American Journal of Respiratory Cell and Molecular Biology

Eotaxin is an eosinophil-specific chemokine associated with the recruitment of eosinophils to the site of allergic inflammation. The aims of this study were to determine the expression of eotaxin in nasal biopsies from allergic and nonallergic individuals with chronic severe sinusitis, and to examine whether the expression of this chemokine is upregulated following allergen challenge in the nasal mucosa of patients with allergic rhinitis. We also undertook to phenotype of inflammatory cells within the submucosa expressing eotaxin mRNA. Nasal turbinate tissue from 16 individuals with allergic or nonallergic chronic sinusitis and 10 normal controls were examined for the presence of eotaxin mRNA and immunoreactivity by in situ hybridization and immunocytochemistry. The numbers of cells expressing eotaxin mRNA were also determined after either allergen or diluent challenge in atopic subjects with a history of allergic rhinitis. There was a constitutive expression of eotaxin-immunoreactivity and the presence of eotaxin mRNA-positive cells in nasal biopsies from normal individuals. Compared with normal controls, the numbers of cells expressing eotaxin mRNA and protein were significantly increased in both allergic and nonallergic sinusitis (P < 0.001). Eotaxin mRNA was expressed by nasal epithelial cells and primarily colocalized to CD68-positive macrophages within the subepithelium. In subjects with allergic rhinitis, allergen challenge markedly increased the numbers of cells expressing eotaxin mRNA and immunoreactivity in the epithelial and subepithelial cell layers (P < 0.05). This could be largely attributed to a local increase in eotaxin production within the nasal tissues. The results of this study demonstrate the constitutive expression of eotaxin and show that the numbers of cells expressing eotaxin mRNA are increased within the epithelial and subepithelial layers of the nasal mucosa in individuals with chronic sinusitis. Furthermore, allergen challenge of the nasal mucosa in atopic subjects results in a local upregulation of eotaxin expression. These data suggest a potential role for this chemokine in the pathogenesis of allergic and nonallergic eosinophilic inflammation characterizing chronic sinusitis and allergic rhinitis.

Persistent infiltration of the nasal mucosa by eosinophils is thought to contribute to the underlying pathophysiology of allergic inflammatory diseases such as chronic sinusitis (CS) and allergic rhinitis (1– 4). Indeed, the activation of eosinophils and the subsequent release of cytotoxic mediators and cytokines, has been proposed to account for the epithelial desquamation, subepithelial fibrosis and hyperresponsiveness characterizing allergic inflammation (2, 5, 6). From their site of production within the bone marrow, eosinophils must transit through the bloodstream to elicit a tissue-specific action. While the interactions between specific adhesion molecules and the migration of eosinophils into the airways is well delineated (7), there is little in vivo information regarding the ability of local tissue factors to induce eosinophil accumulation.

Eotaxin was initially described as the predominant eosinophil chemoattractant in the bronchoalveolar lavage fluid from allergen-challenged guinea pigs (8, 9). This unique 8 kD C-C chemokine is highly selective in its actions on eosinophil recruitment (9) and is upregulated by cytokines such as IL-3, IL-4, IFN-γ, and TNF-α, which have been associated with allergic and nonallergic eosinophilic inflammation (10-12). While there is little information to date regarding the expression of eotaxin in human tissues, preliminary reports suggest that eotaxin-immunoreactivity is present in nasal mucosa and polyp tissue (13). The phenotype of cells producing eotaxin mRNA and its relationship to the degree of eosinophil and other inflammatory cell infiltration remains to be assessed.

Our hypothesis was that the expression of eotaxin would be increased locally in disorders associated with the infiltration of eosinophils, such as CS. Furthermore, since eosinophils are actively recruited to the nasal mucosa during acute allergic inflammation, we hypothesized that the numbers of cells expressing eotaxin mRNA and immunoreactivity would be increased following allergen challenge in patients with allergic rhinitis. The aims of this study were to determine the expression of eotaxin mRNA and immunoreactivity in nasal biopsies from individuals with allergic and nonallergic CS and normal controls, and to investigate the cellular source of eotaxin. We also proposed to investigate the expression of eotaxin mRNA and protein following allergen challenge in nasal mucosa of patients with allergic rhinitis and determine its relationship to eosinophilic inflammation.

Patient Selection

Nasal mucosal biopsies were taken from 16 patients with chronic perennial sinusitis and 10 normal control subjects. The clinical characteristics of the subjects are given in Table 1. Individuals with sinusitis were selected on the basis of a history of more than 1 yr of chronic perennial sinusitis with one or more of the following symptoms: chronic perennial nasal congestion, chronic rhinorrhea, chronic postnasal drip, anosmia, ageusia, and pain or discomfort in the regions of the sinuses. Each patient signed an informed consent form that was approved by the Hospital Review Board. All patients had nasal polyps and radiographic evidence of severe sinusitis on a recently taken sinus computed tomographic scan. On the basis of the results of allergy skin testing using a panel of at least 15 common allergens, patients were subdivided into an allergic or nonallergic subgroup. Patients were excluded if they had received systemic steroids greater than their average daily dose (< 5 mg/day of prednisone or equivalent) during the month preceding the study. Antibiotics were withheld for 4 wk, and inhaled nasal steroids were withheld for 2 wk before the study.

Table 1. Clinical characteristics of patients with allergic and nonallergic sinusitis and normal controls

Allergic Sinusitis (n = 7)Nonallergic Sinusitis (n = 9)Normal Controls (n = 10)
Sex
 Male  4 5  8
 Female  3 4  2
Age (years)
 Median 3133 29
 Range(19–38)(23–48)(22–38)
Serum IgE (U/ml)
 Mean190.095.4* 100.9*
 SEM  9.8 3.7  5.5
Associated asthma  2 2  0

*P < 0.001 compared with allergic sinusitis.

As a control population, normal individuals with no history of allergies, asthma, sinus disease, previous sinus surgery, recent upper respiratory tract infections, or cigarette smoking within the previous 3 yr were biopsied. Nasal mucosal biopsies were obtained from the inferior or middle turbinates.

Nasal biopsies were also taken from 7 individuals with grass pollen seasonal allergic rhinitis who exhibited positive skin prick tests to Timothy grass pollen extract. None had received topical or oral medication in the 6 mo before the study nor immunotherapy in the previous 5 yr. Nasal biopsies were performed outside the grass pollen season at a time when the patients were asymptomatic. Patients were challenged by the application of 7 μl solution of 1,000 biological units (BU) of grass pollen extract or its appropriate diluent on 4-mm filter paper discs to the inferior nasal turbinate, as previously described (14). At 24 h, nasal biopsies (2.5 mm) were taken under local anesthesia. Informed written consent for this study was approved by the Hospital Ethics Committee.

Tissue Preparation

For the individuals with CS and the normal control subjects, biopsy specimens of maxilliary sinus tissue were obtained. Generally, two to six specimens were obtained from both right and left maxilliary sinuses. For in situ hybridization, one set of nasal biopsy specimens was immediately fixed in 4% paraformaldehyde for 4 h, washed in 15% sucrose in phosphate-buffered saline overnight, blocked, and then sectioned at 10-μm thickness onto poly-l-lysine coated slides. Another set of clinical specimens was snap-frozen in liquid nitrogen and stored at −70°C until processing for immunocytochemical analysis.

In Situ Hybridization

To detect eotaxin mRNA, the technique of in situ hybridization (ISH) using a digoxigenin-labeled cRNA probe was used as previously described (15). The cDNA probe was constructed as previously described (11). Briefly, the coding region of the human eotaxin cDNA was amplified by PCR using cDNA generated from colon RNA. The PCR product was subsequently cloned into the PCRII vector (TA vector; Invitrogen Corp., San Diego, CA) and the sequence confirmed by automated sequences of both strands. Probes were generated from cDNA and transcribed in the presence of SP6 or T7 polymerases, and labeled with digoxigenin-11-UTP to generate antisense and sense probes, respectively. The sections were permeabilized with proteinase K and then prehybridized with 50% formamide and 2× standard saline citrate. Following application of the probe, the sections were hybridized at 42°C overnight. Nonspecific binding was removed by posthybridization washing under high stringency conditions and subsequent treatment with RNase. The hybridization signal was visualized by incubating the sections overnight with sheep polyclonal anti-digoxigenin antibodies conjugated with alkaline phosphatase. Color development was achieved by adding the freshly prepared substrate (X-phosphate-5-bromo-4-chloro-3-indoly phosphate [BCIP] and nitroblue tetrazolium [NBT]). To ensure the specificity of our signal, we performed the ISH technique using the sense probe, and following pretreatment of the tissues with RNase.

Immunocytochemistry

Eotaxin-immunoreactivity was detected in 5-μm tissue sections by the use of an eotaxin-specific polyclonal antibody raised against human eotaxin supplied by Dr. A. Luster. This antibody has previously been shown to be specific for eotaxin in dot blot and ELISA assays, and has no cross- reactivity against MCP-1, -2, -3, -4, RANTES, MIP-1α, or MIP-1β. The specificity of the antibody-antigen reaction was tested by preabsorption of the antibody with excess antigen and by using nonspecific rabbit immunoglobulin. The reaction was visualized using the avidin-biotin-peroxidase complex method (16). The identification of inflammatory cells within the nasal biopsies were performed using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique as previously described (14). Specific monoclonal antibodies detecting total eosinophils (anti-MBP; a gift from Dr. Redwan Moqbel, University of Alberta, Edmonton, AB, Canada), macrophages (anti-CD68; Dako Diagnostics, Mississauga, ON, Canada), pan-T cells (anti-CD3; Becton Dickinson Canada, Inc., Mississauga, ON, Canada) and human mast cells (anti-tryptase; Chemicon International Inc., Temecula, CA) were used. For negative control preparations, the primary antibody was replaced by either nonspecific mouse immunoglobulin or Tris-buffered saline.

Simultaneous In Situ Hybridization and Immunocytochemistry

The phenotype of the cells expressing eotaxin mRNA was determined using the technique of simultaneous in situ hybridization and immunocytochemistry as previously described (17). Briefly, the sections were immunostained with monoclonal antibodies to CD3 (T cells), CD68 (macrophages), MBP (eosinophils), and anti-tryptase (mast cells), and hybridized with digoxigenin-labeled cRNA probes coding for eotaxin. The hybridization signal was developed with a freshly prepared solution of BCIP and NBT. Double stained cells were visualized by the end product of immunostaining (red) and in situ hybridization (blue).

Double Immunocytochemistry

To confirm the phenotype of eotaxin-immunoreactive positive cells we undertook double sequential immunocytochemistry as previously described (18). Briefly, endogenous peroxidase activity in cryostat sections of nasal biopsies was blocked using 1% H2O2 (plus 0.02% sodium azide in TBS) for 30 min. A mixture of primary antibodies was then applied consisting of the polyclonal anti-eotaxin antibody used to detect eotaxin-immunoreactivity and the appropriate monoclonal antibody to determine cellular phenotype (CD3, CD68, MBP, and anti-tryptase). After incubating with the appropriate secondary antibodies, a tertiary layer of streptavidin peroxidase and murine APAAP conjugate were then applied. Sections were developed sequentially in Fast Red (the APAAP substrate) and diaminobenzidine (DAB; a peroxide substrate). Eotaxin-immunoreactive cells stained brown, the specific inflammatory cells stained red, and cells of a particular phenotype expressing eotaxin- immunoreactivity stained a reddish brown. To localize cells to the nasal epithelium, sequential immunostaining was performed using a polyclonal anti-keratin antibody (Dako) and a monoclonal antibody specific for eotaxin supplied by Dr. A. Luster. This anti-eotaxin antibody (5A3) has been previously shown to be specific for eotaxin in an immunoblot assay, and shows no cross-reactivity against MCP-1, -2, -3, -4, RANTES, MIP-1α, or MIP-1β. In these experiments, eotaxin-immunoreactive cells were visualized using the APAAP technique and stained red, while anti-keratin positive cells (epithelial cells) were identified using DAB and stained brown. In all immunochemical studies, the appropriate negative controls were included which included TBS alone, omission of the primary antibodies, and the use of an irrelevant mouse IgG antibody.

Quantitation

For immuncytochemical analysis and in situ hybrization, sections were counted by a blinded observer in a coded random order with an Olympus microscope with an eyepiece graticule at ×200 magnification. At least 2 sections were immunostained or hybridized from which six to eight fields were evaluated. Epithelial staining was assessed using morphometry and the results were expressed as the percentage of epithelium that expressed mRNA or immunoreactivity. Subepithelial eotaxin expression was presented as the mean counts of cells expressing mRNA and protein per high-power field (0.202 mm2). For the colocalization studies, the results were expressed as a percentage of the eotaxin mRNA positive cells which coexpressed either CD3, CD68, MBP, or tryptase.

Statistics

To compare the expression of eotaxin in CS versus controls a nonparametric Kruskal-Wallis with subsequent post hoc Mann-Whitney U tests (Systat v5.1, Elvaston, IL) was used. To compare eotaxin expression prior to and after allergen challenge, a Wilcoxon signed rank test was used. Correlation coefficients and significance values were obtained by the use of Pearson's moment correlation with a subsequent Bonferroni correction factor applied for multiple comparisons.

Using in situ hybridization, eotaxin mRNA-positive cells exhibited a dark purple staining within the nasal epithelium and submucosa of individuals with sinusitis and allergic rhinitis (Figure 1). The detection of eotaxin mRNA was observed only with the antisense probe, confirming the specificity of our hybridization technique. Using APAAP immunostaining, eotaxin-immunoreactive positive cells were observed as red cytoplasmic staining overlying cells within the nasal submucosa and could also be localized within the nasal epithelium (Figure 1c). Colocalization of eotaxin-immunostaining using DAB immunostaining, to inflammatory cells indicated macrophages (Figure 1d) and T cells were a source of eotaxin-immunostaining within the nasal submucosa.

Eotaxin mRNA and Immunoreactive Protein Expression in Chronic Sinusitis

The individuals with chronic sinusitis were subdivided into allergic (n = 7) and nonallergic (n = 9) subgroups according to the results of allergy skin testing. Both the allergic and nonallergic patients had an increase in the number of cells within the subepithelial layer expressing eotaxin mRNA when compared with normal controls (Figure 2; P < 0.001). This increase in eotaxin mRNA expression in allergic and non- allergic CS was also observed within the nasal epithelium (Figure 3; P < 0.005). Using immunostaining techniques, the numbers of cells within the subepithelial layer expressing eotaxin-immunoreactivity were also increased in allergic and nonallergic CS compared with normal controls (mean and SEM; allergic CS, 15.3 ± 3.2; n = 7; nonallergic CS, 17.1 ± 2.8, n = 9; normals, 8.5 ± 3.1; n = 10; P < 0.001). This profile of increased eotaxin-immunoreactivity in allergic and nonallergic CS was similarly observed within the airway epithelium (mean and SEM; allergic CS, 1.7 ± 0.4; n = 7; nonallergic CS, 2.0 ± 0.6, n = 9; normals, 0.85 ± 0.5; n = 10; P < 0.001).

Eotaxin Expression and Nasal Mucosal Inflammation in Chronic Sinusitis

In both subgroups, the increase in the number of cells expressing eotaxin mRNA and immunoreactivity was accompanied by elevated numbers of MBP-positive eosinophils and T lymphocytes within the subepithelium (Figure 2; P < 0.05). Tryptase-positive cells were also increased in number within the nasal mucosa of individuals with allergic sinusitis compared with controls (Figure 2; P < 0.05). In contrast, there were no differences in the numbers of CD68-positive cells in the nasal subepithelial layer between individuals with allergic and nonallergic sinusitis and normal controls (mean CD68-positive cells ± SEM; allergic sinusitis, 23.3 ± 6.2; n = 7; non-allergic sinusitis, 28.7 ± 2.8; n = 9; normal controls, 24.3 ± 3.2; n = 10; P > 0.05).

There was a significant association between expression of eotaxin mRNA and immunoreactive protein, and the numbers of resident eosinophils in allergic CS. This correlation was observed for both eotaxin mRNA and protein expression in the subepithelial cell layer (P < 0.05). No such association was observed in the individuals with nonallergic CS, nor were there any other significant associations between eotaxin mRNA and protein expression and the presence of other inflammatory cells in CS.

Eotaxin mRNA Expression and Nasal Mucosal Inflammation in Allergic Rhinitis

In subjects with allergic rhinitis, allergen challenge resulted in an increase in the density of cells, within the subepithelial cell layer, expressing eotaxin mRNA and immunoreactivity compared in diluent challenge (Figure 4; P < 0.05). This was accompanied by an increase in the number of cells exhibiting MBP-immunoreactivity (Figure 4; P < 0.05) and no change in the numbers of inflammatory cells showing positive immunostaining for CD68, CD3, and tryptase (Figure 4; P > 0.05). Within the nasal epithelium, there was a marked increase in the number of cells expressing eotaxin mRNA 24 h after allergen challenge when compared with the diluent challenge (Figure 5; P < 0.05).

To determine if the expression of eotaxin mRNA in the epithelial or subepithelial cell layers was quantitatively associated with the infiltration of inflammatory cells, we performed correlational analyses. There were no significant correlations between the expression of eotaxin mRNA and immunoreactive protein in the subepithelial cell layer following either allergen or diluent exposure, and the numbers of inflammatory cells within the submucosa (P > 0.05). Similarly, there were no significant correlations between the expression of eotaxin mRNA and immunoreactive protein in the epithelial cell layer and the numbers of inflammatory cells within the submucosa (P > 0.05).

Phenotype of Cells Expressing Eotaxin mRNA

Colocalization studies were performed in nasal biopsies from seven nonallergic sinusitis patients using simultaneous in situ hybridization and immunocytochemistry. The majority of eotaxin mRNA within the airways submucosa was associated with CD68-positive macrophages (mean ± SEM, 50.66 ± 0.03%; n = 7). Significant numbers of cells expressing eotaxin mRNA also expressed eosinophil and T-cell markers (mean ± SEM, MBP-positive eosinophils, 24.77 ± 0.01, n = 7; CD3-positive T lymphocytes, 17.26 ± 0.03, n = 7). In addition, a small number of eotaxin mRNA-positive cells were found to stain with the anti-tryptase antibody (mean ± SEM, tryptase-positive cells, 5.23 ± 0.01; n = 7). The remainder of the eotaxin mRNA-positive cells within the submucosa (approximately 2%) did not colocalize to any of the inflammatory markers used.

Eotaxin Expression and Clinical Indices

To determine whether the expression of eotaxin was related to the presence of associated asthma or serum IgE levels, we performed correlational analyses. Neither eotaxin expression (mRNA and protein) in the epithelium, nor in the subepithelium, was significantly correlated to IgE levels (P > 0.05). Similarly in patients with CS, the expression of eotaxin was not significantly different in the 4 individuals with associated asthma from those without associated symptoms (P > 0.05).

The aims of this study were to determine the expression of eotaxin mRNA and immunoreactive protein in the eosinophilic inflammation characterizing both allergic and nonallergic chronic sinusitis, and to examine the expression of this chemokine during acute eosinophil infiltration in allergic rhinitis. Our results demonstrate that the numbers of cells expressing eotaxin mRNA and protein are increased in allergic and nonallergic sinusitis and that gene transcripts are localized to the epithelium and to inflammatory cells within the nasal submucosa. We also showed that there is an increase in the numbers of cells expressing eotaxin mRNA and immunoreactivity following allergen challenge in allergic rhinitis, which could not be accounted for by the infiltration of inflammatory cells alone.

The recruitment of eosinophils to the nasal mucosa is a characteristic of both chronic sinusitis and allergen rhinitis (1– 4). Furthermore, the presence of these cells and their cytotoxic mediators has been associated with pathologic features, namely epithelial injury and desquamation, subepithelial fibrosis and hyperresponsiveness (5, 6). While several chemokines, such as eotaxin, RANTES, IL-8, and MCP-3 are known to induce eosinophil recruitment in vivo (8, 9, 19-21), the factors responsible for the infiltration of these cells in disease states such as sinusitis remain to be clarified. Our studies suggest that the activation of eotaxin gene transcription occurs within the nasal mucosa of individuals with sinusitis and following allergen challenge in allergic rhinitis patients. Furthermore, the significant correlations we observed between eotaxin mRNA and immunoreactive protein expression, and eosinophil infiltration in allergic CS would support a role for this chemokine in the ongoing recruitment of eosinophils to the site of inflammation.

Increased numbers of cells expressing eotaxin mRNA and protein were observed in both allergic and nonallergic sinusitis, suggesting that mechanisms independent of IgE production regulate the expression of eotaxin within the nasal mucosa. Several cytokines have been proposed to enhance eotaxin mRNA and protein production including IL-3, IL-4, IFN-γ, and TNF-α (10-12). Recently, it has been demonstrated that the profile of cytokine expression in allergic sinusitis resembles a Th2-type of response with increased numbers of cells expressing GM-CSF, IL-3, IL-4, and IL-5 (22). In contrast, nonallergic sinusitus is associated with the expression of GM-CSF, IL-3, TNF-α, and IFN-γ mRNA. It is possible that distinct cytokine pathways operate to enhance eotaxin expression in allergic and nonallergic eosinophil accumulation, respectively. Further studies are required to address the factors promoting eotaxin expression in allergic and nonallergic sinusitis.

Eotaxin mRNA expression was also markedly enhanced 24 h after allergen challenge in nasal biopsies from individuals with allergic rhinitis. Unlike the data presented in sinusitis, there were no significant correlations between the infiltration of inflammatory cells and eotaxin expression. These observations may suggest that other chemokines such as MCP-3 and RANTES may play a substantial role in the acute eosinophilic inflammation following allergen challenge (23, 24). In addition, the time course of chemokine production, its influence on the kinetics of eosinophil recruitment, and intersubject variability may also be responsible for the lack of correlation observed.

The increase in eotaxin mRNA and protein seen within the nasal submucosa as a result of allergen exposure may have reflected the infiltration of inflammatory cells. To determine whether eotaxin mRNA was upregulated locally and thus played a role in cellular recruitment, we examined the numbers and phenotype of cells infiltrating the nasal submucosa. Our data demonstrated that although there was significant eosinophil infiltration as a consequence of allergen challenge, the magnitude of this response was insufficient to fully account for the increased expression of eotaxin mRNA seen in our specimens. Thus, the large majority of eotaxin mRNA appeared to be derived from cells resident within the nasal submucosa.

An increase in eotaxin mRNA expression following allergen challenge has been previously reported in several animal models of allergic sensitization (8, 9, 25). The increase in eotaxin mRNA and protein levels 24 h following allergen challenge in allergic rhinitis is in contrast to the acute increase observed within the lungs 3 h after antigen exposure in the guinea pig (25). Whether these differences are attributable to species variation, the pattern of mediator/cytokine production, or organ specificities remains to be determined.

In nasal biopsies from normal individuals, there was a constitutive expression of eotaxin mRNA and immunoreactivity observed both in the epithelial and subepithelial cell layers. From clinical specimens, it has been previously demonstrated that eotaxin mRNA is constitutively expressed in the gastrointestinal tract and to a lesser extent in organs such as the heart and lungs (11). Constitutive expression of eotaxin has also been described in several organs in the guinea pig and mouse, including the lungs, lymph nodes, and skin (25-27). Since eosinophils are not normally found within the tissues of normal individuals, the action of eotaxin within these organs warrants further investigation. It is a distinct possibility that this chemokine acts to recruit eosinophils into the tissues only once they have been primed within the bloodstream by such hemotopoietic factors as IL-5 (28).

In our clinical specimens, we observed eotaxin mRNA and protein associated both with the airways epithelium and resident inflammatory cells in the nasal submucosa. Phenotyping the cells expressing eotaxin mRNA demonstrated that macrophages, eosinophils, and T lymphocytes were significant cellular sources of this chemokine. Previous data has suggested that in nasal mucosa and polyp tissue, eotaxin-immunoreactivity was most strongly localized to the ciliated pseudostratified epithelium (13). Positive immunoreactivity was also associated with leukocytes but no colocalization studies were performed.

In summary, we have shown that the numbers of cells expressing eotaxin mRNA and immunoreactivity are increased in allergic and nonallergic sinusitis, and that the expression of this chemokine is correlated to the eosinophil inflammatory cell infiltrate observed in allergic CS. Eotaxin mRNA and protein were localized both to the nasal epithelium and to inflammatory cells within the submucosa. Furthermore, we have described an increase in the local expression of eotaxin mRNA following allergen challenge in individuals with allergic rhinitis. These data indicate that eotaxin gene transcription may play an important role in the sequalea of events characterizing allergic and nonallergic eosinophilic inflammation. Inhibition of eotaxin production may prove to be favorable in the treatment of disorders associated with eosinophilic infiltration.

Dr. Eleanor Minshall is a recipient of a Medical Research Council/Canadian Lung Association Fellowship and Dr. Andrew Luster is supported by a Cancer Research Institute/Benjamin Jacobson Family Investigator Award. The authors would like to thank Ms. Elsa Schotman and Ms. Zivart Yasruel for their technical assistance.

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Supported by Network Centers of Excellence, MRC Canada, NIH HL36577, RR00051 and AI40618.
Address correspondence to: Dr. Qutayba Hamid, M.D., Ph.D., Meakins-Christie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, PQ, H2X 2P2 Canada. E-mail:

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