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While the current pertussis vaccines protect against severe forms of the disease, many exposed individuals become infected with Bordetella pertussis and develop a milder form of the disease (1, 3, 8, 9, 16, 19). Developing pertussis vaccines that prevent bacterial infection is an important goal, since immune responses leading to bacterial elimination would prevent mild forms of the disease, eliminate carriage, and break the cycle of transmission.

Phagocytosis and killing by neutrophils is an effective bactericidal defense, but until recently it has not received much attention as an immune defense against pertussis. Using a mouse model, McGuirk and Mills have demonstrated that neutrophils can play a role in immunity to pertussis (14). A significant degree of neutrophil infiltration was observed in the lungs of naïve mice and mice immunized with the whole-cell pertussis vaccine following aerosol challenge; however, a neutrophil infiltration was not observed in mice immunized with an acellular pertussis vaccine. Recruitment of neutrophils was correlated with the type of immune response, occurring when Th1 cells were induced in the mice but not when Th2 cells were induced. Unlike mice, humans do not have such a polarized response to pertussis (2, 17, 18), and it is likely that neutrophils would always be recruited during human infection. Harvill et al. demonstrated a role for adenylate cyclase toxin as a bacterial counterdefense to neutrophils (7) when mice were infected with the closely related bacterial pathogen Bordetella bronchiseptica. Wild-type organisms, but not adenylate cyclase toxin mutants, caused a lethal infection in T- and B-cell-deficient mice. However, both wild-type and adenylate cyclase toxin mutants were lethal in neutropenic mice, suggesting that neutrophils play a critical role in resolving the infection, and that adenylate cyclase toxin can block clearance by neutrophils (7).

A method was previously developed to quantify phagocytosis using B. pertussis labeled with green fluorescent protein (GFP) and differential staining to distinguish extracellular bacteria from phagocytosed, intracellular bacteria (13, 20, 21). Using this technique, a previous study found that 98% of phagocytosed B. pertussis bacteria were killed (13); however, wild-type bacteria were phagocytosed very inefficiently by neutrophils, and two virulence factors, filamentous hemagglutinin (FHA) and adenylate cyclase toxin, were shown to influence this process.

Bacterial mutants lacking the adhesin FHA failed to attach to neutrophils and were not phagocytosed. Wild-type strains expressing FHA attached efficiently to neutrophils; however, little phagocytosis was observed. These results suggest that FHA mediates the attachment of B. pertussis to neutrophils in a way that fails to provoke phagocytosis. An unexpected result from these studies was that opsonization with an immune serum containing antibodies to FHA (23) caused a significant reduction in bacterial attachment as well as phagocytosis, suggesting that FHA-mediated attachment may be more efficient than Fc-mediated attachment.

Adenylate cyclase toxin also influenced phagocytosis by neutrophils. Adenylate cyclase toxin is an essential virulence factor of B. pertussis (5, 22) and has been shown to inhibit the ability of neutrophils to phagocytose and kill microorganisms (4). It is primarily located on the surface of the bacteria (10) and acts as a contact toxin (15). It can elevate intracellular cyclic AMP (cAMP) levels in target cells (this is referred to as adenylate cyclase toxin activity), and it can also cause hemolysis (lysis of red blood cells), an activity that is independent of cAMP generation (6). It was found that in the absence of opsonization, phagocytosis of the adenylate cyclase toxin mutant was very inefficient (21). However, following opsonization with human immune serum, 67% of the adenylate cyclase toxin mutants were internalized, compared to only 5% of the wild-type bacteria. These results suggest that a signal, likely generated when the antibody binds to the Fc receptors on neutrophils, is needed for B. pertussis to be recognized as foreign and phagocytosis to proceed. Adenylate cyclase toxin appears to block this process.

The observation that opsonization with an immune serum could result in reduced phagocytosis of the wild-type strain is disturbing and has important implications for vaccine development. However, the same serum could promote phagocytosis of the adenylate cyclase toxin mutant, and in this study we examined the influence of neutralizing monoclonal antibodies to adenylate cyclase toxin on phagocytosis of wild-type B. pertussis. Addition of the monoclonal antibodies alone did not promote phagocytosis. However, neutralizing antibodies to adenylate cyclase toxin promoted phagocytosis of B. pertussis opsonized with the immune serum, which by itself inhibited phagocytosis. These studies suggest that adenylate cyclase could be a useful vaccine antigen.

Monoclonal antibodies. Monoclonal antibody preparations described in a previous study were used (12). Antibodies 3D1 and 5D1 are capable of neutralizing adenylate cyclase toxin activity but have no effect on the hemolytic activity of the toxin (12). Antibodies 2A12 and 6E1 can neutralize hemolysin activity but have variable effects on adenylate cyclase toxin activity. Antibody 2A12 inhibits adenylate cyclase toxin activity, but at a much lower potency than 3D1 and 5D1, while antibody 6E1 has no effect on intracellular cAMP levels (12). The antibodies were purified from ascitic fluids and diluted to the same protein concentration (0.88 µg/ml). Enzyme-linked immunosorbent assay titers were 1:1 × 10⁵ for 3D1, 5D1, and 6E1 and 1:5 × 10⁴ for 2A12 (12). All four monoclonals were of the mouse immunoglobulin G1 subtype.

Phagocytosis. Phagocytosis assays using human neutrophils were performed as previously described (13, 21). A variant of BP338 expressing GFP, BP338(pCW504), was used for the phagocytosis studies. Briefly, 10 µl of each monoclonal antibody was added to 3 × 10⁶ bacteria suspended in 30 µl of HBSA (Hanks' buffer supplemented with 0.25% bovine serum albumin and 2 mM HEPES), followed by the addition of 30 µl of the human immune serum where indicated. The bacteria were incubated at 37°C for 15 min. Following opsonization, bacterial suspensions were adjusted to 400 µl and added to adherent neutrophils (5 × 10⁵). To promote contact, the bacteria were centrifuged onto the adherent neutrophils at 640 × g for 5 min at room temperature. Phagocytosis was allowed to occur for 1 h at 37°C in 5% CO₂. The cells were washed, and ethidium bromide (100 µg/ml) was added for 15 min at room temperature. Cells were washed and fixed, and phagocytosis was quantified by fluorescence microscopy. Intracellular GFP-labeled bacteria resist staining with ethidium bromide and remain green, while extracellular bacteria accumulate ethidium bromide and appear orange by fluorescence microscopy.

Phagocytosis in the presence of monoclonal antibodies to adenylate cyclase toxin. Opsonization with the mouse monoclonal antibodies alone did not alter phagocytosis or adherence of the wild-type strain, compared to the buffer control (data not shown). A previous study had shown that efficient phagocytosis of the adenylate cyclase toxin mutant occurred only when the bacteria were opsonized with human immune serum (21), and in this experiment the mouse monoclonal antibody would have to serve as the opsonizing antibody. Their failure to promote phagocytosis suggests these antibodies cannot perform this function. The mouse antibodies may not interact optimally with human Fc receptors; alternatively, monoclonal antibodies recognize only one epitope, and very little antibody might be present on the bacterial surface.

Phagocytosis in the presence of human immune serum and monoclonal antibodies to adenylate cyclase toxin. In previous studies (13, 20, 21), it was observed that opsonization with a human immune serum inhibited both attachment and phagocytosis of the wild-type strain, compared to the no-serum control. The same results were obtained in this study. Opsonization inhibited attachment of the wild-type strain about fourfold (Fig. 1), a statistically significant decrease (P < 0.004). Opsonization also inhibited phagocytosis about twofold (Fig. 1), a statistically significant decrease (P < 0.02). Monoclonal antibodies were then added to the bacteria opsonized with the human immune serum (Fig. 1). The addition of monoclonal antibodies 3D1 and 5D1 to the opsonized bacteria caused a statistically significant increase in the number of extracellular bacteria attached to the neutrophils as well as in the number of phagocytosed bacteria relative to the number in opsonized controls (Fig. 1). In contrast, two antibodies, 2A12 and 6E1, did not cause a statistically significant change in either attachment or phagocytosis. Mixing all four antibodies together caused a statistically significant increase in phagocytosis compared to that of the opsonized bacteria (P < 0.03) but was not better than using monoclonal antibodies 3D1 and 5D1 alone.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Effect of antibodies to adenylate cyclase toxin on extracellular attachment and internalization in the presence of human immune serum. Bacteria either were not opsonized () or were opsonized (+) by the addition of human immune serum. Monoclonal antibody (MAB) 3D1, 5D1, 2A12, or 6E1 or all four (mix) were added to adherent neutrophils. Centrifugation was performed to promote contact, and phagocytosis was allowed to occur for 1 h. One hundred consecutive neutrophils were examined for the number of adherent extracellular bacteria and the number of phagocytosed bacteria. Data were analyzed by the Student t test. Each bar represents the mean (plus the standard error of the mean) from five independent experiments in duplicate. Extracellular attachment rates of the unopsonized bacteria and the opsonized bacteria incubated with monoclonal antibodies 3D1 and 5D1 were statistically different from those of the opsonized control (P < 0.02 to 0.04). The internalization rates of the unopsonized bacteria and of the opsonized bacteria incubated with monoclonal antibody 3D1 or 5D1 or with all four monoclonals were significantly different from those of opsonized controls without monoclonals (P < 0.02 to 0.004).

View larger version (48K): [in this window] [in a new window]   FIG. 2.   Model depicting the interaction of B. pertussis with human neutrophils. (A) In the absence of antibodies, B. pertussis organisms attach to the neutrophils via FHA but do not appear to stimulate phagocytosis. (B) Following opsonization by a human immune serum, attachment is mediated by the interaction of the antibody and the Fc receptor, but the wild-type strain is not efficiently phagocytosed. (C) Following opsonization by a human serum in the presence of neutralizing antibodies to adenylate cyclase toxin, the binding of the antibody to multiple Fc receptors signals the neutrophil to undergo cytoskeletal rearrangements. The phagosomal membrane zippers around the opsonized organism, increasing the number of receptors bound to the antibody and the affinity of the interaction and ultimately leading to internalization (arrow). Adenylate cyclase toxin appears to inhibit the Fc-mediated signaling step, since strains expressing adenylate cyclase toxin activity remain extracellular, as seen in diagram B. However, the adenylate cyclase mutants or the opsonized wild-type bacteria in the presence of neutralizing antibody to adenylate cyclase toxin are phagocytosed, as depicted in diagram C.