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
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ABSTRACT |
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Key Words: pertussis; dust mite; allergy; IgE; lymphocyte; eosinophil; vaccine; rat.
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INTRODUCTION |
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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., 1970; Lindsay et al., 1994
; Mu and Sewell, 1994
). 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, 2000
). 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, 2002
).
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, 2000; Kemp et al., 1997
; Odent et al., 1994
; 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., 2001
; Destefano et al., 2002
; Gruber et al., 2001
; Henderson et al., 1999
). 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., 1993
) or after vaccination (Torre et al., 1990
). Asthma incidence is also significantly higher in children with proven pertussis infection than noninfected children (Nilsson et al., 1998
).
It is clear that exposure to environmental antigens such as HDM (Shapiro et al., 1999; Sporik et al., 1992
) and the number and type of infant immunizations have increased in recent decades (Schleiss and Dahl, 2000
). 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., 1998
). 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., 1998
; Gilmour et al., 1996
; Lambert et al 1999
; 2000
; Luebke et al., 2001
). 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.
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MATERIALS AND METHODS |
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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 1. 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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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 105 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).
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RESULTS |
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DISCUSSION |
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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., 1999). Elevated levels of IL4 and IL5 and decreased IFN-
levels have also been reported in children following booster immunization with acellular pertussis toxin (Ryan et al., 2000
), 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., 2001
; Gruber et al., 2001
; Henderson et al., 1999
). 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, 1994; Samore and Siber, 1996
) 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., 1998
), 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., 1998
).
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., 1998). In addition, recent studies from our laboratory (Singh et al., 2003
) 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 (14 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, 1983), or irradiated parasites (Menson and Wilson, 1990
), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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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