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Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics
Department of Pathology and Molecular Medicine
Department of Medicine, Firestone Institute for Respiratory Health, McMaster University, Hamilton, Ontario, Canada
ABSTRACT
Rationale: Mouse models of allergic airway disease have greatly contributed to our understanding of disease induction and pathogenesis. Although these models typically investigate responses to a single antigen or allergen, humans are frequently exposed to a myriad of allergens, each with distinct antigenic potential. Objectives: Given that airway exposure to ovalbumin (OVA), a prototypic innocuous antigen, induces inhalation tolerance, we wished to investigate how this response would be altered if OVA were encountered concurrently with a house dust mite extract (HDM), which we have recently shown is capable of eliciting a robust allergic airway inflammatory response that is mediated, at least in part, by granulocyte-macrophage colonyeCstimulating factor. Methods: Balb/c mice were exposed daily to HDM (intranasally) followed immediately by exposure to aerosolized OVA for 5 weeks. To allow the inflammatory response elicited by HDM to subside fully, mice were then allowed to rest, unexposed, for 8 weeks, at which time they were rechallenged with aerosolized OVA for 3 consecutive days. Measurements and Main Results: At this time, we observed a robust eosinophilic inflammatory response in the lung that was associated with an increase in bronchial hyperreactivity. Moreover, we documented significantly elevated serum levels of OVA-specific IgE and IgG1 and increased production of the Th2 cytokines interleukin 4 (IL-4), IL-5, and IL-13 by splenocytes stimulated in vitro with OVA. Conclusion: Our data demonstrate the potential of a potent allergen such as HDM to establish a lung microenvironment that fosters the development of allergic sensitization to otherwise weak or innocuous antigens, such as OVA.
Key Words: allergic sensitization; allergy inflammation; lung; mouse
Mucosal surfaces are under constant exposure to a diverse array of biological and nonbiological entities, each with a distinct pathogenic potential. To survive, the immune system has evolved, over millions of years, sophisticated mechanisms to determine the type of immunologic response that each warrants. However, because humans are exposed simultaneously to more than one agent, it is likely that concurrent exposure has an impact on the response the immune system evolved for each single entity. We wished to explore this concept in the context of aeroallergen exposure.
Aeroallergens comprise a vast collection of nonreplicating entities, with diverse immunogenic potential capable of inducing specific immune-inflammatory responses (1, 2). Whether, and how, they facilitate allergic sensitization and airway inflammation remains a subject of intense research. A great deal has been learned through experimental modeling of aeroallergen exposure, much of it using ovalbumin (OVA) as a surrogate allergen. Because OVA is an archetypic innocuous antigen, and mucosal exposure to aerosolized OVA alone results in inhalation tolerance (3eC5), conventional modeling has relied on the introduction of OVA into the peritoneum in conjunction with an adjuvant, generally aluminum hydroxide, to elicit allergic sensitization. In contrast, we have recently shown that intranasal exposure to a house dust mite (HDM) extract generated acute (6) as well as chronic (7) airway inflammation with the characteristic hallmarks of a Th2-type immune-inflammatory response. Because it is evident that humans are concurrently exposed to various aeroallergens, we were interested in investigating whether HDM could subvert the expected immunologic response to OVA. The issue is of relevance because there is a definite increase in the prevalence of allergic disease, including asthma (reviewed in Reference 8), and most of the increase has been observed with indoor allergens, notably HDM (8).
To our knowledge, only three studies have previously investigated the impact of concurrent allergen exposure on the development of allergic responses (9eC11). The extent of the analysis in these three studies was limited to the assessment of serum levels of OVA-specific Th2-affiliated immunoglobulins after concurrent administration of a bona fide allergen with OVA. Thus, whether exposure to one allergen can facilitate the full development of the "asthmatic phenotype" in response to another remains to be determined.
To address this issue experimentally, we exposed mice to HDM concurrently with OVA for 5 weeks. Subsequently, mice were allowed to rest for 8 weeks, at which time they were then reexposed to aerosolized OVA alone. This in vivo OVA recall elicited a robust airway inflammatory response characterized by eosinophils and Th2 effector cells and was associated with bronchial hyperreactivity. Systemically, we documented increased serum levels of Th2-affiliated OVA-specific immunoglobulins and production of OVA-specific Th2-associated cytokines by splenocytes on in vitro recall. Collectively, these data demonstrate that HDM is able to subvert OVA's intrinsic innocuous nature and to privilege a Th2 inflammatory response over the default tolerogenic bias (12).
METHODS
Animals
Female Balb/c mice (6eC8 weeks old) were purchased from Charles River Laboratories (Ottawa, ON, Canada). The mice were housed under specific pathogen-free conditions and maintained on a 12-hour lighteCdark cycle, with food and water ad libitum. All experiments described in this study were approved by the Animal Research Ethics Board of McMaster University (Hamilton, ON, Canada).
Sensitization Protocols
Allergen administration.
HDM extract (Greer Laboratories, Lenoir, NC) was resuspended in sterile phosphate-buffered saline at a concentration of 5.0 mg (protein)/ml, and 10 e was administered to isofluorane-anesthetized mice intranasally. After reconstitution, we evaluated the proteolytic activity of the HDM extract by zymography (as previously described in Reference 13), and observed that a single dose of HDM was indeed proteolytically active (data not shown). Mice that received OVA (Grade V; Sigma-Aldrich, Oakville, ON, Canada) were placed in a Plexiglas chamber ( 10 x 15 x 25 cm) and were exposed to aerosolized OVA (1% wt/vol in 0.9% saline) for 20 minutes. Aerosolized OVA was produced using a Bennet nebulizer with an airflow rate of 10 L/minute.
Short-term exposure.
Mice were exposed daily to HDM, OVA, or HDM followed 1 to 4 hours later by OVA for 10 consecutive days.
Extended exposure.
Mice were exposed daily to HDM, OVA, or HDM followed 1 to 4 hours later by OVA, for 5 consecutive days a week followed by 2 days of rest for a total of 5 weeks.
Rechallenge.
After extended allergen exposure, mice were rested for a period of approximately 8 weeks at which time they were then rechallenged with OVA (as above) daily for 3 consecutive days.
Collection and Measurement of Specimens
Seventy-two hours after the last challenge, mice were killed and bronchoalveolar lavage (BAL) fluid, lungs, blood, and spleen were collected. BAL was performed as previously described (14). Total and differential cell counts were determined as previously described (14). Where applicable, after BAL, lungs were inflated with 10% formalin. Tissues were then embedded in paraffin, and 3-e-thick sections were cut and stained with hematoxylin and eosin. Peripheral blood was collected by retroorbital bleeding, and serum was obtained and stored at eC20°C. See the online supplement for additional details on the methods used to make these measurements.
Splenocyte Culture
Spleens were harvested, and splenocytes were isolated and resuspended in complete RPMI at a concentration of 8 x 106 cells/ml and cultured in medium alone, or with medium supplemented with OVA (40 e/ml). After 5 days of culture, supernatants were collected for cytokine measurement. See the online supplement for additional details on the methods used to make these measurements.
Cytokine and Immunoglobulin Measurements
Levels of interleukin 4 (IL-4), IL-5, IL-13, and IFN- were measured by ELISA using kits purchased from R&D Systems (Minneapolis, MN). Levels of OVA-specific IgE in the serum were measured using a previously described antigen-capture ELISA method (14), whereas OVA-specific serum IgG1 and IgG2a were measured by sandwich ELISA with OVA in the solid phase, as previously described in detail (15). See the online supplement for additional details on the methods used to make these measurements.
Lung Cell Isolation and Flow Cytometric Analysis of Lung Mononuclear Cells
Lung mononuclear cells were isolated as previously described (16) and subsequently stained with a panel of antibodies. Data were collected using a FACScan (Becton Dickinson, Franklin Lakes, NJ) four-color flow cytometer and analyzed using WinMDI software (Scripps Research Institute, La Jolla, CA). See the online supplement for additional details on the methods used to make these measurements.
Airway Hyperreactivity Measurements
Airway responsiveness was measured on the basis of the response of total respiratory system resistance to increasing intravenous (internal jugular vein) doses of methacholine as previously described (17). See the online supplement for additional details on the methods used to make these measurements.
Data Analysis
Data are expressed as mean ± SEM. Results were interpreted using analysis of variance with a Tukey post hoc test, unless otherwise indicated. Differences were considered statistically significant when p values were less than 0.05.
RESULTS
Impact of HDM on the Induction of OVA-specific Immune Responses
Balb/c mice received daily administrations of HDM, OVA, or both HDM and OVA (HDM/OVA) for 10 consecutive days (Days 0eC9) and were then killed approximately 72 hours (Day 12) after the last challenge (ALC; Figure 1A). Analysis of the BAL revealed no significant differences in the cellular profile of mice exposed to OVA alone compared with untreated (naive) mice (Figure 1B). In direct contrast, and as we have previously demonstrated (6), we observed significant increases in the number of total cells and eosinophils in the BAL of mice repeatedly exposed to HDM when compared with naive and OVA mice (Figure 1B). Furthermore, we noted significant increases in the BAL of HDM/OVA mice in terms of total cells and eosinophils versus naive and OVA mice with no differences between HDM/OVA and HDM mice (Figure 1B).
Because we were unable to differentiate between the OVA-elicited component of the inflammatory response from that of HDM in the BAL of HDM/OVA mice, we examined the in vitro cytokine production by OVA-stimulated splenocytes as a means of assessing OVA-specific sensitization. We measured higher levels of the Th2-associated cytokines IL-5 and IL-13 (Figure 1C) produced by splenocytes isolated from HDM/OVA mice compared with naive and HDM groups after in vitro stimulation with OVA, although when compared with OVA mice, significance was only reached in the case of IL-5.
Impact of Extended Concurrent Allergen Exposure
OVA-specific markers of systemic sensitization immediately after extended concurrent exposure to HDM and OVA.
Because 10-day exposure to HDM concurrently with OVA suggested the incipient generation of OVA-specific sensitization, we proceeded to examine the influence of HDM on the development of OVA-specific immunity in a protocol that involved prolonged exposure to both HDM and OVA. Thus, we exposed mice to daily administrations of HDM, OVA, or HDM/OVA on 5 consecutive days/week for a total of 5 weeks and then killed them shortly afterward ( 72 hours ALC; Figure 2A). We observed significantly increased in vitro OVA-specific production of the Th2-associated cytokines IL-4 and IL-13 (Figure 2B), by splenocytes that were isolated from HDM/OVA mice compared with naive or control mice. In addition, we did not observe any differences in OVA-specific production of the Th1-associated cytokine IFN- between any of the experimental groups (data not shown). Correspondingly, significant increases in the serum levels of OVA-specific IgG1 were also observed in HDM/OVA mice compared with naive and control groups (Figure 2C). Analysis of the BAL revealed no significant differences in the differential cell profile of mice exposed to OVA alone versus naive (data not shown). In contrast, we observed significant increases in the total cell number of HDM/OVA mice compared with naive and OVA mice, which was characterized by increases in both mononuclear cells and eosinophils. No differences between HDM/OVA and HDM-alone mice were noted (data not shown).
OVA-specific recall responses after extended concurrent exposure: cellular profile in the BAL.
To assess conclusively whether an OVA-specific immune response in the lung had developed on extended concurrent exposure to HDM and OVA, a group of mice were allowed to rest after 5 weeks of exposure for a period of approximately 8 weeks (Figure 3A), to allow the lung inflammatory response to resolve completely (Figures 3B and 3C). After this period, mice were reexposed for 3 consecutive days to aerosolized OVA and killed approximately 72 hours after the last challenge (Figure 3A). We observed that mice that initially received either OVA or HDM alone showed no significant changes in the BAL cellular profile compared with naive mice after in vivo recall with OVA (Figures 3B and 3C). In contrast, OVA reexposure to HDM/OVA mice induced a significant increase in the total cell number compared with all other groups (Figure 3B). Moreover, this inflammatory response was characterized by a considerable accumulation of both mononuclear cells and eosinophils (Figures 3B and 3C).
Histologic evaluation of lung tissue.
Histologic examination of the lung tissue confirmed the observations made in the BAL. Indeed, we observed no evidence of inflammation in the lungs of HDM/OVA and HDM mice that were killed just before in vivo recall with OVA, verifying that the lung inflammatory response had fully resolved over the 8-week rest period (data not shown). In addition, after in vivo recall with OVA, the lungs of OVA and HDM-alone mice resembled that of naive mice because there was no evidence of either peribronchial or perivascular inflammation (Figures 4AeC4C). Moreover, no eosinophils could be found among the residing lung cells. In contrast, the lungs of HDM/OVA mice exhibited pronounced perivascular and peribronchial inflammation (Figure 4D), with clear evidence of goblet cell hyperplasia (Figure 4E). Furthermore, in agreement with the BAL data, the inflammatory infiltrate observed in the lungs of HDM/OVA mice after OVA recall was predominantly composed of mononuclear cells and eosinophils (Figure 4F).
Flow cytometric analysis of T1/ST2 expression on lung mononuclear cells.
To further evaluate the composition of the lung mononuclear infiltrate, we performed flow cytometric analysis. Specifically, we wished to ascertain whether the increased influx of mononuclear cells after in vivo OVA recall in HDM/OVA mice was accompanied by an increase in the percentage of CD4+ T cells and, more specifically, in activated Th2 cells. After recall with OVA, we detected an approximate doubling in the percentage (10-fold increase in the number) of CD3+/CD4+ cells in the lung mononuclear cell population of HDM/OVA mice compared with naive and control groups (Figure 5). Moreover, this increase was associated with two- to threefold and four- to fivefold increases in the percentage of CD3+/CD4+ cells expressing CD69 (an early T-cell activation marker) or T1/ST2 (a putative marker of Th2 effector cells [18, 19]), respectively, over naive and control animals, and a five- to sixfold increase in the percentage of CD3+/CD4+ cells expressing both CD69 and T1/ST2.
Further evidence of OVA-specific immune-effector function.
To extend our evaluation of OVA-specific immune-effector function, we examined the cytokine profile in the supernatant of OVA-stimulated splenocyte cultures and the levels of OVA-specific immunoglobulins in the serum. We detected a considerable increase in the production of the Th2 cytokines IL-4, IL-5, and IL-13 from splenocytes obtained from HDM/OVA mice compared with naive and control groups (Figure 6). Furthermore, there were no significant differences in the levels of IFN- among all groups (Figure 6). In addition, we detected significantly increased levels of the OVA-specific Th2-associated immunoglobulins IgE and IgG1 in the sera of HDM/OVA mice versus naive and control animals; no significant differences were observed in Th1-associated OVA-specific IgG2a between OVA and HDM/OVA groups (Figure 7).
Impact of the lung inflammatory response on lung function.
Finally, we investigated the physiologic impact of the observed inflammatory response on airway function of HDM/OVA mice after OVA recall in vivo. We evaluated the respiratory resistance after increasing doses of methacholine in naive and HDM/OVA mice. HDM/OVA mice that were killed after the rest period but just before in vivo OVA recall (HDM/OVA before recall) demonstrated, as expected (7), a residual increase in airway resistance over naive animals (Figure 8). This is attributable to prolonged HDM exposure because HDM-alone mice demonstrated similar increases after the same period of cessation (data not shown and Reference 7). After in vivo recall with OVA (HDM/OVA after recall), we observed a marked increase in airway resistance, which was statistically significant at all doses of methacholine when compared with naive mice and HDM/OVA mice before recall.
DISCUSSION
There is increasing recognition of the concept that the immunologic milieu in which an antigen is first encountered largely determines the type of immune response that subsequently evolves. In the context of allergic airway sensitization, a series of experimental studies provide supporting evidence for this concept. Indeed, although repeated exposure to aerosolized OVA alone resulted in inhalation tolerance (3eC5), exposure in a granulocyte-macrophage colonyeCstimulating factor (GM-CSF)eCenriched microenvironment elicited the generation of an OVA-specific Th2 response (16), whereas conditioning with GM-CSF and IL-12 induced a Th1-type response (20). Although the airway microenvironment, in these studies, was altered by the overexpression of proinflammatory cytokines, the recent observation that intranasal administration of HDM extract elicits, without additional adjuvants, both acute (6) and chronic (7) airway inflammation intimates that HDM, a common environmental allergen, is intrinsically capable of creating an airway milieu conducive to allergic sensitization. Here, we investigated whether an HDM-conditioned lung microenvironment could subvert the expected immunologic response to aerosolized OVA.
We observed that a short exposure (10 days) to HDM alone or to HDM and OVA resulted in equivalent levels of airway inflammation. This was not surprising given that HDM alone elicits a robust inflammatory response in the lung. That we also observed an increase, albeit modest, in the production of OVA-specific Th2-associated cytokines by splenocytes harvested from HDM/OVA mice suggested that a degree of OVA-specific sensitization had occurred. In other words, short concurrent exposure may have been sufficient to subvert the development of OVA-specific inhalation tolerance.
Impelled by this observation, we explored whether extending the period of concurrent exposure to 5 weeks would strengthen the degree of OVA-specific sensitization. Indeed, production of OVA-specific Th2 cytokines by splenocytes harvested from mice concurrently exposed to HDM and OVA was considerably enhanced. In addition, we documented elevated serum levels of OVA-specific IgG1 under these protracted exposure conditions. Of interest, this observation is in agreement with that of Gough and coworkers (10) who demonstrated an enhancement of OVA-specific IgE after concurrent intraperitoneal injection of the major dust mite allergen Der p 1 with OVA and with that of Deplazes and colleagues (9) who made a similar observation in dogs. Notably, the serum level of OVA-specific immunoglobulins was the only outcome evaluated in these studies. Nevertheless, our findings of increased levels of OVA-specific IgG1 and Th2 cytokine production by OVA-stimulated splenocytes on extended concurrent exposure to HDM and OVA told us nothing about whether an inflammatory response would be triggered in the lung/airway compartment on subsequent exposure to OVA (21).
Thus, to evaluate the impact of extended concurrent allergen exposure on the development of OVA-specific memory more conclusively and, particularly, to ascertain whether this would result in airway inflammation on subsequent OVA exposure, mice were rested and then rechallenged, in vivo, with OVA. Our data demonstrate that in vivo recall with OVA led to a robust Th2-type airway inflammatory response only in mice that had initially been exposed to OVA in the context of an HDM-conditioned airway. Importantly, this inflammatory response was associated with significantly increased bronchial hyperreactivity. Furthermore, we also observed elevated serum levels of OVA-specific IgE and IgG1 as well as Th2 cytokine production by splenocytes. Collectively, our data convincingly demonstrate that exposure to HDM subverts the expected immunologic outcome of passive OVA airway exposure (i.e., inhalation tolerance) and facilitates instead a response that immunologically and functionally resembled asthma.
Mechanistically, it has been proposed that HDM may contribute to allergic sensitization in a number of ways. For example, there is evidence that HDM disrupts cell monolayers and degrades tight junctions in the airway epithelium in vitro (22eC25). This would facilitate its penetration across the airway epithelial barrier (26) and, presumably, increase its accessibility to antigen presenting cells located in the subepithelial compartment (26). In addition, it has been suggested that HDM may privilege the generation of a Th2-polarized response by cleaving CD25 (IL-2R chain [27]) and by modulating the balance between IL-4 and IFN- (28). It has also been suggested that cleavage of CD23 (low-affinity IgE receptor) may promote and enhance an IgE immune response (29, 30). Notably, these various effects are related to HDM's intrinsic proteolytic activity, an attribute shared by a number of common environmental aeroallergens (31eC35) and fungi (11, 36, 37). In this regard, Kheradmand and colleagues (11) have recently shown that the proteolytic activity of Aspergillus fumigatus is necessary and sufficient to elicit airway eosinophilic inflammation. However, it has become increasingly clear that proteases can have more direct proinflammatory effects. Indeed, it has been shown that various dust mite allergens act on bronchial epithelial cells in vitro to elicit the production of a number of cytokines (38eC40), including GM-CSF, a powerful maturation and activating signal for dendritic cells, and that such an effect is elaborated via proteolytic activity (39, 40). We suggest that HDM-induced GM-CSF may play a significant role in the findings that we report here. First, there is evidence that exogenous administration of GM-CSF to the airway generates a Th2 immune-inflammatory response not only to OVA (16) but also to allergens with weak proteolytic activity, such as ragweed (21). Moreover, we have recently shown that intranasal administration of HDM daily for 10 days induces allergic sensitization and Th2 airway inflammation, both of which are substantially reduced by concurrent treatment with antieCGM-CSF antibodies (6). Thus, we would propose that HDM may elicit a cascade that conditions the airway microenvironment with cytokines such as GM-CSF, and likely others, to facilitate the generation of a long-lived Th2-polarized immune-inflammatory response to OVA and itself. Unfortunately, a protocol that involves continuous exposure to HDM and OVA for 5 weeks disallowed the use of GM-CSF or GM-CSF receptoreCdeficient mice due to their short survival span; moreover, GM-CSF neutralization was prohibitive, economically, and ill-advised, immunologically, due to the likelihood of triggering a type III hypersensitivity reaction with such prolonged antibody treatment.
In summary, our data show that exposure to HDM extract establishes a mucosal environment that facilitates the generation of a bona fide Th2-polarized memory response to an otherwise innocuous antigen, OVA. We believe that the clinical significance of these findings is apparent. Indeed, it is likely that humans are simultaneously exposed to an assortment of different aeroallergens with different antigenic potential. From this perspective, exposure to the pervasive aeroallergen HDM may facilitate or amplify the response to other allergens with a lower intrinsic ability to cause disease. By extrapolation, our data may highlight the importance of controlling exposure to HDM, and other indoor allergens, in the overall management of asthma and specifically to curtail the development of poli-allergen sensitization. Moreover, the implications of concurrent exposure can probably be extended beyond the domain of aeroallergens. For example, the potential of certain pollutants to elicit allergic airway disease has been established in both experimental models (41, 42) and in humans (43), and perhaps the ability of certain viruses, such as respiratory syncytial virus (RSV), to facilitate allergic airway disease stems from a similar mechanism (44). Ultimately, the findings that we report here compel us to consider that whether a certain aeroallergen will generate allergic airway disease may depend, at least in part, not on simply being exposed to it but rather the context in which that exposure occurs.
Acknowledgments
The authors gratefully acknowledge the secretarial assistance of Mary Kiriakopoulos and the technical help of Jennifer Wattie. They also thank Gordon Gaschler, Clinton S. Robbins, and Ryan E. Wiley for critical review of the manuscript and Dr. Anthony J. Coyle (Millennium Pharmaceuticals, Inc.) for the anti-T1/ST2 Ab.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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