Systemic Administration of Bordetella pertussis Enhances Pulmonary Sensitization to House Dust Mite in Juvenile Rats

Wumin Dong, MaryJane K. Selgrade and M. Ian Gilmour1

Immunotoxicology Branch, MD-92, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

Received September 16, 2002; accepted December 11, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of allergies and asthma has increased significantly in the past few decades. The objectives of this study were to establish an allergy model in weanling rats to more closely reflect the developing immune system of children, and to determine whether systemic administration of inactivated Bordetella pertussis could enhance pulmonary and systemic immune responses to locally administered house dust mite antigen (HDM). Three-week old female Brown Norway rats were sensitized with 10 µg HDM intratracheally or intraperitoneally, with or without a simultaneous injection of 108 whole killed B. pertussis organisms. Ten days later, all the rats were challenged with 5 µg HDM via the trachea. Bronchial lymph nodes and bronchoalveolar lavage fluid (BAL) were collected 0, 2, and 4 days post-challenge. Coadministration of pertussis and intratracheal instillation of HDM enhanced HDM-specific lymphoproliferative responses and increased BAL levels of total protein, lactate dehydrogenase, HDM-specific IgE and IgG antibodies, and the number of eosinophils in BAL to the same extent as had occurred in the systemically immunized animals. The data show that intratracheal instillation of HDM induces a mild allergic sensitization in juvenile rats, and that ip injection with B. pertussis enhances this sensitization process to levels seen in animals injected with antigen and B pertussis together. These results suggest that simultaneous exposure to Th2-inducing vaccine components and allergenic proteins may be a risk factor for allergic sensitization and the development of asthma in susceptible individuals.

Key Words: pertussis; dust mite; allergy; IgE; lymphocyte; eosinophil; vaccine; rat.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of asthma has increased dramatically over the last 20 years in the U.S. and other industrialized nations as a result of ill-defined changes in living conditions in modern western society (Cookson, 1999Go; Salvi, 2001Go; Weitzman et al., 1992Go). One hypothesis proposes that decreased exposure to microbes reduces stimulation of the cell mediated (Th1) component of the immune response with a corresponding increase in Th2 responsiveness during development (Shirakawa et al., 1997Go). It has also been argued however, that other factors including decreased exercise, more numerous respiratory viral infections (e.g., from day care facilities), increased incidence of obesity and changes in diet, and exposure to air pollutants, including environmental tobacco smoke and diesel exhaust, contribute to the higher sensitization rate (Frick et al., 1979Go; Hu et al., 1997Go; Peat et al., 1992Go).

Bordetella pertussis and its associated toxins are an integral component of the diphtheria/pertussis/tetanus (DPT) vaccine, but have also been used to elicit IgE antibody responses in experimental animals (Clausen et al., 1970Go; Lindsay et al., 1994Go; Mu and Sewell, 1994Go). The original pertussis vaccine consisted of a killed whole-cell preparation, which was replaced with an acellular (subunit) vaccine that causes fewer side effects, in Japan and Scandinavia in the early 1980s and in the U.S. in 1998 (Schleiss and Dahl, 2000Go). The original whole-cell formulation is still recommended in the U.K. for primary immunizations, although the acellular component is now used in booster immunizations (Girard, 2002Go).

It has been difficult to establish whether there is more allergy and asthma in vaccinated populations of western countries because immunization rates are high, and nonvaccinated children may have significantly different lifestyles from vaccinated children. Some small cross-sectional studies from several countries have reported significant increases in childhood asthma and other allergic illnesses in whole-cell preparation DPT-immunized children, compared to individuals who did not receive the vaccine (Hurwitz and Morhenstern, 2000Go; Kemp et al., 1997Go; Odent et al., 1994Go; Yoneyama et al., 2000). Other larger prospective cohort studies, which examined individuals immunized with either the whole cell or the acellular preparation, however, have not shown any associations between pertussis vaccinations and wheezing illnesses (Anderson et al., 2001Go; Destefano et al., 2002Go; Gruber et al., 2001Go; Henderson et al., 1999Go). While the role of vaccines in promoting allergic disease remains controversial, there is evidence that B. pertussis does enhance IgE production. Studies have demonstrated that total serum IgE levels are elevated in children with pertussis infection (Schuster et al., 1993Go) or after vaccination (Torre et al., 1990Go). Asthma incidence is also significantly higher in children with proven pertussis infection than noninfected children (Nilsson et al., 1998Go).

It is clear that exposure to environmental antigens such as HDM (Shapiro et al., 1999Go; Sporik et al., 1992Go) and the number and type of infant immunizations have increased in recent decades (Schleiss and Dahl, 2000Go). Thus, the opportunity exists for these two factors to act synergistically to increase the incidence of allergic sensitization. Our laboratory has developed an adult rat model of respiratory hypersensitivity to HDM antigen that provides the opportunity to explore this issue in a controlled experimental setting (Lambert et al., 1998Go). The rats exhibit many of the hallmarks of allergic asthma in humans, including allergen-specific IgE antibody, immediate airway responses, airway hyperresponsiveness, and pulmonary eosinophilia. Furthermore, we have used this model to evaluate the impact of various chemical exposures on allergic responses to HDM (Dong et al., 1998Go; Gilmour et al., 1996Go; Lambert et al 1999Go; 2000Go; Luebke et al., 2001Go). The objectives of this study were to adapt this animal model of allergic responses to HDM to weanling rats in order to mimic the developing human immune system, and to assess the effect of pertussis immunization on allergen-specific immune responses and immune-mediated lung disease. Our hypothesis was that coadministration of B. pertussis would enhance the development of allergic immune responses to HDM.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Three-week-old female Brown Norway rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN), housed 2 per cage in an environmentally controlled room with a 12-h light/dark cycle, and fed Laboratory Rodent Diet 5001 (Purina Mills, St. Louis, MO). Randomly selected sentinel animals were tested serologically to ensure that rats were free of Sendai virus, murine pneumonia virus, and a variety of other rodent viruses, as well as Mycoplasma sp. Rats were also monitored for and found to be free of ectoparasites and endoparasites. All procedures were approved by the Animal Care and Use Committee of the National Health and Environmental Effects Research Laboratory, U.S. EPA.

Antigen preparation.
Purified extracts of Dermatophagoides farinae Group I allergen were obtained under contract from Greer Laboratories, Inc. (Lenoir, NC). D. farinae mites were cultivated, harvested, and extracted using standard procedures. The group I allergen, Der f1, was purified to >75% using a combination of ammonium sulfate precipitation, DEAE ion-exchange chromatography, and gel filtration. Freeze dried preparations were supplied in glass vials containing 4 mg of protein, of which 3.57 mg was calculated to be Der f1 by commercial double bind ELISA. Pyrogen testing by the vendor, using the limulus amebocyte-lysate assay test, indicated each vial held 89 ng/vial (22.25 ng/mg protein) of lipopolysaccharide (LPS). The antigen was rehydrated in pyrogen-free sterile saline to 1 mg/ml and stored in aliquots at –20°C until use.

Experimental design and antigen sensitization.
Preliminary studies had shown that a single intratracheal instillation of 10 µg HDM, with or without coadministration of pertussis, did not result in appreciable sensitization in this age rat. Thus, for the mucosal immunization, 3 daily instillations of antigen were chosen, with the principal comparison being made by the presence or absence of an initial pertussis injection. Systemically immunized animals which showed sensitization after a single injection of antigen, were also included in order to provide perspective on the strength of immune responses generated by ip injection of the same total amount of antigen, and to determine whether this could be augmented by inclusion of pertussis. The rats were randomly divided into 7 groups of 15 rats each, as listed in Table 1Go. Control groups were injected with either B. pertussis or saline ip, and were not sensitized with antigen. Antigen-only groups received a single systemic ip immunization of 10 µg antigen or 3 daily intratracheal instillations of 3.3 µg antigen. Test groups of animals (pertussis and HDM) were injected ip with the pertussis adjuvant along with the systemic antigen, or were injected with pertussis immediately before the first intratracheal instillation. For the intratracheal procedures, rats were anesthetized by halothane inhalation (Aldrich, Milwaukee, WI), and given 3 daily intratracheal instillations of 3.3 µg HDM in 0.1 ml of saline (total of 10 µg). On the first day of either immunization regime, selected groups of rats were injected ip with 108 heat inactivated B. pertussis organisms (IAF Biovac, Inc., Montreal, Quebec) in 0.25 ml saline. Ten days after the sensitization phase, all the animals (including saline and pertussis controls) were challenged by intratracheal instillation of 5 µg antigen.


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TABLE 1 Effect of B. pertussis on Cell Populations in Rat Lungs After Pulmonary Challenge with House Dust Mite
 
Pulmonary function testing.
Prior to antigen challenge, 5 rats from each group were placed in a Buxco whole-body plethysmograph (Buxco Electronics, Troy, NY) and measurements of respiratory rate and enhanced pause were made for 10 min, as described previously (Dong et al., 1998Go). The animals were then instilled with 5 µg of HDM in 0.1 ml saline, returned to the plethysmograph and antigen-induced changes in these respiratory parameters were monitored for a further 20 min. Enhanced pause measurements taken every half-second were averaged for the pre- and posttreatment periods and compared for statistical significance before and after challenge and between experimental treatment groups.

Necropsy and BAL.
At each time point, animals were euthanized with sodium pentobarbital (180 mg/kg) (Abbott Laboratories, North Chicago, IL). Serum was obtained through cardiac puncture and analyzed for HDM-specific IgE, IgG, and IgA. The bronchial lymph nodes were removed from the right main stem bronchus and processed into a single cell suspension. Bronchoalveolar lavage (BAL) fluid was collected by lavaging the lungs 3 times with a single volume of saline (35 ml/kg) via a tracheal cannula, and total and differential cell counts were obtained. The lavage supernatant was then assessed for HDM-specific IgE, IgG, and IgA as well as total protein, lactate dehydrogenase (LDH) and eosinophil peroxidase (EPO) for markers of pulmonary edema, acute lung injury, and eosinophil activation, respectively. The entire study was repeated and data from the two experiments were pooled and analyzed in toto.

Biochemical and cytological analyses.
Cell counts in BAL fluid were obtained with a hemocytometer and cell viability was assessed by trypan blue dye exclusion. Following dilution, 50,000 cells from each sample were centrifuged in duplicate onto microscope slides using a Cytospin Model II (Shandon, Pittsburgh, PA) and stained with Diff Quick (American Scientific, Sewickley, PA). Differential counts were performed on 200 cells per specimen for identification of alveolar macrophages, polymorphonuclear leukocytes (PMN), lymphocytes, and eosinophils. After centrifugation at 425g for 10 min at 4°C, supernatant BAL fluid was assessed for LDH, using a commercially prepared kit and controls (Sigma, St. Louis, MO). Total protein in BAL fluid was detected with Pierce Coomassie Plus protein assay reagent (Pierce, Rockford, IL) using bovine serum albumin (BSA; Sigma) as a standard. Eosinophil peroxidase (EPO), as a measure of eosinophil activation, was determined using OPD Tablet Set (Sigma). All biochemical assays were modified for automated analysis by a Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche, Branchburg, NJ).

Antibody analysis.
Serum and BAL fluid were assessed for HDM-specific immunoglobulins by ELISA assays. Prior to assay, pooled serum and BAL samples were tested to determine the appropriate dilution factor for the linear range of the reaction curve. For the assay, 96-well polystyrene plates (Costar, Cambridge, MA) were coated overnight with 17.6 µg HDM/ml of 0.05 M carbonate buffer (pH 9.6) at 4°C. The plates were washed five times with 0.1 M phosphate-buffered saline (PBS) containing 0.05% Tween 20. Each well was filled with a blocking solution of 1% BSA in PBS, and incubated for 2 h prior to washing. Diluted serum samples (1:100 for IgG, 1:10 for IgE and IgA) or BAL fluid (1:10 IgG, 1:2 for IgE and IgA) were added to the wells and incubated overnight with the coated HDM at 4°C. Following another wash step, biotin-labeled mouse antirat IgG, IgE, or IgA (Accurate, Westbury, NY) was added to the plates at optimal dilutions and incubated for 2 h at room temperature. Excess antibody was removed by washing, and HRP-streptavidin (Vector Laboratories, Burlingame, CA) was added to the plates. After another 2-h incubation the plates were washed, and a solution of TM blue enzyme reagent (TSI-CDP, Milford, MA) was added and the color change measured at a wavelength of 650 nm with an ELISA plate reader (Molecular Devices, Menlo Park, CA). Background values were subtracted from all sample values by the instrument software (Softmax) to correct for nonspecific binding effects. The ELISA assays were optimized, using a checkerboard titration, to provide optimal concentrations of antigen and antibodies for the reaction. Reaction times and OD values were compared to historical data, obtained during the development of this assay, for quality assurance.

Lymphoproliferative responses.
Lung-associated lymph nodes from the main stem bronchus were collected and homogenized in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 20 mM HEPES buffer, 5 x 10–5 M 2-mercaptoethanol, 100 units/ml penicillin, and 100 mg/ml streptomycin (GIBCO, Grand Island, NY). After two lysing steps with 0.89% NH4Cl and medium, cells were washed, counted on a hemocytometer in the presence of trypan blue, and resuspended to a concentration of 5 x 106 viable cells/ml. Five x 105 cells were incubated in the presence of 10 µg/ml of HDM or medium alone for 4 days. Eighteen h before the end of the incubation period, 0.5 µCi [3H] thymidine (DuPont, Wilmington, DE) in 20 µl of medium was added to each well. Cells were harvested automatically onto filters (Skatron, Sterling, VA) and the [3H]thymidine incorporation was measured by scintillation counting (Packard, Meriden, CT). Lymphocyte responsiveness was expressed as the difference in radiotracer counts between cells incubated with HDM (stimulated) and those incubated with medium alone (unstimulated).

Statistical analysis.
The data were analyzed using a two-way analysis of variance (ANOVA). The two independent variables were pertussis and HDM exposures. Pair-wise comparisons were performed as subtests of the overall ANOVA. The significance levels for these pair-wise comparisons were established using a modified Bonferroni correction (p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibody and immediate airway responses.
To assess the effect of the HDM sensitization and the combination of HDM and pertussis sensitization on the allergic responses to HDM challenge, HDM-specific IgE, IgG, and IgA in BAL fluid and serum were measured. No significant changes were found in BAL IgE and IgG, 0 and 2 days after HDM challenge (Figs. 1 and 2GoGo). However, 4 days after the HDM challenge, BAL IgE and IgG were synergistically elevated in both systemic and local HDM plus pertussis-sensitization groups, compared to their corresponding saline and pertussis controls and to groups sensitized with HDM alone. No significant changes were found for BAL IgA or for any of the serum immunoglobulins (data not shown). No differences were found between baseline airway responses and those after antigen challenge in any of the treatment groups, indicating that the level of immunity was insufficient to induce an immediate airway response (data not shown).



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FIG. 1. Effect of pertussis and house dust mite on HDM-specific IgE in BAL. Three panels represent BAL samples collected before HDM challenge (Day 0), 2 days after (Day 2), and 4 days after HDM challenge (Day 4). Data are means and SE (n = 10) of optical density (OD). Two-way ANOVA was applied and pair-wise comparisons were performed, following a significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 


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FIG. 2. Effect of pertussis and house dust mite (HDM) on HDM-specific IgG in BAL. Three panels represent BAL samples collected before HDM challenge (Day 0), 2 days (Day 2), and 4 days after HDM challenge (Day 4). Data are means and SE (n = 10). Two-way ANOVA was applied and pair-wise comparisons were performed following any significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 
Lavage fluid constituents.
Levels of LDH, total protein, and eosinophil peroxidase (EPO) in BAL fluid were measured to evaluate pulmonary cell membrane integrity, edema, and eosinophil activation, respectively. There were no significant changes in any of these parameters prior to dust mite challenge (Day 0). LDH levels were unchanged 2 days post-challenge in groups sensitized with HDM alone but were significantly increased in both systemic and local HDM sensitization groups treated with pertussis (Fig. 3Go). By 4 days the LDH levels were high with both sensitization regimes, regardless of pertussis coadministration. Similarly, BAL protein was not significantly altered between the groups prior to antigen challenge (Fig. 4Go), but was higher in the locally sensitized animals given pertussis compared to all other groups 2 days post-challenge. While this effect had subsided by 4 days, the protein levels in the systemic group with pertussis had reached statistical significance compared to all other groups. No significant changes were found in BAL EPO (data not shown).



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FIG. 3. Effect of pertussis and house dust mite (HDM) on lactate dehydrogenase (LDH in BAL. Three panels represent BAL samples collected before HDM challenge (Day 0), 2 days (Day 2), and 4 days after HDM challenge (Day 4). Data are means and SE (n = 10). Two-way ANOVA was applied and pair-wise comparisons were performed following any significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 


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FIG. 4. Effect of pertussis and house dust mite (HDM) on total protein in BAL. Three panels represent BAL samples collected before an HDM challenge (Day 0), 2 days (Day 2), and 4 days after the HDM challenge (Day 4). Data are means and SE (n = 10). Two-way ANOVA was applied and pair-wise comparisons were performed following any significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 
Pulmonary inflammatory cells.
Injection of B. pertussis caused an increase in alveolar macrophages, which was evident prior to challenge (Table 1Go). This effect persisted in the pertussis control and systemically immunized animals after allergen challenge, but declined in the locally sensitized animals given pertussis. No significant changes were found in any other cell type on Day 0 before HDM challenge. The total number of BAL eosinophils after challenge significantly increased in rats systemically immunized with HDM (Fig. 5Go). This was only significant in the pertussis animals 2 days post-challenge, but was also significant from controls in the HDM-alone group at 4 days, although the inclusion of pertussis as an adjuvant tripled the numbers compared to injection of antigen alone. The total number of BAL eosinophils was also significantly increased in rats locally sensitized with HDM 2 and 4 days after HDM challenge and these values were significantly higher in the animals coinjected with pertussis. The numbers of neutrophils and lymphocytes also followed this same response pattern, being highest in the pertussis groups from both the systemically and locally immunized animals (Table 1Go)



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FIG. 5. Effect of pertussis and house dust mite (HDM) on eosinophil numbers in BAL. Three panels represent BAL samples collected before an HDM challenge (Day 0), 2 days (Day 2), and 4 days after the HDM challenge (Day 4). Data are means and SE (n = 10). Two-way ANOVA was applied and pair-wise comparisons were performed following any significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 
Local lymph node effects.
Lymphocyte proliferation at Day 0 was highest in the rats injected with pertussis and instilled intratracheally with HDM (Fig. 6Go). This effect was still significantly higher 2 days after challenge and further increased by 4 days. Animals instilled with antigen without pertussis had no response at day 0, a moderate (though significantly lower) response at day 2 and an equal maximal response at day 4. A similar (though less robust) pattern was seen in the systemically immunized animals, whereby inclusion of pertussis resulted in a modest level of activity at Day 0 which gradually rose over time after antigen challenge. Animals injected with HDM alone had no response at days 0 or 2, but had similar levels to antigen-plus-pertussis treatment group at day 4. No significant lymphocyte activity was seen in the pertussis or saline-primed animals that also received intratracheal instillation of HDM at challenge.



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FIG. 6. Effect of pertussis and house dust mite (HDM) on HDM-specific lymphocyte proliferation in pulmonary lymph nodes in BAL. Three panels represent BAL samples collected before an HDM challenge (Day 0), and 2 days (Day 2), and 4 days after the HDM challenge (Day 4). Data are means and SE (n = 10). Two-way ANOVA was applied and pair-wise comparisons were performed following any significant interaction. *Significant differences between HDM and saline controls; **significant differences between HDM with and without pertussis. (n = 10; p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bordetella pertussis and its associated toxins have long been considered as effective adjuvants for eliciting IgE responses to a variety of antigens in experimental animals (Clausen et al., 1969Go, 1970Go; Lehrer, 1977Go; Lindsay, et al., 1994Go). Because of this specific property, pertussis has also been utilized in animal models of hypersensitivity to boost allergic responses to HDM in Brown Norway rats (Waserman et al., 1992Go), to ovalbumin in guinea pigs (Heuer et al., 1996Go), and to ragweed pollen in mice (Chang et al., 1974). After subsequent pulmonary antigen challenge, presensitized animals display many features of allergic asthma including increased mucus production, bronchial hyperresponsiveness, and eosinophilic inflammation (Lambert et al., 1998Go). The results from this present study confirm the adjuvant capabilities of B pertussis, and extend them by showing that systemic administration of whole killed cells augments allergic sensitization to HDM antigen delivered to the mucosal surface of the lung. This scenario is relevant to human allergic sensitization, since infants receive several immunizations with either whole killed pertussis cells or, more recently, a subunit vaccine toxoid during the first two years or life (Girard, 2002Go), when initial exposures to allergens also occur (Holt et al., 1990Go).

A potential reason we observed that enhanced mucosal sensitization to HDM with concurrent systemic immunization with B. pertussis is that this treatment may have caused a broad immunological activation, which extended to the lung and associated lymph nodes. In a similar study where pertussis toxin was administered intramuscularly to mice subsequently infected via the nose with RSV virus, IgG1 and IgE levels in serum and IL4 in lung supernatants were increased compared to either injection or virus infection alone (Fischer et al., 1999Go). Elevated levels of IL4 and IL5 and decreased IFN-{gamma} levels have also been reported in children following booster immunization with acellular pertussis toxin (Ryan et al., 2000Go), although, in this study there was no apparent effect on IgE antibody against common allergens. Thus, it would seem that under certain experimental conditions where pertussis and antigen are administered simultaneously, a vaccine component promoting a Th2 type response could augment allergic sensitization in susceptible individuals, although this may not occur often enough to be statistically significant in population studies (Anderson et al., 2001Go; Gruber et al., 2001Go; Henderson et al., 1999Go). Alternatively, concomitant immunization with components of other vaccines that promote Th1 responses may mask or even counteract the Th2 immune priming effects, and the effect reported here might only occur in real life with the selective use of Th2-inducing vaccines.

The mechanism for enhancing Th2 responses as characterized by IgE antibody is thought to lie in the ability of pertussis toxin to upregulate IL4 (Mu and Sewell, 1994Go; Samore and Siber, 1996Go) although how this occurs at the molecular level has not been elucidated. The toxin also augments expression of the co-stimulatory molecules B7.1 and B7.2 on macrophages and B cells, and CD28 on T cells (Ryan et al., 1998Go), which presumably enhances antigen presentation and the development of pertussis specific immunity. Analysis of mutant forms of the S1 and B oligomer components of the toxin has demonstrated that loss of enzymatic activity does not affect cytokine induction, but that binding to the glycoprotein receptors on the surface of eukaryotic cells is essential for adjuvant activity (Ryan et al., 1998Go).

The systemically and locally sensitized rats received the same amount of HDM, with the only difference being that rats were given 10 µg of HDM once, systemically, or 3.3 µg of HDM three times locally. Systemic sensitization induced relatively higher levels of HDM-specific IgE in BAL fluid, while local sensitization induced more severe pulmonary eosinophilia and increased HDM-specific lymphocyte proliferation in pulmonary lymph nodes. It would thus appear that the local sensitization was more effective at activating mucosal lymphocytes (as evidenced by greater lymphoproliferative activity), resulting in increased eosinophil recruitment and pulmonary injury. In support of this concept, we have previously reported that adoptive transfer of activated lymphocytes from pulmonary lymph nodes confer the ability to evoke airway hyperresponsiveness and eosinophilia after antigen challenge (Lambert et al., 1998Go). In addition, recent studies from our laboratory (Singh et al., 2003Go) have shown that local sensitization in adult animals produces relatively weak IgE levels and correspondingly low immediate airway responses after antigen challenge, while pulmonary eosinophils were as high as rats systemically immunized with HDM and aluminum hydroxide adjuvant.

We were unable to sensitize neonatal (1–4 days old) Brown Norway rats to HDM in preliminary experiments (data not shown), but succeeded in sensitizing juvenile rats at 3 weeks-of-age, although the responses were stronger with coadministration of pertussis as an adjuvant. These animals did not experience immediate bronchoconstriction following antigen challenge however, and the pulmonary eosinophil responses in juveniles were higher at four days than 2 days. The lack of immediate airway responses and delayed pulmonary inflammation may reflect the immaturity of the immune system in these young animals and suggest it may take longer, or require multiple exposures to generate substantial allergic responses following antigen challenge. The synergistic effect of pertussis on allergic responses to HDM in juvenile Brown Norway rats was most apparent with the BAL IgE and IgG four days after antigen challenge, and with the pulmonary eosinophils and lymphocytes, two and four days after antigen challenge. Interestingly, however, the simultaneous injection of pertussis and HDM instillation resulted in maximal HDM-specific lymphocyte proliferation before the antigen challenge at Day 0, indicating that these animals had a greater primary immune response, which may have resulted in increased boosted levels of antibody and allergic lung disease post-challenge.

Because B. pertussis is a gram-negative organism and is coated with LPS, which was also present in trace (noninflammatory) concentrations in the HDM antigen, our controls were important in demonstrating that the responses were not caused by immune-mediated reactions to LPS. Pertussis injection alone did not produce antigen-specific lymphocyte proliferation or granulocyte infiltration into the lung, indicating that priming with LPS in the pertussis preparation was not responsible for the immune-mediated inflammation after HDM challenge. Pertussis immunization did, however, cause a marked increase in alveolar macrophage number, suggesting that the lung was indeed affected by this treatment. Although the reasons for this are not entirely clear, previous studies have reported an increase in number and activity of alveolar macrophages after immunization with Freunds adjuvant or BCG (Petit and Leclerc, 1983Go), or irradiated parasites (Menson and Wilson, 1990Go), and have speculated that systemic priming results in mucosal activation of host defenses through the release of immunostimulatory cytokines.

The juvenile Brown Norway rat model of allergy to HDM described here provides an experimental system that could be used to further investigate the potential role of vaccine components on the development of allergy and asthma. The synergistic effect of whole killed pertussis cells and HDM in induction of allergic manifestations was quite apparent, and even though this vaccine formulation is less frequently used, the results demonstrate that a Th2 adjuvant can augment allergic immune responses to antigens delivered by another route. While the benefits of vaccination far outweigh the risks of coincidental sensitization to environmental allergens, the use of strong Th2 adjuvants during periods of high allergen exposure may increase the possibility of sensitization in susceptible individuals.


    ACKNOWLEDGMENTS
 
We thank Stephen Gavett and Mike Viana for their helpful comments, Judy Richards for her technical support, and Donald Doerfler for his statistical assistance. This work was performed while Wumin Dong held a National Research Council–U.S. EPA Senior Research Associateship.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 541-4284. E-mail: gilmour.ian{at}epa.gov. Back

The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor the mention of trade names or commercial products constitute endorsement or recommendation for use.


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 RESULTS
 DISCUSSION
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