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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-α) and gamma interferon (IFN-γ) increased CR3 surface expression, but only TNF-α 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 phagocytosed 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 conditions and cause unnecessary damage. To resolve these problems, neutrophil activation, or priming, occurs through multiple 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 cytokines (e.g., tumor necrosis factor alpha [TNF-α] or gamma interferon [IFN-γ]) (4, 11, 23), immune effectors (e.g., immunoglobulin or complement), and bacterial components (e.g., lipopolysaccharide [LPS] or peptides containing formyl methionine). Adherence to a solid surface can also influence neutrophil activation (3). Furthermore, many bacterial pathogens express virulence factors that directly influence cellular signaling pathways, either positively or negatively. While their role in neutrophil activation has not been extensively studied, the toxins of Bordetella pertussis interfere with well-defined cytoplasmic 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 hemagglutinin (FHA) (1, 21), pertactin (10), and fimbriae (15) have been shown to influence mammalian cellular responses, presumably by binding to and activating surface-expressed receptors that control signaling pathways. FHA is especially interesting 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 removed 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 important 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 neutrophils. However, neutrophils possess large intracellular pools of these receptors in secretory vesicles. When appropriate activation signals are received, the vesicles fuse with the plasma membrane, rapidly increasing surface receptor expression. Priming can also alter neutrophil responsiveness by increasing the affinity of receptors for their targets or by coupling receptors to new intracellular signaling pathways.
The Fc receptor family (16, 26), which mediates the phagocytosis 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 αMβ2 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 ultimately influences cell adherence, migration, and generation of the oxidative burst. CR3 can mediate the phagocytosis of complement-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 β-glucan (33).
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 sensitive 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.
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 mutant, BPM409(pCW504); and BPLC5(pCW504), obtained from Francoise Jacob-Dubuisson (8).
ACT, FHA, pertussis toxin, and the B subunit of pertussis toxin were obtained from List Biological Laboratories (Campbell, CA); TNF-α and IFN-γ 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 minutes prior to the addition to 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 × 105 per 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 μl of Hanks' buffer supplemented 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-μg/ml FHA, 1- or 10-μg/ml ACT, 10-μg/ml TNF-α, or 10 units/ml IFN-γ. 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 statistical 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 incubation at 37°C to allow phagocytosis to occur. In control experiments, phagocytosis was allowed to occur for 2 h at 37°C.
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 overnight, and adherent and internalized bacteria were counted as described above. Student's t test was used to determined statistical significance at Plevels of <0.05.
The amount of CR3 expressed on the surfaces of neutrophils was quantified by an enzyme-linked immunosorbent assay (ELISA). In brief, neutrophils (2 × 105 per 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 100μl of 5-ng/ml purified ACT, FHA, pertussis toxin, or the B subunit of pertussis toxin, 100 μl of 5-ng/well TNF-α, or 10 units of IFN-γ 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 developed 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.
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. (Fig.1).1). Membrane fusion events are sensitive to temperature, and the surface expression of CR3 on cells incubated 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 cytokines known to activate neutrophils (e.g., TNF-α and IFN-γ) has not previously been reported. Treatments with TNF-α and IFN-γ both resulted in significantly increased CR3 expression for neutrophils incubated at 37°C (Fig. (Fig.1).1). These results suggest 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 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), suggesting 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.
To confirm the role of the CR3 receptor in the binding and phagocytosis of B. pertussis, neutrophils were incubated in the presence or absence of a monoclonal antibody that blocks CR3 action, and adherence and internalization were quantified microscopically. As observed previously (22, 32), at 37°C wild-type B. pertussis attached efficiently to neutrophils (Fig. (Fig.2A),2A), and about 10% of the bacteria were phagocytosed (Fig. (Fig.2B).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. (Fig.2A)2A) and phagocytosis (Fig. (Fig.2B)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 neutrophils.
Both TNF-α and IFN-γ increased the surface expression of CR3 (Fig. (Fig.1),1), and their ability to promote the phagocytosis of B. pertussis by neutrophils was examined. TNF-α activates inflammatory leukocytes to kill microbes and is especially potent at activating neutrophils. Treatment with TNF-α significantly increased both the attachment and internalization of wild-type strain BP338 (Fig. (Fig.3A).3A). IFN-γ caused the greatest increase in the surface expression of CR3 (Fig. (Fig.1).1). IFN-γ activates neutrophils and up-regulates the respiratory burst but is a less potent activator of neutrophils than TNF-α. IFN-γ 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. (Fig.3B3B).
The ability of FHA to up-regulate CR3 expression (Fig. (Fig.1)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 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 neutrophils (Fig. (Fig.4A)4A) and reduced internalization (Fig. (Fig.4B)4B) compared to wild-type B. pertussis. The addition of purified FHA promoted the attachment and internalization of the FHA mutant and the internalization of wild-type B. pertussis in a dose-dependent manner (Fig. (Fig.44).
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 susceptibility 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 consequence, reducing the susceptibility to phagocytosis. The role of SphB1 on adherence and phagocytosis was examined (Fig. (Fig.5).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 phagocytosed by neutrophils. In contrast, the mutant lacking the SphB1 protease was able to attach to neutrophils, and phagocytosis of the SphB1 mutant was significantly greater than that of the wild-type strain.
The ability of exogenous ACT to protect the ACT-deficient mutant from phagocytosis was examined. Pretreatment of the neutrophils with purified ACT at 10 μg per well protected the ACT-deficient mutant (BPM3183) from phagocytosis by neutrophils (Fig. (Fig.66).
Phagocytosis is a sequential process, and attachment must precede internalization. A common technique to uncouple the process of attachment from internalization involves incubating the cells at low temperatures, which prevents internalization 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. (Fig.1)1) is sensitive to temperature, and in addition it has been reported that treatments that affect bacterial viability or metabolism 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 influences phagocytosis, the effect of temperature shifts on phagocytosis was examined (Fig. (Fig.7).7). The attachment of wild-type B. pertussis and the ACT mutant was similar at 37°C or with a temperature shift (Fig. (Fig.7A).7A). This result differs from that seen in Fig. Fig.2,2, where significantly reduced attachment of the wild-type strain was observed at 4°C. The reduced attachment observed at 4°C in Fig. Fig.22 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. (Fig.7B).7B). Increased phagocytosis of the wild-type strain was observed when the assay was performed with a temperature shift, and phagocytosis of the wild-type strain was similar to that of the ACT mutant when the experiment was performed with a temperature 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 compromised 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. (Fig.2)2) or FHA (Fig. (Fig.4).4). Attachment 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 phagocytosis 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 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 human body.
The surface expression of CR3 is highly regulated by host and bacterial factors. Treatment with either TNF-α or IFN-γ 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-α promoted an increased uptake of B. pertussis. Cytokines affect many aspects of cellular signaling, and the enhanced phagocytosis observed following TNF-α treatment could occur at several levels. For example, TNF-α 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 expression; 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 activity.
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 susceptibility 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 neutrophil defenses.
This work was supported by NIH, Institute of Allergy and Infectious Disease, grant RO1 AI45715 to A.A.W.
Editor: J. T. Barbieri