INTRODUCTION

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Asthma is characterized by mucosal inflammation of the airways and is closely related to atopy, which is characterized by enhanced IgE responses to common environmental allergens. Epidemiologic studies link total serum IgE concentrations with the incidence of asthma symptoms and bronchial hyperresponsiveness (1, 2). An important characteristic of IgE is its ability to bind to mast cells and basophils with high affinity through its Fc portion to the IgE receptor (Fc RI) (3, 4). FcRI comprises an  chain, a  chain, and two disulphide-linked  chains. Alpha chain is involved in IgE binding and receptor internalization, resulting in the allergic mediator release process, whereas chain plays a role in signal transduction (3, 4). Cross-linking of the Fc RI, even in the absence of IgE, results in mast-cell triggering (5). In sensitized persons, the interaction of FcRI-bound IgE with the relevant antigen elicits an immediate reaction characterized by mast-cell degranulation and release of preformed mediators and cytokines.

Apart from mediating the immediate response, the allergen-induced late phase skin reaction (LPR) has also been shown to be IgE-dependent (6, 7). Biopsies of allergen-induced cutaneous late phase reactions have demonstrated IgE-bound antigen-presenting cells such as epidermal Langerhans' cells and dermal dendritic cells (8, 9). More recently, it has been demonstrated that Langerhans' cells (10, 11), dermal dendritic cells (10, 12), peripheral blood monocytes (13), and eosinophils (14) may all express the high affinity receptor for IgE (FcRI). FcRI, therefore, is being considered as a critical component of the effector arm of the allergic response in mediating both early and late phase responses. We have recently demonstrated elevated numbers of FcRI+ cells in the bronchial submucosa of patients with stable, mild asthma compared with control subjects (17). Colocalization studies revealed that the majority of FcRI+ cells were identified as mast cells and macrophages and a much smaller percentage were eosinophils. However, a variable proportion (0 to 40%) of these eosinophils were FcRI+. Because of the relatively small numbers of eosinophils in this baseline study, we felt it important to reevaluate expression of Fc RI by bronchial eosinophils after allergen challenge.

	METHODS

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Patients

Nine patients (four male and five female) with a history of house-dust-mite-sensitive asthma and four nonatopic, normal subjects (three male and one female) took part in the study (Table 1). Subjects were recruited from the Allergy Clinic and from the general public after an advertisement in the local newspaper. All subjects were nonsmokers. Asthmatic subjects were chosen on the basis of (1) a clear history of asthma symptoms, (2) histamine PC₂₀ value of < 16.0 mg/ml in the previous 2 wk, (3) a strongly positive skin weal response (> 5 mm) to prick testing with Dermatophagoides pteronyssinus extract (Soluprick; ALK, Horsholm, Denmark), and (4) positive RAST test for the same house dust mite extract (Table 1). Normal volunteers were asymptomatic, had negative skin responses to a panel of 11 common aeroallergens, including house dust mite, animal danders, moulds, and grass pollen (Soluprick; ALK), and negative RAST tests for the same panel of allergen extracts. None of the subjects was receiving oral steroids or theophylline preparations during the previous 6 mo, and inhaled steroids were withheld for 72 h prior to the study. None of the subjects had a history of lower respiratory tract infection in the preceding 4 wk or of any current medical illness other than atopic asthma. Subjects with FEV₁ of less than 80% predicted and histamine PC₂₀ < 1.0 mg/ml were excluded. None of the patients had received immunotherapy in the previous 5 yr. The study was approved by the Ethics Committee of the Royal Brompton National Heart and Lung Hospital, London, and informed written consent was obtained from each subject.

Study Design

Subjects were asked to attend the laboratory to undergo two fiberoptic bronchoscopies separated by 24 h. Fiberoptic bronchoscopy was undertaken as previously described (17). In brief, all subjects received nebulized albuterol (2.5 mg) and intravenous atropine (0.6 mg) and midazolam (0 to 10 mg) 10 min prior to the procedure. Oxygen was delivered via the nasal canulae throughout the procedure, and oxygen saturation was monitored by pulse oximetry. Local anaesthesia of the upper airways was induced with 2 to 4% lidocaine. The bronchoscope (BFP20; Olympus Corp., Lake Success, NY) was then passed through the nares or mouth, and as much as 10 ml of lidocaine were introduced through the bronchoscope to anaesthetize the vocal cords and lower airways. After inspection of the bronchial tree, the bronchoscope was wedged in the lateral subsegment of the right middle lobe to undertake a control challenge with 5 ml of sterile saline solution prewarmed to 37° C. The bronchoscope was then passed into the lateral subsegment of lingula of the left lung, and 5 ml of prewarmed saline containing 100 BU of allergen (Der p) were instilled. Allergen and control challenge sites were randomly chosen for individual subjects. The appearance of the airways was then observed for 5 min. If there was no visible reaction to 100 BU of allergen, a further 5 ml of saline containing 400 BU of allergen were then instilled into the allergen challenge site and was observed for 5 min to confirm an airway response (18). The subjects were then observed closely, and a second bronchoscopy was performed with the same premedications and oxygenation at 24 h after segmental allergen challenge. After inspection of the bronchial tree, BAL was performed using 120 ml of prewarmed 0.9% saline solution at each challenge site. All subjects were then observed closely for 2 h.

Processing of Bronchoalveolar Lavage Fluid

BAL fluid was passed through two layers of sterile gauze to remove debris and then centrifuged at 500 × g for 10 min at 4° C. The pellet was then resuspended in RPMI 1640 (Chester Beatty Laboratories, London, UK) supplemented with 2 U/ml sodium heparin. The fluid was then centrifuged again at 500 × g for 10 min. The cells were then resuspended at 0.25 to 0.5 × 10⁶ cells/ml in RPMI-1640 medium (GIBCO, Paisley, UK) supplemented with 100 IU/ml penicillin/streptomycin (GIBCO), 2 mM L-glutamine (GIBCO), and 5% human AB serum (Sigma, Poole, Dorset, UK); 100 µl of cells were used per poly- L-lysine-coated slide and mounted on Shandon 190005 filter cards and then centrifuged in a Shandon 2 machine for 5 min at 500 rpm (Shandon Ltd, Runcorn, Cheshire, UK). Slides were then air-dried and fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature, and this procedure was repeated again. Cytospins were then immersed in 15% sucrose for 30 min and then rinsed in PBS to remove sucrose. The cytospins were then dried overnight at 37° C incubator. The slides were stored at 80° C pending analysis.

Differential Cell Count

A differential cell count was performed on unfixed cytospins of BAL cells using May-Grunwald Giemsa stain.

In Situ Hybridization

The cDNA fragment encoding FcRI  (bp 25-936) was kindly provided by Dr. J.-P. Kinet (Molecular Allergy and Immunology Section, National Institute of Allergy and Infectious Diseases, Rockville, MD) (19). This cDNA segment was inserted into the RNA expression vector (pGEM7) (Promega, Southampton, UK) and linearized to produce antisense and sense riboprobes. ³⁵S-labeled riboprobes were prepared with Sp6 or T7RNA polymerase (Promega) to generate antisense or sense probes, respectively. In situ hybridization (ISH) of cytospins prepared from BAL cells were performed according to a protocol that has been validated previously (16), with some modifications. In brief, cytospins were made permeable by immersion in 0.3% Tritron × 100 in PBS for 10 min. After a brief washing in PBS, slides were further made permeable by exposure to proteinase K (Promega) solution (1 mg/ml in 20 mM TRIS-HCl and 1 mM EDTA at pH 7.2) for 20 min at 37° C, the activity of which was then terminated by immersion in 4% paraformaldehyde/PBS for 5 min. To inhibit nonspecific binding of ³⁵S-labeled probes, slides were treated with 10 mM iodoacetamide and N-ethyl-malemide (Sigma) for 30 min at 37° C and then treated with 0.5% acetic anhydride and 0.1 M triethanolamine for 10 min before air-drying. Slides were hybridized with ³⁵S-labeled riboprobes (either antisense or sense at 10⁶ cpm per slide) at 55° C overnight. After hybridization, the slides were washed under high-stringency conditions (65° C, 0.1 SSC). After dehydration, slides were immersed in K-5 emulsion (Ilford Ltd, Ilford, Essex, UK) and exposed for 2 weeks at 4° C. The autoradiographs were developed in developing solution (D-19; Eastman, Kodak Co., Rochester, NY), fixed with Hypam (Ilford), counterstained with hematoxylin and chromotrope 2R (BDH, Poole, UK) for 30 min. For negative controls, slides were hybridized with a sense probe or pretreated with RNase (Sigma) prior to hybridization with the antisense probe.

Immunocytochemistry

A noncompetitive murine monoclonal antibody 22E7 (a kind gift from Drs. R. Chizzonite and J. P. Kochan, Hoffman La Roche Inc., Nutley, NJ; concentration, 10 µg/ml) directed against the  chain of Fc RI was used for immunocytochemistry using the APAAP method (17). As a negative control, the primary antibody was replaced with nonspecific mouse IgG.

Cells on cytospin slides were first treated with 1% hydrogen peroxide and 0.02% sodium azide in PBS to block endogenous peroxidase. Immunocytochemistry was then performed using a vector kit (Vector, Peterborough, UK) consisting of rabbit serum, noncompetitive murine monoclonal antibody 22E7 (10 µg/ml), biotinylated rabbit antimouse antibody, and avidin biotin complex. The reaction was developed in SG substrate (Vector). 22E7+ cells stained grey/black. Slides were then washed in water after immunocytochemistry and counterstained with chromotrope 2R (BDH) for 45 min. The slides were then dehydrated and mounted in DPX.

Quantification

Slides were counted blind in a random coded fashion using a BH2 microscope (Olympus Corp.) fitted with an eyepiece graticule. For in situ hybridization studies, hybrids between FcRI chain mRNA and cRNA probes were localized as dense collections of silver grains in photographic emulsion overlying individual cells. Cells expressing Fc RI chain-specific mRNA were quantified in terms of the number of cells with overlying silver grains. For each subject (control and allergen challenged), at least 200 total BAL cells were counted. Chromotrope 2R+ cells (red color) were considered as eosinophils. For immunocytochemistry, counts were performed at magnification ×200 and cells double-stained with FcRI+/chromotrope 2R+ were confirmed at magnification ×1,000 under oil immersion. For both techniques, random fields on slides were counted. Within-observer mean coefficient of variation for cell counts was always less than 5%.

Statistical Analysis

Data were analyzed using a Microsoft Excel statistical package. Wilcoxon's matched-pairs signed-rank test was used for within-group comparisons. Between groups, the net increases in cell counts (allergen minus control) were compared using the Mann-Whitney U test. Correlation coefficients were obtained using Spearman's rank analysis; p values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Clinical

Relevant details of the patients and normal volunteers studied are shown in Table 1. The two groups were well matched for age and sex. All asthmatics had circulating Der p specific IgE, as demonstrated by positive RAST tests (mean, 38.5 IU/ml), whereas this was not detectable in the nonatopic control subjects (Table 1). The asthmatics also demonstrated airway hyperresponsiveness to inhaled histamine; mean value being 2.02 mg/ml (p < 0.05). Seven of the nine asthmatic subjects reacted to the instillation of Der p allergen by visible constriction of the challenged bronchus. This was accompanied by local wheezing determined by auscultation. At 24 h, slight mucosal swelling was visible in some of these patients, but the airway caliber appeared normal. No change in airway caliber was visible after saline control challenge. On the other hand, nonatopic normal subjects demonstrated no visible changes in airway caliber or hyperemia after both allergen and saline challenges.

BAL Differential Cell Counts

The absolute numbers of macrophages, lymphocytes, neutrophils, and eosinophils in lavaged bronchial segments after control saline and allergen challenge of atopic asthmatics and normal control subjects are shown in Figure 1. After segmental challenge with Der p, there was a significant increase in the absolute numbers of BAL eosinophils, median values of 0.15 × 10⁶ (0 to 0.55 × 10⁶) and 11.1 × 10⁶ (0.5 to 46.5 × 10⁶) cells (p = 0.007) for control and allergen, respectively (Figure 1). BAL lymphocytes and neutrophils also showed significant increases after allergen (Figure 1). On the other hand, no significant changes in BAL macrophages was observed after allergen provocation (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Composition of cells in bronchoalveolar lavage in atopic asthmatic (n = 9) and normal (n = 4) subjects after saline and allergen challenge. Between groups, the net increases in cell counts (allergen minus saline) are compared. Horizontal bars represent median values.

BAL FcRI+ Cell Count

When compared with a control (diluent) challenge, in situ hybridization studies demonstrated a 6-fold increase in FcRI mRNA+ cells after allergen; median values were 2 (0.7 to 7.2) and 11.5 (0.6 to 65.0) × 10⁶ cells for control and allergen, respectively (Figure 2A). Net increases in FcRI mRNA+ cells correlated with the net increases in BAL eosinophils (r = 0.98, p = 0.0001). Similarly, the total numbers of FcRI chain protein-bearing cells also increased after allergen; median values were 0 (0 to 0.30 × 10⁶) and 3.1 × 10⁶ (0.45 to 162.5 × 10⁶) cells (p = 0.007) (Figure 2B). Net increases in FcRI+ cells correlated with the net increases in BAL eosinophils (r = 0.72, p = 0.02). There was no significant change in FcRI+ cells in BAL after allergen in normal control subjects (Figure 2).

View larger version (14K): [in this window] [in a new window]   Figure 2.   Expression of FcRI (A) mRNA and (B) protein in BAL cells in atopic asthmatic (n = 9) and normal (n = 4) subjects after saline (open circles) and allergen (closed circles) challenge. Horizontal bars represent median values.

Colocalization of FcRI mRNA and Protein to BAL Eosinophils

In situ hybridization studies in nine asthmatics demonstrated that only 4% of FcRI mRNA+ cells were colocalized to chromotrope 2R+ eosinophils after saline (control) challenge (Table 2). On the other hand, 85% of FcRI mRNA+ cells colocalized to eosinophils after segmental allergen challenge (Table 2 and Figure 3A and B). The eosinophil numbers were very low in BAL after control challenge (1 to 2 per whole fields counted), and 75% of them expressed Fc RI chain mRNA. Segmental allergen challenge resulted in significant BAL eosinophilia (Figure 1), and 80% of these eosinophils showed positive hybridization signals for FcRI mRNA.

View larger version (139K): [in this window] [in a new window]   Figure 3.   BAL cytospin from an atopic subject 24 h after segmental allergen challenge. A and B: Hybridization in situ with FcRI chain antisense probe and counterstained with chromotrope 2R (solid arrow) and single mRNA+ cells (open arrowhead ) (A: bright field; B: dark field). C and D: Cytospins immunostained with 22E7 and counterstained with chromotrope 2R. Double-stained cells are indicated by solid arrowhead; single-stained cells against FcRI (C ) and eosinophils (D) are indicated by open arrowhead.

In order to confirm colocalization of FcRI chain protein to eosinophils, colocalization studies with 22E7 and chromotrope 2R were performed in six asthmatic subjects who had appreciable numbers of 22E7+ cells by single staining. These studies revealed that 95% (93 to 96%) of Fc RI+ cells stained with chromotrope 2R (Table 3 and Figure 3C and D). We also examined the percentage of chromotrope 2R+ cells in BAL that were immunostained with 22E7; 91% (26 to 100%) of 2R+ cells coexpressed the FcRI subunit. There were no changes in FcRI+ cells, eosinophils, or other cell populations in normal control subjects after allergen challenge (Figures 1 and 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated increased numbers of FcRI mRNA and protein-bearing cells in BAL of subjects with atopic asthma after segmental allergen challenge. We confirmed that the predominant FcRI+ cells (85 and 93%) in BAL after allergen exposure were eosinophils and 80 to 91% of the eosinophils expressed both mRNA and protein for FcRI+. The fact that similar changes were not observed in the nonatopic control subjects suggests that these changes are dependent upon the presence of allergen-specific IgE and not upon some nonspecific effect of locally instilled allergen.

Allergen-induced increases in BAL Fc RI+ cells were observed in the asthmatics within the time frame of the late phase bronchoconstrictor response to global allergen bronchial challenge, which is well known to be associated with a dose-dependent influx of inflammatory cells, particularly eosinophils, lymphocytes, and neutrophils, into the bronchial lumen 18 to 24 h after allergen exposure (18, 20, 21). The advantage of the segmental challenge model is that it allows simultaneous challenge with allergen and diluent in remote bronchial segments, thus ensuring that each subject acts as his or her own control. The disadvantage is that global bronchoconstrictor responses cannot be measured. Nevertheless, seven of the nine atopic subjects in our study clearly demonstrated an immediate segmental bronchial response 5 to 10 min after allergen challenge, and this observation coupled with the characteristic inflammatory cell influx 24 h later provide compelling evidence that the observed allergen-induced increases in FcRI+ cells in the asthmatics occurred within the context of a late phase inflammatory response to allergen challenge.

In this study we took mRNA and protein expression of the  chain of the high affinity receptor for IgE (FcRI) as an indicator of their potential to be activated by IgE-mediated mechanisms. Because cytospins were used in this study some immunostaining may reflect intracellular rather than membrane staining. The biologic importance of FcRI in mediating IgE-dependent immune reactions has been demonstrated in a number of different studies (5, 14, 22). It has been speculated that eosinophils may also be involved in antigen presentation in allergic diseases via IgE linked to FcRI (28). FcRI has also been implicated in IgE-dependent schistosomal killing by eosinophils (14).

Our data show that a median of 80 to 91% of eosinophils recruited to the bronchial lumen of atopic asthmatics 24 h after local allergen challenge expressed the  chain of FcRI, although there was some variability in this figure. Furthermore, eosinophils comprised a median of 85 to 95% of the total FcRI+ cells so recruited (Tables 2 and 3). Our results are consistent with those observed by Barata and colleagues (16) in the skin after allergen challenge. These findings contrast strikingly with the distribution of FcRI chain expression on cells within the bronchial mucosa and peripheral blood of stable, atopic asthmatics at "baseline" (i.e., in the absence of allergen challenge). We found some FcRI mRNA expression after control challenge in both asthmatics and normal subjects, but no significant protein expression was seen (Figure 2). This dichotomy might be due to the difference in sensitivity of the two methods employed. However, only a few eosinophils were present after control challenge expressing Fc RI mRNA, and only 4% of FcRI mRNA+ cells were eosinophils at "baseline" (control challenge). Consistent with this, three recent studies on such patients have suggested that, in both the bronchial mucosa and the peripheral blood, mast cells/basophils and monocytes/macrophages constituted the majority of FcRI+ cells, whereas eosinophils made up only a small percentage (17, 29, 30). The remainder of the FcRI+ cells recovered from the bronchial lumen of the atopic asthmatics after allergen challenge in the present study were presumably other cells (monocyte/macrophages, mast cells/basophils and dendritic cells) known to express FcRI constitutively (12, 13, 17, 29, 30). Our data do not permit accurate quantification of the distribution of FcRI+ expression on these cells. Because the numbers of FcRI+ cells and eosinophils in the bronchial lumen of the atopic asthmatics after control challenge were in general very low, our data considered with the data referred to the above concerning FcRI expression in the blood and bronchial mucosa of these patients suggest a process whereby eosinophils expressing both mRNA and protein for FcRI are preferentially recruited to the bronchial lumen of these patients. The mechanism of this process remains to be defined, but it might include synthesis and upregulation of FcRI expression on eosinophils in transit from the peripheral blood to the bronchial lumen after interaction with cytokines such as IL-5, IL-3, or GM-CSF (31), selective recruitment of FcRI-expressing eosinophils from the blood or bronchial mucosa, or both. Again, this process would appear to be IgE-dependent since it was not observed in the nonatopic control subjects in the present study after allergen challenge under identical conditions.

There are early indications that FcRI expression on eosinophils may play an important role in mediating eosinophil function in inflammatory processes. In peripheral blood monocytes, allergen-specific IgE bound to surface FcRI receptors has been shown to enhance allergen presentation to T cells, and it has been speculated that eosinophils may also present antigen in this way (26). Gounni and colleagues (14) provided the first evidence that cross-linking of Fc RI receptors on eosinophils obtained from patients with hypereosinophilic syndrome resulted in eosinophilic degranulation and participated in eosinophil-mediated cytotoxicity against Schistosoma mansoni. Release of cationic proteins such as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO) are believed to mediate, at least in part, the bronchial epithelial damage characteristic of asthma (31).

We therefore propose that, in vivo, allergen exposure in sensitized asthmatics not only upregulates eosinophilopoesis and eosinophil recruitment to the site of allergen exposure but also upregulates expression of FcRI mRNA and protein by these cells. Our study indicates that, in contrast to mast cells, macrophages, and dendritic cells, which constitutively express FcRI, eosinophils may be the predominant cells in BAL expressing FcRI after endobronchial allergen exposure. This raises the possibility that FcRI-expressing eosinophils may therefore participate in IgE-mediated allergic inflammatory responses. Further studies should focus on the regulation and functional significance of FcRI expression by eosinophils and their IgE-dependent activation.