| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
vß3 Integrin and CR31
*
Université de la Méditerranée, Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Propre de Recherche de l’Enseignement Supérieur Associée 6020, Faculté de Médecine, Marseille, France; and
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 integrin)
and CR3 (Mß2 integrin), as demonstrated by
using specific Abs and RGD sequence-containing peptides. The phagocytic
efficiency of CR3 depends on its activation via
vß3 integrin and integrin-associated
protein. Indeed, CR3-mediated phagocytosis of avirulent C.
burnetii was abrogated in macrophages from integrin-associated
protein-/- mice. In contrast, the internalization of
virulent C. burnetii organisms involved the engagement
of vß3 integrin but not that of CR3. The
pretreatment of monocytes with virulent C. burnetii
organisms prevented the CR3-mediated phagocytosis of zymosan particles
and CR3 activation assessed by the expression of the 24 neo-epitope. We
conclude that the virulence of C. burnetii is associated
with the engagement of vß3 integrin and
the impairment of CR3 activity, which probably results from uncoupling
vß3 integrin from integrin-associated
protein. This study describes a strategy not previously reported of
phagocytosis modulation by intracellular
pathogens. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subdivision
of Proteobacteria (3). Their survival strategy is based on
multiplication in mature phagosomes of monocytes/macrophages
(4). C. burnetii virulence is mainly related to
the expression of LPS. Upon serial passage in culture, C.
burnetii undergoes an irreversible transition from virulent to
avirulent form, which is similar to smooth-to-rough LPS transition of
enterobacteria. Hence, phase I bacteria expressing a smooth-type LPS
are virulent, whereas phase II variants exhibit a truncated rough-type
LPS and are avirulent (5).
The entry of C. burnetii into monocytes and macrophages is
likely to be critical for its adaptation to host cells and the
development of Q fever. So far, no convincing evidence about the type
of receptors engaged by C. burnetii has been provided
(6). During the past few years, it has become increasingly
clear that some receptors of eukaryotic cells including macrophages are
especially important in bacterial recognition and probably determine
the fate of internalized pathogens (7, 8). Numerous
microorganisms use integrins, heterodimeric glycoproteins comprised of
various combinations of several and ß subunits (9),
as specific receptors to invade host cells (10). A
leukocyte ß2 integrin, CR3
(Mß2, CD11b/CD18), is
involved in the recognition of iC3b-coated particles and ß glucan
(11), as well as pathogens such as Escherichia
coli, Mycobacteria sp., Leishmania sp.,
Legionella pneumophila, Bordetella pertussis,
Rhodococcus equi, and Histoplasma capsulatum
(12). Nevertheless, CR3-mediated uptake of targets by
macrophages usually requires an additional activation signal
(13). ß1 and
ß3 integrins, which are involved in the
interaction of leukocytes with extracellular matrix, can also recognize
microorganisms such as B. pertussis, Mycobacteria
avium/intracellulare, Yersinia pseudotuberculosis,
Yersinia enterocolitica, and Borrelia burgdorferi
(10, 14, 15, 16). Leukocyte response integrin
(LRI;3
vß3 integrin) and
integrin-associated protein (IAP; CD47), a member of the Ig family, can
be functionally associated (17) and are involved in the
binding of filamentous hemagglutinin of B. pertussis by
monocytes (18). Recently, it has been reported that
IAP-deficient mice exhibited decreased resistance to bacterial
infection (19). Nevertheless, the way used by
intracellular pathogens to govern leukocyte integrin activity and their
survival inside host macrophages remains largely unknown.
In this report, we demonstrated that virulent C. burnetii
organisms entered into monocytes through
vß3 integrin and
survived inside the cells. Avirulent variants were more easily ingested
than virulent bacteria but were eliminated by monocytes. Their
phagocytosis was mediated by
vß3 integrin-IAP
complex and CR3, suggesting that the efficiency of C.
burnetii phagocytosis mainly results from the activation of CR3
and the availability of IAP. Virulent bacteria interfered with CR3
activation and CR3-dependent phagocytosis, thus preventing CR3
engagement. We suggest that C. burnetii-induced impairment
of CR3 function results from uncoupling
vß3 integrin from
IAP.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
mAbs directed to monocyte surface Ags were purchased from
Immunotech (Marseille, France). They included: the common
ß1 integrin chain (CD29, IgG1), the common
ß2 integrin chain (CD18, IgG1),
Lß2 (CD11a, IgG1),
Mß2 (CD11b, IgG1),
Xß2 (CD11c, IgG1), and
IgG1 controls. OKM1 mAb and BM104 mAb directed against
Mß2 (CD11b) were IgG1,
obtained from Ortho Diagnostic Systems (New Jersey) and Bender
Medsystem (Austria), respectively. mAbs against LRI (7G2, IgG1) and IAP
(B6H12, 2D3, IgG1), and F(ab')2 of anti-LRI
and anti-IAP mAb were obtained as previously described
(17). The mAb 24 was IgG1 specific for the leukocyte
integrin subunit (20). Rabbit Ab directed to C.
burnetii recognized virulent and avirulent bacteria.
FITC-conjugated F(ab')2 anti-rabbit IgG were
obtained from Immunotech. SRBC and specific immune serum were provided
by BioMérieux (Marcy l’Etoile, France). Synthetic hexapeptides
KGAGDV, KGRGDV, and KGALEV were prepared as described
(21). Lysophosphatidylcholine (LPC), zymosan and
cytochalasin D were purchased from Sigma (St. Louis, MO).
Monocytes and macrophages
PBMCs were isolated from healthy volunteers on Ficoll-Hypaque (Eurobio, Les Ulis, France) and suspended in RPMI 1640 containing 20 mM HEPES (Life Technologies, Eragny, France), 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies) as previously described (22). Monocytes were purified by incubating 5 x 105 PBMCs in a glass Labtek chamber/slide (Miles, Naperville, IL) for 60 min at 37°C. Nonadherent cells were removed by washing, and remaining cells were designated as monocytes because >90% of them were CD14+ and had phagocytic characteristics. In some experiments, monocytes were incubated with mAb or peptides for 60 min at 37°C before infection.
Bone marrow-derived macrophages (BMDMs) were isolated from wild-type mice (IAP+/+) and gene-targeted knockout mice deficient in IAP expression (IAP-/-) and incubated in a medium consisting of high-glucose DMEM with 15% L929 cell supernatant (19). BMDMs were pipetted off after addition of EDTA, and suspended cells were plated on glass Labtek chamber/slides before infection.
Bacteria
Virulent C. burnetii (Nine Mile strain, VR-615; American Type Culture Collection, Manassas, VA) were injected into mice and 10 days later were recovered from spleens, then cultured in mouse L929 fibroblasts maintained in antibiotic-free MEM (Life Technologies) supplemented with 4% FCS and 2 mM L-glutamine for two passages. Avirulent variants were cultured in L929 cells by repeated passages of Nine Mile strain as previously described (23). After 1 wk, L929 cells were sonicated, and the homogenates were centrifuged at 10,000 rpm for 10 min. Bacteria were layered on 25–45% linear Renograffin gradient, the gradients were spun down, then the bacteria were collected, washed, and suspended in serum-free medium before being stored at -80°C. The concentration of C. burnetii was determined by Gimenez staining (24).
Bacterial phagocytosis
Adherent monocytes (5 x 104
cells/assay) were incubated with C. burnetii at different
bacterium-to-cell ratios in 0.2 ml HBSS. After different periods of
time, the cells were washed and fixed with 1% formaldehyde, and
bacteria were revealed by immunofluorescence (IF). Bacterial labeling
was obtained by incubating cell preparations with rabbit Ab directed
against C. burnetii or control serum at 1:250 in PBS
containing 1% BSA for 30 min at room temperature. After washing, 1:200
dilution of FITC-conjugated F(ab')2
anti-rabbit IgG was added to cells for 30 min. The number of
bacteria associated with monocytes was determined by fluorescence
microscopy with a Zeiss microscope. To discriminate between extra and
intracellular bacteria, monocytes were permeabilized with 0.1 mg/ml
LPC. In the absence of LPC, only monocyte-bound organisms were detected
(Fig. 1
A), whereas LPC allowed
the detection of attached and internalized bacteria (Fig. 1B). Phagocytosis was quantified as follows. The product
of the number of bacteria per positive monocyte in the presence of
LPC and the percentage of positive cells was designated as the
association index. The difference between indexes in the presence or
the absence of LPC quantified the uptake of C. burnetii and
was named as the phagocytosis index. This parameter is a specific
measurement of phagocytosis because cytochalasin D dramatically
decreased the phagocytosis index but not the association index (Fig. 1, C and D). The phagocytosis of C.
burnetii was also expressed as the phagocytosis efficiency, which
was defined as the ratio of phagocytosis index/association index
(25).
|
Monocytes (5 x 104 cells/assay) were incubated with C. burnetii at a 200:1 bacterium-to-cell ratio for 24 h at 37°C in RPMI 1640. Cells were then washed to remove free bacteria (corresponding to day 0) and cultured for 12 days in RPMI 1640 supplemented with 10% FCS. Cellular infection was quantified by IF every 3 days. Results were expressed as a relative infection index compared with day 0. The viability of bacteria was determined as previously described (24). Briefly, infected monocytes were sonicated and the homogenates were added to human embryonic lung (HEL) cell monolayers in shell vials. After 7 days, C. burnetii replication was studied by indirect IF as described above.
Particle phagocytosis
In some experiments, monocytes (5 x
104 cells/assay) were pretreated with C.
burnetii at a 200:1 bacterium-to-cell ratio for 15 min at 37°C,
washed, and incubated with different particles. Unopsonized zymosan and
zymosan opsonized by 50% human serum were used to assess the function
of CR3 lectin sites and iC3b-binding sites, respectively. SRBC were
opsonized by 1:250 dilution of specific immune serum. IgG-SRBC were
used to assess FcR activity. Monocytes were incubated with
107 IgG-opsonized SRBC, unopsonized zymosan or
iC3b-coated zymosan (1 mg/ml) in RPMI 1640 containing 10%
heat-inactivated FCS for 60 min at 37°C. The cells were washed to
remove unbound particles, and extracellular IgG-coated SRBC were lyzed
by distilled water. Monocytes were then fixed with 1% formaldehyde and
examined microscopically. Phagocytosis results were expressed as the
product of the percentage of cells having phagocytozed at least one
particle and the number of phagocytozed particles per cell x 100
(phagocytosis index).
Flow cytometry
Adherent monocytes were treated with C. burnetii for 15 min at 37°C, washed, and then gently scraped with a rubber policeman. In some experiments, suspended PBMC were incubated with C. burnetii for 15 min at 37°C. Then, cells were incubated with anti-CD18, anti-CD11b, anti-LRI mAb, mAb 24, or isotypic controls at 1/100 dilution for 30 min at 4°C and stained with FITC-tagged F(ab')2 anti-mouse Igs. The cell fluorescence was analyzed by an EPICS XL (Coulter Electronics, Hialeah, FL). Gating was established using forward and side scatters and fluorescence recorded on the log scale.
Statistical analysis
Results are given as the mean ± SE and compared with Mann Whitney U test. Differences were considered as significant if p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Monocyte infection by virulent C. burnetii organisms
steadily increased from 3 days to 12 days postinfection as determined
by IF (Fig. 2A). After 12
days, 70% of infected cells contained each three to four bacteria. In
contrast, the number of avirulent organisms rapidly decreased after 3
days, and they were undetectable after 9 days. The decrease in the
number of avirulent organisms resulted from monocyte-mediated bacterial
killing. The viability of intracellular organisms was assessed by
culture of infected cell homogenates on HEL cells. Virulent bacteria
were viable (152 ± 20 at day 12 vs 104 ± 24 vacuoles per
shell vial at day 0), whereas avirulent variants were completely
eliminated by monocytes (24 ± 12 at day 12 vs 450 ± 80
vacuoles per shell vial at day 0). The distinct intracellular behavior
of virulent and avirulent C. burnetii may result from
different mechanisms of entry into monocytes. The uptake of virulent
organisms (bacterium-to-cell ratio of 200:1) required 60 min to become
detectable whereas intracellular avirulent bacteria were detected after
30 min (Fig. 2B). After 2 h, the phagocytosis of
virulent bacteria was 10 times lower than that of avirulent organisms
and phagocytosis efficiency did not exceed 33 ± 4%. In contrast,
the phagocytosis efficiency of avirulent C. burnetii was
84 ± 8%, demonstrating that almost all the bacteria that bound
to monocytes were ingested. A prolonged incubation of monocytes (>4 h)
with a bacterium-to-cell ratio of 500:1 did increase the phagocytosis
of virulent C. burnetii, but again not to a level close to
that seen with avirulent variants (data not shown). We also excluded
the hypothesis that virulent organisms settled less well than avirulent
variants during the course of phagocytosis assay. Bacteria were added
to monocytes and spun down at 1500 rpm for 10 min before a 2-h
incubation at 37°C. This procedure increased the phagocytosis index
of virulent C. burnetii (113 ± 31 vs 41 ± 5 in
the absence of centrifugation). But, it remained markedly lower than
the phagocytosis index of avirulent bacteria (569 ± 81). The
restriction of phagocytosis is a property of C. burnetii
virulence.
|
vß3 integrin and CR3 in
C. burnetii phagocytosis
The difference between the phagocytosis of virulent and avirulent
C. burnetii may be related to the engagement of specific
receptors on monocytes. Chelation of divalent cations completely
inhibited the interaction of C. burnetii with monocytes
(83 ± 8% inhibition), as did lowering of the incubation
temperature to 4°C (86 ± 7% inhibition), indicating a role for
monocyte integrins. Therefore, we investigated the role of
ß1, ß2, and
ß3 integrins in the uptake of C.
burnetii using specific mAbs. Phagocytosis of virulent bacteria
was not impaired by mAbs directed against ß1
chain (CD29), ß2 chain (CD18), and -chains
of ß2 integrins (CD11a, CD11b, CD11c) (Fig. 3A), whatever the
concentration of mAbs used (Fig. 4,
A and B, and data not shown). Blocking
vß3 integrin with mAb
7G2 markedly decreased the phagocytosis index in a dose-dependent
manner (Fig. 3A and Fig. 4C) with a maximum
inhibition at a mAb concentration of 5 µg/ml (75 ± 11%).
F(ab')2 of mAb 7G2 (5 µg/ml) also decreased the
phagocytosis by 73 ± 6%. Taken together, these results suggest
that the uptake of virulent C. burnetii by monocytes depends
on the engagement of
vß3 integrin.
|
|
B). The concentrations of
vß3 integrin-blocking
mAb 7G2 necessary to inhibit the uptake of avirulent C.
burnetii were similar to those required to prevent the
phagocytosis of virulent bacteria (Fig. 3B and Fig. 4C). The use of F(ab')2 of mAb 7G2
yielded the same results as those obtained with the complete mAb. In
contrast to virulent organisms, CR3 (CD11b/CD18) was involved in the
phagocytosis of avirulent bacteria by monocytes. First, a mAb directed
against CD18 (used at 5 µg/ml) decreased the phagocytosis index by
61 ± 6% (Fig. 3B). Second, the OKM1 mAb directed
against the lectin domain of CD11b inhibited the uptake of avirulent
C. burnetii by 55 ± 7% (Fig. 3B). The
BM104 mAb, directed against the I domain of CD11b, inhibited the
phagocytosis of avirulent bacteria to the same extent (data not shown).
It is noteworthy that mAbs against CD18 and CD11b exhibited a
superimposable pattern of dose-dependent inhibition (Fig. 4, A and B). Clearly, our results demonstrate that
the phagocytosis of avirulent C. burnetii depends on both
vß3 integrin and
CR3. KGAGDV and KGRGDV peptides and C. burnetii phagocytosis
To confirm the role of integrins in the uptake of C.
burnetii, we used peptides containing RGD-related sequences.
KGRGDV inhibits vß3
integrin function independently of its activation state, whereas KGAGDV
is selective for its activated state and the control peptide KGALEV is
without effect (21). KGALEV did not affect the
phagocytosis of C. burnetii whatever the peptide
concentration used (Fig. 5). KGAGDV
inhibited the uptake of virulent (Fig. 5A) and avirulent
(Fig. 5B) C. burnetii in a dose-dependent manner.
The inhibition was evident with 1 µM peptide (Fig. 5, A
and B, insets) and reached maximum value with 20
µM (80% inhibition). KGRGDV displayed a distinct pattern of
inhibition. Although as potent as KGAGDV at inhibiting the phagocytosis
of avirulent bacteria (Fig. 5B), it was less potent at
inhibiting the internalization of virulent C. burnetii (Fig. 5A, inset). Maximum inhibition required 10–20
times higher concentrations of KGRGDV than those of KGAGDV. Hence, the
phagocytosis of virulent C. burnetii is particularly
sensitive to KGAGDV peptide.
|
The difference between phagocytosis of virulent and avirulent
organisms could be due to C. burnetii-mediated effect on CR3
function. First, adherent monocytes were treated with virulent or
avirulent bacteria for 15 min (this time enabled C. burnetii
to induce F-actin reorganization (23)). After scraping of
monocytes, the expression of CR3 was assessed by flow cytometry. The
levels of CD18 and CD11b expression remained similar in the presence or
the absence of C. burnetii (Fig. 6). PBMC were also incubated with
C. burnetii for 15 min, and the CR3 expression was studied
in suspended monocytes. This expression was similar in control
monocytes and monocytes treated by virulent or avirulent organisms
(data not shown). The increase in the incubation time with C.
burnetii up to 2 h did not change the expression of CR3 (data
not shown). Second, we investigated the ability of C.
burnetii to activate CR3 by measuring the expression of activation
neo-epitopes, such as the 24 epitope, on ß2
integrins. While no detectable binding of mAb 24 to monocytes was
observed in the absence of divalent cations, the addition of 200 µM
Mn2+ dramatically increased its binding to
monocytes (Fig. 7). Virulent C.
burnetii organisms were unable to induce the expression of the 24
epitope (Fig. 7A), whereas avirulent bacteria stimulated its
expression after 15 min (Fig. 7B). The 24 epitope was not
expressed by monocytes even after 2 h of incubation with virulent
bacteria (data not shown). It is noteworthy that the preincubation of
monocytes for 15 min or 2 h with virulent organisms did not
interfere with Mn2+-stimulated expression of 24
epitope (data not shown). Third, we assessed the effect of
bacterium-monocyte interaction on CR3-dependent phagocytosis (Table I). In monocytes treated with virulent
C. burnetii for 15 min, the phagocytosis of unopsonized
zymosan (which binds lectin sites on CR3) and avirulent bacteria was
down-modulated, whereas the uptake of serum-opsonized zymosan (which
binds iC3b sites on CR3) and virulent bacteria was not affected. The
effect of C. burnetii on CR3-mediated phagocytosis was
sustained because CR3-dependent uptake was not corrected after 2 h
of monocyte pretreatment with virulent organisms (data not shown). The
pretreatment of monocytes with avirulent C. burnetii for 15
min (Table I) or 2 h (data not shown) did not modify their
phagocytic ability. Indeed, virulent C. burnetii organisms
specifically prevented the activation of CR3 and reduced the phagocytic
activity of CR3 dependent on its lectin sites.
|
|
|
As CR3 activation requires a cosignal provided by
vß3 integrin and its
transductional unit, IAP (17), we investigated the role of
IAP in monocyte uptake of C. burnetii using two mAb: mAb
B6H12 recognizes a site on the extracellular domain of IAP necessary
for functional interaction with
vß3 integrin, whereas
mAb 2D3 binds to a distinct IAP site (26). mAb B6H12
significantly depressed the phagocytosis index of virulent and
avirulent C. burnetii (Fig. 8A). The inhibition was
evident with 1 µg/ml of B6H12 (36 ± 7%) and reached a maximum
value with 5 µg/ml (65 ± 10% inhibition). Similar data were
obtained with F(ab')2 of the anti-IAP mAb
B6H12 used at 5 µg/ml (60 ± 10% inhibition). mAb 2D3 was
inactive whatever the concentrations of mAb used (Fig. 8A
and data not shown). Taken together, these results suggest that the
phagocytosis of C. burnetii is sensitive to mAb directed to
a site of IAP involved in the interaction with
vß3 integrin.
|
vß3 integrin function,
or be due to a requirement for IAP per se. To discriminate between
these hypotheses, we used BMDMs from normal and IAP-deficient mice
(19). In IAP+/+ BMDMs, avirulent
variants of C. burnetii were more efficiently internalized
than virulent bacteria (Fig. 8B), thus supporting the
results we obtained using human monocytes. In
IAP-/- BMDMs, the uptake of avirulent C.
burnetii was reduced to the level of that of virulent bacteria.
These results emphasize an essential role for IAP in the phagocytosis
of avirulent bacteria. Thus, both IAP and CR3 are necessary for the
high-level phagocytosis of avirulent C. burnetii, but both
are dispensable for the low-level phagocytosis of virulent
organisms. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We show here a selective use of monocyte receptors according to the
virulence of C. burnetii. The phagocytosis of virulent
organisms was independent of ß1 and
ß2 integrins but could be largely inhibited by
mAb directed against
vß3 integrin and
peptides KGAGDV and KGRGDV known to inhibit
vß3 integrin-dependent
phagocytosis. Thus, independent of other integrins, the activated form
of vß3 integrin
allowed phagocytosis of virulent C. burnetii, as
demonstrated by the potent inhibitory effect of KGAGDV. The use of
vß3 integrin as a
receptor for viral infection has been previously reported. The
infection with pathogenic hantavirus is inhibited by Abs to
ß3 integrin and by vitronectin, the ligand of
vß3 integrin, whereas
the infection with the nonpathogenic virus was prevented by
ß1 integrin-specific Abs and fibronectin
(31). Virulent foot-and-mouth virus uses
vß3 integrin as a
primary receptor for infection. The adaptation of the virus to cell
culture results in its ability to use heparan sulfate instead of
vß3 integrin and a
concomitant loss of virulence (32). In contrast to that of
virulent C. burnetii, phagocytosis of avirulent C.
burnetii by monocytes involves both
vß3 integrin and CR3.
Again, anti-vß3
integrin mAbs as well as KGAGDV and KGRGDV induced a significant
inhibition of the phagocytosis of avirulent organisms, suggesting that
it requires vß3
integrin. The uptake of avirulent bacteria was also inhibited by
anti-CD11b or anti-CD18 Abs, demonstrating an essential role
for CR3. The mAbs directed against the I domain or the lectin sites of
CD11b inhibited the uptake of avirulent C. burnetii to the
same extent. These domains exhibit distinct specificities of
recognition. I domain ligands include ICAM-1, fibrinogen, and
neutrophil-inhibiting factor, while lectin domain binds ß-glucan and
mannan polysaccharides (33, 34). Recently, it has been
reported that Candida albicans, a fungal pathogen, uses I
domain and lectin domain to efficiently interact with CR3
(35). It is likely that both regions of CD11b are involved
in the internalization of avirulent C. burnetii by
monocytes.
The virulence of C. burnetii is associated with the
inhibition of cross-talk between
vß3 integrin and CR3.
The efficient phagocytosis of avirulent organisms requires the
activation of CR3, as demonstrated by using mAb 24 initially described
to be specific for the leukocyte integrin subunits. Manganese is
known to directly alter the conformation of ß2
integrins and to strongly promote the expression of the 24 epitope
(36). This mAb inhibits monocyte-dependent T cell
proliferation and CR3-mediated neutrophil chemotaxis to FMLP, each
event being dependent on the activation of ß2
integrins (20). Avirulent C. burnetii organisms
caused an increase in mAb 24 expression, which seems to be associated
with high affinity of CR3 (N. Hogg, unpublished observation). The
activated state of CR3 is regulated by several cosignals including the
engagement of other integrins (12, 13). Hence, the
ligation of vß3
integrin increases the avidity of CR3 (37). Similarly, the
binding of B. pertussis filamentous hemagglutinin to
vß3 integrin leads to
the activation of CR3 (18). As the phagocytosis of
avirulent C. burnetii is associated with the engagement of
both vß3 integrin and
CR3, the integrin cross-talk should result in the efficient
internalization of avirulent bacteria via activated CR3. In contrast,
the interaction of virulent C. burnetii with monocytes
actively inhibits this integrin cross-talk. Virulent organisms did not
induce the expression of 24 epitope on CR3, suggesting that they did
not enable CR3 to become activated and competent for phagocytosis. In
addition, bacterium-monocyte interaction affected CR3-mediated
phagocytosis. The preincubation of monocytes with virulent C.
burnetii inhibited the phagocytosis mediated by lectin sites of
CR3 (unopsonized zymosan and avirulent C. burnetii) whereas
the uptake involving iC3b recognition site (serum-opsonized zymosan)
was preserved. It is likely that the impairment of lectin site activity
of CD11b prevents both conformational changes of I domain and exposure
of activation epitopes required for CR3 activation (38).
Thus, virulent C. burnetii organisms appear to actively
prevent CR3 activation by interfering with its lectin sites. As the
impairment of CR3 activation occurred when the interaction of virulent
C. burnetii with monocytes was very low, it is likely that
some fluid phase bacterial products play a role in this impairment. We
found that LPS from virulent organisms was able to interfere with CR3
(data not shown), suggesting that the interaction of LPS expressed or
released by virulent C. burnetii is involved in the
inhibition of CR3-dependent functions.
As the ability of vß3
integrin to mediate CR3 activation depends on IAP, the inhibitory
mechanism mediated by virulent bacteria may be directed to IAP. IAP is
physically and functionally associated with ß3
integrins. The mAbs that recognize IAP inhibit some
ß3 integrin-mediated functions such as binding
of vitronectin-coated beads to cells, neutrophil activation by
RGD-containing ligands, and calcium increase during adhesion of
endothelial cells to fibronectin or vitronectin (17, 39).
We demonstrated that IAP-blocking mAb inhibited the phagocytosis of
virulent and avirulent C. burnetii. The inhibitory effect on
virulent C. burnetii uptake was likely steric because only
the phagocytosis of avirulent bacteria was down-modulated in
macrophages from IAP-/- mice. As the uptake of
virulent and avirulent C. burnetii was similar in
IAP-/- macrophages, it is likely that
CR3-mediated phagocytosis of avirulent bacteria is under the control of
IAP. Conversely, virulent bacteria prevent CR3-dependent phagocytosis
by interfering with the cosignal activity of IAP but not by
down-modulating its expression (Fig. 6).
In conclusion, C. burnetii interacts with the monocyte
vß3 integrin.
Normally, this would result in IAP-dependent activation of CR3, rapid
phagocytosis, and death of the bacterium. However, virulent organisms
via a novel mechanism interfere with this host defense signal, allowing
them to be taken up via a nonmicrobicidal pathway allowing
intracellular survival.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to: Dr. Jean-Louis Mege, Université de la Méditerranée, Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Propre de Recherche de l’Ensignement Supérieur Associée 6020, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. E-mail address:
3 Abbreviations used in this paper: LRI, leukocyte response integrin; BMDM, bone marrow-derived macrophage; IF, immunofluorescence; IAP, integrin-associated protein; LPC, lysophosphatidylcholine; HEL, human embryonic lung.
Received for publication April 2, 1999. Accepted for publication September 8, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 on human monocytes and monocyte-derived macrophages. Infect. Immun. 61:663.vß3 and 5ß1 mediate the attachment of Lyme disease spirochetes to human cells. Infect. Immun. 66:1946.-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF. J. Immunol. 162:2259.. J. Infect. Dis. 162:932.[Medline]
-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol. 152:743.[Abstract]
vß3 as its receptor. Infect. Immun. 72:3587.
mß2, Mac-1). J. Immunol. 151:3324.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  J. Bedke, E. Kiss, C.-L. Behnes, Z. V. Popovic, M. Heuser, T. Stojanovic, T. Sijmonsma, P. Huber, S. Domhan, S. Muschal, et al. Anti-Inflammatory Effects of {alpha}v Integrin Antagonism in Acute Kidney Allograft Rejection Am. J. Pathol., October 1, 2007; 171(4): 1127 - 1139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-D. Sauer, J. G. Shannon, D. Howe, S. F. Hayes, M. S. Swanson, and R. A. Heinzen Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection Infect. Immun., August 1, 2005; 73(8): 4494 - 4504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Shannon, D. Howe, and R. A. Heinzen Virulent Coxiella burnetii does not activate human dendritic cells: Role of lipopolysaccharide as a shielding molecule PNAS, June 14, 2005; 102(24): 8722 - 8727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Zamboni and M. Rabinovitch Phagocytosis of Apoptotic Cells Increases the Susceptibility of Macrophages to Infection with Coxiella burnetii Phase II through Down-Modulation of Nitric Oxide Production Infect. Immun., April 1, 2004; 72(4): 2075 - 2080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Honstettre, E. Ghigo, A. Moynault, C. Capo, R. Toman, S. Akira, O. Takeuchi, H. Lepidi, D. Raoult, and J.-L. Mege Lipopolysaccharide from Coxiella burnetii Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4 J. Immunol., March 15, 2004; 172(6): 3695 - 3703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wetzel, T. Chavakis, K. T. Preissner, M. Sticherling, U.-F. Haustein, U. Anderegg, and A. Saalbach Human Thy-1 (CD90) on Activated Endothelial Cells Is a Counterreceptor for the Leukocyte Integrin Mac-1 (CD11b/CD18) J. Immunol., March 15, 2004; 172(6): 3850 - 3859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Seshadri, I. T. Paulsen, J. A. Eisen, T. D. Read, K. E. Nelson, W. C. Nelson, N. L. Ward, H. Tettelin, T. M. Davidsen, M. J. Beanan, et al. Complete genome sequence of the Q-fever pathogen Coxiellaburnetii PNAS, April 29, 2003; 100(9): 5455 - 5460. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Capo, A. Moynault, Y. Collette, D. Olive, E. J. Brown, D. Raoult, and J.-L. Mege Coxiella burnetii Avoids Macrophage Phagocytosis by Interfering with Spatial Distribution of Complement Receptor 3 J. Immunol., April 15, 2003; 170(8): 4217 - 4225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Zamboni and M. Rabinovitch Nitric Oxide Partially Controls Coxiella burnetii Phase II Infection in Mouse Primary Macrophages Infect. Immun., March 1, 2003; 71(3): 1225 - 1233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Hoover, D. W. Culp, M. H. Vodkin, J. C. Williams, and H. A. Thompson Chromosomal DNA Deletions Explain Phenotypic Characteristics of Two Antigenic Variants, Phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile Strain Infect. Immun., December 1, 2002; 70(12): 6726 - 6733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dellacasagrande, E. Ghigo, D. Raoult, C. Capo, and J.-L. Mege IFN-{gamma}-Induced Apoptosis and Microbicidal Activity in Monocytes Harboring the Intracellular Bacterium Coxiella burnetii Require Membrane TNF and Homotypic Cell Adherence J. Immunol., December 1, 2002; 169(11): 6309 - 6315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ghigo, C. Capo, C.-H. Tung, D. Raoult, J.-P. Gorvel, and J.-L. Mege Coxiellaburnetii Survival in THP-1 Monocytes Involves the Impairment of Phagosome Maturation: IFN-{gamma} Mediates its Restoration and Bacterial Killing J. Immunol., October 15, 2002; 169(8): 4488 - 4495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ghigo, C. Capo, M. Aurouze, C.-H. Tung, J.-P. Gorvel, D. Raoult, and J.-L. Mege Survival of Tropheryma whipplei, the Agent of Whipple's Disease, Requires Phagosome Acidification Infect. Immun., March 1, 2002; 70(3): 1501 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ghigo, C. Capo, D. Raoult, and J.-L. Mege Interleukin-10 Stimulates Coxiella burnetii Replication in Human Monocytes through Tumor Necrosis Factor Down-Modulation: Role in Microbicidal Defect of Q Fever Infect. Immun., April 1, 2001; 69(4): 2345 - 2352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. Dellacasagrande, E. Ghigo, S. Machergui-El, Hammami, R. Toman, D. Raoult, C. Capo, and J.-L. Mege alpha vbeta 3 Integrin and Bacterial Lipopolysaccharide Are Involved in Coxiella burnetii-Stimulated Production of Tumor Necrosis Factor by Human Monocytes Infect. Immun., October 1, 2000; 68(10): 5673 - 5678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dellacasagrande, P. A. Moulin, C. Guilianelli, C. Capo, D. Raoult, G. E. Grau, and J.-L. Mege Reduced Transendothelial Migration of Monocytes Infected by Coxiella burnetii Infect. Immun., June 1, 2000; 68(6): 3784 - 3786. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
