INFECTION AND IMMUNITY, Nov. 2005, p. 7317–7323
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 11
Influence of CR3 (CD11b/CD18) Expression on Phagocytosis of
Bordetella pertussis by Human Neutrophils
Paula S. Mobberley-Schuman and Alison A. Weiss*
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati,
231 Albert Sabin Way, Cincinnati, Ohio 45267-0524
Received 11 May 2005/Returned for modification 23 June 2005/Accepted 28 July 2005
CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3
serves as the receptor for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and for the
adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the
phagocytosis of B. pertussis by human neutrophils was examined. The surface expression and function of CR3
are regulated. Tumor necrosis factor alpha (TNF-?) and gamma interferon (IFN-?) increased CR3 surface
expression, but only TNF-? increased the ability of neutrophils to phagocytose B. pertussis, suggesting that
elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also
increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not
affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of
ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3
blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and
phagocytosis of wild-type B. pertussis and FHA mutants. Mutants lacking the SphB1 protease, which cleaves
FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than
wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-type B. pertussis or ACT mutants plus
exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3,
FHA expression, and the efficiency of ACT internalization all influence whether B. pertussis will be phagocy-
tosed and ultimately killed by neutrophils.
Neutrophils are key players in the innate immune defense.
Armed with potent killing mechanisms, they present a threat
not only to microbes but also to the host, and the activation of
neutrophils must be carefully balanced. Neutrophils must be
sensitive to low microbial numbers, or they will be ineffective in
the face of rapid microbial replication. However, if they are too
sensitive, they will be diverted by minor inflammatory condi-
tions and cause unnecessary damage. To resolve these prob-
lems, neutrophil activation, or priming, occurs through multi-
ple signals. Neutrophils integrate multiple inputs, and while
the activation signal for any single stimulus may be small,
signaling pathways overlap, and priming through one agent
facilitates activation by others (for a review, see reference 20).
Neutrophils can be activated by many factors, including cy-
tokines (e.g., tumor necrosis factor alpha [TNF-?] or gamma
interferon [IFN-?]) (4, 11, 23), immune effectors (e.g., immu-
noglobulin or complement), and bacterial components (e.g.,
lipopolysaccharide [LPS] or peptides containing formyl methi-
onine). Adherence to a solid surface can also influence neu-
trophil activation (3). Furthermore, many bacterial pathogens
express virulence factors that directly influence cellular signal-
ing pathways, either positively or negatively. While their role in
neutrophil activation has not been extensively studied, the tox-
ins of Bordetella pertussis interfere with well-defined cytoplas-
mic signaling pathways; for example, adenylate cyclase toxin
(ACT) elevates cyclic AMP (cAMP) (6), pertussis toxin alters
signaling through G proteins (19), and dermonecrotic toxin
alters signaling through the Rho-GTP binding protein (17). In
addition, B. pertussis adhesins such as filamentous hemagglu-
tinin (FHA) (1, 21), pertactin (10), and fimbriae (15) have
been shown to influence mammalian cellular responses, pre-
sumably by binding to and activating surface-expressed recep-
tors that control signaling pathways. FHA is especially inter-
esting in this regard, since it is an adhesin that is both surface
localized and secreted. FHA is synthesized as a single 3,590-
amino-acid (367-kDa) precursor (25). The precursor protein is
processed at two sites. The N-terminal 71 amino acids are part
of an unconventional secretion signal sequence and are re-
moved by proteolysis (18), and the amino acids up to position
322 are thought to play an additional role in secretion and
protein folding (5). FHA also undergoes proteolytic processing
at the C terminus (7, 8) by a bacterial protease, SphB1, to
create the mature 220-kDa form of FHA. The 220-kDa form of
FHA is both secreted and found on the surfaces of bacteria,
while the 160-kDa C-terminal fragment remains associated
with the bacteria. Interestingly, mutants lacking the SphB1
protease responsible for cleaving FHA are less virulent in mice
(7), suggesting that the secreted form of FHA plays an impor-
tant role in pathogenesis.
The activation state of neutrophils changes their ability to
respond to microbial pathogens in several ways (for a review,
see reference 9). Priming can increase the receptor density on
the cell surface. Surface expression of the receptors involved in
cell adhesion and opsono-phagocytosis is low in unprimed neu-
trophils. However, neutrophils possess large intracellular pools
of these receptors in secretory vesicles. When appropriate ac-
tivation signals are received, the vesicles fuse with the plasma
* Corresponding author. Mailing address: Department of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati,
231 Albert Sabin Way, Cincinnati, OH 45267-0524. Phone: (513) 558-
2820. Fax: (513) 558-8474. E-mail: Alison.Weiss@uc.edu.
membrane, rapidly increasing surface receptor expression.
Priming can also alter neutrophil responsiveness by increasing
the affinity of receptors for their targets or by coupling recep-
tors to new intracellular signaling pathways.
The Fc receptor family (16, 26), which mediates the phago-
cytosis of antibody-opsonized microbes, and the CR3 receptor
(26, 27) have been shown to play a role in the phagocytosis of
B. pertussis. CR3 (reviewed in reference 2) is also known as
CD11b/CD18, Mac1, and ?M?2 integrin. CR3 expression is
limited to phagocytic cells such as macrophages, dendritic cells,
and neutrophils. CR3 regulates signaling pathways involved in
gene expression and cytoskeletal rearrangements and ulti-
mately influences cell adherence, migration, and generation of
the oxidative burst. CR3 can mediate the phagocytosis of com-
plement-opsonized, and in some cases, unopsonized microbes.
CR3 is a promiscuous receptor and has been shown to bind to
?30 substrates, including LPS. Most substrates bind to sites
contained within an “inserted” I domain at the N terminus of
the CD11b subunit that is induced to express a high-affinity
metal ion-dependent adhesion site following cell activation;
however, a few substrates bind to sites other than the I domain,
including a lectin-like binding domain involved in binding
Two B. pertussis virulence factors bind to CR3, namely, the
adhesin FHA (24) and ACT (14). ACT is an enzyme that
exerts its toxic action on neutrophils by elevating cytoplasmic
cAMP levels (6). cAMP levels in ACT-treated cells surpass
normal physiologic levels, ultimately paralyzing intracellular
communication, and neutrophils appear to be particularly sen-
sitive to ACT. While ACT is capable of gaining entry and
elevating the cAMP level in many types of mammalian cells via
a low-affinity receptor, recent studies have shown that CR3 can
serve as a high-affinity receptor for ACT (14). FHA and ACT
appear to bind to different domains of CR3. FHA binds via its
RGD motif (24), while ACT lacks an RGD motif. This result
suggests that the two bacterial factors would not compete for
binding sites and could simultaneously bind to CR3. ACT is
not efficiently secreted by many strains of B. pertussis, and
FHA-mediated binding to cells has been proposed to deliver
ACT to cellular targets (13, 34).
For this study, we examined the interplay between the host
factors and the bacterial factors that influence the ability of
human neutrophils to phagocytose B. pertussis, particularly
with regard to CR3.
MATERIALS AND METHODS
Bacterial strains and growth conditions. B. pertussis strains were grown on
Bordet-Gengou agar (BD Diagnostic Systems, Sparks, MD) supplemented with
15% defimbrinated sheep’s blood (Lampire Biological Laboratories, Pipersville,
PA) and appropriate antibiotics as previously described (32). The following
B. pertussis strains expressing green fluorescent protein from a plasmid-carried
gene (29, 32) were used: wild-type B. pertussis, BP338(pCW504); ACT-deficient
mutant, BPM3183(pCW504); Bvg mutant, BP347(pCW504); FHA-deficient mu-
tant, BPM409(pCW504); and BPLC5(pCW504), obtained from Francoise Jacob-
ACT, FHA, pertussis toxin, and the B subunit of pertussis toxin were obtained
from List Biological Laboratories (Campbell, CA); TNF-? and IFN-? were
obtained from Sigma (St. Louis, MO); and anti-CR3 (mouse anti-human CD11b)
was obtained from Accurate Chemical and Scientific Corp. (Westbury, NY). To
verify that the observed responses were due to the activity of the proteins, control
experiments were performed using protein factors that were boiled for 10 min-
utes prior to the addition to neutrophils.
Phagocytosis by neutrophils. Phagocytosis assays were performed as described
previously (22). In brief, neutrophils were purified from human peripheral blood
by dextran sedimentation and Ficoll-Paque centrifugation, and 1 ml of cells (5 ?
105per ml) was allowed to adhere to glass coverslips in tissue culture plates.
B. pertussis cells expressing green fluorescent protein were suspended to a final
multiplicity of infection of approximately 10 in 30 ?l of Hanks’ buffer supple-
mented with 0.25% bovine serum albumin and 2 mM HEPES and were pelleted
onto the adherent neutrophils to facilitate contact. In some studies, neutrophils
were treated with 1 ml of 1- or 10-?g/ml FHA, 1- or 10-?g/ml ACT, 10-?g/ml
TNF-?, or 10 units/ml IFN-?. Phagocytosis was allowed to occur for 1 h at 37°C.
Ethidium bromide was used to counterstain adherent bacteria, and cells were
fixed overnight in 1% paraformaldehyde. Each sample was examined in three or
more independent experiments. Student’s t test was used to determined statis-
tical significance, at P values of ?0.05, in all phagocytosis experiments.
For temperature shift experiments, neutrophils were incubated with wild-type
BP338 or the ACT mutant BP3183 at 4°C for 1 h, followed by a 1-hour incuba-
tion at 37°C to allow phagocytosis to occur. In control experiments, phagocytosis
was allowed to occur for 2 h at 37°C.
Antibody blocking of CR3. Neutrophils were incubated with mouse anti-CR3
at a 1:1,000 dilution (as recommended by the manufacturer) for 30 min at 37°C
after adherence to coverslips as described above. Wells were washed, bacteria
were added, and phagocytosis was allowed to occur for 1 hour at 37°C in 5%
CO2. Wells were washed, counterstained with ethidium bromide, and fixed over-
night, and adherent and internalized bacteria were counted as described
above. Student’s t test was used to determined statistical significance at
P levels of ?0.05.
CR3 expression levels. The amount of CR3 expressed on the surfaces of
neutrophils was quantified by an enzyme-linked immunosorbent assay (ELISA).
In brief, neutrophils (2 ? 105per well) were plated in 96-well microtiter plates
and incubated at 37°C or 4°C for 1 h. The neutrophils were then treated with
100 ?l of 5-ng/ml purified ACT, FHA, pertussis toxin, or the B subunit of
pertussis toxin, 100 ?l of 5-ng/well TNF-?, or 10 units of IFN-? per well for
another hour at the appropriate temperature. Antibody to CR3 was added at
1:1,000, and the cells were incubated for 1 h at 4°C. The wells were aspirated, and
the cells were fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Fort
Washington, PA) for half an hour. Alkaline phosphatase-conjugated goat anti-
mouse antibody (Serotec Inc., Raleigh, NC) was added at 1:20,000 and incubated
for 1 h at room temperature. The wells were washed, and the color was devel-
oped with Sigma-fast p-nitrophenyl phosphate tablet sets (Sigma) following the
manufacturer’s recommendations. Absorbance was read at 405 nm. Each sample
was examined in five or more independent experiments. Student’s t test was used
to determined statistical significance at P values of ?0.05.
CR3 receptor expression. The surface expression of CR3 is
highly regulated, and the influence of temperature, bacterial
factors, and human cytokines on CR3 expression was examined
by ELISA (Fig. 1). Membrane fusion events are sensitive to
temperature, and the surface expression of CR3 on cells incu-
bated at a low temperature (4°C) was compared to that for
cells incubated at the physiological temperature, 37°C. Low-
level expression of CR3 was observed for untreated control
neutrophils at both 4°C and 37°C. None of the conditions
tested resulted in significantly increased CR3 expression for
neutrophils incubated at 4°C. The regulation of CR3 by cyto-
kines known to activate neutrophils (e.g., TNF-? and IFN-?)
has not previously been reported. Treatments with TNF-? and
IFN-? both resulted in significantly increased CR3 expression
for neutrophils incubated at 37°C (Fig. 1). These results sug-
gest that the untreated neutrophils were not fully activated and
that neutrophils incubated at 37°C could respond to priming by
increasing the surface expression of CR3.
The ability of bacterial factors to activate neutrophils was
also examined. Incubation with FHA at 37°C resulted in an
eightfold increase in CR3 surface expression, but incubation
with ACT did not significantly alter CR3 expression. The
7318 MOBBERLEY-SCHUMAN AND WEISSINFECT. IMMUN.
coadministration of FHA and ACT also resulted in increased
CR3 expression, suggesting that ACT did not block the FHA-
mediated increase in CR3 expression. Treatment with pertussis
toxin also resulted in increased CR3 expression; however,
treatment with the B subunit of pertussis toxin, which lacks S1,
the subunit that catalyzes the covalent transfer of the ADP-
ribosyl group to regulatory GTP-binding proteins, did not alter
CR3 levels. Cells treated with boiled proteins did not display
an elevated surface expression of CR3 (data not shown), sug-
gesting that elevated CR3 expression was due to the activity of
the cytokine or bacterial factor and not due to heat-stable
contaminants such as LPS.
Antibody to CR3 blocks attachment and phagocytosis. To
confirm the role of the CR3 receptor in the binding and phago-
cytosis of B. pertussis, neutrophils were incubated in the pres-
ence or absence of a monoclonal antibody that blocks CR3
action, and adherence and internalization were quantified mi-
croscopically. As observed previously (22, 32), at 37°C wild-
type B. pertussis attached efficiently to neutrophils (Fig. 2A),
and about 10% of the bacteria were phagocytosed (Fig. 2B).
Pretreatment with antibody to CR3 significantly reduced both
attachment and internalization. Since the surface expression
of CR3 is influenced by temperature, phagocytosis was also
monitored at 4°C. Significantly reduced bacterial attachment
(Fig. 2A) and phagocytosis (Fig. 2B) were observed at 4°C
compared to those at 37°C, and attachment was reduced even
further in the presence of the antibody to CR3. The lack of
adherence and phagocytosis at 4°C was likely due to a lack of
CR3 expression. These results confirm the role of CR3 in the
adherence to and phagocytosis of B. pertussis by human neu-
Influence of TNF-? and IFN-? on attachment and phago-
cytosis. Both TNF-? and IFN-? increased the surface expres-
sion of CR3 (Fig. 1), and their ability to promote the phago-
cytosis of B. pertussis by neutrophils was examined. TNF-?
activates inflammatory leukocytes to kill microbes and is espe-
cially potent at activating neutrophils. Treatment with TNF-?
significantly increased both the attachment and internalization
of wild-type strain BP338 (Fig. 3A). IFN-? caused the greatest
increase in the surface expression of CR3 (Fig. 1). IFN-?
activates neutrophils and up-regulates the respiratory burst but
is a less potent activator of neutrophils than TNF-?. IFN-?
treatment resulted in significantly increased attachment of
wild-type strain BP338 but did not cause a significant increase
in the internalization of BP338 by neutrophils (Fig. 3B).
Influence of FHA on phagocytosis by neutrophils. The abil-
ity of FHA to up-regulate CR3 expression (Fig. 1) suggests that
in addition to mediating the attachment of B. pertussis to CR3
(27), FHA also has the potential to influence CR3 expression
and signaling. We examined the influence of surface-localized
FIG. 1. CR3 expression by neutrophils. A sandwich ELISA was
used to determine the level of CR3 expression. Neutrophils isolated
from the blood of human donors were allowed to attach to 96-well
tissue culture plates for 1 h at either 37°C or 4°C, followed by an
incubation with the indicated bacterial protein or cytokine for another
hour at the same temperature. The CR3-specific monoclonal antibody
was added for 1 h at 4°C, the cells were fixed with paraformaldehyde,
a secondary antibody and the indicator were added, and the absor-
bance was read at 405 nm. The mean absorbance of the untreated
bacteria incubated at 37°C was assigned a value of 1, and the other
results are reported relative to that value. Solid bars, neutrophils
incubated at 37°C; white bars, neutrophils incubated at 4°C. Ninety-
five-percent confidence intervals are shown. ?, significantly different
from CR3 expression of untreated neutrophils incubated at the same
FIG. 2. Antibody to CR3 blocks adherence and phagocytosis of
B. pertussis. Neutrophils were incubated at 37°C or 4°C without or with
a monoclonal antibody that blocks CR3 activity. Bacteria were added
and incubated with the neutrophils for 1 h, maintaining the original
temperature. One hundred consecutive neutrophils were scored for
adherent and internalized bacteria. (A) Adherent, extracellular bacte-
ria; (B) internalized bacteria. Solid bars indicate bacteria associated
with control neutrophils; white bars indicate bacteria associated with
neutrophils pretreated with anti-CR3 antibody. Ninety-five-percent
confidence intervals are shown. ?, significantly different from control
neutrophils incubated at 37°C by Student’s t test; #, significantly dif-
ferent from control neutrophils incubated at 4°C by Student’s t test.
VOL. 73, 2005 CR3 AND PHAGOCYTOSIS OF B. PERTUSSIS7319
and secreted FHA on adherence and phagocytosis. FHA has
been shown to mediate binding to neutrophils (22, 32), and
similar results were observed in this study. The FHA mutant
BPM409 displayed significantly reduced attachment to neutro-
phils (Fig. 4A) and reduced internalization (Fig. 4B) compared
to wild-type B. pertussis. The addition of purified FHA pro-
moted the attachment and internalization of the FHA mutant
and the internalization of wild-type B. pertussis in a dose-
dependent manner (Fig. 4).
The ability of exogenous FHA to promote the attachment
and phagocytosis of B. pertussis suggests that the amount of
FHA expressed by the bacterium influences bacterial suscep-
tibility to phagocytosis. Proteolysis of FHA by SphB1 could
serve as a mechanism to reduce the amount of surface-bound
FHA, reducing FHA-mediated adherence, and as a conse-
quence, reducing the susceptibility to phagocytosis. The role of
SphB1 on adherence and phagocytosis was examined (Fig. 5).
Strains lacking FHA, either due to mutation of the FHA gene
(BPM409) or due to mutation of the Bvg virulence regulatory
gene locus (BP347), were unable to attach and were not phago-
cytosed by neutrophils. In contrast, the mutant lacking the
SphB1 protease was able to attach to neutrophils, and phago-
cytosis of the SphB1 mutant was significantly greater than that
of the wild-type strain.
Influence of ACT on phagocytosis by neutrophils. The ability
of exogenous ACT to protect the ACT-deficient mutant from
phagocytosis was examined. Pretreatment of the neutrophils
with purified ACT at 10 ?g per well protected the ACT-
deficient mutant (BPM3183) from phagocytosis by neutrophils
Influence of temperature shifts on attachment and internal-
ization. Phagocytosis is a sequential process, and attachment
must precede internalization. A common technique to uncou-
ple the process of attachment from internalization involves
incubating the cells at low temperatures, which prevents inter-
nalization but not attachment, followed by a shift to 37°C,
which restores conditions permissive for internalization. In one
study (26), efficient phagocytosis of B. pertussis was reported
for an assay that uncoupled attachment and phagocytosis by
using a temperature shift. However, the expression of CR3
(Fig. 1) is sensitive to temperature, and in addition it has been
reported that treatments that affect bacterial viability or me-
tabolism reduce the ability of ACT to intoxicate cells (13),
suggesting that the entry of ACT may also be sensitive to
temperature. Since the expression of both CR3 and ACT in-
fluences phagocytosis, the effect of temperature shifts on
phagocytosis was examined (Fig. 7). The attachment of wild-
type B. pertussis and the ACT mutant was similar at 37°C or
with a temperature shift (Fig. 7A). This result differs from that
seen in Fig. 2, where significantly reduced attachment of the
wild-type strain was observed at 4°C. The reduced attachment
FIG. 3. Influence of TNF-? and IFN-? on adherence and internal-
ization of B. pertussis. Wild-type strain BP338 was added to neutro-
phils, and phagocytosis was allowed to occur for 1 h. The numbers of
adherent and internalized bacteria were determined for 100 consecu-
tive neutrophils. (A) Neutrophils were incubated without or with
TNF-? at 10 ng per well; (B) neutrophils were incubated without or
with IFN-? (10 units per well). Solid bars, control neutrophils; white
bars, cytokine-treated neutrophils. Data are means ? standard errors.
?, significantly different from untreated neutrophils by Student’s t test.
FIG. 4. FHA mediates adherence and phagocytosis of B. pertussis.
Neutrophils were incubated without or with purified FHA (1 or 10 ?g
per well) for 1 hour. The wild type, BP338 (solid bars), or an FHA
mutant, BPM409 (white bars), was added, and phagocytosis was al-
lowed to occur for 1 h. The number of bacteria was determined for 100
consecutive neutrophils. (A) Adherent bacteria; (B) internalized bac-
teria. Data are means ? standard errors. ?, significantly different from
no-FHA control of the same strain by Student’s t test.
7320 MOBBERLEY-SCHUMAN AND WEISSINFECT. IMMUN.
observed at 4°C in Fig. 2 is likely due to the low-level CR3
expression at 4°C, but the shift to 37°C could allow elevated
CR3 surface expression.
In contrast to attachment, the susceptibility to phagocytosis
was influenced by temperature. When the phagocytosis assays
were performed at 37°C, the ACT mutant was internalized
more efficiently than the wild-type strain (Fig. 7B). Increased
phagocytosis of the wild-type strain was observed when the
assay was performed with a temperature shift, and phagocyto-
sis of the wild-type strain was similar to that of the ACT
mutant when the experiment was performed with a tempera-
ture shift, suggesting that the incubation at 4°C compromised
the ability of ACT to prevent phagocytosis. Interestingly,
phagocytosis of the ACT mutant was more efficient at 37°C
than at 4°C, suggesting that the temperature shift also com-
promised the phagocytic capacity of the neutrophils.
B. pertussis plays a dangerous game with neutrophils. B.
pertussis uses FHA to mediate its own binding to the CR3
receptor expressed by activated neutrophils. No association
occurs in the absence of CR3 (Fig. 2) or FHA (Fig. 4). At-
tachment to the CR3 receptor allows bacteria to efficiently
deliver ACT, a potent inhibitor of neutrophil function (6).
However, even in the presence of ACT, binding to the CR3
receptor can mediate low-level phagocytosis, and mutants that
lack FHA fail to attach and are totally resistant to phagocytosis
by neutrophils (32).
While it seems counterintuitive for bacteria to risk phago-
cytosis by binding to CR3, this risk may be balanced by the
ability of ACT to block both CR3-mediated phagocytosis and
antibody-mediated phagocytosis via the Fc receptor (30, 31).
We have only observed efficient phagocytosis of B. pertussis by
FIG. 5. Phagocytosis of wild-type and mutant B. pertussis. The wild-
type strain BP338, FHA mutant BPM409, Bvg mutant BPM347, and
SphB1 mutant BPLC5 were added to neutrophils, and phagocytosis
was allowed to occur for 1 h. The number of bacteria was determined
for 100 consecutive neutrophils (A) Adherent bacteria; (B) internal-
ized bacteria. Data are means ? standard errors. ?, significantly dif-
ferent from wild-type bacteria by Student’s t test.
FIG. 6. Exogenous ACT protects ACT mutants. Neutrophils were
incubated without or with purified ACT (1 or 10 ?g per well) for
1 hour. The ACT mutant BPM3183 was added, and phagocytosis was
allowed to occur for 1 h. The numbers of adherent and internalized
bacteria were determined for 100 consecutive neutrophils. Data are
means ? standard errors. ?, significantly different from no treatment
(ACT control) by Student’s t test.
FIG. 7. Adherence and internalization of B. pertussis at 37°C and
4°C. Neutrophils were incubated with the wild-type strain (BP338,
black bars) or the ACT mutant (BPM3813, white bars) at a multiplicity
of infection of approximately 10, and phagocytosis was allowed to
occur for 2 h at 37°C or for 1 h at 4°C followed by 1 h at 37°C
(4°C337°C). The number of bacteria was determined for 100 consec-
utive neutrophils. (A) Adherent bacteria; (B) internalized bacteria.
Data are means ? standard errors. ?, significantly different from wild-
type bacteria at 37°C by Student’s t test.
VOL. 73, 2005CR3 AND PHAGOCYTOSIS OF B. PERTUSSIS 7321
neutrophils when ACT was inactivated chemically (29), by
mutagenesis (32), or by neutralizing antibodies (22, 31). In
contrast, one study reported efficient phagocytosis of antibody-
opsonized B. pertussis (26). In this study, phagocytosis was
monitored by a flow cytometry assay that involved incubation
at 4°C to allow bacterial attachment followed by a shift to 37°C
to permit phagocytosis. Previous studies have shown that while
ACT can generate cAMP in vitro during incubation at low
temperatures, ACT cannot generate intracellular cAMP at low
temperatures, suggesting that low temperatures prevent the
entry of ACT into target cells (12). Our results suggest that
incubation at 4°C adversely affects both CR3 expression and
the efficiency of internalization of ACT, and the ability to
internalize ACT takes more time to recover than the ability
to express CR3 when the temperature is returned to 37°C.
Experiments involving an incubation at 4°C may not reflect
the events that occur at physiologic temperatures in the
The surface expression of CR3 is highly regulated by host
and bacterial factors. Treatment with either TNF-? or IFN-?
up-regulated CR3 expression, and the enhanced attachment of
B. pertussis observed following cytokine treatment was likely
due to increased CR3 expression. However, only TNF-? pro-
moted an increased uptake of B. pertussis. Cytokines affect
many aspects of cellular signaling, and the enhanced phagocy-
tosis observed following TNF-? treatment could occur at sev-
eral levels. For example, TNF-? could improved CR3 receptor
signaling or counter the block in phagocytosis due to cAMP
produced by ACT.
Bacterial factors also influenced CR3 expression. Treatment
with pertussis toxin resulted in increased CR3 surface expres-
sion; however, the B subunit of pertussis toxin, which lacks the
enzymatic activity needed to disrupt signaling through GTP-
binding proteins, did not up-regulate CR3 expression. While
the B subunit has been shown to influence mammalian cellular
processes independent of the S1 enzyme activity (28), the up-
regulation of CR3 appears to require ADP-ribosyl transferase
Treatment with FHA can also increase CR3 expression.
Interestingly, elevated FHA expression can tip the balance
toward phagocytosis. The addition of exogenous FHA to either
the FHA mutant or the wild-type strain increased attachment
to neutrophils as well as phagocytosis. Mutants lacking the
SphB1 protease, which mediates the cleavage and release of
FHA from the bacterial surface, have been shown to be less
virulent than wild-type bacteria (7). Our data suggest that
SphB1 mutants are more susceptible to phagocytosis than wild-
type bacteria, and this may be the reason for the reduced
virulence seen in animal models of disease. SphB1 may serve to
regulate the amount of FHA present on the bacterial surface,
and the failure to do so could result in an increased suscepti-
bility to CR3-mediated phagocytosis. In summary, neutrophils
are potent effector cells that could play a role in the clearance
of B. pertussis, but FHA and ACT cooperate to disarm the
This work was supported by NIH, Institute of Allergy and Infectious
Disease, grant RO1 AI45715 to A.A.W.
1. Abramson, T., H. Kedem, and D. A. Relman. 2001. Proinflammatory and
proapoptotic activities associated with Bordetella pertussis filamentous hem-
agglutinin. Infect. Immun. 69:2650–2658.
2. Agramonte-Hevia, J., A. Gonzalez-Arenas, D. Barrera, and M. Velasco-
Velazquez. 2002. Gram-negative bacteria and phagocytic cell interaction
mediated by complement receptor 3. FEMS Immunol. Med. Microbiol.
3. Berton, G., S. R. Yan, L. Fumagalli, and C. A. Lowell. 1996. Neutrophil
activation by adhesion: mechanisms and pathophysiological implications. Int.
J. Clin. Lab. Res. 26:160–177.
4. Brandhorst, T. T., M. Wuthrich, B. Finkel-Jimenez, T. Warner, and B. S.
Klein. 2004. Exploiting type 3 complement receptor for TNF-alpha suppres-
sion, immune evasion, and progressive pulmonary fungal infection. J. Im-
5. Clantin, B., H. Hodak, E. Willery, C. Locht, F. Jacob-Dubuisson, and V.
Villeret. 2004. The crystal structure of filamentous hemagglutinin secretion
domain and its implications for the two-partner secretion pathway. Proc.
Natl. Acad. Sci. USA 101:6194–6199.
6. Confer, D. L., and J. W. Eaton. 1982. Phagocyte impotence caused by an
invasive bacterial adenylate cyclase. Science 217:948–950.
7. Coutte, L., S. Alonso, N. Reveneau, E. Willery, B. Quatannens, C. Locht, and
F. Jacob-Dubuisson. 2003. Role of adhesin release for mucosal colonization
by a bacterial pathogen. J. Exp. Med. 197:735–742.
8. Coutte, L., R. Antoine, H. Drobecq, C. Locht, and F. Jacob-Dubuisson. 2001.
Subtilisin-like autotransporter serves as maturation protease in a bacterial
secretion pathway. EMBO J. 20:5040–5048.
9. Edwards, S. W. 1995. Cell signalling by integrins and immunoglobulin re-
ceptors in primed neutrophils. Trends Biochem. Sci. 20:362–367.
10. Everest, P., J. Li, G. Douce, I. Charles, J. De Azavedo, S. Chatfield, G.
Dougan, and M. Roberts. 1996. Role of the Bordetella pertussis P.69/pertactin
protein and the P.69/pertactin RGD motif in the adherence to and invasion
of mammalian cells. Microbiology 142:3261–3268.
11. Forsberg, M., R. Lofgren, L. Zheng, and O. Stendahl. 2001. Tumour necrosis
factor-alpha potentiates CR3-induced respiratory burst by activating p38
MAP kinase in human neutrophils. Immunology 103:465–472.
12. Gray, M. C., G. M. Donato, F. R. Jones, T. Kim, and E. L. Hewlett. 2004.
Newly secreted adenylate cyclase toxin is responsible for intoxication of
target cells by Bordetella pertussis. Mol. Microbiol. 53:1709–1719.
13. Gray, M., G. Szabo, A. S. Otero, L. Gray, and E. Hewlett. 1998. Distinct
mechanisms for K? efflux, intoxication, and hemolysis by Bordetella pertussis
AC toxin. J. Biol. Chem. 273:18260–18267.
14. Guermonprez, P., N. Khelef, E. Blouin, P. Rieu, P. Ricciardi-Castagnoli, N.
Guiso, D. Ladant, and C. Leclerc. 2001. The adenylate cyclase toxin of
Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin
(CD11b/CD18). J. Exp. Med. 193:1035–1044.
15. Hazenbos, W. L., B. M. van den Berg, C. W. Geuijen, F. R. Mooi, and R. van
Furth. 1995. Binding of FimD on Bordetella pertussis to very late antigen-5 on
monocytes activates complement receptor type 3 via protein tyrosine kinases.
J. Immunol. 155:3972–3978.
16. Hellwig, S. M., A. B. van Spriel, J. F. Schellekens, F. R. Mooi, and J. G. van
de Winkel. 2001. Immunoglobulin A-mediated protection against Bordetella
pertussis infection. Infect. Immun. 69:4846–4850.
17. Horiguchi, Y., T. Senda, N. Sugimoto, J. Katahira, and M. Matsuda. 1995.
Bordetella bronchiseptica dermonecrotizing toxin stimulates assembly of actin
stress fibers and focal adhesions by modifying the small GTP-binding protein
rho. J. Cell Sci. 108:3243–3251.
18. Jacob-Dubuisson, F., C. Buisine, N. Mielcarek, E. Clement, F. D. Menozzi,
and C. Locht. 1996. Amino-terminal maturation of the Bordetella pertussis
filamentous haemagglutinin. Mol. Microbiol. 19:65–78.
19. Katada, T., and M. Ui. 1982. Direct modification of the membrane adenylate
cyclase system by islet-activating protein due to ADP-ribosylation of a mem-
brane protein. Proc. Natl. Acad. Sci. USA 79:3129–3133.
20. Ley, K. 2002. Integration of inflammatory signals by rolling neutrophils.
Immunol. Rev. 186:8–18.
21. McGuirk, P., and K. H. Mills. 2000. Direct anti-inflammatory effect of a
bacterial virulence factor: IL-10-dependent suppression of IL-12 production
by filamentous hemagglutinin from Bordetella pertussis. Eur. J. Immunol.
22. Mobberley-Schuman, P. S., B. Connelly, and A. A. Weiss. 2003. Phagocytosis
of Bordetella pertussis incubated with convalescent serum. J. Infect. Dis.
23. Propper, D. J., D. Chao, J. P. Braybrooke, P. Bahl, P. Thavasu, F. Balkwill,
H. Turley, N. Dobbs, K. Gatter, D. C. Talbot, A. L. Harris, and T. S.
Ganesan. 2003. Low-dose IFN-gamma induces tumor MHC expression in
metastatic malignant melanoma. Clin. Cancer Res. 9:84–92.
24. Relman, D., E. Tuomanen, S. Falkow, D. T. Golenbock, K. Saukkonen, and
S. D. Wright. 1990. Recognition of a bacterial adhesion by an integrin:
macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hem-
agglutinin of Bordetella pertussis. Cell 61:1375–1382.
7322MOBBERLEY-SCHUMAN AND WEISSINFECT. IMMUN.
25. Relman, D. A., M. Domenighini, E. Tuomanen, R. Rappuoli, and S. Falkow. Download full-text
1989. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence
and crucial role in adherence. Proc. Natl. Acad. Sci. USA 86:2637–2641.
26. Rodriguez, M. E., S. M. Hellwig, D. F. Hozbor, J. Leusen, W. L. van der Pol,
and J. G. van de Winkel. 2001. Fc receptor-mediated immunity against
Bordetella pertussis. J. Immunol. 167:6545–6551.
27. Saukkonen, K., C. Cabellos, M. Burroughs, S. Prasad, and E. Tuomanen.
1991. Integrin-mediated localization of Bordetella pertussis within macro-
phages: role in pulmonary colonization. J. Exp. Med. 173:1143–1149.
28. Strnad, C. F., and R. A. Carchman. 1987. Human T lymphocyte mitogenesis
in response to the B oligomer of pertussis toxin is associated with an early
elevation in cytosolic calcium concentrations. FEBS Lett. 225:16–20.
29. Weingart, C. L., G. Broitman-Maduro, G. Dean, S. Newman, M. Peppler,
and A. A. Weiss. 1999. Fluorescent labels influence phagocytosis of Bordetella
pertussis by human neutrophils. Infect. Immun. 67:4264–4267.
30. Weingart, C. L., W. A. Keitel, K. M. Edwards, and A. A. Weiss. 2000.
Characterization of bactericidal immune responses following vaccination
with acellular pertussis vaccines in adults. Infect. Immun. 68:7175–7179.
31. Weingart, C. L., P. S. Mobberley-Schuman, E. L. Hewlett, M. C. Gray, and
A. A. Weiss. 2000. Neutralizing antibodies to adenylate cyclase toxin promote
phagocytosis of Bordetella pertussis by human neutrophils. Infect. Immun.
32. Weingart, C. L., and A. A. Weiss. 2000. Bordetella pertussis virulence factors
affect phagocytosis by human neutrophils. Infect. Immun. 68:1735–1739.
33. Xia, Y., G. Borland, J. Huang, I. F. Mizukami, H. R. Petty, R. F. Todd III,
and G. D. Ross. 2002. Function of the lectin domain of Mac-1/complement
receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion. J. Immu-
34. Zaretzky, F. R., M. C. Gray, and E. L. Hewlett. 2002. Mechanism of association
of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for
toxin-filamentous haemagglutinin interaction. Mol. Microbiol. 45:1589–1598.
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