of Rho proteins (16). CSF-1 stimulates macrophage
spreading apparently via the activation of Cdc42 and Rac GTPases (17),
but the role of Rho GTPases in phagocytosis, a major
mechanical function of professional phagocytes, has not been
determined. Several receptors mediate the binding and internalization
of particles, opsonized or not. The best characterized receptors are
the different types of receptors for the Fc portion of IgG (FcR) and
CR type 3 (CD11b/CD18), a ß2 integrin involved in the
recognition of iC3b and several determinants expressed by bacteria and
parasites (18). Particle internalization requires actin polymerization
and its remodeling at the contact area between phagocytes and targets
(19).
In this report we studied the role of Rho GTPases in cytoskeleton organization and the phagocytosis of human monocytes by using a bacterial toxin active on the GTPase Rho. Indeed, cytotoxic necrotizing factor-1 (CNF1),2 isolated from Escherichia coli strains, covalently interacts with Rho, resulting in its activation through the deamidation of a glutamine residue at position 63 (20, 21). CNF1 has been reported to induce membrane ruffling, stress fiber assembly, and phagocytic competence in epithelial cells (20, 22). In human monocytes and the myelomonocytic cell line THP-1, CNF1 induced dramatic morphologic alterations and increased filamentous actin (F-actin) content and reorganization. CNF1 also affected some features of CR3-dependent phagocytosis and impaired the codistribution of CR3 with actin cytoskeleton. We suggest that Rho-mediated modulation of actin cytoskeleton might negatively regulate the phagocytic activity of integrins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CNF1 from pathogenic E. coli was purified as previously described (22) and filtered on sulfone to retain endotoxins (23). RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, and HBSS without phenol red were purchased from Life Technologies (Eragny, France). All cell preparations and media were checked for the absence of endotoxins by using Limulus amebocyte lysate (Boehringer Ingelheim, Gagny, France). SRBC and the specific rabbit IgG were purchased from BioMérieux (Marcy l’Etoile, France). Bodipy phallacidin and calcein-AM were obtained from Molecular Probes (Eugene, OR). [32P]nicotinamide adenine dinucleotide (NAD; sp. act., 1.11 TBq/mmol) was obtained from New England Nuclear Products (Les Ulis, France). mAb directed against CD11b (IgG1), CD18 (IgG1), CD32 (IgG2a), controls (IgG1 and IgG2a), and secondary Ab were obtained from Immunotech (Marseille, France). mAb 24 was provided by Dr. N. Hogg (London, U.K.). Other reagents were purchased from Sigma (St. Louis, MO).
Monocytic cells
Blood from healthy adult volunteers was collected in heparinized tubes. PBMC were isolated by Ficoll gradient centrifugation (Nycomed, Oslo, Finland) as previously described (24). Then, they were suspended in RPMI 1640 containing 25 mM HEPES, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (supplemented RPMI 1640). For cytoskeletal determination and phagocytosis assay, 5 x 105 PBMC in a 0.5-ml volume were incubated in Lab-Tek glass chambers (Nunc, Naperville, IL). For superoxide assay, 106 PBMC in a 1-ml volume were cultured in 24-well culture plates (Nunc). After 1 h at 37°C, nonadherent cells were removed by washing. Remaining adherent cells consisted of about 95% monocytes as assessed by morphology, CD14 expression, and latex phagocytosis.
The human myelomonocytic cell line THP-1 was provided by the European Collection of Animal Cell Cultures (Cerdic, Sophia-Antipolis, France). Suspended cells were propagated at an initial density of 5 x 105 cells/ml in supplemented RPMI 1640 by biweekly passages.
Morphologic changes in monocytic cells
Morphologic changes in monocytic cells were studied with scanning electron microscopy (25). Cells were incubated with CNF1 for 24 h and fixed for 30 min in 0.1 M cacodylate buffer (pH 7.2) containing 4% glutaraldehyde. After extensive washings, cells were dehydrated through graded ethanols and critical point-dried in CO2. THP-1 cells and monocytes were examined with a scanning electron microscope (JEOL 35CF, Croissy-sur-Seine, France).
Flow cytometry
Determination of F-actin. After treatment with CNF1, monocytic cells were fixed with 3.7% formaldehyde and incubated for 20 min with PBS containing 10 U/ml bodipy phallacidin, a specific fluorescent probe of F-actin, and 100 µg/ml lysophosphatidylcholine, as previously described (25). After being washed in PBS, the cellular content in F-actin was determined. The cell fluorescence was analyzed by an EPICS XL (Coulter, Hialeah, FL) equipped with an argon laser (488 nm excitation and 525 nm fluorescence emission). Linear fluorescence intensities of 10,000 cells were expressed as the mean of arbitrary units ± SD as provided by the data processing software.
Expression of membrane Ags.
Monocytes and THP-1 cells were incubated with anti-CD11b,
anti-CD18, anti-CD32 (FcRII) mAb, or isotypic controls at
1/100 dilution for 30 min at 4°C. After washing, they were incubated
with FITC-tagged F(ab')2 anti-mouse Igs for 30 min at
4°C. After fixation with 1% formaldehyde, cells were analyzed by
flow cytometry. Gating was established using forward and side scatters
and fluorescence recorded on the log scale.
Fluorescence microscopy
The cytoskeletal organization of monocytes and THP-1 cells was observed with a Labophot microscope (Nikon, Tokyo, Japan) equipped for epifluorescence. The quantification of cell deformations rich in F-actin was determined by comparing the large and the small axes in fluorescence micrographs of single cells. The intracellular distribution of F-actin was also examined with a laser scanning confocal fluorescence microscope (Leica, DMIRBE, Lyon, France) equipped with a x100 (NA 1.4) oil-immersion lens. Serial optical sections of images were collected at 1-µm intervals, analyzed using Adobe Photoshop 3.0, and printed with a Mavigraph color video printer (Sony, Tokyo, Japan).
The colocalization between F-actin and membrane receptors was determined with laser scanning confocal fluorescence microscopy. Monocytes and THP-1 cells were incubated first with mAb anti-CD11b, anti-CD18, anti-CD32, or isotypic controls at a 1/100 dilution for 30 min at 4°C and, second, with rhodamine-tagged F(ab')2 anti-mouse Igs for 30 min at 4°C. After fixation with 1% formaldehyde, cells were incubated with bodipy phallacidin as described above. The specimens were mounted in slowfade solution (Molecular Probes) and examined with the confocal fluorescence microscope equipped with separate filters for each fluorochrome. Monocytes and THP-1 cells were individually labeled with each fluorochrome to evaluate their contribution to fluorescent images under the given confocal conditions. The intensities of bodipy and rhodamine images were adjusted to be roughly equal, then respectively converted into green and red images and merged to synthesize a yellow color.
Rho ADP-ribosylation assay
Rho activation was determined as previously described (21). PBMC and THP-1 cells at 107 cells/assay were incubated with 12.5 nM CNF1 for 18 h in supplemented RPMI 1640. Adherent monocytes and THP-1 cells were scraped; washed twice with 20 mM Tris buffer, pH 7.4, containing 5 mM MgCl2, 10 mM DTT, and protease inhibitors; and harvested in 100 µl of Tris containing MgCl2 and DTT. Then, cells were frozen and thawed at 37°C four times, and spun down (21,500 x g) for 30 min at 4°C. Supernatants were saved, and their protein concentrations were adjusted. Twenty-five microliters of supernatant from control and CNF1-treated cells was added to 2 µg of C3 exoenzyme and [32P]NAD for 1 h at 37°C. RhoA fusion protein (1.5 µM) was also incubated with or without CNF1 and [32P]NAD as controls. Samples were then subjected to 12% SDS-PAGE electrophoresis, and autoradiograms of the gels were performed.
Superoxide assay
Superoxide production was monitored by measuring the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, as previously described (26). Briefly, THP-1 cells or adherent monocytes were incubated in HBSS containing 120 µM ferricytochrome c and 2 mM sodium azide. The reaction was conducted for 1 h at 37°C in the presence of 50 ng/ml of PMA and was stopped by the addition of 1 mM N-ethylmaleimide. Supernatants were collected, centrifuged, and assayed for absorbance at 550 nm. The generation of superoxide was calculated by subtracting the change absorbance in the presence of SOD (300 U/ml) from that in its absence. The results are expressed as nanomoles of superoxide released by 106 monocytic cells in 1 h with an extinction coefficient of 21,000 M-1/cm.
Phagocytosis determination
After CNF1 treatment, THP-1 cells and adherent monocytes were incubated for 1 h at 37°C with unopsonized or opsonized particles. The phagocytosis of unopsonized zymosan is largely mediated by CR3 (24). The opsonization of zymosan particles by complement was conducted as previously described (27). Briefly, zymosan particles were incubated with 50% human serum for 15 min at 37°C. These conditions result in a maximum deposition and conversion of C3 into iC3b. SRBC were opsonized with specific IgG at 1/2000 dilution (IgG-SRBC). One milligram per milliliter of unopsonized zymosan, iC3b-coated zymosan, or 2 x 107 IgG-SRBC were added to cells in RPMI 1640 containing 10% heat-inactivated FCS. Then cells were washed to remove unbound particles and examined microscopically after lysis of opsonized SRBC by distilled water. For the determination of zymosan phagocytosis, cell preparations were stained with Diff Quik (Baxter, Maurepas, France) before microscopic examination. More than 200 cells were counted in each assay. Phagocytosis results are expressed as the product of the percentage of cells having phagocytosed at least one particle and the number of phagocytozed particles per cell.
Determination of monocyte adherence to HUVEC monolayers
PBMC and THP-1 cells were incubated with 10 mM calcein-AM for 30 min. Calcein-labeled cells (105 cells/assay) were added to HUVEC monolayers as previously described (28). To determine CR3-dependent adherence, monocytic cells were incubated with HUVEC monolayers in the presence of mAb directed against CD11b. After 1 h of incubation at 37°C and washing, cell-associated fluorescence was measured with a fluorescence multiwell plate reader (Cytofluor, PerSeptive Biosystems, Framingham, MA). Assays were performed in triplicate, and results were expressed as relative mean adherence corresponding to the ratio of fluorescence values before and after washing.
Data analysis
Results are given as the mean ± SEM and compared with Mann-Whitney U test. Differences were considered significant if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The morphology of control and CNF1-treated THP-1 cells and
monocytes was first assessed by scanning electron microscopy. Control
THP-1 cells were perfectly spherical, with very few small pseudopodal
extensions (Fig. 1
A). The
changes in cell shape of CNF1-stimulated THP-1 cells were detectable
after 12 h of treatment, but were greatest 24 h after the
addition of CNF1 (data not shown). Indeed, after 24 h of treatment
with 500 pM CNF1, THP-1 cells showed deformations consisting of a
polarized shape (Fig. 1B). Another cell projection was
sometimes found on the opposite side of the cell (Fig. 1C).
Control monocytes were round with discrete deformations caused by their
adherence to substrate (Fig. 1D). The duration of
morphologic changes induced by CNF1 in monocytes was the same as that
observed in THP-1 cells. After 24 h of treatment, CNF1 increased
spreading of monocytes, with a thin circumferential lamellipodium and
circular swirls at the top of the cells (Fig. 1E).
Lamellipodia were also associated with major cell deformations
consisting of knob-like protuberances (Fig. 1F).
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As morphologic changes probably resulted from cytoskeletal
reorganization, we studied F-actin content and its distribution. THP-1
cells were incubated with 500 pM CNF1 for different periods of time.
F-actin was labeled with bodipy phallacidin, and the cell content in
F-actin was determined by cytofluorographic measurements. The F-actin
content of CNF1-treated cells reached a plateau between 8 and 24
h, and declined after 48 h of treatment (Fig. 2A). After 24 h of
treatment, the content of F-actin was significantly higher
(p < 0.02) than that in control cells. It is
noteworthy that this increase in F-actin was equivalent to that induced
by 10-7 M FMLP, a chemoattractant known to elicit actin
polymerization in phagocytic cells (data not shown). We also studied
the effects of different concentrations of CNF1 on the F-actin content
in THP-1 cells treated for 24 h (Fig. 2B). A
progressive increase in the F-actin content was observed for
concentrations of CNF1 between 30 pM and 1 nM. Subsequent experiments
were performed by incubating cells with CNF1 at 500 pM for 24 h.
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A and D). The diameter ratio was 1.05 ±
0.03, thus emphasizing the spherical shape of control cells. After CNF1
treatment, two patterns of F-actin reorganization were found. Using the
procedure of diameter comparison, a ratio >1.10 was assigned to
polarized cells. A fraction of treated cells (58 ± 7%) remained
spherical (diameter ratio, 1.06 ± 0.04) but exhibited numerous
peripheral foci of F-actin organized as microspikes (Fig. 3, B and E). The other fraction of THP-1 cells
(42 ± 8%) lost the rounded shape and displayed a polarized
morphology (diameter ratio, 1.32 ± 0.17) with a large membrane
expansion rich in F-actin (Fig. 3, C and F). CNF1
induced more dramatic reorganization of F-actin in adherent monocytes
than in suspended THP-1 cells. In control cells, the staining of
F-actin was homogeneous with peripheral reinforcement. As monocytes
were cultured for 24 h, they already displayed a moderate
spreading (Fig. 4, A and
C). Monocyte treatment with CNF1 induced an increase in cell
size and the formation of large folds and swirls on their surface and
at their periphery. The organization of the microfilament network was
lost; F-actin was concentrated in several foci, and actin cables were
present (Fig. 4, B and D).
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It has been reported that CNF1 stimulates Rho activation in
several cell types (21). Monocytes and THP-1 cells were incubated with
CNF1, and the C3 exoenzyme known as ADP-ribosylate Rho was added to
cell extracts. As a control, RhoA fusion protein was incubated in the
presence of CNF1 and the C3 exoenzyme. CNF1 caused a clear shift in the
apparent m.w. of [32P]ADP-ribosylated RhoA on SDS-PAGE
(Fig. 5, compare lanes 5 and
6). In control monocytes (lane 1) and
THP-1 cells (lane 3) ADP-ribosylated target of C3
exoenzyme migrated with the same m.w. as the RhoA protein. The
treatment of monocytes (lane 2) and THP-1 cells
(lane 4) by CNF1 resulted in a change in the mobility
of Rho. Thus, CNF1 elicited Rho activation in monocytes and THP-1
cells.
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Some GTPases, such as Rac1 and Rac2, are involved in activation of
the NADPH oxidase. The effect of CNF1 on superoxide release was tested
by using the SOD-inhibitable reduction of ferricytochrome c.
Adherent monocytes (Table I) and THP-1
cells did not release superoxide after 24 h of incubation with
CNF1. PMA-stimulated THP-1 cells released low amounts of superoxide
(
2–3 nmol/106 cells), which were not affected by CNF1
treatment (data not shown). PMA at 50 ng/ml stimulated an efficient
oxidative response in control monocytes. The CNF1 treatment of
monocytes did not modify superoxide production (Table I). Thus, the
activation of GTPase Rho is not involved in the activity of NADPH
oxidase.
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As phagocytosis requires the reorganization of cytoskeleton, we
investigated the effect of CNF1 treatment on FcR- and CR3-dependent
phagocytosis using IgG-SRBC and unopsonized zymosan or iC3b-coated
zymosan, respectively. CNF1 treatment did not significantly modify
IgG-mediated phagocytosis or the uptake of iC3b-coated zymosan in
monocytes and THP-1 cells (Table II). In
contrast, CNF1 significantly decreased the ingestion of unopsonized
zymosan by monocytes (81 ± 8% inhibition; p <
0.01) and THP-1 cells (58 ± 5% inhibition; p <
0.05). These findings suggest that CNF1 specifically affects some
features of CR3-mediated functions.
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We also studied the CR3-dependent adherence of monocytes and THP-1
cells to HUVEC monolayers (Table III).
The adherence of control cells to HUVEC did not exceed 20% and was
partly dependent on CR3, as demonstrated using specific mAb. CNF1
slightly enhanced the adherence of THP-1 cells to HUVEC, but had no
effect on the interaction of monocytes with HUVEC. CNF1 did not affect
the adherence between monocytes or THP-1 cells and HUVEC under CR3
control.
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CNF1-mediated impairment of the phagocytosis of unopsonized
zymosan may be related to the modulation of the expression of phagocyte
receptors and/or some activation epitopes. The expression of CD11b,
CD18, and CD32 (FcRII) was studied in THP-1 cells and monocytes by
flow cytometry. CNF1 treatment did not modify the expression of CD11b,
CD18, and CD32 (Fig. 6). We also
investigated the expression of the -subunit epitope of
ß2 integrins recognized by mAb 24, which is critical for
the activation of these integrins. In the absence of divalent cations,
no significant binding of mAb 24 to monocytes was found. When 200 µM
Mn2+ was added, the binding of mAb 24 to monocytes
increased dramatically. CNF1 treatment of monocytes did not affect the
Mn2+-induced expression of epitope 24 (data not shown). The
modulation of CR3 activity may also be related to its distribution. We
studied the effect of CNF1 on the distribution of CR3 and CD32 by
confocal microscopy. CD11b and CD32 were detected at the periphery of
control monocytes as a fluorescent ring. In CNF1-treated cells,
fluorescence staining was concentrated in peripheral patches (Fig. 7). Similar results were obtained for
CD18 (data not shown). Thus, CNF1 clearly induced the clustering of
CD11b, CD18, and CD32. We then evaluated the relationship between
clusterized distribution of the receptors and F-actin. Monocytes were
incubated with mAb directed to CD11b or CD32 (in red) and then with
bodipy phallacidin (in green). CD11b and CD32 were colocalized with
F-actin (in yellow) at the periphery of resting monocytes. In
CNF1-treated monocytes, CD32 and F-actin were colocalized as in control
cells. In contrast, CNF1 markedly decreased the association of CD11b
with F-actin (Fig. 8). Similarly, the
colocalization of CD18 with F-actin was altered by CNF1 treatment (data
not shown). The CNF1-stimulated impairment of CR3/F-actin
colocalization did not result from monocyte adherence. We found that
CR3 was colocalized with F-actin in control THP-1 cells as in adherent
monocytes, and that again its distribution with F-actin was altered by
CNF1 treatment (data not shown). Hence, CNF1 specifically modulated the
activation of CR3 and its colocalization with actin cytoskeleton.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chemokines were not secreted by
monocytes in response to CNF1 (data not shown), suggesting that
CNF1-induced cytoskeleton reorganization did not depend on cytokine
release. The cell shape changes and cytoskeletal reorganization induced by CNF1 directly involve the GTPase Rho but not Cdc42 or Rac proteins. The injection of constitutively active Cdc42 into Bac1 macrophages elicited the formation of long filopodia (17) that were not observed in CNF1-treated monocytes or THP-1 cells (our results). CNF1-stimulated shape changes in adherent monocytes and in THP-1 cells may involve the activation of Rho or Rac proteins. The morphologic modifications after microinjection of constitutively active Rac1 in Bac1 cells (17) evoke some features of actin reorganization in CNF1-treated monocytes. We took advantage of the property that Rac has of activating phagocytic NADPH oxidase (12, 13) to assess the effect of CNF1 on Rac proteins in monocytic cells. Treating monocytes and THP-1 cells only with CNF1 did not induce any oxidative response and did not modify the superoxide generation stimulated by phorbol ester. Hence, CNF1-induced morphologic changes and actin reorganization did not depend on the activation of Rac proteins but required Rho GTPase activity. Three series of data further support this statement. CNF1 decreased the electrophoretic mobility of Rho but not that of Rac (21). CNF1 stimulated a shift in the electrophoretic mobility of ADP-ribosylated Rho in both monocytes and THP-1 cells (our results). Moreover, pretreatment of monocytic cells with C3 exoenzyme before addition of CNF1 prevented morphologic changes (data not shown).
The activation of Rho affected the CR3-dependent phagocytosis of
unopsonized zymosan but did not modify the ingestion of IgG-SRBC. These
results were not related to changes in receptor expression, since the
expression of CR3 and FcRII (CD32) was similar in control and
CNF1-treated cells. There is a growing body of evidence that Rho is
important for the clustering of receptors including integrins and
FcR (8, 32). We also found that CNF1 induced the clustering of CR3
and FcRII. The clustering of integrins may occur through the
modulation of actin-myosin tension and cell contractility (33). Indeed,
lysophosphatidic acid-mediated activation of Rho resulted in contracted
morphology, phosphorylation of myosin light chain, and aggregation of
integrins in fibroblasts (21). CNF1 stimulates the relocalization of
myosin in stress fibers of epithelial cells; butanedione monoxime, an
inhibitor of myosin ATPase and contractility, prevents stress fibers
and the relocalization of myosin (30). As butanedione monoxime
inhibited morphologic changes in CNF1-treated monocytes (data not
shown), contractility may account for the cytoskeletal rearrangements
and receptor clustering. Nevertheless, the clustering of CR3 and
FcRII cannot account for the selective down-modulation of
CR3-dependent phagocytosis.
Rho activation affected only some features of CR3 functions. Since CNF1
did not modify the monocyte-HUVEC adherence dependent on CR3 or the
uptake of iC3b-coated zymosan, it is probable that the activation of
Rho modifies the functional state of CR3. Indeed, lymphocyte
aggregation is inhibited by C3 exoenzyme through avidity change of
LFA-1 (34). Similarly, C3 exoenzyme blocks agonist-induced neutrophil
ß2 integrin adherence to fibrinogen (9). Thus, one
potential mechanism of the CR3 impairment may be the prevention of an
active conformation. Divalent cations induce the expression of an
activation epitope recognized by mAb 24 on the subunit of
ß2 integrins (35). This mAb inhibits LFA-1-dependent,
Ag-specific T cell proliferation and CR3-mediated neutrophil chemotaxis
to FMLP (36). We found that the treatment of monocytes with CNF1 did
not affect Mn2+-induced expression of epitope 24. It has
been demonstrated that some lectin sites on CR3 are able to bind
zymosan polysaccharides and ß-glucan. The fact that these sites are
located outside the CD11b I domain that contains the binding sites for
iC3b, ICAM-1, and fibrinogen (37) means that CNF1 selectively affects
the CR3 functional state, probably through lectin sites.
As the interaction of integrins, including ß2
integrins, with the cytoskeleton modulates their activity (38, 39),
Rho-mediated cytoskeletal reorganization may affect the activity of
CR3. The cytoskeletal distribution of CR3 was clearly distinct from
that of FcRII. First, the association of FcRII with F-actin in
resting monocytes probably resulted from their adhesion to substrate,
since FcRII was not localized with F-actin in suspended THP-1 cells.
Second, the activation of the GTPase Rho did not affect the
cytoskeletal distribution of FcRII in monocytes and THP-1 cells.
Thus, Rho-mediated F-actin reorganization did not impair FcRII
distribution and FcRII-dependent phagocytosis. This may be related
to the inability of cytoskeletal inhibitors to affect FcR diffusion in
adherent macrophages (40). We show here that CNF1 decreased the
colocalization of CR3 with F-actin. Since CNF1 treatment is also
associated with the impairment of unopsonized zymosan phagocytosis, we
suggest that the distribution of CR3 with actin cytoskeleton is
necessary for zymosan phagocytosis.
In conclusion, CNF1 dramatically affects actin cytoskeleton in
monocytes and specifically impairs some features of CR3-dependent
phagocytosis. It also acts on both the activation of CR3 and its
codistribution with actin cytoskeleton. The GTPase Rho
differentially controls CR3 and FcR pathways of phagocytosis.
Although the activation of Rho creates the driving strength for
pseudopodal formation and the ingestion process in epithelial cells, it
appears to restrict ingestion in professional phagocytes. By limiting
integrin-mediated uptake, CNF1 may prevent the entry of several other
pathogens and maintain a favorable ecologic niche for pathogenic
strains of E. coli.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CNF1, cytotoxic necrotizing factor-1; F-actin, filamentous actin; NAD, nicotinamide adenine dinucleotide; SOD, superoxide dismutase.
Received for publication December 1, 1997. Accepted for publication June 11, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by human monocytes: down-modulation by phorbol ester. J. Immunol. 148:2229.[Abstract]
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K. E. Rippere-Lampe, M. Lang, H. Ceri, M. Olson, H. A. Lockman, and A. D. O'Brien Cytotoxic Necrotizing Factor Type 1-Positive Escherichia coli Causes Increased Inflammation and Tissue Damage to the Prostate in a Rat Prostatitis Model Infect. Immun., October 1, 2001; 69(10): 6515 - 6519. [Abstract] [Full Text] [PDF]
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K. E. Rippere-Lampe, A. D. O'Brien, R. Conran, and H. A. Lockman Mutation of the Gene Encoding Cytotoxic Necrotizing Factor Type 1 (cnf1) Attenuates the Virulence of Uropathogenic Escherichia coli Infect. Immun., June 1, 2001; 69(6): 3954 - 3964. [Abstract] [Full Text] [PDF]
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S. Meconi, C. Capo, M. Remacle-Bonnet, G. Pommier, D. Raoult, and J.-L. Mege Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis Infect. Immun., April 1, 2001; 69(4): 2520 - 2526. [Abstract] [Full Text] [PDF]
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 P. Hofman, G. Le Negrate, B. Mograbi, V. Hofman, P. Brest, A. Alliana-Schmid, G. Flatau, P. Boquet, and B. Rossi 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 J. Leukoc. Biol., October 1, 2000; 68(4): 522 - 528. [Abstract] [Full Text]
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S. Fournout, C. M. Dozois, M. Odin, C. Desautels, S. Peres, F. Herault, F. Daigle, C. Segafredo, J. Laffitte, E. Oswald, et al. Lack of a Role of Cytotoxic Necrotizing Factor 1 Toxin from Escherichia coli in Bacterial Pathogenicity and Host Cytokine Response in Infected Germfree Piglets Infect. Immun., February 1, 2000; 68(2): 839 - 847. [Abstract] [Full Text] [PDF]
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C. Capo, F. P. Lindberg, S. Meconi, Y. Zaffran, G. Tardei, E. J. Brown, D. Raoult, and J.-L. Mege Subversion of Monocyte Functions by Coxiella burnetii: Impairment of the Cross-Talk Between {alpha}v{beta}3 Integrin and CR3 J. Immunol., December 1, 1999; 163(11): 6078 - 6085. [Abstract] [Full Text] [PDF]
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V. Vouret-Craviari, D. Grall, G. Flatau, J. Pouyssegur, P. Boquet, and E. Van Obberghen-Schilling Effects of Cytotoxic Necrotizing Factor 1 and Lethal Toxin on Actin Cytoskeleton and VE-Cadherin Localization in Human Endothelial Cell Monolayers Infect. Immun., June 1, 1999; 67(6): 3002 - 3008. [Abstract] [Full Text] [PDF]
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