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Departments of 1Physiology, 2Medicine, and 3Comparative Medicine, East Carolina University, Greenville, North Carolina 27858; 4LABS of Virginiac, Yemassee, South Carolina 29945; and 5EpiGenesis Pharmaceuticals, Cranbury, New Jersey 08520
Submitted 17 October 2003 ; accepted in final form 5 December 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization. airway hyperresponsiveness; airway remodeling; cytokines; histamine; adenosine; compliance; resistance
Historically, nonhuman primate models of asthma and airway hyperresponsiveness have been based on two paradigms: identification of animals exhibiting natural allergic sensitivity and induction of sensitivity through systemic exposure. Over 25 years ago, Patterson and Harris (15) began studying rhesus monkeys that exhibited natural sensitivity to Ascaris suum antigens. In 1983, Richards and colleagues (17, 18) demonstrated the feasibility of inducing sensitivity by infecting non-sensitive animals with Ascaris suum ova. This model has been used effectively by the pharmaceutical industry to screen novel candidate asthma drugs, although the allergen is not normally associated with human asthma. In 1997, Yasue and colleagues (26) demonstrated that periodic subcutaneous injection of dust mite allergen in rhesus monkeys elevated serum levels of allergen-specific immunoglobulins and induced cutaneous and conjunctival allergic responses, but the response to aerosolized allergen was not reported. In 2001, Schelegle and colleagues (20) used aerosolized dust mite to induce progressive inflammation, decline in pulmonary function, and structural changes in the airway wall of rhesus monkeys. This model has revealed exciting new information on potential neural adaptation and changes in airway innervation in response to allergen and toxins (3). The model involves a single injection of allergen and adjuvant followed by repeated intranasal and aerosol exposures to allergen. Although this model closely resembles the human disease, it is labor intensive, and the dependence on repeated aerosol challenge limits its practicality for routine drug screening.
Previous work in rabbits has demonstrated that periodic injection of allergen and adjuvant from the time of birth and then challenging with aerosolized allergen will induce pulmonary inflammation, early- and late-phase responses, and airway hyperresponsiveness characteristic of asthma. This technique, which was originally developed using Alternaria tenius (21) and later adapted to house dust mite (11) and ragweed (4), has been useful for testing conventional and genomic-based drugs, such as EPI-2010, a respirable antisense against the adenosine A1 receptor (13). Limitations in the number of immunologic reagents compatible with this species and genetic homology with humans restrict the use of rabbits in biomedical research, but the approach used to induce asthmatic responses in this species could be applicable to other, more relevant species.
In the present study, cynomolgus monkeys were sensitized to house dust mite, either as adults or from the time of birth, and challenged with aerosolized dust mite. The allergen-induced immune response, pulmonary inflammation, airway dysfunction, and tissue remodeling were subsequently characterized.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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All animals used in this study were born and raised for at least 6 mo at LABS of Virginia, Yemassee, SC. On arrival at East Carolina University, the animals were group housed in the Department of Comparative Medicine. Animal husbandry was conducted under US Department of Agriculture guidelines. All protocols were approved by the Institutional Animal Care and Use Committee of East Carolina University.
Protocols
Three- to 4-yr-old (adult) animals. Seven animals, 3-4 yr old, were evaluated for sensitivity to aerosolized histamine and airway inflammation via differential cell counts of bronchoalveolar lavage (BAL) fluid. The animals were then sensitized by bimonthly subcutaneous injections of allergen adsorbed to alum [312 arbitrary units (AU) of Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) extract, Greer Laboratories, Lenoir, NC; Imject alum, Pierce, Rockford, IL] for 4 mo. As originally conceived, the protocol called for the animals to receive three aerosol dust mite challenges at 1-mo intervals to establish chronic inflammation before the asthmatic response was characterized. However, the animals exhibited significant distress during the first aerosol dust mite challenge, so the protocol was modified as follows. Six weeks after the first dust mite challenge, the subjects were boosted by subcutaneous injection with allergen extract in alum. Two weeks later, responses to aerosolized histamine were determined, the lungs were lavaged, and differential cell counts of BAL fluid were acquired. Twenty-four hours later, the animals were challenged with aerosolized dust mite, and the early asthmatic response was measured. Forty-eight hours after the dust mite challenge, the histamine challenge and BAL were repeated.
Neonatal animals. Fifteen animals were injected with allergen (312 AU of Dp and Df extract adsorbed to alum) within 24 h of birth, once per week for an additional 4 wk, and then twice per month until 6 mo of age. At 8 mo of age, the subjects were boosted by subcutaneous injection with allergen extract in alum. Two weeks later, responses to aerosolized histamine were determined and the lungs were lavaged for differential cell counts. Twenty-four hours later, the animals were challenged with aerosolized dust mite, and the early asthmatic response was measured. Forty-eight hours after the dust mite challenge, the histamine challenge and BAL were repeated. Eight animals were sham-sensitized by subcutaneous injection of saline on the same schedule used for sensitizing to allergen.
After the initial characterization, the animals were allowed to recuperate for 9 wk before being separated into cohorts for additional characterization of late-phase responses to dust mite and responses to steroid and 
-adrenergic agonist therapies. Responsiveness to steroid therapy was assessed by use of a sequential protocol in which all subjects were treated with vehicle in the first leg and dexamethasone in the second leg. This ensured that inflammation and pulmonary function had returned to baseline at the time the protocol began. The subjects were boosted by inhalation of allergen (200 AU/ml for 4 min). Two weeks later, sensitivity to aerosolized histamine was determined and BAL samples were collected. The animals were treated daily for 3 days with vehicle or dexamethasone (1 mg twice/day orally). On the fourth day, the animals received the morning treatment via intramuscular injection, were intubated, and then were challenged with 200 AU/ml (1:50 dilution) of dust mite delivered through a Devilbiss ultrasonic nebulizer for 4 min. Dynamic lung compliance (Cdyn) and airway resistance (Raw) were monitored for 6 h. Oral treatments were continued until the morning of the sixth day, when the treatment was given by intramuscular injection. Sensitivity to aerosolized histamine was reassessed and the lungs lavaged. A 3-wk washout period separated the vehicle and dexamethasone legs of the study.
Responsiveness to -adrenergic receptor agonist therapy was assessed by treating the animals with albuterol (2.5 mg/ml, 2 ml delivered by aerosol) 60 min before provocation with aerosolized methacholine or dust mite.
Pulmonary Function Testing
Subjects were anesthetized with 2.5 mg/kg telazol by intramuscular injection and maintained on propofol delivered by intravenous infusion at 10-15 or 20-25 mg·kg-1·h-1 for dust mite or histamine provocation, respectively. An endotracheal tube and esophageal balloon were inserted. Cdyn and Raw were measured by standard computer analyses of the flow and pressure signals, digitalized by a MuMed PR800 physiological recorder (MuMed, London, UK). Saline was delivered via a Devilbiss ultrasonic nebulizer for 2 min, and pulmonary function was monitored for 2 min to establish baseline parameters. Aerosolized histamine or adenosine was delivered for 2 min. The animals were allowed to rest for 1 min, and then the response to the challenge was monitored for 1 min. Cumulative doses were delivered until a 30% decrease in Cdyn was observed with adenosine or 50% decrease in Cdyn was observed with histamine.
Animals were challenged with aerosolized dust mite for 4 min, and pulmonary function was monitored. Increasing concentrations of allergen were delivered at 15-min intervals until a 50% change in Cdyn was observed. Initially, dilutions of 1:500 (20 AU/ml), 1:100 (100 AU/ml), and 1:4 (2,500 AU/ml) were delivered. In later experiments, the 1:4 dilution was replaced with 1:20 and 1:50 dilutions to protect against excessive responses.
After dust mite challenge, pulmonary function was monitored for up to 6 h. The data were expressed as a percent of the saline control period.
Bronchoalveolar Lavage and Differential Cell Counts
After the histamine challenge, a polyethylene catheter (PE 190) was advanced through the endotracheal tube into the lungs and gently wedged into an airway. The segment beyond the tip of the catheter was lavaged with 2.5 ml of sterile saline, which was collected by gentle aspiration. Total cell counts were determined by using a Coulter Counter (Beckman Coulter Instruments, Miami, FL). Cytospin preparations were prepared and stained with DiffQuik (VWR Scientific, South Plainfield, NJ). Differential cell counts were acquired by counting 200 cells per sample.
ELISA for Allergen-Specific IgE and IgG
Ninety-six-well plates were coated with dust mite allergen (10 µg/ml per well, overnight at 4°C in 0.01 M sodium bicarbonate buffer, pH 9.6). The plates were washed and blocked with buffer containing 10% newborn calf serum. After washing was completed, serum samples (1:5 to 1:100,000 dilution) were added to the wells (100 µl/well). The plates were incubated overnight at 4°C.
IgG was detected by incubating the plate with 1 µg/ml horseradish peroxidase-conjugated anti-monkey IgG for 1 h at room temperature [GAMon/IgG(H+L), Nordic Immunological Laboratories, Tilburg, The Netherlands]. IgE was detected by incubating the plates with 1 µg/ml biotinylated goat anti-human IgE for 1 h at room temperature (Vector Lab, Burlingame, CA). The plates were developed by use of streptavidin-conjugated horseradish peroxidase (BD PharMingen, San Diego, CA) and 3,3',5,5'-tetramethylbenzidine (BD PharMingen). The reaction was stopped by addition of sulfuric acid, and the absorbance at 450 nm was determined.
Cytokine Recall Assay
Lymphocytes were isolated from 1-3 ml of EDTA-treated blood diluted up to fourfold with saline. The diluted blood was fractionated on Ficoll-Isopaque (Histopaque 1.077, Sigma Chemical) by centrifugation at 800 g for 30 min. Mononuclear cells were collected from the top of the gradient. When necessary, contaminating red blood cells were lysed by hypotonic shock. The cells were washed three times and resuspended in complete media [RPMI 1640 plus 10% FBS plus antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml amphotericin B)] to a density of 1 x 106 cells/ml. Cell suspensions were incubated with either dust mite allergen (10 µg/ml Dp, Greer Laboratories) or a combination of 1 µM calcium ionophore (A23187
[GenBank]
) and 1 µM phorbol myristic acid (PMA) for 1 day at 37°C, 95% CO2, and 99% humidity. Supernatants were collected, and concentrations of IL-4, IL-5, IL-10, and IFN- were determined by ELISA.
For IL-4 detection, purified mouse anti-human IL-4 monoclonal antibody (BD PharMingen) was used for capturing IL-4, and biotinylated rat anti-human IL-4 (BD PharMingen) was used for detection. Recombinant human IL-4 (BD PharMingen) was used as a standard.
For IL-5 detection, purified rat anti-human IL-5 monoclonal antibody (BD PharMingen) was used for capture, and biotinylated rat anti-human IL-5 (BD PharMingen) was used for detection. Recombinant human IL-5 (BD PharMingen) was used as a standard.
IL-10 was detected by use of purified rat anti-human IL-10 monoclonal antibody (BD PharMingen) for capture and biotinylated rat anti-human IL-10 (BD MPharmingen) for detection. Recombinant human IL-10 (BD PharMingen) was used as a standard.
IFN- was measured by using murine anti-human IFN- monoclonal antibody (MD-1 Biosource) for capture, anti-human IFN- monoclonal antibody (DiaPharma Group, West Chester, OH) for detection, and recombinant human IFN- as a standard (BDPharMingen).
Pathology
Tissues were fixed in 10% neutral buffered formalin, paraffin embedded, cut into 10-µm sections, and stained with hematoxylin and eosin, alcian blue and periodic acid-Schiff, and Masson's trichrome to evaluate inflammation, smooth muscle hyperplasia, submucosal fibrosis, and mucous cell hyperplasia, respectively. Tissue sections from the trachea, lungs, and nasal passages were evaluated by a pathologist who was blinded to the subject identification.
Statistics
Means ± SE are reported. Statistical significance was determined by using Student's t-test for paired observations and ANOVA with Fisher's least significant difference post hoc test for comparison of multiple groups (P 
0.05).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Serum dust mite-specific IgE and IgG levels were determined in four cohorts to assess effectiveness of the sensitization protocols: animals sensitized to allergen starting at 3-4 yr of age, 6- to 8-mo-old animals that had been sham-sensitized or sensitized to allergen from birth, and 82 animals of various ages from the colony where the study subjects originated. For the sham- and dust mite-sensitized animals, blood samples were collected within 2 wk of subcutaneous injection with saline or allergen in adjuvant. Sham- and nonsensitized animals exhibited significantly lower levels of dust mite-specific IgE and IgG than allergen-sensitized animals (Fig. 1, P < 0.03). Animals sensitized as neonates exhibited higher levels of dust mite-specific IgG in serum than animals sensitized as adults (P < 0.001), but the level of dust mite-specific IgE in serum was not significantly different between these two group.
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Interestingly, the nonsensitized animals, which had been raised in outdoor corrals, exhibited higher levels of dust mitespecific IgG than either the sham-sensitized animals or the adult animals before sensitization. In contrast to the former group, both of the latter groups were housed indoors under conditions in which the animals would be expected to have minimal contact with dust mite allergen.
Pulmonary Function Changes in Response to Initial Aerosol Allergen Exposure
Pulmonary sensitivity to allergen was assessed in sham-sensitized and dust mite-sensitized animals. Animals were exposed to decreasing dilutions of aerosolized allergen until a 50% decrease in compliance was observed. All sham-sensitized animals were exposed to the lowest dilution (i.e., 1:4 dilution) and exhibited minimal responses in Cdyn and Raw (Fig. 2, A and D). Two adult animals exhibited a 50% decrease in Cdyn after exposure to the 1:100 dilution, and four remaining animals exhibited decreases in Cdyn after exposure to the 1:4 dilution that did not reach the 50% level of inhibition (Fig. 2C). One animal did not complete the protocol because of technical complications. Only a small increase in resistance was observed in the adult animals (Fig. 2F).
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In contrast to the sham-sensitized and adult animals, animals sensitized from birth exhibited larger, more consistent changes in both Cdyn and Raw (Fig. 2, B and E). Of the 12 animals entering the protocol, six animals exhibited a >50% drop in Cdyn after exposure to the 1:100 dilution of allergen, and the remaining animals exhibited a >50% drop in Cdyn after exposure to the 1:4 dilution of allergen. Comparison of the area under the curves indicated that the dust mite-induced changes in Cdyn and Raw were significantly greater in the neonatally sensitized group compared with the sham- and adult-sensitized groups (P < 0.007).
Responses to Aerosolized Histamine and Adenosine
Responses to aerosolized histamine provocation were measured in sham-sensitized animals, animals sensitized to dust mite as adults, and animals sensitized from birth (Fig. 3). Sham-sensitized animals exhibited a slight decrease in the provocative dose of histamine that caused a 50% decrease in Cdyn (PC50) after exposure to aerosolized dust mite, but the change was not statistically significant. In contrast, animals sensitized to allergen as adults exhibited a statistically significant decrease in the PC50 to histamine after allergen challenge (P = 0.003). Neonatally sensitized animals exhibited the lowest PC50 to histamine before allergen challenge. Challenging with aerosolized dust mite yielded a PC50 that was statistically different from the sham-sensitized animals challenged with dust mite (P = 0.034) but was not statistically different from the prechallenge PC50 recorded for the neonatally sensitized group. These observations indicate that neonatally sensitized cynomolgus monkeys are hyperresponsive to aerosolized histamine, and at least a portion of the responsiveness can be attributed systemic exposure of allergen during the sensitization and priming procedures.
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Neonatally sensitized animals exhibited greater sensitivity to adenosine than animals sensitized as adults. Only one of seven adult animals responded to adenosine aerosolized at concentrations 10 mg/ml. The one adenosine-sensitive animal exhibited a PC30 to adenosine of 0.02 mg/ml. In contrast, 7 of 12 neonatally sensitized animals exhibited sensitivity to aerosolized adenosine (mean PC30 to adenosine = 2.8 ± 1.0 mg/ml, range of PC30 values = 0.01-10 mg/ml, n = 7).
Allergen-Induced Airway Inflammation
Sham-sensitized animals exhibited minimal inflammation either before or after aerosol allergen challenge (Table 1). Initial exposure of neonatally sensitized animals to aerosolized dust mite increased the percentage of eosinophils in the BAL fluid from 1 to 6% of the total cells recovered. Rechallenging with allergen 2 wk after priming with aerosolized allergen (5) further increased the eosinophils in the BAL fluid to 20% of the total recovered cells. Adult animals sensitized to dust mite also exhibited an increase in eosinophils in the BAL fluid to 
5%, but rechallenging with aerosolized had minimal effect. These observations indicate that systemic sensitization of cynomolgus monkeys to dust mite induces susceptibility to allergic airway inflammation, and neonatal sensitization is more effective than sensitization of adult animals. However, even in neonatally sensitized animals, repeated allergen challenge may be required to establish peak inflammation.
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To verify that 48 h corresponded with the time of peak eosinophilic inflammation, four neonatally sensitized animals were pretreated with the histamine H1 receptor antagonist diphenhydramine (50 mg/ml, 12.5 mg/animal im) and challenged with allergen. BAL fluid was subsequently collected from one animal at each of the following time points: 6, 24, 48, and 96 h. At the 6-h time point, no eosinophils were observed in the BAL fluid. At 24, 48, and 96 h, the eosinophils accounted for 6, 22.5, and 7.5% of the total cells, respectively. The findings indicated that the 48 h after challenge was an appropriate time point for BAL collection. Furthermore, the observation that the eosinophil counts at the latter three time points were higher than the average of 6.3% recorded in the absence of diphenhydramine indicated that a large early-phase histamine response may oppose the subsequent development of eosinophilic inflammation. In all subsequent experiments, animals were pretreated with diphenhydramine (50 mg/ml, 12.5 mg/animal im) before dust mite challenge to compensate for this possibility.
Early- and Late-Phase Responses With Repeat Allergen Challenges
To better simulate asthma in humans, adult and neonatally sensitized animals were primed by inhalation of allergen in lieu of injection of allergen and adjuvant. Three weeks later, the challenge was repeated, and the early- and late-phase responses were measured. The repeat allergen challenge induced a greater decrease in Cdyn and increase in Raw in neonatally sensitized animals than in the adults (Fig. 4). Adult-sensitized animals exhibited minimal change in resistance during the early- and late-phase responses. Both groups exhibited late declines in compliance starting 5 h after allergen challenge. However, only the neonatally sensitized animals exhibited increased resistance late in the protocol.
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Pathology
Tissues from three animals were evaluated for histological correlates of asthma. One animal (animal 1) had been sensitized as an adult, challenged with aerosolized allergen nine times over the course of 2 yr, and euthanized 2 days after the last allergen challenge. A second adult animal (animal 2) was a naive age-matched control. The third animal (animal 3) was a 3.5-yr-old female that had been sensitized as a neonate and challenged with aerosolized dust mite eight times over 2 yr, with the last exposure occurring 7 days before tissue collection.
Sections from the naive control animal (animal 2) exhibited mild, submucosal tracheal edema and minimal pleocellular tracheitis that was presumed to be a nonspecific effect of inflammation resulting from intubation (Fig. 5). No significant lesions were observed in the airways or nasal passages, although a mild increase in cellularity (primarily mononuclear cells) was observed in alveolar septa.
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Animal 1, which was sensitized and challenged as an adult, exhibited moderate, mucous cell hyperplasia in the tracheal and bronchial epithelium (Fig. 5D). In the trachea, moderate, diffuse, submucosal edema was noted, with mild lymphoplasmacytic and eosinophilic tracheitis along with thickening of the basement membrane (Fig. 5B). Prominent submucosal blood vessels containing increased numbers of marginated and circulating eosinophils were also evident. Pulmonary changes consisted of bronchial mucous cell hyperplasia, bronchial and bronchiolar submucosal edema, and mild to moderate lymphoplasmacytic and eosinophilic bronchitis. Minimal peribronchiolar and perivascular lymphoid follicular hyperplasia was observed (Fig. 5F). Within the nasal passages, submucosal vessels contained increased numbers of marginated and circulating eosinophils. Nasal passages exhibited rhinitis (lymphoplasmacytic, eosinophilic, and neutrophilic) and submucosal vascular ectasia (Fig. 5H).
Animal 3 exhibited bronchial mucous cell hyperplasia (Fig. 6A) and eosinophilic bronchitis (Fig. 6B). Bronchioles and terminal bronchioles exhibited hyperplasia of smooth muscle (Fig. 6, C and D).
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These results demonstrate that systemic sensitization of either adult or neonatal cynomolgus monkeys followed by exposure to aerosolized dust mite induces immunologic, inflammatory, physiological, and morphological features consistent with early phases of asthma. However, animals sensitized at an early age exhibit more robust functional and morphological responses to aerosolized allergen.
Early and Late Responses to Aerosolized Dust Mite Allergen After 90 Days
To evaluate the longevity of airway sensitization to allergen, four neonatally sensitized animals were allowed to rest for 90 days after the initial provocation with aerosolized dust mite and were then subjected to a second aerosol challenge without another allergen boost. Animals were pretreated with diphenhydramine before allergen challenge as a protective measure. Even in the presence of diphenhydramine, one animal exhibited an excessive early-phase response and had to be resuscitated. The other three animals exhibited early- and late-phase responses. A representative tracing of the response is presented in Fig. 7. These results demonstrate that allergic sensitization and aerosol allergen challenge induce a prolonged sensitivity of the airways to aerosolized allergen, which negates the need for monthly systemic boosts with allergen.
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Th2 Cytokine Production
Evidence that systemic sensitization induced a Th2 cytokine profile was obtained by isolating mononuclear cells from peripheral blood of sensitized animals and stimulating in vitro with dust mite allergen or calcium ionophore with PMA. Addition of dust mite to the medium induced IL-5 release (Fig. 8A), but no IL-4 or IFN- release was detected. Stimulation with A-23187 and PMA induced release of both IL-4 and IL-5, but not the Th1 cytokine, IFN- (Fig. 8B). Interestingly, the cells exhibited a relatively high unstimulated rate of IL-10 release, but the rate of IL-10 release was not increased by exposure to dust mite and A-23187 + PMA. These results confirmed that subcutaneous injection of dust mite and alum induced a Th2 phenotype in the cynomolgus monkey.
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Response to Albuterol and Dexamethasone
The ability of inhaled -adrenergic agonists to dilate the airways and inhibit allergen-induced airway constriction was assessed. Responsiveness of the cynomolgus monkey to inhaled -agonist was confirmed by treating animals with aerosolized albuterol and determining the dose response to inhaled methacholine 60 min later. A log rightward shift in the PC40 to methacholine was observed after treatment with albuterol, demonstrating that the animals were responsive to the -agonist (Fig. 9). The effect of albuterol on the earlyphase dust mite response was then measured in four neonatally sensitized monkeys. Albuterol reduced the maximum change in Cdyn during the early phase by 63%. Raw was also improved, but variability in the control group and small sample size kept the difference from being statistically significant (Table 2).
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Sensitivity to corticosteroid treatment was assessed by using dexamethasone. The protocol was a linear design in which all animals received vehicle first. After a washout period of 3 wk, the animals were treated with dexamethasone (1 mg bid). Dexamethasone was delivered orally, except in the morning on days 3 and 6, when the animals underwent pulmonary function testing and the dexamethasone was delivered intramuscularly. Allergen challenge was performed on day 3 of treatment, and BAL fluid was collected 48 h after the allergen challenge. The treatment inhibited allergen-induced eosinophilia (Table 3) and attenuated the changes in Cdyn and Raw induced by inhalation of allergen (Fig. 10). In addition, treatment with dexamethasone appeared to inhibit lymphocytic inflammation, although statistical significance was not demonstrated by ANOVA (Table 3). The repetitive allergen challenge induced a progressive rise in the percentage of lymphocytes in BAL, as indicated by the increase in percentage of lymphocytes between the prevehicle, postvehicle, and predexamethasone time points. This trend was reversed by dexamethasone treatment as indicated by a decrease in the percentage of lymphocytes between the predexamethasone and postdexamethasone time points.
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No change in airway hyperresponsiveness to histamine was observed with steroid treatment. The PC50 values before and after vehicle and dexamethasone were 0.014 ± 0.008, 0.010 ± 0.003, 0.017 ± 0.007, and 0.016 ± 0.004 mg/ml, respectively.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The choice of allergen, the species used, and the overall approach to establishing the model reflected the stated goal of the research. Dust mite is commonly associated with human asthma (10) and has been shown to be effective in inducing symptoms of asthma in common laboratory animals, including mice (14, 19), rats (8), rhesus monkeys (20, 26), and rabbits (11). In addition, natural sensitivity to dust mite had been documented in monkeys (7), increasing the relevance of the allergen. Research conducted by Turner and colleagues at Pfizer (23-25), Gundel et al. at Boehringer Ingelheim (6), Mauser et al. at Schering-Plough (9), Bell et al. at Abbott Laboratories (2), and Baugh et al. at Merrell Dow (1) provided a strong precedent for using cynomolgus monkeys for testing novel pulmonary therapeutics, and the cynomolgus monkeys exhibit 95% homology with humans (27), making this a candidate species suitable for genomic studies. The protocols for sensitizing the adult and neonatal animals were based on the work of Yasue et al. (26) and Metzger (11), who demonstrated the feasibility in rhesus monkeys and rabbits, respectively. The ability to prime an animal systemically and then precipitate pulmonary symptoms at a defined point in time should facilitate chronological studies of gene-expression changes associated with asthma pathogenesis and be suitable for high-throughput screening of targets emerging from genomic studies.
Nonspecific hyperresponsiveness is a key feature of human asthma (10). In this model, both adult and neonatally sensitized animals exhibited hyperresponsiveness to histamine and aeroallergen. Early- and late-phase responses to inhaled allergen were observed in both adult and neonatally sensitized animals, but allergen-induced increase in Raw was more prominent in neonatally sensitized animals (Figs. 2 and 4). Enhanced Raw is often attributed to constriction of the conducting airways, which would indicate greater involvement of the bronchi in animals sensitized from birth. However, this interpretation may not be valid because significant closure of the small airways or changes in tissue mechanics can also be reflected in increase total Raw (22). At present, there is no technique for assessing pulmonary function that definitively differentiates the involvement of proximal and distal regions in the asthmatic response. Forced oscillation is becoming more common (22), but even this technique relies on homogeneity within the lungs, which is not present in asthma. Thus the relative involvement of proximal vs. distal lung regions in the asthmatic response remains unclear. Regardless, the data indicate that the asthmatic response is accentuated in neonatally sensitized animals.
A key distinguishing clinical feature of asthma is a high degree of reversibility of airway obstruction by bronchodilators and anti-inflammatory agents (10), and this feature was demonstrated in the present model by treating neonatally sensitized animals with albuterol and dexamethasone. Treatment with either of these agents attenuated the pulmonary function changes after inhalation of allergen (Figs. 9 and 10). Ideally, an inhaled corticosteroid would have been used for this purpose. However, animal welfare concerns precluded that experiment because the animals would have been anesthetized twice per day for multiple days for proper dosing of the inhaled steroids.
One benefit of using nonhuman primates as a model of asthma is that the animals are long-lived and thus chronic aspects of the disease can be studied. In the long term, elimination of systemic boosts with allergen would be more representative of the disease in humans. Therefore, boosting with aerosolized allergen was investigated as an alternative to the systemic allergen boost used to sensitize the animals. Both adult and neonatally sensitized animals exhibited some response to the allergen challenge under these conditions, but the younger animals were clearly more responsive, exhibiting nearly a 50% reduction in Cdyn and 150% increase in resistance in response to a 1:50 dilution of dust mite allergen (Fig. 4). The cohorts were separated in age by 3 yr, and thus the differences could have been due to the age difference at the time of challenge. Work with rabbits indicates that age at the time of sensitization determines the degree of severity and the chronicity of the asthmatic response. Sensitization of adult rabbits leads to a more diverse immunoglobulin profile (16, 21), milder allergen-induced pulmonary function changes (11), and lower degree of hyperresponsiveness to histamine (12). The heightened asthmatic responses observed in the neonatally sensitized monkeys throughout the present study are consistent with the differences observed in rabbits sensitized at different ages.
An adult cynomolgus monkey subjected to the systemic sensitization protocol and challenged three to four times per year for 2 yr exhibited histological alterations consistent with a chronic antigenic stimulus and suggestive of early asthma associated with mild chronic antigenic stimulation. A lack of appreciable submucosal mucous glands, mucous plugs in the airways, and smooth muscle hyperplasia precluded diagnosis of fully developed asthma in this animal. In contrast, a neonatally sensitized animal challenged an equivalent number of times over a 2-yr period exhibited significant increases in the amount of bronchial and bronchiolar smooth muscle (Fig. 6). This finding was consistent with the elevated resistance responses to allergen observed in this cohort (Figs. 2 and 4). These findings indicate that the neonatally sensitized animals more closely model the human asthmatic condition than adult-sensitized animals.
There is a possibility that the adult-sensitized animal in the pathology evaluation was exposed to allergen early in life, which initiated remodeling. However, this subject was raised in HEPA-filtered air from 1 yr of age. Even if exposed to an environmental allergen during the first year of life, it is unlikely that the animal would have experienced exposure after that time sufficient to drive the remodeling. This argument is supported by pathology of one animal that was sensitized as an adult but died prematurely after receiving only one aerosol allergen challenge. That animal did not exhibit basement membrane thickening in the airways at the time of death (data not shown).
This report constitutes the first phase of a longitudinal study of cynomolgus monkeys sensitized to dust mite allergen. The results indicate that sensitization of either adult or neonatal animals establishes an allergic background on which inhaled allergen induces pulmonary responses. Animals sensitized from birth exhibit inflammatory, functional, and morphological features similar to those observed in allergic asthma, including airway eosinophilia, increases in lung compliance and airway resistance, and smooth muscle goblet cell hyperplasia. The model offers control over the sensitization process and initiation of the lung disease and therefore should be useful in genomic studies and drug screening activities.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and 10 neonatally sensitized (
) animals.
), and 60 min later the response to methacholine was reassessed. Values are means ± SE; n = 4. Only doses delivered to all animals are included in the plots.