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Department of Pathology and Molecular Medicine, Division of Respiratory Diseases and Allergy and Center for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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That allergen exposure to perennial allergens, such as house dust mite or cat dander, is rather continuous overall seems to be at variance with the "intermittent" nature of asthma. Indeed, if exacerbation of inflammation were attributed solely to allergen exposure, one would predict unabated symptoms among individuals presenting with allergic asthma. While cellular and molecular mechanisms underlying allergic sensitization and acute inflammation are subjects of intense research, comparatively little is known with regard to the impact of persistent Ag exposure on immune inflammatory processes in the airway. This understanding is further limited by the fact that research examining the effect of chronic allergen exposure on airway inflammation has produced controversial results. Studies showing that chronic allergen exposure does not lead to persistence of airway inflammation (8, 9) suggest that allergen alone is insufficient in perpetuating the inflammatory response. Others have documented persistent airway eosinophilia (10, 11); however, the focus of these studies was on airway remodeling and lung physiology rather than on immune inflammatory processes.
The objective of this study was to investigate the impact of chronic OVA exposure on immune inflammatory processes in the lung. We have recently established a model of mucosal allergic sensitization, which, like the conventional models, elicits some of the important features of asthma (12, 13, 14). In this model, mice are exposed to OVA in the context of a GM-CSF-enriched airway microenvironment for 10 consecutive days. While we argue that this is a better reflection of the route in which sensitization occurs in humans, Ag exposure is transient. Hence, to investigate immune inflammatory processes in the airway associated with chronic Ag exposure, we sensitized mice mucosally and exposed them to OVA for up to 4 wk. Expression of GM-CSF is required to allow for allergic mucosal sensitization, because exposure to OVA alone induces inhalation tolerance, as we and others have previously shown (15, 16). Importantly, GM-CSF on its own does not elicit airway eosinophilia (12); hence, its effects are likely due to its adjuvant-like properties (17, 18, 19, 20, 21, 22, 23). After 2 wk of exposure, we observed marked eosinophilia in the bronchoalveolar lavage (BAL),3 lung tissue and peripheral blood but, despite the continuous presence of the Ag, eosinophilia was substantially diminished by 3 wk and resolved after 4 wk. In contrast, similar OVA-specific IgE and increased IgG1 and IgG2a levels were observed after 4 wk, compared with 2 wk, of exposure. Next, we exposed mice to OVA for either 2 or 4 wk, rested them for 28 days, and subsequently re-exposed them to OVA only. We observed a significant decrease in airway eosinophilia as well as BAL and serum IL-5 in animals initially exposed to 4 wk, compared with 2 wk, of OVA. Nonetheless, the diminished airway eosinophilia was associated with a robust humoral response as well as activated dendritic cells (DC) and T cells. Delivery of rGM-CSF in the context of OVA recall challenge reestablished airway eosinophilia. The data demonstrate that chronic exposure to OVA does not lead to sustained airway inflammation but to a state of unresponsiveness that is overcome by GM-CSF. Therefore, we suggest that additional factors, other than Ag, are required to elicit persistent airway inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (6–8 wk old) were purchased from Harlan Breeders (Indianapolis, IN). Mice were maintained in a 12-h light-dark cycle with unlimited access to food and water. Cages, food, and bedding were autoclaved and all animal manipulations were conducted in a laminar flow hood by gloved, gowned, and masked personnel. All experiments were approved by the Animal Research Ethics Board of McMaster University.
Allergic mucosal sensitization
Mice were exposed to OVA in the context of a GM-CSF-enriched airway microenvironment. Prolonged expression of GM-CSF was achieved using an adenovirus-mediated gene transfer approach, as previously described (24). Briefly, a replication-deficient human type 5 adenoviral construct carrying the transgene for GM-CSF in the E1 region of the viral genome (Ad/GM-CSF) was delivered intranasally. A dose of 3 x 107 PFU Ad/GM-CSF construct was delivered in a total volume of 30 µl of PBS vehicle (two 15-µl administrations 5 min apart) into anesthetized animals. Subsequently, mice were placed in a Plexiglas chamber (10 x 15 x 25 cm) and exposed to aerosolized OVA (1% w/v in 0.9% saline; Sigma-Aldrich, St. Louis, MO) for 20 min daily. Mice were exposed 5 days per week from Monday to Friday. The aerosol is produced by a Bennett/Twin nebulizer (Puritan-Bennett, Carlsbad, CA) at a flow rate of 10 L/min.
Delivery of rGM-CSF
In a limited number of experiments, we delivered 1 µg of rGM-CSF (BioSource International, Camarillo, CA) in 10 µl PBS intranasally over five consecutive days to achieve sustained levels of GM-CSF in the airways.
Collection and measurement from specimens
BAL was performed as previously described (25). In brief, the lungs were dissected and the trachea was cannulated with a polyethylene tube (BD Biosciences, Sparks, MD). The lungs were lavaged twice with PBS (0.25 ml followed by 0.2 ml). Approximately 0.25 ml of the instilled fluid was consistently recovered. Total cell counts were determined using a hemocytometer. After centrifugation cell pellets were resuspended in PBS and slides were prepared by cytocentrifugation (Thermo Shandon, Pittsburgh, PA) at 300 rpm for 2 min. HEMA 3 Stain set (Biochemical Sciences, Swedesboro, NJ) was used to stain all smears. Differential counts of BAL cells were determined from at least 500 leukocytes using standard hemocytological procedures to classify the cells as mononuclear cells, neutrophils, or eosinophils. Peripheral blood was obtained using heparin-coated capillaries (Fisher Scientific, Pittsburgh, PA). Total white blood cell counts were determined after lysing RBCs and cell differentials were assessed on smears stained with the HEMA 3 Stain set. For serum, animals were bled with nonheparinized capillary tubes. Serum was prepared by incubating whole blood for 30 min at 37°C. Finally, lung tissue was fixed in 10% formalin and embedded in paraffin. Three-micrometer-thick sections were stained with H&E.
Cytokine and Ig measurements
IL-5 was detected using a commercially available ELISA kit (Amersham, Little Chalfont, U.K.). The threshold of detection was 5 pg/ml. Levels of OVA-specific IgE were detected with an ELISA that has been described in detail previously (25). For OVA-specific IgG1 and IgG2a, Maxi-Sorb plates (Nunc, Roskilde, Denmark) were coated with 5 µg OVA in borate buffer (pH 8.3–8.5) overnight at 4°C. Subsequently, coated wells were blocked with 1% BSA in PBS for 2 h at room temperature. After washing, serum samples were incubated overnight at 4°C, washed, and developed with biotin-labeled, anti-mouse IgG1 and IgG2a (Southern Biotechnology Associates, Birmingham, AL) overnight at 4°C. Plates were washed and incubated with alkaline-phosphatase streptavidin for 1 h at room temperature. The color reaction was developed with p-nitrophenyl phosphate tablets. Samples were compared with a standard serum containing OVA-specific IgG1 and IgG2a. Units correspond to maximal dilution that results in an OD that is greater than the blank + 2 SD.
Lymph node and lung cell isolation
Hilar, mediastinal, and tracheobronchial lymph nodes were dissected, ground between the frosted ends of slides, and filtered through a nylon mesh (BSH Thompson, Scarborough, Ontario, Canada). The cell suspension was centrifuged at 1200 rpm for 10 min at 4°C and resuspended in PBS. Cells were resuspended in flow cytometric analysis buffer (PBS supplemented with 0.2% BSA).
For isolation of lung cells, lungs were flushed via the right ventricle
of the heart with 10 ml of warm (37°C) HBSS (calcium and magnesium
free) containing 5% FBS (Sigma-Aldrich), 100 U/ml penicillin, and 100
mg/ml streptomycin (Invitrogen Life Technologies, Burlington, Ontario,
Canada). The lungs were then cut into small (
2 mm in diameter)
pieces and shaken at 37°C for 1 h in 15 ml of 150 U/ml
collagenase III (Worthington Biochemical, Freehold, NJ) in HBSS. Using
a plunger from a 5-ml syringe, the lung pieces were triturated through
a metal screen into HBSS, and the resulting cell suspension was
filtered through nylon mesh. Cells were washed twice and mononuclear
cells were isolated by density centrifugation over a 30 and 60%
Percoll gradient (Pharmacia Biotech, Uppsala, Sweden).
Flow cytometry
Panels of mAbs were selected to study the phenotype of cells in the lymph nodes and lung. To minimize nonspecific binding, 106 cells were incubated with 0.5 µg Fc Block (CD16/CD32; BD PharMingen, Mississauga, Canada) at 0–4°C for 10 min and subsequently with first-stage mAbs at 0–4°C for 30 min. Cells were then washed and treated with second-stage reagents. Data were collected using a FACScan and analyzed using WIN-MDI software (BD Biosciences, Sunnyvale, CA). The following Abs and reagents were used: anti-CD3, biotin-conjugated 145-2C11 (BD PharMingen); anti-CD4, FITC-conjugated L3T4 (BD PharMingen); anti-CD69, PE-conjugated H1 2F3 (BD PharMingen); anti-CD25, PE-conjugated PC61 (BD PharMingen); anti-T1/ST2, FITC-conjugated 3E10 (kindly provided by A. J. Coyle, Millennium Pharmaceuticals, Cambridge, MA) labeled in-house according to a standard protocol (24); anti-MHC class II (MHCII), FITC-conjugated 25-9-17 (BD PharMingen); anti-CD11c, PE-conjugated HL3 (BD PharMingen); anti B7.1, biotin-conjugated 16-10AI (BD PharMingen); anti-B7.2, biotin-conjugated GLI (BD PharMingen); and streptavidin PerCP (BD Biosciences, San Jose, CA). Titration was performed to determine the optimal concentration of each Ab.
Data analysis
Data are expressed as mean ± SEM. Statistical interpretation of results is indicated in the figures. Differences were considered statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were infected intranasally with 3 x
107 PFU Ad/GM-CSF. We previously reported that
GM-CSF is expressed in the airways for 10 days, with peak expression
of 80–100 pg/ml in the BAL at day 7. GM-CSF was undetectable in the
serum (26). Three days later, mice were exposed to
aerosolized OVA daily for 1, 2, 3, or 4 wk. Fig. 1
A shows that we observed only
few eosinophils in the BAL after the first week of OVA exposure. After
2 wk of OVA exposure, mice developed significant eosinophilia in the
BAL. Despite continued exposure to OVA, airway eosinophilia was
decreased by 95% after 3 wk and was completely resolved after 4
wk.
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B). These levels were
significantly decreased and were similar to naive levels after 4 wk of
exposure. Impact of 2 and 4 wk of OVA exposure on Ig production
Next, we assessed the impact of continuous OVA exposure on
Ag-specific Ig production (Table I). Mice
were subjected to OVA exposure in the context of GM-CSF
according to the protocol outlined above. Serum Ig levels of
OVA-specific IgE increased significantly during the first 2 wk but
remained similar after 4 wk. In contrast, we observed significantly
increased levels of OVA-specific IgG1 and IgG2a after 2 wk, and these
were further elevated after 4 wk of exposure.
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Given that continuous exposure to OVA did not result in
persistence of eosinophilia and airway inflammation, we investigated
whether continuous exposure to Ag impacts the ability of the lung to
respond to subsequent long-term Ag exposure. To this end, we first
exposed mice to either 2 or 4 wk of OVA in the context of GM-CSF.
Animals exposed to OVA for only 2 wk were subsequently subjected to
saline aerosol to control for the handling. All groups were then rested
for 28 days and exposed to OVA on three consecutive days.
Forty-eight hours after the last exposure, mice were sacrificed and the
inflammatory infiltrate in the BAL was assessed. We observed a robust
mononuclear and eosinophilic response in animals initially exposed to 2
wk of OVA (Fig. 2A). This
response was significantly greater than that observed in naive mice and
similar in magnitude to the inflammation observed after 2 wk of OVA
exposure, as depicted in Fig. 1. In contrast, animals initially exposed
to OVA for 4 wk exhibited a significant decrease in total cell number
and negligible eosinophilia following OVA recall. Indeed, the cellular
profile in these animals was statistically not different from
naive mice.
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B, i
and ii), animals initially exposed to 4 wk of OVA had
dramatically reduced tissue inflammation, with no eosinophilia (Fig. 2B, iii and iv).
Next, we assessed IL-5 expression in the BAL and serum after recall
challenge. Twenty-four hours after the first OVA exposure, we observed
significantly reduced levels of IL-5 in both the BAL and serum in
animals initially exposed to 4 wk of OVA when compared with animals
initially exposed to 2 wk only (Fig. 3).
A similar trend was observed 24 h after the second OVA exposure,
but the levels of IL-5 expression were lower (data not shown). No IL-5
was detected in naive animals (data not shown).
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). Levels of all Igs were markedly
higher than those observed in naive mice (Table I).
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Given that our histological assessment showed residual
mononuclear inflammation in mice initially exposed to 4 wk of OVA, we
quantified tissue mononuclear cells in the lungs and draining lymph
nodes following in vivo recall to OVA. As Table III shows, lungs from animals
initially exposed to 2 wk of OVA had approximately twice the
number of mononuclear cells compared with animals initially exposed to
4 wk of OVA (11.8 x 106 vs 4.94
x 106 cells per mouse in experiment 1 and
16.2 x 106 vs 7.8 x
106 cells per mouse in experiment 2). In naive
mice, we previously documented only 2.2 x
106 lung cells per mouse (27).
Similarly, in the draining lymph nodes we observed substantially fewer
cells at 4 wk compared with 2 wk (6.89 x
106 vs 3.64 x 106
cells per mouse in experiment 1 and 10 x
106 vs 4.5 x 106
cells per mouse in experiment 2). Naive mice had only 1.2 x
106 lymph node mononuclear cells per mouse
(27).
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shows increased
expression of B7.2 on lung DCs in both groups. We observed 19.5 and
28.7% of B7.2 in animals initially exposed to 2 and 4 wk,
respectively, while historical data shows that only 5.4% of lung DCs
express this molecule in naive animals (27). Similarly, we
observed increased expression of B7.2 in the lymph nodes. Compared with
naive mice (3.4%) (27), 27.5 and 33% of lymph node DCs
expressed B7.2 in animals exposed to 2 and 4 wk of OVA,
respectively. No differences in the expression of B7.1 were observed
between the groups in either the lung or the lymph nodes (Fig. 4 and
Ref. 27). Fig. 5 shows that
the levels of CD69 and CD25 in animals exposed to OVA for 2 or 4 wk
were similar, and were substantially higher than the levels we observed
and previously documented in naive mice (15, 27).
Specifically, in the lungs 20.2 and 27.8% of
CD4+ T cells expressed CD69 in animals exposed to
2 and 4 wk of OVA, respectively. Likewise, CD25 was expressed at 17.8
and 20% in 2- and 4-wk exposed animals, respectively, with <2%
expression in naive animals. A similar pattern of expression was
observed in the lymph nodes (data not shown). Finally, T1/ST2 was
expressed on 12% of lung CD4+ T cells in animals
exposed to OVA for 2 wk, and on 12.7% of lung
CD4+ T cells in mice exposed for 4 wk,
while typically 3.6% of CD4+ T cells of naive
animals express this molecule (27).
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Next, we investigated whether the expression of GM-CSF at the time
of long-term recall could reconstitute airway eosinophilia. Mice were
exposed to OVA for 4 wk and then rested for 28 days. Subsequently, we
delivered rGM-CSF intranasally on five consecutive days and exposed the
animals concurrently to OVA daily for nine consecutive days, with the
first challenge coinciding with the day of the first rGM-CSF delivery.
We observed that animals exposed to OVA in the context of rGM-CSF, but
not PBS, had significantly higher levels of eosinophilia in the airway
(Fig. 6). The total cell number and level
of airway inflammation was similar to that observed in animals
initially exposed to OVA for 2 wk, as depicted in Fig. 2A.
In contrast, inflammation could not be recapitulated in control animals
receiving OVA and PBS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, we investigated the cellular changes in the airway during
chronic exposure to OVA. We found that 2 wk of exposure resulted in
peak inflammation, both eosinophilic and mononuclear (Fig. 1). That we
observed eosinophilic airway inflammation at this time point
corroborates our previous findings (12). Note that GM-CSF
is expressed in the airway for 10 days (12) and
precedes peak inflammation by 1 wk. After 3 and 4 wk of OVA exposure
airway eosinophilia was dramatically reduced. Similarly, peripheral
blood eosinophilia peaked after 2 wk and was resolved after 4 wk. That
both airway eosinophilia and peripheral blood eosinophilia were
decreased after 4 wk of exposure argues against an impairment in
eosinophil recruitment.
While we observed decreased cellular responses in the lung during
prolonged exposure, the increased levels of Igs in the serum (Table I),
particularly IgG1 and IgG2a, indicate that immune responsiveness was
not fully silenced. This finding suggests that processes involved in
isotype switching and Ig production were not affected. That we did not
observe preferential up-regulation of the Th1-associated IgG2a, over
IgG1, which is Th2 associated (31), argues against a
Th2
Th1 skew during prolonged exposure to Ag.
Our observation in the airway is in agreement with studies by Haczku et al. (8) and Cui et al. (9), who have shown that chronic allergen exposure in rats does not lead to persistence of inflammation. In sharp contrast, other studies have documented persistent airway eosinophilia (10, 11). With respect to Igs, it has been shown in one study that prolonged exposure leads to persistent IgE production (11), while in another, prolonged exposure leads to transient IgG and IgE expression (9). While these discrepancies may reflect differences in the experimental models, our observations demonstrate that, in the mouse, chronic exposure to Ag does not result in persistence of airway and peripheral blood eosinophilia but does result in increased or sustained Ig expression.
The diminished airway eosinophilia but elevated Ig levels during
prolonged Ag challenge led us to investigate whether such exposure
influenced in vivo memory recall responses. To this end, following
either 2 or 4 wk of OVA challenge, mice were rested for 28 days and
rechallenged with OVA. Animals initially exposed to 4 wk of OVA and
subsequently rechallenged had significantly reduced airway eosinophilia
(Fig. 2) and reduced BAL and serum IL-5 levels (Fig. 3) compared with
2-wk exposed animals. In contrast, Ig levels were similar (IgE and
IgG2a) or increased (IgG1) in the 4-wk, compared with the 2-wk, exposed
mice (Table II). These findings may suggest that levels of IgE may be
of limited predictive value for inflammatory responses in the lung. We
are currently pursuing studies assessing the impact of chronic Ag
exposure on airway hyperresponsiveness.
Airway eosinophilia is a terminal event that relies on DC presenting Ag
to T cells (32) in the context of the appropriate
costimulatory signals (33). To investigate whether chronic
exposure altered the phenotype of DCs in 4-wk exposed animals at the
time of in vivo recall challenge, we assessed the number and activation
status of these cells. We observed elevated levels of B7.2 on DCs in
the lymph nodes and lungs of animals initially exposed to 4 wk of OVA
(Fig. 4), suggesting DC activation.
To investigate whether the changes observed in the APC compartment were
associated with T cell activation, we evaluated the phenotype of
CD4+ T cells in the lungs at the time of in vivo
recall challenge (Fig. 5). CD4+ T cells from
animals initially exposed to 2 and 4 wk of OVA expressed not only
similar levels of the early activation marker CD69 (34, 35) and the IL-2R CD25 (36, 37), but also similar
levels of T1/ST2, a marker of Th2 differentiation and a necessary
factor in the development of eosinophilic airway inflammation
(38, 39, 40). The level of expression of these molecules was
substantially higher than previously documented in naive animals.
Interestingly, these phenotypic observations did not translate into
expression of the Th2-associated cytokine, IL-5. Our data suggest that
chronic exposure to OVA does not alter T cell activation while
preventing the generation of airway eosinophilia upon in vivo recall.
Importantly, that a seemingly differentiated Th2
CD4+ T cell is incapable of eliciting airway
eosinophilia may argue for the presence of regulatory mechanisms in the
airway microenvironment, as has previously been suggested (41, 42).
The fact that animals were first exposed to OVA in a GM-CSF-enriched
environment led us to investigate whether re-exposure to OVA with
GM-CSF at the time of in vivo recall could recapitulate the
eosinophilic airway inflammation. To this end, animals initially
exposed to OVA for either 2 or 4 wk were rechallenged with the Ag in
the context of GM-CSF. To avoid an immune response to the adenovirus
that may confound the interpretation of our data, we opted to use the
recombinant protein rather than the adenovirally encoded GM-CSF. We
found that animals initially exposed to OVA for 4 wk and subsequently
re-exposed to the Ag in the context of GM-CSF developed robust airway
eosinophilia. Our data suggest that persistent airway inflammation is
dependent on not only Ag, but also additional factors such as GM-CSF
(Fig. 6). Therefore, GM-CSF not only is required for sensitization and
development of airway eosinophilia in a protocol that otherwise leads
to inhalation tolerance (12), but it may also be required
for the persistence of airway inflammation in the context of continued
Ag exposure.
The ability to reconstitute eosinophilic airway inflammation with the help of GM-CSF in seemingly unresponsive animals is likely of clinical relevance. It has been shown that exposure to environmental pollutants, as well as viral and bacterial agents, up-regulates GM-CSF production (43, 44, 45, 46). Indeed, these agents have been associated with exacerbation of asthma. Therefore, we hypothesize that exacerbation of symptoms among asthmatics may require not only Ag, but also additional agents that, along with Ag, generate sustained Th2-mediated eosinophilic airway inflammation. This may serve to explain, at least in part, why exacerbations are intermittent even if the Ag is continuously present. Therefore, we suggest that our experimental protocol provides a good model system to study mechanisms that regulate inflammation in the context of chronic exposure to innocuous Ag.
In summary, we show that prolonged exposure to OVA in the context of GM-CSF leads to abrogated eosinophilic airway inflammation, which is nevertheless associated with a robust humoral response and an activated CD4+ T cell and DC phenotype. This unresponsiveness is reversible with GM-CSF. Understanding the principles that lead to or prevent chronic inflammation elicited by innocuous Ag is key to our understanding of allergic diseases. We propose that the elucidation of these principles and mechanisms may help us to reveal intrinsic protective mechanisms and design new ways of controlling allergic diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martin R. Stämpfli, Department of Pathology and Molecular Medicine, McMaster University, Health Sciences Center, Room 4H21A, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail address: stampfli{at}mcmaster.ca
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; DC, dendritic cell; MHCII, MHC class II; LSD, least significant difference.
Received for publication March 28, 2002. Accepted for publication July 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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