1 North Carolina State University, College of Veterinary Medicine, Raleigh 27606; and 2 National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Brown Norway (BN) rats develop a robust response to antigens in the lung, characterized by a large increase in allergen-specific immune function and pulmonary eosinophilia. The objective of this study was to investigate alternative models by determining whether other rat strains could be sensitized to house dust mite (HDM) antigen and whether the allergic disease process could be worsened with repeated allergen exposure. In general, BN rats sensitized by either subcutaneous or intratracheal routes exhibited increased pulmonary allergy compared with Sprague-Dawley (SD) and Lewis (L) rats. Multiple intratracheal allergen exposures incrementally increased HDM-specific immune function in BN rats but progressively decreased eosinophil recruitment and markers of lung injury. SD rats had more moderate responses, whereas L rats were relatively unresponsive. Because BN rats developed stronger clinical hallmarks of allergic asthma under various immunization regimes compared with SD and L rats, we conclude that the BN is the most appropriate strain for studying allergic asthma-like responses in rats. Phenotypic differences in response to HDM were associated with differences in the Th1/Th2 cytokine balance and antioxidant capacity.
genetic variability; atopy; asthma
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INTRODUCTION |
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ANIMAL MODELS of
allergic airway disease are useful tools for studying susceptibility
factors and pathophysiological processes associated with human allergic
asthma. Numerous genetic differences among rat and mouse strains in IgE
responsiveness and other phenotypes of respiratory allergy have been
reported. Studies in allergy-susceptible (A/J strain) and
allergy-resistant (C3H strain) mice suggest that the genes associated
with a Th2-type cytokine profile and its regulation by other cytokines,
like IL-12 and IFN-, may determine phenotypic responses to allergen
challenge (12, 31). Brown Norway (BN) rats have
consistently demonstrated high IgE responsiveness to allergen
provocation and significantly greater constitutive IgE production
(22, 26) compared with other rat strains
(28). BN rats are also high responders in pulmonary
pathologies associated with parainfluenza virus infection
(25), although both Lewis (L) and Sprague-Dawley (SD) rats
have demonstrated susceptibility to experimentally induced airway
hyperresponsiveness (18) as well as experimental allergic
neuritis (9) and neurogenic inflammation (11). It would then seem likely that all three rat strains
would have potential for use in models of inflammatory and allergic airway diseases. However, studies of ovalbumin (OVA)-induced
respiratory allergy have shown that L and SD rats do not develop
pulmonary eosinophilia (21) and airway hyperresponsiveness
(3) following OVA challenge. Pulmonary responsiveness to
house dust mite (HDM) antigen has not been compared among these three
rat strains, and an integrated assessment of immune responses, altered
pulmonary function, and pathological processes would create a more
complete view of the similarities and differences among the three phenotypes.
The tendency toward high baseline responses in untreated BN rats and
interindividual variability in the intensity of allergic responses to
allergen challenge are two recurrent problems that can confound
investigations of allergic pathophysiology. This paper examines whether
or not a different rat strain could provide an alternative model of
human allergic airway disease with lower baseline levels and less
variability than observed in BN rats. Because bronchoconstriction,
eosinophilic inflammation of the airways, and mucus production are
three of the hallmarks of human allergic asthma, these responses should
be included in an animal model of the disease. In addition, although
decreased plasma levels and dietary deficiencies in ascorbic acid
(vitamin C) have been associated with asthma (7), it is
not known whether strain-dependent variations in antioxidant capacity
affect susceptibility to allergic sensitization. The present study
tested the hypothesis that the BN is the most appropriate rat strain
for investigating allergic airway disease and asthma because of its
pro-Th2 and proeosinophilic phenotype. We compared response profiles
among BN, L, and SD rats sensitized either subcutaneously (SC) or
intratracheally (IT) to better understand associations among lung
injury and inflammation, immune function, and a key symptomatic
indicator of disease, allergen-induced bronchoconstriction.
Additionally, we investigated the effect of multiple allergen
challenges to determine whether immunological tolerance or chronic
disease would develop. This integrated evaluation of pulmonary allergy
included measurements of immediate airway responses (IARs) to HDM
challenge and allergen-specific serum IgG and IgE levels, HDM-specific
proliferation of lymphocytes in bronchial lymph nodes, antioxidant
levels, inflammatory cell infiltrate, and IL-13 (Th2 cytokine) and
IFN- (Th1 cytokine) concentrations in bronchoalveolar lavage fluid (BALF).
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MATERIALS AND METHODS |
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Animals. Inbred, female BN (strain BN/Ss Nhsd; 160-180 g); inbred, female L (210-240 g); and outbred, female SD (250-280 g) rats were purchased from Charles River Laboratories (Wilmington, MA) and used at 8-10 wk of age. Rats were housed in American Association for Accreditation of Laboratory Animal Care-approved animal facilities with high-efficiency particulate air filters, and their use was reviewed by the U. S. Environmental Protection Agency's Animal Care and Use Committee. All rats were fed rat chow and water ad libitum. All rats that were selected randomly and serologically tested upon arrival, as well as sentinels monitored throughout the study, were free of Sendai virus, pneumonia virus, and a variety of other rodent viruses and Mycoplasma sp.
Antigen. HDM antigen derived from Dermatophagoides farinae (Der f) was purified from ground, whole-bodied mites following defatting, extraction in 0.125 M ammonium bicarbonate, and dialysis with distilled water (Greer Laboratories, Lenoir, NC). Purification of the extract was achieved by a combination of DEAE ion exchange chromatography and gel filtration, with the final preparation containing >75% of group 1 allergen (Der f 1), as determined by the vendor.
Experimental design. BN, L, and SD rats were sensitized with HDM by either systemic (SC) or local (IT) routes. Systemic injection was assessed for maximal immunization; however, we were also interested in mucosal sensitization as a more relevant exposure route and because of the potential for different sensitization regimes to produce different immunological effects. IT instillation is a quick, easily reproducible procedure and shows results comparable with those based on the inhalation route of administration (8). On days 1 and 3 systemically sensitized rats received an SC injection of 5 µg of HDM in 0.5 ml of aluminum hydroxide adjuvant (Alhydrogel containing 1.3% Al2O3 as dry matter; Accurate Chemical & Scientific, Westbury, NY) or 0.5 ml of aluminum hydroxide adjuvant alone as a sham sensitization. Two weeks later these rats were challenged IT with 10 µg of HDM in 250 µl of saline. Airway responses to HDM were measured on the day of challenge, and rats were euthanized and bled by cardiac puncture 2 days later for assessment of allergic responses, pulmonary inflammation, and lung injury in tissue samples. Postchallenge day 2 was selected as the time point for evaluation of responses to allergen, because it is known that pulmonary inflammation and injury peak 2-3 days postchallenge (21). Locally sensitized BN, L, and SD rats received 5 µg of HDM in 250 µl of saline by IT instillation on days 1 and 3. Two weeks later, separate groups of these rats were IT challenged with 10 µg of HDM in 250 µl of saline either one, two, or five times, each successive challenge separated by 1 wk. Airway responses to HDM were evaluated in all locally sensitized rats on the last day of allergen challenge. These rats were euthanized and bled by cardiac puncture 2 days after the final IT challenge for assessment of allergic responses, pulmonary inflammation, and lung injury.
IARs to HDM. Two weeks after sensitization, animals were placed in a whole body plethysmograph (Buxco Electronics, Troy, NY) equipped with a pneumotachograph and pressure transducer to monitor pulmonary ventilation responses as previously described (15-17). Baseline ventilatory readings were measured for 10 min before challenge. Animals were then removed from the plethysmograph, anesthetized with halothane, IT instilled with 10 µg of HDM in 250 µl of saline, and then placed in the plethysmograph for an additional 20 min to evaluate IARs following challenge. These responses are expressed as the enhanced pause (Penh), which is a derived value that provides an indicator of changes in specific airway resistance (20). Penh values were averaged during the baseline (control) periods and the postchallenge periods to obtain mean values for each event and are represented as change from the mean during the baseline period to the mean during the postchallenge period.
Bronchoalveolar lavage and lavage cell differentials. The trachea of each rat was surgically exposed, cannulated, and tied off with a silk thread suture. The left lobe was tied off to prepare for histopathology, and the right lobe was lavaged three times with a single volume of warmed saline (0.035 × body wt × 0.55 ml/kg). Total white blood cell counts were obtained with a hemocytometer, and cell viability was assessed by trypan blue exclusion. Approximately 50,000 cells from each sample were centrifuged onto duplicate glass slides in a Cytospin (Shandon, Pittsburgh, PA) and stained with Diff Quik (American Scientific, Sewickley, PA) for identification of eosinophils, macrophages, neutrophils, and lymphocytes. We counted at least 200 cells for each duplicate slide to obtain percent values of each leukocyte subpopulation. If the difference between duplicate slides was >2%, then the slides were recounted until agreement in the differential was reached.
BALF biochemical analysis.
BALF was centrifuged (1,500 rpm, 10 min, 4°C), and the supernatant
was analyzed. Lactate dehydrogenase (LDH) and total protein levels were
determined with kit 228 (Sigma) and Coomassie Plus reagent (Pierce,
Rockford, IL), respectively. We adapted both assays for automated
analysis using a Cobas Fara II centrifugal spectrophotometer
(Hoffman-La Roche, Branchburg, NJ). Perchloric acid (PCA) was added to
a separate aliquot of BALF for antioxidant analysis at a final
concentration of 3%, and samples were stored at 80°C. Before
analysis for reduced glutathione (GSH), ascorbic acid (AA), uric acid
(UA), and nonprotein sulfhydryls (NPSH), PCA-treated samples were
centrifuged at 20,000 g for 30 min at 4°C
(24). AA and UA analyses were performed by liquid
chromatography with electrochemical detection as described in Kutnink
et al. (14), and total GSH was measured in the presence of
GSH reductase and 5,5'-dithiobis-(2-nitrobenzoic acid) by enzymatic
recycling (1) by a Cobas Fara II centrifugal
spectrophotometer. The detection limit for both AA and UA was 0.2 nmol/ml. NPSH concentrations in BALF were determined with a reagent
containing 2.0 mM ethylene-diaminetetraacetic acid and 0.21 mM
5,5'-dithiobis-2-nitrobenzoic acid in a 0.4 M Tris · HCl buffer, pH 8.9 (27). We
determined the sample concentration of NPSH from a standard curve using
reduced glutathione. The limit of detection for both GSH and NPSH was
0.21 µg/ml.
BALF cytokine analysis.
An aliquot of BALF (stored at 80°C) was used for analysis of
IFN-
and IL-13 cytokines as representative markers of Th1 and Th2
phenotypes, respectively. We assessed concentrations by enzyme-linked immunosorbent assay (ELISA) using rat Cytoscreen kits purchased from
Biosource International (Camarillo, CA). Selection of these cytokines
was based upon IFN-
's recognition as the prototypic Th1 cytokine
and the fact that IL-13 has been conclusively linked to the development
of respiratory allergy (29, 30).
Lymphocyte proliferation assay. Lung-associated lymph nodes were removed from the right main stem bronchus of each rat 2 days after (final) HDM challenge. We prepared single cell suspensions of lymph node tissues using ground-glass homogenizers, and the in vitro lymphocyte proliferation response to HDM was assessed by cellular incorporation of [3H]thymidine as described in Gilmour et al. (5). Allergen-specific lymphocyte proliferation was expressed as the difference in radiotracer counts (disintegrations per min) between lymphocytes from a given sample incubated with HDM (1 µg/well) and lymphocytes from the same sample incubated with media alone.
Antigen-specific serum IgE and serum IgG.
Antigen-specific serum immunoglobulin production was measured by ELISA
as described previously (5). Rats were euthanized with 200 mg/kg pentobarbital sodium and bled by cardiac puncture. Serum was
prepared and kept frozen at 80°C until assay. Briefly, for the
HDM-specific IgE assay, 96-well flat-bottom ELISA plates were coated
with 100 µl/well of mouse anti-rat IgE heavy chain antibody (Serotec,
Oxford, UK) at a concentration of 2.5 µg/ml in coating buffer
(Pierce) and incubated overnight at 4°C. The following day, after a
blocking step and washing, 100 µl of each serum sample (diluted 1:5
in blocking buffer) were added in duplicate wells to the plates. After
an overnight incubation at 4°C and washing, the plates were treated
successively with 100 µl/well of biotinylated HDM (2 µg/ml,
prepared using Sulfo-NHS-LC-Biotinylation kit; Pierce) and horseradish
peroxidase-streptavidin (diluted 1:1,500), with washes and incubation
for 1 h at room temperature between each of these steps. For the
HDM-specific serum IgG assay, 96-well flat-bottom plates were coated
with 100 µl of purified HDM (containing >75% Der f 1 antigen) at a concentration of 1.6 µg/ml in coating buffer and
incubated overnight at 4°C. The next day, after washing, 200 µl of
blocking buffer were added to each well followed by a 2-h incubation at
37°C. Diluted serum (1:50) was added (100 µl/well) after washing
followed by overnight incubation at 4°C. Wells were then washed and
treated successively with 100 µl of mouse anti-rat biotinylated IgG
(diluted 1:5,000; Serotec) and horseradish peroxidase-streptavidin
(diluted 1:1,500) separated by washes and an incubation for 1.5 h
at 37°C. In the final step, 100 µl/well TM Blue (Dako, Carpinteria,
CA) was added as a substrate for horseradish peroxidase, and reactions
were allowed to develop at room temperature for at least 10 min. Plates
were read at 650 nm by a Spectromax ELISA plate reader (Molecular
Devices, Menlo Park, CA).
Histopathology. The left lobe of the lung was inflated and fixed with a volume (0.035 × body wt × 0.40 ml/kg) of paraformaldehyde (4% wt/vol in deionized water) (Sigma), immersed in 4% paraformaldehyde for 24 h then transferred into Tris-buffered saline (pH 7.4) (Pierce). Samples were then sent to Experimental Pathology Laboratories (Research Triangle Park, NC) for processing and histopathological evaluation. Midsagittal lung sections were stained with hematoxylin and eosin to determine inflammatory changes and periodic acid Schiff/Alcian blue (pH 2.5) to determine goblet cell hypertrophy and hyperplasia and mucus production. Lung sections were microscopically evaluated in seven different histopathological lesion categories by open examination (10). These categories included perivascular lymphocytic infiltration, terminal bronchiolar lymphocytic infiltration, peribronchiolar eosinophilic infiltration, terminal bronchiolar eosinophilic infiltration, perivascular eosinophilic infiltration, focal/multifocal pneumonitis, and goblet cell hypertrophy/hyperplasia with mucus in the airways. In each of these seven different categories, lesions were scored for both severity of inflammatory cell infiltrate and distribution: 0 (normal), 1 (minimal), 2 (mild), 3 (moderate), or 4 (marked). We derived the total lesion scores (Fig. 8) by adding up the combined lesion scores (0-4) from all seven categories for each sample and calculating the mean total lesion score for each treatment group.
Statistical analysis.
The data were analyzed (SAS version 6.02; SAS Institute, Cary, NC) by
analysis of variance (ANOVA) models. We used one-way (by strain) ANOVA
for the locally sensitized groups and two-way (by strain and treatment)
ANOVA for the systemically sensitized groups. After an overall
statistically significant finding, pairwise comparisons were performed
among strains and various strain-treatment combinations. Significance
levels were adjusted for multiple comparisons by a Bonferroni
technique. The level of significance was set at 0.05.
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RESULTS |
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IARs.
After systemic immunization and challenge, only the BN rats developed a
strong IAR compared with sham-immunized, challenged controls (Fig.
1A). With local sensitization,
BN rats that were challenged two or more times developed significant
IARs, although these responses were not as strong as with systemic
immunization. Local immunization also resulted in the development of a
significant IAR in SD rats after five challenges, which was of the same
magnitude as the BN response. L rats did not develop IARs under any
conditions.
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BALF cell differentials.
Among systemically sensitized rats, there were no significant strain
differences in numbers of macrophages, polymorphonuclear cells
(PMNs)/neutrophils, or lymphocytes in the BALF (Table
1). However, there were much higher
numbers of eosinophils in the BALF of both control and
antigen-challenged BN rats compared with the same groups of L and SD
rats on postchallenge day 2 (Fig. 2A). Systemic sensitization to
HDM significantly increased total numbers of eosinophils in BALF
(11-fold higher) over sham sensitization in BN rats (Fig.
2A).
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BALF biochemical analysis.
All three strains demonstrated significant increases in total protein
levels postchallenge in the BALF compared with their sham-sensitized
controls (Fig. 3A).
Systemically sensitized BN and SD rats had significantly greater
amounts of total protein in the BALF on postchallenge day 2 than L rats that received the same treatment (Fig. 3A).
There were no significant differences in levels of LDH in BALF of
HDM-sensitized rats compared with their respective sham-sensitized
controls in any strain (data not shown). However, the LDH levels of BN
rats were significantly higher than those of SD and L rats with both
systemic and local sensitization (data not shown). Among all locally
sensitized rats, LDH (data not shown) and total protein (Fig.
3B) levels in BALF steadily decreased with increasing IT
challenges. After five IT challenges, total protein levels in L rats
dropped more than in BN and SD rats, accounting for a significant
difference between BN and SD compared with L rats with this treatment
(Fig. 3B).
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HDM-specific lymphoproliferative responses.
Systemic sensitization to HDM induced significantly higher HDM-specific
lymphoproliferative responses in BN rats compared with L rats that
received the same treatment (Fig.
5A). SD rats had intermediate
responses. Local sensitization to HDM also induced significantly higher
responses in BN rats compared with L rats following two or more
challenges (Fig. 5B). Responses in SD rats were initially
low but developed to the same level as BN rats with successive IT
challenges (Fig. 5B).
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HDM-specific serum IgG and serum IgE.
Among systemically sensitized rats, both BN and SD had significantly
higher HDM-specific serum IgG than L rats on postchallenge day
2 (Fig. 6A). Significant
increases in allergen-specific serum IgG levels were observed in the
sensitized rats of all strains compared with their controls.
HDM-specific IgG titers developed more slowly in the locally immunized
rats of each strain but with successive challenges increased above the
levels of the systemically immunized animals challenged one time (Fig.
6B).
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Histopathology.
Lung histopathology was evaluated two days after challenge in
systemically sensitized rats and in locally sensitized rats that
received a single IT challenge. Both BN and SD rats that were
systemically sensitized to HDM had significantly more lung lesions than
L rats with the same treatment (Fig. 8).
HDM-sensitized rats of all three strains had greater severity of lung
lesions than their respective sham-sensitized controls; however,
control levels in BN rats were significantly higher than in L and SD
rats. Among locally sensitized rats, BN had significantly more lung lesions than both SD and L, and SD had significantly greater numbers of
lesions than L (Fig. 8). Although the number of lesions differed between SD and L, the characteristics of the lesions were identical in
these two strains. By contrast, BN rats were distinct from SD and L
rats in their histopathology. Unlike sham-sensitized SD and L rats,
sham-sensitized BN rats presented atypical multifocal granulomatous
pneumonia, characterized by the presence of eosinophils, lymphocytes,
macrophages, and multinucleated giant cells in the alveolar spaces. In
BN rats, granulomatous pneumonitis accounted for a significant portion
of the lesions, whereas eosinophilic and lymphocytic infiltration
around the small blood vessels primarily characterized lesions observed
in SD and L rats. In general, the degree of inflammatory lesions was
more severe in systemically sensitized rats than in locally sensitized
rats in both SD and L, whereas in BN rats, local sensitization produced
more severe lesions than systemic sensitization (Fig. 8). Goblet cell
hyperplasia and hypertrophy in both locally and systemically sensitized
BN rats was more pronounced than in either SD or L rats with the same
treatment. Mast cells were not found in significant numbers, regardless
of strain.
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DISCUSSION |
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The genetic basis of allergic asthma is becoming better understood through animal studies, which show how genotypic variations result in characteristic phenotypic outcomes. It is well supported in experimental rodent literature that susceptibility to allergic sensitization and physiological responsiveness to allergen challenge in the lung are strain dependent (12, 13, 18, 21, 31). The observed differences among both rat and mouse strains in responsiveness to allergens and in their phenotypes of allergic airway disease have provided opportunities to select strains that best simulate allergic asthma and to identify genes that are causally linked to the disease. However, there has been little integration of immune function, airway responses, subsequent pathology, and antioxidant responses in experimentally induced respiratory allergy. In this study we report striking strain differences in immune responses to allergen, which were associated with Th2 cytokine polarization, increased pulmonary inflammation, and altered antioxidant balance.
One of the hallmarks of allergic asthma is pulmonary eosinophila, which
has been described kinetically in OVA-allergic BN rats
(21). Our data showing increased BALF eosinophils in
HDM-challenged BN rats agree with earlier studies with OVA, which
demonstrated greater responsiveness among BNs in antibody production,
eosinophilic inflammation (21), and increased mucus
production (19) compared with other rat strains. The
minimal pulmonary inflammation and virtually no allergen-specific IgE
titers in L rats confirm previous observations by Schneider et al.
(21), who reported that two rat strains of a Th1 phenotype
demonstrate relatively weak responses to allergen challenge. In this
paper we have extended these findings to include antigen-induced IARs,
changes in allergen-specific T cell function, Th1/Th2 cytokine
measurements, and antioxidant levels to gain an integrated perspective
of strain differences in allergic phenotypes. In addition, the two
presumed phenotypic Th1 rat strains (SD and L) could be separated by
differences in their IAR and HDM-specific lymphocyte proliferation.
Specifically, following multiple allergen challenges, it was found that
SD rats became responsive to allergen, whereas L rats did not. SD rats were more like BNs in their levels of HDM-specific IgG and intermediate between BN and L rats with regard to IFN- levels in the BALF, suggesting a range of Th1/Th2 allergic phenotypes among rat strains.
It has been previously demonstrated that pulmonary resistance in response to dry-gas hyperpnea challenge is increased in BN rats compared with ACI, L, and Fischer 344 rats and is associated with the release of tachykinins and cysteinyl leukotrienes (32). Although levels of these proteins were not measured in the present study, we have observed significantly elevated levels of cysteinyl leukotrienes in HDM-allergic BN rats exposed to diesel exhaust particles (unpublished observation). Other mediators released from alveolar macrophages, such as nitric oxide, IL-10, and TNF, have also been suggested to explain the differences in allergic susceptibility between SD and BN rats (3).
Earlier studies in rats sensitized and challenged with Der p
1 (from Dermatophagoides pteronyssinus) (26)
demonstrated the high IgE responder status of BN compared with Wistar
and Lou/M rats and that the low IgE responder WAG (derivative of the
Wistar rat) rat was 1,000 times more sensitive to tolerance induction by repeated aerosol challenges with OVA than BN rats (22).
In the present study, increased production of HDM-specific serum IgE
and IgG was observed in all three strains with successive IT
challenges, and the magnitude of the IgE response reflected the
responder status of each strain (BN > SD > L). This trend was also observed in T cell function and the IAR. In contrast, eosinophil recruitment and IL-13 (in BN), IFN- (in L and SD), and
total protein and GSH concentrations in BALF of all strains decreased
over time, indicating altered response kinetics or some form of
tolerance building with repeated IT challenge. Together the results
show that a sustained cellular and humoral immune response over
multiple allergen challenges is not necessarily accompanied by a
sustained and progressive release of Th1 or Th2 cytokines, eosinophil
recruitment, or tissue damage. A prolonged study, including additional
weeks of allergen challenge, would more clearly illustrate strain
differences in tolerance induction and immune regulation under a
repeated exposure regime.
Hall et al. (6) demonstrated that SD and BN rats had increased serum IgG responses to intrabronchial infection with Bordetella pertussis compared with L and Hooded Lister rats. In that study, SD and L rats had the lowest baseline total serum IgE levels and showed the most increase in total serum IgE after infection (6). Although these results show the advantage of low baseline responses in models of infection and immunity, IgE responsiveness among BN, SD, and L rats was demonstrated as high, intermediate, and low, respectively (6), indicating a relative weakness in humoral immune responses among L rats. Rat strain differences in the predominance of humoral vs. cellular immunity have been reported in a model of autoimmune disease induced by immunization with human myeloperoxidase (2). In that model, BN rats have stronger humoral immune responses (antibody responses), whereas L rats have more potent cellular immune responses, as demonstrated by (Th1-mediated) delayed type hypersensitivity responses to myeloperoxidase (2). The same strain-specific tendencies toward humoral and cellular immunity have been shown in the present model of allergic airways disease.
L and SD rats had lower baseline responses (in nonsensitized rats) than
BN rats with respect to histopathology lesions, IL-13 levels, and
inflammatory cells (especially eosinophils) in the BALF. On the other
hand, L and SD rats had higher baseline levels of BALF IFN-, and SD
rats had higher baseline BALF GSH levels. These differences in Th1
cytokine levels and antioxidant reserves in the BALF of nonsensitized
rats may have accounted for the observed differences in responsiveness
to allergen and lung injury among sensitized rats. Oral administration
of the antioxidant taurine has been shown to significantly reduce
eosinophil influx as well as vascular leakage into the BALF of BN rats
after allergen challenge (4). Shvedova et al.
(23) demonstrated that OVA challenge in sensitized guinea
pigs (Hartley strain) induced a significant increase in eosinophils and
lipid peroxidation products with a coincident decrease in AA and
-tocopherol concentrations in BALF, suggesting that allergen
challenge induces an oxidative stress response in the lung. The present
paper is the first report indicating strain-specific differences in
endogenous antioxidant levels in an allergic rat model. Peak
antioxidant levels were generally lower in BN rats than in L and SD
rats, among both allergen-sensitized and nonsensitized groups. These
reduced antioxidant levels may be indicative of a difference in the
ability of BN rats to adapt to oxidative stress induced by allergen
challenge. It is possible that the differences in endogenous
antioxidant levels may play a role in the initiation of immune
responses and subsequent hypersensitivity disease. Further
investigation of the development of nonatopic and atopic immune
responses to allergens in different rat strains (and F1 hybrids between
responder and nonresponder strains) will help identify individual genes
that confer susceptibility to allergic airway disease.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Linda Birnbaum, Ralph Smialowicz, and Michael Viana for reviewing this manuscript and for providing valuable comments. We are also grateful to Kay Crissman for performing antioxidant analyses on the BALF samples and to Jim Lehmann for advice on rat IT instillations.
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FOOTNOTES |
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This work was supported by Environmental Protection Agency/North Carolina State University Cooperative Training Agreement CT826512010.
Disclaimer: 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 does the mention of trade names or commercial products constitute endorsement or recommendation for use.
Address for reprint requests and other correspondence: M. I. Gilmour, National Health and Environmental Effects Research Laboratory, U.S. EPA, MD-92, Research Triangle Park, NC 27711 (E-mail: gilmour.ian{at}epa.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 22, 2002;10.1152/ajplung.00287.2002
Received 21 August 2002; accepted in final form 19 November 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, ME.
Determination of glutathione and glutathione disulfide in biological samples.
Methods Enzymol
113:
48-55,
1985.
2.
Brouwer, E,
Weening JJ,
Klok PA,
Huitema MG,
Tervaert JWC,
and
Kallenberg CGM
Induction of an humoral and cellular (auto) immune response to human and rat myeloperoxidase (MPO) in Brown-Norway (BN), Lewis and Wistar Kyoto (WKY) rat strains.
Adv Exp Med Biol
336:
139-142,
1993[Medline].
3.
Careau, E,
Sirois J,
and
Bissonnette EY.
Characterization of lung hyperresponsiveness, inflammation, and alveolar macrophage mediator production in allergy resistant and susceptible rats.
Am J Respir Cell Mol Biol
26:
579-586,
2002
4.
Cortijo, J,
Blesa S,
Martinez-Losa M,
Mata M,
Seda E,
Santangelo F,
and
Morcillo EJ.
Effects of taurine on pulmonary responses to antigen in sensitized Brown-Norway rats.
Eur J Pharmacol
431:
111-117,
2001[ISI][Medline].
5.
Gilmour, MI,
Park P,
and
Selgrade MJK
Increased immune and inflammatory responses to dust mite antigen in rats exposed to 5 ppm NO2.
Fundam Appl Toxicol
31:
65-70,
1996[ISI][Medline].
6.
Hall, E,
Parton R,
and
Wardlaw AC.
Differences in coughing and other responses to intrabronchial infection with Bordetella pertussis among strains of rats.
Infect Immun
65:
4711-4717,
1997[Abstract].
7.
Hatch, GE.
Vitamin C and asthma.
In: Vitamin C in Health and Disease, edited by Packer L,
and Fuchs J.. New York: Dekker, 1997, p. 279-294.
8.
Henderson, RF,
Driscoll KE,
Harkema JR,
Lindenschmidt RC,
Chang IY,
Maples KR,
and
Barr EB.
A comparison of the inflammatory response of the lung to inhaled versus instilled particles in F344 rats.
Fundam Appl Toxicol
24:
183-197,
1995[ISI][Medline].
9.
Hoffman, PM,
Powers JM,
Weise MJ,
and
Brostoff SW.
Experimental allergic neuritis. I. Rat strain differences in the response to bovine myelin antigens.
Brain Res
195:
355-362,
1980[ISI][Medline].
10.
House, DE,
Berman E,
Seely JC,
and
Simmons JE.
Comparison of open and blind histopathologic evaluation of hepatic lesions.
Toxicol Lett
63:
127-133,
1992[ISI][Medline].
11.
Karalis, K,
Crofford L,
Wilder RL,
and
Chrousos GP.
Glucocorticoid and/or glucocorticoid antagonist effects in inflammatory disease-susceptible Lewis rats and inflammatory disease-resistant Fischer rats.
Endocrinology
136:
3107-3112,
1995[Abstract].
12.
Keane-Myers, A,
Wysocka M,
Trinchieri G,
and
Wills-Karp M.
Resistance to antigen-induced airway hyperresponsiveness requires endogenous production of IL-12.
J Immunol
161:
919-926,
1998
13.
Knippels, LM,
Penninks AH,
van Meeteren M,
and
Houben GF.
Humoral and cellular immune responses in different rat strains on oral exposure to ovalbumin.
Food Chem Toxicol
8:
881-888,
1999.
14.
Kutnink, MA,
Hawkes WC,
Schaus EE,
and
Omaye ST.
An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components.
Anal Biochem
166:
424-430,
1987[ISI][Medline].
15.
Lambert, AL,
Dong W,
Selgrade MJK,
and
Gilmour MI.
Enhanced allergic sensitization by residual oil fly ash particles is mediated by soluble metal constituents.
Toxicol Appl Pharmacol
165:
84-93,
2000[ISI][Medline].
16.
Lambert, AL,
Dong W,
Winsett DW,
Selgrade MJK,
and
Gilmour MI.
Residual oil fly ash exposure enhances allergic sensitization to house dust mite.
Toxicol Appl Pharmacol
158:
269-277,
1999[ISI][Medline].
17.
Lambert, AL,
Winsett DW,
Costa DL,
Selgrade MJK,
and
Gilmour MI.
Transfer of allergic airway responses with serum and lymphocytes from rats sensitized to dust mite.
Am J Respir Crit Care Med
157:
1991-1999,
1998[ISI][Medline].
18.
Matsubara, S,
Nakata A,
Kikuchi M,
Kikkawa H,
Ikezawa K,
and
Naito K.
Strain-related differences in Sephadex bead-induced airway hyperresponsiveness and inflammation in rats.
Inflamm Res
46:
299-305,
1997[ISI][Medline].
19.
Ohtsuku, R,
Doi K,
and
Itagaki S.
Histological characteristics of respiratory system in Brown Norway rat.
Exp Anim
46:
127-133,
1997[ISI][Medline].
20.
Pennock, BE,
Cox CP,
Rogers RM,
Cain WA,
and
Wells JH.
A noninvasive technique for measurement of changes in specific airway resistance.
J Appl Physiol
46:
399-406,
1979
21.
Schneider, T,
van Velzen D,
Moqbel R,
and
Issekutz AC.
Kinetics and quantitation of eosinophil and neutrophil recruitment to allergic lung inflammation in a Brown Norway rat model.
Am J Respir Cell Mol Biol
17:
702-712,
1997
22.
Sedgwick, J,
and
Holt PG.
Suppression of IgE responses in inbred rats by repeated respiratory tract exposure to antigen: responder phenotype influences isotype specificity of induced tolerance.
Eur J Immunol
14:
893-897,
1984[ISI][Medline].
23.
Shvedova, AA,
Kisin ER,
Kagan VE,
and
Karol MH.
Increased lipid peroxidation and decreased antioxidants in lungs of guinea pigs following an allergic pulmonary response.
Toxicol Appl Pharmacol
132:
72-81,
1995[ISI][Medline].
24.
Slade, R,
Crissman K,
Norwood J,
and
Hatch G.
Comparison of antioxidant substances in bronchoalveolar lavage cells and fluid from humans, guinea pigs and rats.
Exp Lung Res
19:
469-484,
1993[ISI][Medline].
25.
Sorden, SD,
and
Castleman WL.
Brown Norway rats are high responders to bronchiolitis, pneumonia, and bronchiolar mastocytosis induced by parainfluenza virus.
Exp Lung Res
17:
1025-1045,
1991[ISI][Medline].
26.
Stewart, GA,
and
Holt PG.
Immunogenicity and tolerogenicity of a major house dust mite allergen, Der p I from Dermatophagoides pteronyssinus, in mice and rats.
Int Arch Allergy Appl Immunol
83:
44-51,
1987[ISI][Medline].
27.
Sullak, J,
and
Lindsay RH.
Estimation of total protein bound and nonprotein sulfhydryl groups in tissue with Ellman's reagent.
Anal Biochem
25:
192-205,
1968[ISI][Medline].
28.
Wilkes, LK,
McMenamin C,
and
Holt PG.
Postnatal maturation of mast cell subpopulations in the rat respiratory tract.
Immunology
75:
535-541,
1992[ISI][Medline].
29.
Wills-Karp, M.
The gene encoding interleukin-13: a susceptibility locus for asthma and related traits.
Respir Res
1:
19-23,
2000[Medline].
30.
Wills-Karp, M.
IL-12/IL-13 axis in allergic asthma.
J Allergy Clin Immunol
107:
9-18,
2001[ISI][Medline].
31.
Wills-Karp, M,
and
Ewart SL.
The genetics of allergen-induced airway hyperresponsiveness in mice.
Am J Respir Crit Care Med
156:
S89-S96,
1997
32.
Yang, XX,
Powell WS,
Xu LJ,
and
Martin JG.
Strain dependence of the airway response to dry-gas hyperpnea challenge in the rat.
J Appl Physiol
86:
152-158,
1999