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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in the airway allows allergic
sensitization to ovalbumin (OVA) in an experimental protocol that others have shown to induce inhalation
tolerance. The ensuing response is characterized by T helper (Th)2 cytokines, marked eosinophilia in the
bronchoalveolar lavage fluid (BALF) and the tissue, and goblet-cell hyperplasia. These findings, which
underscore the importance of the airway microenvironment in the development of immune responses to
airborne antigens, prompted us to investigate whether a Type 1 polarized cytokine milieu in the airway
would modulate the allergic sensitization. To this end, we concurrently expressed GM-CSF and interleukin (IL)-12 in the airway, using an adenovirus-mediated gene transfer approach. Coexpression of IL-12
did not prevent the development of an antigen-specific immune inflammatory response, but altered its phenotype. Whereas a similar total cell number was observed in the BALF, airway eosinophilia was abrogated. Histologic evaluation of the tissue corroborated the findings in the BALF and demonstrated that
IL-12 coexpression prevented goblet-cell hyperplasia. Expression of IL-12 decreased IL-4 and IL-5 content in the BALF by about 80 and 95%, respectively, and IL-5 in the serum by approximately 80%. In contrast, interferon (IFN)-
was increased in both BALF and serum. Similarly, we observed a Th2/Th1 shift in
OVA-specific cytokine production in vitro. Recall challenge with OVA in vivo after resolution of the initial inflammatory response demonstrated that the effect of IL-12 was persistent. IL-12-mediated inhibition of airway eosinophilia was mainly IFN--independent, whereas inhibition of OVA-specific IgE synthesis
was IFN--dependent. Our data underscore the importance of the airway microenvironment in the elicitation of immune responses to environmental antigens.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The immune system is confronted with two critical decisions upon antigen encounter: first, whether to respond; and second, how to respond (1). Understanding the basis of these decisions impinges on one of the most controversial issues concerning allergy and asthma: why some individuals mount harmful immune responses to otherwise innocuous environmental allergens (2). The increasing prevalence of allergic airway diseases in developed countries over the last 20 yr makes this issue particularly relevant (6). We surmise that the cytokine milieu in the airway at the time of initial exposure to allergens critically influences whether an immune response is elicited and, if so, the nature of the ensuing response. We further propose that this milieu is largely influenced by an individual's exposure to environmental agents such as viruses, bacteria or air pollutants. That these agents can initiate diverse immune responses with complex cytokine profiles (9) may help to explain apparently conflicting results from epidemiologic studies examining the prevalence of allergy and allergic diseases in connection with exposure to infectious agents in early childhood (12).
Studies in experimental animals have shown that repeated passive exposures to allergen favor long-lasting immunosuppression over priming (17), a phenomenon known as inhalation tolerance (20). We have recently demonstrated that expression of a single cytokine, granulocyte macrophage colony-stimulating factor (GM-CSF), in the airway overrides this outcome. That is, ovalbumin (OVA) aerosolization in the context of GM-CSF expression allows mucosal sensitization, and results in T helper (Th)2-driven eosinophilic inflammation and goblet-cell hyperplasia (21). Using this model, we investigated whether the introduction of a Type 1 polarized airway microenvironment at the time of antigen exposure would modulate the ensuing immune- inflammatory response to this antigen. We chose to express interleukin (IL)-12 as a key signal driving Type 1 responses elicited by a number of viruses, bacteria, and mycobacteria (22). The extremely short half-life of recombinant cytokines precludes an adequate recapitulation of the airway milieu in vivo. Thus, we elected to use an adenoviral (Ad)-mediated gene transfer approach that would permit sustained yet transient expression of biologically relevant levels of IL-12 in the airway.
Expression of IL-12 in the airway did not inhibit the immune-inflammatory response elicited by OVA exposure
in the context of GM-CSF, but remarkably altered its nature. Although the number of mononuclear cells and neutrophils in the bronchoalveolar lavage fluid (BALF) was
enhanced, airway eosinophilia was abrogated. Histologic
assessment of the tissue corroborated the findings in the
BALF. A long-term recall challenge in vivo demonstrated that the effect of IL-12 on GM-CSF-driven mucosal sensitization to OVA was persistent. The inhibition of airway
eosinophilia was mainly IL-12-dependent and interferon
(IFN)--independent, and occurred as a result of deviation of the primary allergic sensitization. In contrast, inhibition of OVA-specific immunoglobulin (Ig)E synthesis was IFN--dependent and only transient in nature. These
findings lend experimental support to the hypothesis that
the cytokine milieu in the airway at the time of antigen exposure determines the nature of the immune-inflammatory response to this antigen.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
Female Balb/c mice (6 to 8 wk old) were purchased from
Harlan (Indianapolis, IN). Female IFN- knockout (KO)
mice on a Balb/c background were obtained from Jackson
Laboratories (Bar Harbor, ME). Mice were housed under
specific pathogen-free conditions following a 12-h light-
dark cycle. All experiments described in this study were
approved by the Animal Research Ethics Board of McMaster University.
Aerosolization Protocol
As previously described (21), mice were exposed for 20 min daily to aerosolized OVA (1% wt/vol in 0.9% saline) over a period of 10 consecutive days (Days 0 to 9) in a Plexiglas chamber (10 × 15 × 25 cm). The OVA aerosol was produced by a Bennet/Twin nebulizer at a flow rate of 10 liters/min. For the recall challenge experiments, mice were exposed to a 1% OVA aerosol twice, 4 h apart, for two 1-h periods at Day 42 of the protocol.
Administration of Ad Constructs
To achieve prolonged expression of IL-12 and GM-CSF in the airway, we used an adenovirus-mediated gene transfer approach. Briefly, replication-deficient human Type 5 Ad constructs carrying the transgenes for GM-CSF (25) or the IL-12 heterodimer (26) in the E1 region of the viral genome were delivered intranasally. As a control, we included an E1-deleted replication-deficient human Type 5 Ad construct carrying no transgene (27). All Ad constructs were delivered in a total volume of 30 µl of phosphate-buffered saline (PBS) vehicle (two 15-µl administrations 5 min apart) into anesthetized animals. Unless otherwise stated, we used a dose of 3 × 107 pfu Ad/GM-CSF, 1 × 107 pfu Ad/IL-12, and 1 × 107 pfu of an empty control virus.
Collection and Measurement of Specimens
At various time points during and after the aerosolization
protocol, mice were killed and BAL was performed according to a standard protocol (28). Briefly, the lungs were
dissected and the trachea was cannulated with a polyethylene tube (Becton Dickinson, Sparks, MD). The lungs were
lavaged twice with PBS (0.25 ml followed by 0.2 ml); approximately 0.3 ml of the instilled fluid was consistently recovered. Total cell counts were determined using a hemocytometer. After centrifugation, supernatants were stored at
20°C for cytokine measurements by enzyme-linked immunosorbent assay (ELISA); cell pellets were resuspended in PBS and smears were prepared by cytocentrifugation (Shandon, Inc., Pittsburgh, PA) at 300 rpm for 2 min.
Diff-Quik (Baxter, McGraw Park, IL) was used to stain all
smears. Differential counts of BALF cells were determined from at least 500 leukocytes using standard
hemocytologic criteria to classify the cells as neutrophils,
eosinophils, or mononuclear cells (MNC). Additionally, blood was collected by retro-orbital bleeding. Serum was
obtained by centrifugation after incubating whole blood
for 30 min at 37°C. Finally, lung tissue was fixed in 10%
formalin and embedded in paraffin. Sections 3 µm thick
were stained with hematoxylin and eosin (H&E) or Congo
red to identify eosinophils further.
Lung Cell Isolation and Culture
Lungs were cut into small (~ 2 mm diameter) pieces and agitated at 37°C for 1 h in 15 ml 150 U/ml collagenase III (Worthington Biochemical Corporation, NJ) in Hanks' balanced salt solution (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. After lysing red blood cells with ACK lysis buffer (0.5 M NH4Cl, 10 mM KHCO3, and 0.1 nM Na2-ethylenediaminetetraacetic acid at pH 7.2-7.4), cells were washed once and MNC were isolated by density centrifugation in 30% Percoll and subsequently washed twice. Lung cells were plated at 2 × 105 cells/well in a flat-bottom 96-well plate (Becton Dickinson, Franklin Lakes, NJ) in RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO, Burlington, ON, Canada), 1% L-glutamine, and 1% penicillin/streptomycin alone, or with 40 µg OVA/well. After 5 d of culture, supernatants were harvested for cytokine measurement.
Cytokine Measurement
ELISA kits for IL-4, IFN-, and IL-12 were purchased from
R&D Systems (Minneapolis, MN), and the kit for IL-5 was
obtained from Amersham (Buckinghamshire, UK). Each of
these assays has a threshold of detection of 5 pg/ml. Serum
levels of OVA-specific mouse IgE were measured using a
previously described antigen-capture ELISA method (28).
Units of OVA-specific IgE were calculated according to a
standard serum derived from intraperitoneally sensitized mice 3 d after antigen challenge (28). The amount of 1,000 U/ml corresponds to undiluted standard serum.
Data Analysis
Data are expressed as means ± standard deviation (SD). Statistical interpretation of results is indicated in figure legends. Differences were considered statistically significant when P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Transgene Products of IL-12 and GM-CSF in the BALF
An initial series of studies was carried out to determine the dose response and time course of transgene expression for the Ad/IL-12 construct administered intranasally. Administration of 105, 106, and 107 pfu Ad/IL-12 resulted in approximately 50, 200, and 4,000 pg/ml, respectively, of IL-12 in the BALF at Day 4. IL-12 expression was detected in the BALF as early as 24 h, and up to approximately 10 d (data not shown). For the Ad/GM-CSF construct, we used a dose of 3 × 107 pfu. At this dose, GM-CSF was detected in the BALF for approximately 10 d, with a peak level of approximately 80 pg/ml in the BALF at Day 7 (29).
BALF Cellular Profile in Mice Exposed to OVA in the Context of IL-12
First, we examined the effect of expressing IL-12 in the airway on the response to aerosolized OVA. We have previously shown that aerosolization of OVA alone does not elicit any appreciable changes in either the BALF or the tissue (21). Table 1 shows that expression of IL-12 alone resulted in a doubling of the total number of cells in the BALF compared with naive mice. This increase consisted of MNC and neutrophils. Similar changes were observed in the BALF of mice exposed to aerosolized OVA in the context of IL-12 expression.
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Effect of IL-12 on OVA-Specific, GM-CSF-Driven Airway Eosinophilia
To examine the effect of IL-12 expression on OVA-induced, GM-CSF-driven airway eosinophilia, we administered different doses of Ad/IL-12 (ranging from 105 to 107 pfu), concurrently with Ad/GM-CSF intranasally, 24 h before daily exposures to OVA over a period of 10 d. Mice were killed 48 h after the last exposure, because we observed maximal airway inflammation at this time in mice exposed to OVA in the context of GM-CSF (21), and the cellular profile in the BALF was assessed. In agreement with our previous data, exposure to aerosolized OVA in the context of GM-CSF expression resulted in an inflammatory response comprised primarily of MNC and eosinophils with some transient neutrophilia (21). Expression of IL-12 inhibited airway eosinophilia in a dose-dependent manner with maximal effect at 107 pfu (Figure 1). Coexpression of GM-CSF and IL-12 (1 × 107 pfu Ad/IL-12) caused a slight, but significant, increase in the number of MNC and neutrophils, and complete abrogation of airway eosinophilia (Figure 2). No significant changes in the BALF cellular profile and, specifically, no abrogation of airway eosinophilia was observed in mice that had received 107 pfu of an empty control virus (data not shown). We conducted a detailed histologic evaluation to corroborate the BAL findings. Figure 3A shows that mice exposed to OVA in the context of GM-CSF developed extensive peribronchial and perivascular inflammation. Higher-power fields reveal that this inflammation is associated with goblet-cell hyperplasia (Figure 3C) and is eosinophilic in nature (Figure 3E). In mice exposed to OVA in the context of both GM-CSF and IL-12, the extent of the peribronchial and perivascular inflammation is comparatively milder (Figure 3B), and is comprised of MNC and neutrophils but no eosinophils (Figure 3F). It is interesting to note that the epithelium of these mice appears ciliated with no evidence of goblet-cell hyperplasia (Figure 3D).
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Cytokine Levels in BALF and Serum of Mice Exposed to OVA in the Context of GM-CSF and IL-12
Next, we examined whether IL-12 expression altered the
cytokine profile in the BALF and serum of mice exposed
to OVA in the context of GM-CSF. To this end, mice were
killed on Day 7, because IL-5 reached peak levels at this
time (21). Figure 4 shows that IL-12 expression decreased
the levels of IL-4 and IL-5 in the BALF by about 80 and
95%, respectively, and IL-5 in the serum by approximately
80%. No IL-4 could be detected in the serum of mice exposed to OVA in the context of GM-CSF alone or with
IL-12. Expression of IL-12 in the airway markedly increased the level of IFN- both in BALF and serum.
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In Vitro OVA-Specific Cytokine Production by Lung Cells
We wished to investigate whether the decrease in the production of Th2 cytokines induced by IL-12 was the result
of nonspecific inhibition or, alternatively, deviation of the
OVA-specific T-cell response. Thus, dispersed cells from
lungs harvested at Day 11 were stimulated with OVA in
vitro. Figure 5 shows that cells derived from mice exposed
to OVA in the context of IL-12 and GM-CSF in vivo produced increased amounts of IFN- in response to OVA stimulation in vitro, whereas production of IL-4 and IL-5 was
completely abrogated. Hence, these findings suggest that
IL-12 expression in the airway deviated GM-CSF-driven
mucosal sensitization to OVA toward a Th1 phenotype.
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Effect of IL-12 Expression on OVA Recall Challenge In Vivo
We then examined whether airway expression of IL-12 during the primary exposure to OVA in the context of GM-CSF would affect memory responses in vivo. As we demonstrated peviously, the inflammatory response in the airway of mice exposed to aerosolized OVA in the context of GM-CSF is resolved approximately by Day 28 (21). In the present study, mice were re-exposed to aerosolized OVA at Day 42, and the BALF and tissue response was assessed 3 d later. Figure 6 shows that mice exposed to OVA and GM-CSF readily developed eosinophilic airway inflammation after OVA recall challenge. In contrast, no airway eosinophilia developed in those mice in which we had expressed IL-12 at the time of the primary immune response to OVA. Histologically, Figure 7A shows that airway inflammation in mice exposed to OVA in the context of GM-CSF was largely resolved at Day 42. Few inflammatory cells can be observed in the peribronchial and perivascular space, whereas goblet-cell hyperplasia was completely resolved and the epithelium appeared ciliated (Figure 7B). Re-exposure to OVA induced a marked peribronchial and perivascular inflammation (Figure 7C) that was eosinophilic in nature (Figure 7D). In mice concurrently expressing GM-CSF and IL-12 at the time of primary exposure to OVA, we observed a mononuclear and neutrophilic inflammatory infiltrate after recall challenge with no eosinophilia (Figure 7E). Again, tissue eosinophilia was completely abrogated (Figure 7F). Moreover, the epithelium appeared normal with no signs of goblet-cell hyperplasia (Figure 7F).
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Effect of Concurrent IL-12 Expression
in IFN- KO Mice
Having shown that IL-12 expression in the airway induced
IFN- production both in vivo and in vitro, we examined
whether IFN- was in fact required to abrogate airway
eosinophilia. Thus, the Ad/IL-12 construct was delivered
to IFN- KO mice under the same experimental conditions described earlier. We found that IL-12 expression
caused a marked (80%) reduction in BAL eosinophilia in
the mutant mice (Figure 8). Total cell number and the
number of MNC were not affected by the treatment.
These findings indicate that the IL-12-mediated inhibition
of airway eosinophilia is largely IFN--independent.
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Effect of IL-12 Expression on OVA-Specific IgE Production
Finally, we investigated whether IL-12 expression in the
airway would modify OVA-specific IgE synthesis in mice
exposed to OVA in the context of GM-CSF (Table 2). In
agreement with our previous data, no IgE was detected in
naive mice, whereas exposure to OVA in the context of
GM-CSF induced a significant OVA-specific IgE response
(Table 2). Concurrent exposure to IL-12 led to significantly lower OVA-specific IgE levels at Day 11. However, this
effect was transient as similar levels of OVA-specific IgE
were detected at later time points (Days 34 and 45). IL-12 expression did not inhibit IgE synthesis in IFN--KO mice, indicating that, in contrast to airway eosinophilia, inhibition
of OVA-specific IgE synthesis was IFN--dependent.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have recently shown that expression of GM-CSF in the airway allows mucosal sensitization to aerosolized OVA (21). The ensuing inflammatory response is characterized by a Th2 cytokine profile, marked eosinophilia, and goblet-cell hyperplasia. Therefore, GM-CSF creates a milieu in the airway that causes mucosal sensitization in an antigen-exposure protocol that others have shown to induce inhalation tolerance (17). This observation underlies the hypothesis that the cytokine microenvironment in the airway at the time of initial allergen exposure determines whether an immune response is elicited and, if so, the nature of that response. In our view, this hypothesis warrants further exploration, as it may help to explain current controversies concerning the impact of exposure to infectious agents early in childhood on the development of allergic disease (12).
Viral and bacterial infections are not homogeneous and can elicit fundamentally different immune responses characterized by complex cytokine profiles (9, 10). We employed an adenovirus-mediated gene transfer approach to create a defined cytokine milieu in the airway. The advantage of this approach over administration of recombinant cytokines lies in the ability to express a relatively high level of specific transgenes over a sustained, albeit transient, period of time. Because these Ad constructs are replication-deficient, infection remains localized within the airway/lung and, consequently, cytokine expression is compartmentalized (29). The ability to express specific cytokines within the airway in a novel protocol of mucosal sensitization involving exposure only to OVA aerosol (21) provides unique opportunities to investigate the interaction between antigens and the airway microenvironment in vivo. We created distinct airway milieus by expressing GM-CSF alone or GM-CSF together with IL-12. We chose to express IL-12 because of its pivotal role in the development of Th1 cells (30) and its biologic relevance, as a number of intracellular pathogens (22) and biologic stimuli, such as lipopolysaccharide (31) or bacterial DNA (32), induce IL-12 synthesis.
Expression of IL-12 alone in the airway elicited a moderate inflammatory reaction characterized by MNC and
neutrophils. Concurrent exposure to OVA did not significantly change the degree or the type of this reaction. However, expression of IL-12 dramatically altered the inflammatory reaction elicited by OVA exposure in the context
of GM-CSF expression. Although the increases in total cells in the BALF at Day 11 of mice expressing GM-CSF
alone and GM-CSF and IL-12 were similar, IL-12 expression completely abrogated eosinophilia in both BALF and
tissue (Figures 1-3). As shown in Figures 4 and 5,
expression of IL-12 in the airway changed the profile of
cytokines produced in response to OVA in vivo and in vitro. Indeed, the concurrent expression of IL-12 resulted
in a remarkable decrease in IL-4 and IL-5 levels, and in a
similarly marked increase in IFN-, this being consistent
with a shift from a Th2 to a Th1 profile. Thus, these findings indicate that IL-12 expression in the airway altered
the nature of, rather than inhibited, the GM-CSF-mediated immune-inflammatory response to OVA. It could be
argued that IL-12 replaced a specific immune response
with a nonspecific inflammatory reaction. However, lung
cells from mice exposed to OVA, in which we concurrently expressed GM-CSF and IL-12, produced high levels
of IFN- and negligible amounts of IL-4 and IL-5 when
stimulated with OVA in vitro (Figure 5). Moreover, expression of IL-12 in the airway at the time of initial antigen
exposure in the context of GM-CSF also inhibited airway eosinophilia after recall challenge in vivo at Day 42 (Figures 6 and 7), indicating that the IL-12 effect was antigen-specific and persistent.
It is well known that IL-12 is a potent inducer of IFN-.
Therefore, it is important to clarify the relative roles of
these two molecules. As it relates to antigen-induced airway eosinophilia, this issue remains controversial. Brusselle and colleagues (33) and Gavett and associates (34)
have reported that inhibition of airway eosinophilia by recombinant IL-12 given before challenge is IFN--independent. On the other hand, Iwamoto and coworkers (35)
and Hogan and colleagues (36), using recombinant IL-12 and a vaccinia virus-mediated gene-transfer approach, respectively, have claimed that IFN- plays a central role in
IL-12-mediated inhibition of airway eosinophilia. A partial explanation for these discrepancies may most likely be
found in the differences between the protocols used to
elicit sensitization. It is important to note that in these
studies IL-12 was administered to mice sensitized systemically (intraperitoneally); that is, to mice that had already developed a memory/effector T-cell population specific for
OVA. Consequently, the question addressed was whether
IL-12 could inhibit a Th2 recall response in the airway. In
our experimental setting, mice were subjected to a protocol of exclusive mucosal sensitization, and IL-12 was expressed in the airway constantly throughout this process.
Consequently, the question that we addressed was whether expression of IL-12 in the airway milieu could abrogate or
deviate a Th2 immune response to OVA. Under these
conditions, our data suggest that inhibition of airway eosinophilia was largely IFN--independent. Although the
mechanisms responsible for this inhibitory effect have not
yet been elucidated, they could involve direct effects of
IL-12 on T-cell development (37), indirect effects on
CD16+ natural killer cells (31), or an as-yet-undefined
pathway. Experiments investigating the target of IL-12 in
our model of mucosal sensitization are being intensively
pursued in our laboratory at present.
IL-12 expression inhibited OVA-specific IgE synthesis
at Day 11, an observation that is in agreement with previous reports (33, 38). However, we demonstrate here that
IL-12-mediated inhibition of OVA-specific IgE synthesis
is transient and IFN--dependent (Table 2). These findings suggest differential regulation of the cellular and the
humoral immune responses against an environmental antigen. While the deviation of the T-cell response was persistent, IgE synthesis was only transiently inhibited. The
mechanisms underlying these observations remain to be
elucidated as well. However, our data clearly demonstrate
that the presence of OVA-specific IgE can be dissociated
from the development of airway eosinophilia after antigen challenge.
We observed increased numbers of eosinophils and
higher levels of OVA-specific IgE in IFN- KO mice as
compared with wild-type mice (Figure 8 and Table 2).
These findings could be due to a slightly different genetic
background of the two mouse strains. Alternatively, it has
previously been shown that IFN- inhibits the development of a secondary allergic response in mice (39). Therefore, endogenous IFN- may partially inhibit IgE synthesis and airway eosinophilia development. Indeed, we observed
approximately 100 pg/ml IFN- in the BALF of wild-type
mice exposed to OVA in the context of GM-CSF (Figure
4). Moreover, lung cells from these mice produce substantial levels of IFN- in vitro after OVA stimulation. However, investigating whether the differences between wild-type and IFN- KO mice were due to endogenous IFN-
production is beyond the scope of this report.
Our study illustrates the importance of the airway microenvironment at the time of the initial exposure to an allergen in the elicitation of an immune response, the nature of the ensuing inflammatory response, and the phenotype of the antigen-specific memory. Although exposure to OVA in the context of GM-CSF induced a Th2-polarized immune response, concurrent expression of IL-12 promoted the development of a Th1-polarized response. It is notable that in both instances, exposure to allergen induced specific immunity. Our data are in agreement with findings indicating that peripheral blood MNC derived from both allergic and nonallergic subjects proliferate in culture when stimulated with allergen (40), although the associated cytokine profiles are Th2- and Th1-polarized, respectively (41, 42). Our findings may also have a bearing on explaining some apparently conflicting epidemiologic data. Although viral infections such as Rous sarcoma virus or rhinovirus in early childhood are associated with increased prevalence of allergy and asthma later in life (12, 13), it has been suggested that exposure to other infectious agents such as Mycobacterium tuberculosis, hepatitis A virus, and measles may prevent allergic sensitization (14- 16). We suggest that the debate about the role of infection in the development of allergic diseases should take into account the biologic diversity of infectious agents and the distinct immune responses that these agents can elicit. In our view, it is not an issue of whether infectious agents cause or prevent allergic disease, but of how the conditions they create in the airway microenvironment affect the type of immune response to allergens.
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Footnotes |
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Address correspondence to: Manel Jordana, M.D., Ph.D., McMaster University, Health Sciences Centre, Room 4H21, Dept. of Pathology and Molecular Medicine, 1200 Main St. West, Hamilton, ON, L8N 3Z5 Canada. E-mail: jordanam{at}fhs.csu.McMaster.CA
(Received in original form November 13, 1998 and in revised form February 24, 1999).
Abbreviations: adenoviral, Ad; analysis of variance, ANOVA; bronchoalveolar lavage, BAL; bronchoalveolar lavage fluid, BALF; enzyme-linked immunosorbent assay, ELISA; granulocyte macrophage colony-stimulating factor, GM-CSF; hematoxylin and eosin, H&E; interferon, IFN; immunoglobulin, Ig; interleukin, IL; knockout, KO; mononuclear cells, MNC; ovalbumin, OVA; phosphate-buffered saline, PBS; standard deviation, SD; T helper, Th.Acknowledgments: The authors thank Drs. Jack Gauldie and Frank L. Graham for providing the Ad constructs used in our experiments. The technical help of Susanna Goncharova, Duncan Chong, and Xueya Feng, and the secretarial assistance of Mary Kiriakopoulos, are gratefully acknowledged. One author (M.R.S.) held a Fellowship from the Medical Research Council/Canadian Lung Association; one author (S.A.R.) holds an Ontario Graduate Scholarship; one author (Z.X.) is a scholar of the Medical Research Council (Canada); and one author (M.J.) is a Career Scientist of the Ontario Ministry of Health. This study was funded in part by the Medical Research Council (Canada) and Astra Draco AB (Sweden).
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References |
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Materials and Methods
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Discussion
References
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