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The Journal of Immunology, 1999, 162: 6901-6911.
Copyright © 1999 by The American Association of Immunologists

Does IgE Bind to and Activate Eosinophils from Patients with Allergy?1

Hirohito Kita2, Masayuki Kaneko, Kathleen R. Bartemes, Deborah A. Weiler, Andrew W. Schimming, Charles E. Reed and Gerald J. Gleich

Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (Fc{epsilon}RI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the Fc{epsilon}RI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (Fc{gamma}RII, CD32), but no expression of Fc{epsilon}RI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-Fc{epsilon}RI mAb, suggesting that eosinophils do express minimal amounts of Fc{epsilon}RI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express ~0.5% of the Fc{epsilon}RI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophils play important roles in the pathophysiology of bronchial asthma and other allergic diseases (reviewed in 1). In such diseases, mediators released by T cells, epithelial cells, and other inflammatory cells induce migration of eosinophils from blood into the affected tissues (reviewed in 2). Subsequently, various stimuli trigger eosinophil activation, resulting in the local release of a series of inflammatory mediators, such as lipid metabolites (2, 3), superoxide anion (4), and toxic cationic granule proteins (5). Indeed, eosinophilic infiltration in the epithelium and submucosa of the airways has been a hallmark of the pathology of asthma and allergic rhinitis (6, 7). Correlations have been observed between the number of infiltrating eosinophils and asthma disease severity (6). In addition, pulmonary segmental allergen challenge in allergic individuals causes eosinophil recruitment into the airways; this is associated with the release of eosinophil granule proteins and an increase in vascular permeability (8, 9). In spite of this strong association between eosinophils and allergic diseases, the activation mechanism(s) of eosinophils in vivo are largely unknown.

A critical feature of the allergic response is the release of histamine and other inflammatory mediators from mast cells and basophils after allergen cross-links specific IgE Ab occupying high affinity IgE receptors (Fc{epsilon}RI) (10). Furthermore, epidemiological studies show a close correlation between serum IgE levels and the prevalence and severity of the bronchial asthma (11). Thus, there is converging evidence to support a role for IgE in the pathophysiology of allergic diseases in humans. Earlier, eosinophils were considered to express only low affinity IgE receptor (Fc{epsilon}RII) (12); however, recent studies by Gounni et al. (13) demonstrated that eosinophils express Fc{epsilon}RI and that they release inflammatory mediators and exert cytotoxicity to helminths through this receptor. Subsequent immunohistochemical and immunocytochemical studies on patients with allergic diseases showed that local allergen provocation induces expression of Fc{epsilon}RI by eosinophils infiltrating into the airways (14) and skin (15, 16). Furthermore, Fc{epsilon}RI was detected in blood eosinophils from patients with various allergic diseases (17). Therefore, it is reasonable to speculate that Fc{epsilon}RI plays a role in the eosinophil secretory process in allergic diseases.

There are caveats to this conception. Most of the patient studies were performed by histologic examination using in situ hybridization and immunocytochemical techniques. Therefore, little is known about the actual expression of Fc{epsilon}RI protein on the eosinophil surface. For example, in human T cells, the low affinity IgG receptor proteins (Fc{gamma}RII, CD32) are stored within the cytoplasm but are not expressed on the cell surface (18), and their surface expression was detected only by a highly sensitive biotin-avidin flow cytometric technique (19). Furthermore, the functional significance of the Fc{epsilon}RI expression by eosinophils from patients with allergic diseases is also unknown. To address these questions, we studied blood eosinophils from patients with ragweed hay fever during the peak of the allergy season. We had three goals. First, we examined whether Fc{epsilon}RI proteins are expressed on the surface of the eosinophil, and we compared the expression levels to known Fc{epsilon}RI-bearing cells, such as basophils. Second, we examined whether Fc{epsilon}RI confers IgE binding to eosinophils. Finally, we examined whether IgE receptors stimulate mediator release by these eosinophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Thirty patients, 18–60 yr of age, with moderate to severe ragweed allergic rhinitis and 14 healthy nonatopic subjects were studied. The patients had a history of ragweed hay fever, a positive skin test to ragweed extract, and an elevated level of IgE Ab for short ragweed (305–2190% of normal, median 1114%). Patients were excluded if they were receiving any forms of glucocorticoid treatment within the preceding 6 mo or immunotherapy within the preceding 12 mo. Each patient kept a diary of daily symptom scores for nasal congestion, pruritus or discharge, ocular pruritus, eye watering, eye redness, and pruritis of the ear or palate to validate their clinical reaction to airborne ragweed allergen. Normal control subjects had no clinical or laboratory evidence of atopy. Blood samples were obtained from patients and control subjects during the peak of hay fever season in Rochester, MN (end of August) and used as described below. The study was approved by the Institutional Review Board of the Mayo Clinic and Mayo Foundation.

Antibodies

Anti-Fc{epsilon}RI {alpha} subunit mAb (15-1, mouse IgG1 (mIgG1))3 and biotinylated chimeric mouse/human IgE monoclonal protein (cIgE) were kind gifts from Dr. J.-P. Kinet, Beth Israel Deaconess Medical Center, Harvard Medical School (Boston, MA). Concentration-response experiments to establish saturating concentrations for cIgE to sensitize human eosinophils in vitro and for 15-1 to inhibit the sensitization of cIgE were performed before this study began (our unpublished results); the saturating concentrations were ~25 µg/ml for cIgE and 10 µg/ml for 15-1 per 1 x 106 eosinophils. Anti-CD11b mAb (Bear1, mIgG1), anti-Fc{epsilon}RII (CD23) mAb (9P25, mIgG1), anti-CD25 mAb (B1.49.9, mIgG2a), anti-CD32 mAb (2E1, mIgG2a), and anti-CD69 mAb (TP1.55.3, mIgG2b) were purchased from Immunotech (Westbrook, ME). Control mouse myeloma Ig, including mIgG1, mIgG2a, and mIgG2b, were obtained from ICN Pharmaceuticals (Costa Mesa, CA). PE-conjugated F(ab')2 fragments of sheep IgG anti-mIgG and PE-conjugated streptavidin were purchased from Sigma Chemical (St. Louis, MO) and Becton Dickinson (Mountain View, CA), respectively. FITC-conjugated goat IgG anti-human IgG, FITC-conjugated goat anti-human IgE, and FITC-conjugated goat IgG anti-mouse IgM were from BioSource International (Camarillo, CA).

Reagents for cell functional analyses

We used myeloma IgE purified from serum from a patient with multiple myeloma as described elsewhere (5). Purified human IgE was also purchased from Cortex Biochem (San Leandro, CA). We performed all the experiments with both of the IgE preparations and found that the results were virtually identical. Therefore, only results from our myeloma IgE preparation(s) are shown. Purified human serum IgG was purchased from ICN Pharmaceuticals. F(ab')2 fragments of goat IgG anti-human IgG and F(ab')2 fragments of goat IgG anti-human IgE were purchased from ICN Pharmaceuticals and DiaMed (Windham, ME), respectively. F(ab')2 fragment of goat IgG was obtained from Jackson ImmunoResearch Laboratories, West Chester, PA.

Cell preparation

Eosinophils were isolated from six patients and six normal subjects by a magnetic cell separation system (MACS, Becton Dickinson, San Jose, CA) as described previously with minor modifications (20). Briefly, 60 ml of venous blood anticoagulated with 50 U/ml heparin were diluted with PBS at a 1:1 ratio. Diluted blood was overlaid on isotonic Percoll solution (density, 1.085 g/ml, Sigma) and centrifuged at 1000 x g for 30 min at 4°C. The supernatant and mononuclear cells at the interface were carefully removed, and erythrocytes in sediment were lysed by two cycles of hypotonic water lysis. Isolated granulocytes were washed twice with PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) containing 1% alpha calf serum (HyClone Laboratories; Logan, UT). An approximately equal volume of anti-CD16-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) was added to the cell pellet. After 60 min of incubation on ice, cells were loaded onto the separation column positioned in the strong magnetic field of the MACS. Cells were eluted three times with 5 ml of PIPES buffer with defined calf serum. The purity of eosinophils counted by Randolph’s stain was regularly >98%. Purified eosinophils were washed and suspended in reaction medium and then used immediately.

Basophils were obtained as the mononuclear cell layer after density gradient centrifugation from the same donor used for eosinophil isolation. Briefly, heparinized venous blood was centrifuged over Histopaque 1077 (Sigma), following the procedure recommended by the manufacturer. The mononuclear cell layer was collected, washed with PIPES buffer, suspended in reaction medium, and used immediately. The purity of basophils counted by Wright-Giemsa staining of cytospin preparations was between 0.5% and 2%.

Flow cytometric analysis

Eosinophil activation markers. Expression of CD25, CD69, and CD11b on eosinophils was measured by flow cytometry as an indicator of eosinophil activation (21, 22, 23). To avoid purification-induced activation or inadvertent subset selection, total leukocyte samples were stained with mAb and eosinophils were electronically gated during FACS analysis. Briefly, 5 parts of heparinized whole blood from patients and normal subjects were incubated with 1 part of 6% hetastarch in 0.9% NaCl (Hespan, DuPont Pharmaceuticals, Wilmington, DE) for 45 min at 37°C to sediment erythrocytes. The buffy coat was collected, and the remaining erythrocytes were lysed by erythrocyte lysing solution (Becton Dickinson). Buffy coat leukocytes were washed with PBS containing 0.1% NaN3 and 1% BSA (PAB buffer). The cells were resuspended in PAB buffer at 2 x 107 cells/ml and added to 96-well plates at 100 µl/well. One microgram of anti-CD11b, anti-CD25, anti-CD69, or isotype-matched control mouse Ig was added separately to individual wells and incubated for 30 min at 4°C. After washing, cells were resuspended with 100 µl of PE-conjugated F(ab')2 fragments of sheep anti-mouse IgG diluted in PAB buffer at 1:400 and incubated in the dark for 30 min. The samples were washed and fixed with 1% paraformaldehyde in PAB. FACS analysis was performed within 2 h using a FACScan flow cytometer (Becton Dickinson). Established programs were used to ensure consistent instrument settings (e.g., laser power, compensation, gain, scaling, etc.) among experiments. Two-dimensional dot-plot analysis of the green fluorescence intensity and side scatter revealed four distinct populations of leukocytes, including lymphocytes, monocytes, neutrophils, and eosinophils. Eosinophils were identified as a cell population with the strongest green autofluorescence and side scatter as previously described (24). Appropriateness of the identification of the eosinophil population was confirmed by positive staining for anti-CD9 mAb (Immunotech) as well as morphological analysis of cytospin preparation of sorted cells. The cells were electronically gated on this eosinophil population, and the expression of activation markers was measured by examining the intensity of PE fluorescence. Fluorescence intensity was determined on 50,000 total cells from each sample using logarithmic amplification and presented as the differences in the mean fluorescence channel ({Delta}MFC) between the test mAb (e.g., anti-CD11b) and isotype-matched control mouse Ig by Becton Dickinson Lysis II software.

Receptor expression

Expression of receptors for IgE and IgG, including Fc{epsilon}RI, Fc{epsilon}RII (CD23), and Fc{gamma}RII (CD32), by eosinophils was examined by a standard FACS technique using isolated eosinophils. To release putative in vivo bound IgE molecules from the surface of the eosinophil, eosinophils were subjected to acid stripping as described previously (25). Briefly, isolated eosinophils were washed with cold 0.15 M NaCl. The pellets were treated with lactic acid buffer (130 mM NaCl, 5 mM KCl, 10 mM lactic acid, pH 3.9) for 3.5 min. Cells were washed with PAB buffer and suspended in the same buffer at 5 x 106 cells/ml. Aliquots (100 µl) of cell suspension were added to 15-ml polypropylene conical tubes, mixed with saturating amounts of anti-Fc{epsilon}RI mAb (15-1), anti-Fc{epsilon}RII mAb (9P25), or anti-Fc{gamma}RII mAb (2E1), or the same amount of control mouse Ig. After an incubation for 30 min on ice, the cell pellets were washed twice with cold PAB buffer and mixed with 100 µl of 1:400 dilution of PE-conjugated F(ab')2 fragments of sheep anti-mouse IgG. After an additional incubation on ice for 30 min in the dark, the cell pellets were washed twice with PAB buffer and fixed with 1% paraformaldehyde. Fluorescence analysis was conducted in a FACScan flow cytometer. Fluorescence intensity was determined on 10,000 cells from each sample using logarithmic amplification and presented as the differences in the mean fluorescence channel ({Delta}MFC) between the test mAb (e.g., 15-1) and isotype-matched control mouse Ig by Becton Dickinson Lysis II software.

Ig binding

Binding of IgE or IgG to eosinophils and other leukocyte populations was examined before and after in vitro passive sensitization with these Ig. Isolated eosinophils or buffy coat leukocytes were obtained as described above. One portion of cells was stained immediately for the analysis of cell-bound IgE or IgG in vivo (see below). The other portion of cells was passively sensitized in vitro with IgE or IgG. First, cells were treated with lactic acid buffer to remove in vivo bound IgE molecules as described above. Eosinophils and other leukocytes were then washed with sensitization buffer (RPMI 1640 medium supplemented with 25 mM HEPES, 1% BSA, 0.1% NaN3, pH 7.3) and resuspended in the medium at 5 x 106 cells/ml and 2 x 107 cells/ml, respectively. Aliquots (100 µl) of each cell suspension were added to 15-ml polypropylene conical tubes and mixed with human IgE (50 µg/ml), human IgG (50 µg/ml), or biotinylated cIgE (25 µg/ml). In some experiments, to block the binding of IgE to Fc{epsilon}RI, cells were pretreated with a saturating concentration of anti-Fc{epsilon}RI mAb (15-1, 10 µg/ml) for 30 min on ice before in vitro sensitization with cIgE. Cells were incubated for 2 h at 4°C with gentle mixing and washed twice with PAB buffer. To detect IgE or IgG bound on the cell surface, cells were incubated with saturating amounts of FITC-conjugated goat anti-human IgE or FITC-conjugated goat anti-human IgG, or the same amounts of control (FITC-conjugated goat anti-mouse IgM). Alternatively, cell-bound biotinylated cIgE was visualized by PE-conjugated streptavidin. After incubation on ice for 30 min in the dark, cell pellets were washed twice with PAB buffer and fixed with 1% paraformaldehyde. Fluorescence analysis was conducted in a FACScan flow cytometer. To analyze IgE, cIgE, or IgG bound to isolated eosinophils, fluorescence intensity was determined on 10,000 cells from each sample using logarithmic amplification and presented as the {Delta}MFC between the test Ab (e.g., anti-human IgE) and control Ab by Becton Dickinson Lysis II software. To analyze binding of cIgE to individual cell populations of buffy coat leukocytes, cells were electronically gated based on their unique green autofluorescence and side scatter characteristics. Two-dimensional dot-plot analysis of the green fluorescence intensity and side scatter of buffy coat leukocytes showed four distinct populations, including lymphocytes, monocytes, neutrophils, and eosinophils (see Results for detail). Basophils fell within the population of lymphocytes. However, when basophils were stained with biotinylated cIgE and PE-conjugated streptavidin, they formed one unique population in green fluorescence/side scatter dot-plot analysis due to their extremely strong PE fluorescence. Thus, individual cell populations were easily identified by their unique green autofluorescence and side scatter characteristics; appropriateness of the identification of each population had been confirmed by positive staining for appropriate Ab (CD9 for eosinophils and basophils, CD16 for neutrophils, and CD14 for monocytes) as well as morphological analysis of cytospin preparations of sorted cells. The cells were electronically gated, and the binding of cIgE was measured by examining the intensity of PE fluorescence. Fluorescence intensity was determined on 50,000 total cells from each sample using logarithmic amplification, which was converted to the linear equivalent by Becton Dickinson Lysis II software. Data are presented as the {Delta}MFI between test samples (e.g., biotinylated cIgE plus PE-conjugated streptavidin) and control (PE-conjugated streptavidin alone).

Leukotriene (LT) C4 production

Eosinophil production of LTC4 was performed in 96-well tissue culture plates as described previously with minor modifications (26). Isolated eosinophils were stimulated by cross-linking cell surface IgE or IgG receptors by human IgE, human IgG, anti-human IgE or anti-human IgG, immobilized onto the wells of tissue culture plates. Wells of 96-well flat-bottom Costar 3596 tissue culture plates (Costar, Cambridge, MA) were coated overnight at 4°C with 50 µl of serial dilutions of human IgE or human IgG, or 50 µg/ml of F(ab')2 fragments of goat anti-human IgG, goat anti-human IgE or goat IgG (control). After aspiration of the solution, 50 µl of 2.5% human serum albumin (HSA, Sigma) was added to each well to block the nonspecific protein binding sites. After incubation for 2 h at 37°C and 5% CO2, wells were washed twice with 0.9% NaCl before use. Freshly isolated eosinophils were washed with HBSS supplemented with 10 mM HEPES, 20 mM L-serine and 5 mM glutathione and resuspended in the same medium at 5 x 105 cells/ml. Aliquots of cell suspensions (200 µl) were added to the wells and incubated for 1 h at 37°C and 5% CO2. The supernatants from each well were collected and frozen at -70°C or assayed immediately. Concentrations of LTC4 in the sample supernatants were measured by ELISA using an LTC4 kit (Cayman Chemical, Ann Arbor, MI) following the procedure recommended by the manufacturer. The sensitivity of the assay was 4.8 pg/ml. All experiments were conducted in duplicate.

Superoxide anion production

Eosinophil superoxide production was induced by Ig or anti-Ig immobilized onto tissue culture plates and measured by reduction of cytochrome c as previously described (27, 28). Briefly, IgE, IgG, anti-IgE, or anti-IgG were immobilized onto polystyrene 96-well flat-bottom tissue culture plates as described above. Freshly isolated eosinophils were washed and resuspended in HBSS with 10 mM HEPES and 100 µM cytochrome c at 5 x 105 cells/ml. Cell suspension (100 µl) was dispensed onto the wells, followed by 100 µl of medium alone. Immediately after addition of stimuli, the reaction wells were measured for absorbance at 550 nm in a microplate autoreader (Thermomax, Molecular Devices, Menlo Park, CA), followed by repeated readings. Between absorbance measurements, the plate was incubated at 37°C. Superoxide anion generation was calculated with an extinction coefficient of 21.1 x 103cm-1 M-1 for reduced cytochrome c at 550 nm and was expressed as nanomols of superoxide produced per 105 cells.

Eosinophil and basophil degranulation

Eosinophils and basophils were stimulated with anti-human IgE or anti-human IgG, either immobilized onto the wells of tissue culture plates or in solution, or FMLP (Calbiochem-Novabiochem, San Diego, CA) in solution. Polystyrene 96-well flat-bottom tissue culture plates were coated with 50 µg/ml F(ab')2 fragments of goat anti-human IgE, goat anti-human IgG, or goat IgG (control); blocked with 2.5% HSA; and washed with saline as described above. Plates used for anti-IgE, anti-IgG, or FMLP in solution were blocked with HSA. Freshly isolated eosinophils or basophil preparations were suspended in RPMI 1640 supplemented with 10 mM HEPES and 0.1% HSA at 5 x 105 cells/ml and 1 x 106 cells/ml, respectively. Aliquots of cell suspensions (100 µl) were added to the wells and stimulated with serial dilutions of F(ab')2 fragments of goat anti-human IgE, goat anti-human IgG or goat IgG (control), or 1 µM FMLP (100 µl). Cells incubated in the wells coated with anti-Ig received 100 µl of medium alone. Cells were incubated for 60 min (basophil preparations) or 180 min (eosinophils) at 37°C and 5% CO2. After incubation, supernatants were collected and stored at -20°C until assayed. To quantitate basophil degranulation, the concentration of histamine in the sample supernatants was measured by histamine enzyme immunoassay (EIA) kit (Immunotech) following the procedure recommended by the manufacturer. To quantitate eosinophil degranulation, the concentration of eosinophil-derived neurotoxin (EDN) in the sample supernatants was measured by RIA. The RIA is a double-Ab competition assay in which radioiodinated EDN, rabbit anti-EDN, and burro anti-rabbit IgG are used, as reported elsewhere (5). Total cellular histamine and EDN contents were measured simultaneously using supernatants from cells lysed with 0.5% Nonidet P-40 detergent. The sensitivities of histamine EIA and EDN RIA were 0.2 nM and 2.0 ng/ml, respectively. All assays were done in duplicate.

Statistical analysis

Data are presented as mean ± SEM from the numbers of experiments indicated. Statistical significance was assessed using the Mann-Whitney U test or paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of activation markers by eosinophils from patients with hay fever

Generally, in Rochester, MN, ragweed hay fever season starts in early August, peaks at the end of August, and continues until the end of September. In the current study, patients’ symptoms increased in parallel to the pollen counts and peaked during the peak pollination period (late August through early September). Similarly, patients’ peripheral blood eosinophil counts increased significantly during the peak hay fever season from the values before the season (284 ± 41/µl at peak season vs 141 ± 19/µl before the season (early July); n = 30, p < 0.01). Initial FACS analyses of peripheral blood eosinophils validated in vivo "activation" of the patients’ eosinophils. These cells expressed significantly higher levels of several eosinophil cell surface activation markers, including CD25, CD69, and CD11b (21, 22, 23), compared with eosinophils from normal subjects (Fig. 1Go). The increased expression of CD11b was especially pronounced. In addition, when stimulated in vitro with 1 µM FMLP, patients’ eosinophils released significantly larger amounts of the eosinophil granule protein, EDN, than normal subjects’ eosinophils (27.3 ± 5.6 and 7.8 ± 1.1% of total EDN for patients and normal subjects, respectively; n = 6, p < 0.05). These findings suggest that blood eosinophils from patients with hay fever are "activated" compared with those from normal subjects, presumably due to exposure to cytokines and other inflammatory mediators in vivo.



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  		FIGURE 1. Expression of cell surface activation markers by eosinophils. Leukocytes from patients with hay fever or normal subjects were stained with anti-CD25, anti-CD69, or anti-CD11b mAb or isotype-matched control mouse Ig followed by PE-conjugated F(ab')2 fragments of sheep anti-mouse IgG. Cells were analyzed by FACScan flow cytometry, and eosinophils were electronically gated as described in Materials and Methods. Data are presented as the differences in the mean fluorescence channel ({Delta}MFC) between the test mAb (e.g., anti-CD11b) and isotype-matched control mouse Ig. Each dot represents the result from an individual subject. Significant differences between patient and normal groups are shown.

 
Expression of IgE and IgG receptors by eosinophils from patients with hay fever

We next examined whether patients’ eosinophils can bind IgE or IgG on their surfaces and whether they express receptors for these Ig. FACS analysis of cell-bound IgE with FITC-conjugated anti-IgE Ab showed that none or minimal amounts of IgE were present on the surface of freshly isolated eosinophils (Figs. 2Go and 3A). When these eosinophils were incubated with IgE in vitro for 2 h, the amounts of bound IgE significantly increased (p < 0.01, Fig. 3GoA), suggesting that these eosinophils have the capacity to bind IgE. Freshly isolated eosinophils bound detectable amounts of IgG on their surface (Figs. 2Go and 3GoA), and the levels increased further by in vitro incubation with IgG (Fig. 3GoA, p < 0.01). Thus, patients’ eosinophils were able to bind both IgE and IgG in vitro although the amounts of IgE bound in vivo were negligible.



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  		FIGURE 2. FACS analyses of the Ig bound on the eosinophil cell surface and the expression of Fc receptors. To analyze the Ig bound on the surface of the eosinophil (sIgE and sIgG), freshly isolated eosinophils were incubated with saturating amounts of FITC-conjugated goat anti-human IgE (B) or FITC-conjugated goat anti-human IgG (C), or the same amounts of control (FITC-conjugated goat anti-mouse IgM). To analyze the expression of Fc receptors (Fc{epsilon}RI, Fc{epsilon}RII, and Fc{gamma}RII), isolated eosinophils were treated with lactic acid to remove cell surface IgE. Cells were then stained with saturating amounts of anti-Fc{epsilon}RI mAb (15-1 (D)), anti-Fc{epsilon}RII mAb (9P25 (E)), anti-Fc{gamma}RII (2E1 (F)), or isotype-matched control mouse Ig followed by PE-conjugated F(ab')2 fragments of sheep anti-mouse IgG. Fluorescence analysis used a FACScan flow cytometer. The forward scatter/side scatter dot-plot (A) and histograms of FITC or PE fluorescence (B–F) are shown.

 


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  		FIGURE 3. Binding of Ig to eosinophils (A) and expression of Fc receptors by eosinophils (B). In A, freshly isolated eosinophils were divided into two portions. One portion of cells (presensitization) were stained immediately with FITC-conjugated goat anti-human IgE or FITC-conjugated goat anti-human IgG, or FITC-conjugated control Ab. The other portion of cells (postsensitization) were stripped with lactic acid and passively sensitized in vitro with human IgE (50 µg/ml) or human IgG (50 µg/ml) for 2 h at 4°C. Cells were then stained with FITC-conjugated goat anti-human IgE or FITC-conjugated goat anti-human IgG, or FITC-conjugated control Ab. Fluorescence analysis was conducted in a FACScan flow cytometer. Data are presented as the differences in the mean fluorescence channel ({Delta}MFC) between the test mAb (e.g., anti-IgE) and control Ab. Each dot represents the result from an individual subject, and the results from the same subject are connected by a line. Significant differences between the groups are shown. In B, isolated eosinophils were treated with lactic acid to remove cell surface IgE. Cells were then stained with saturating amounts of anti-Fc{epsilon}RI mAb (15-1), anti-Fc{epsilon}RII mAb (9P25), anti-Fc{gamma}RII (2E1), or isotype-matched control mouse Ig followed by PE-conjugated F(ab')2 fragments sheep anti-mouse IgG. Fluorescence analysis used a FACScan flow cytometer. Data show the differences in the mean fluorescence channel ({Delta}MFC) between the test mAb (e.g., 15-1) and isotype-matched control Ab. Each dot represents the result from an individual subject.

 
Another question remains. Do eosinophils express Fc receptors accountable for binding these Ig? To detect IgE receptors, eosinophils were pretreated with lactic acid to remove any bound IgE and incubated with anti-receptor mAb and PE-conjugated secondary Ab. Surprisingly, no receptors for Fc{epsilon}RI or Fc{epsilon}RII were detected by 15-1 or 9P25 mAb, respectively, in any of the six patients (Figs. 2Go and 3GoB). In contrast, Fc{gamma}RII was easily detectable on the same eosinophils (Figs. 2Go and 3GoB), consistent with the IgG binding capacity of eosinophils. The experiments were repeated without removing IgE from eosinophils; neither Fc{epsilon}RI nor Fc{epsilon}RII could be detected on these nonstripped eosinophils (data not shown).

Analysis of Fc{epsilon}RI expression by biotinylated cIgE

Perhaps we were unable to detect Fc{epsilon}RI on eosinophils because of the sensitivity limitations of the standard FACS technique. Therefore, to enhance the signals for FACS analysis, we removed any IgE bound to the cells by lactic acid treatment, then incubated them in vitro with biotinylated cIgE, and visualized them with PE-conjugated streptavidin. Fig. 4GoA shows that the binding of cIgE to eosinophils was clearly detectable. Furthermore, the cIgE binding was inhibited by pretreatment of cells with anti-Fc{epsilon}RI mAb (15-1) (Fig. 4GoB); the isotype-matched control Ig for anti-Fc{epsilon}RI did not affect cIgE binding to eosinophils. Similar analyses of eosinophils from six patients showed that this observation is reproducible and that the binding of cIgE was significantly blocked by anti-Fc{epsilon}RI mAb (Fig. 5GoA, p < 0.01). The inhibitory effects of anti-Fc{epsilon}RI mAb were variable among the patients, suggesting a wide variance in the expression levels of Fc{epsilon}RI. Interestingly, eosinophils from normal subjects also bound cIgE, and the binding was also inhibited partially but significantly by anti-Fc{epsilon}RI mAb (Fig. 5GoA, p < 0.05). The amounts of bound cIgE levels as well as the amounts of cIgE-binding inhibited by anti-Fc{epsilon}RI mAb tended to be higher in eosinophils from patients compared with eosinophils from normal subjects. Recent studies on mast cells and basophils show that the density of cell surface Fc{epsilon}RI is regulated by blood IgE levels (29, 30). In eosinophils, we also found that the expression of Fc{epsilon}RI, as estimated by anti-Fc{epsilon}RI-inhibitable binding of cIgE, showed a strong linear correlation with serum levels of total IgE (Fig. 5GoB, r2 = 0.941).



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  		FIGURE 4. FACS analysis of cIgE binding to eosinophils. Isolated eosinophils were treated with lactic acid to remove in vivo bound IgE molecules. Fluorescence analysis used a FACScan flow cytometer, and a histogram of PE fluorescence is shown. A shows the binding of cIgE to eosinophils. Cells were passively sensitized with medium alone (filled gray area) or 25 µg/ml biotinylated cIgE (solid black line) for 2 h at 4°C and stained with PE-conjugated streptavidin. B shows the inhibitory effect of anti-Fc{epsilon}RI on eosinophil cIgE binding. Cells were preincubated without Ab (gray area), with 10 µg/ml anti-Fc{epsilon}RI mAb (15-1, bold black line), or with 10 µg/ml isotype-matched control mouse Ig (thin black line) for 30 min at 4°C. Cells were then sensitized with 25 µg/ml biotinylated cIgE for 2 h at 4°C and stained with PE-conjugated streptavidin.

 


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  		FIGURE 5. Binding of cIgE to eosinophils. In A, isolated eosinophils were treated with lactic acid to remove in vivo bound IgE molecules. Cells were preincubated with or without 10 µg/ml anti-Fc{epsilon}RI mAb (15-1), sensitized with 25 µg/ml biotinylated cIgE, and stained with PE-conjugated streptavidin. Fluorescence analysis used a FACScan flow cytometer. Data are presented as the differences in the mean fluorescence channel ({Delta}MFC) between test sample (e.g., biotinylated cIgE plus PE-conjugated streptavidin) and control (PE-conjugated streptavidin alone). Each dot represents the result from one subject, and data from the same subject are connected by a line. Significant differences with vs without anti-Fc{epsilon}RI mAb pretreatment are shown. B shows the correlation between serum IgE concentrations and anti-Fc{epsilon}RI (15-1)-inhibitable binding of cIgE. Anti-Fc{epsilon}RI-inhibitable binding of cIgE was calculated as follows: ({Delta}MFC of cells preincubated without anti-Fc{epsilon}RI mAb and then incubated with biotinylated cIgE and PE-conjugated streptavidin) - ({Delta}MFC of cells preincubated with anti-Fc{epsilon}RI mAb and then incubated with biotinylated cIgE and PE-conjugated streptavidin). Each dot represents the result from one subject; {circ}, normal subjects; •, data from patients with hay fever.

 
Whole blood analysis of Fc{epsilon}RI expression

Although we could detect Fc{epsilon}RI expression by the cIgE-biotin-streptavidin technique (Figs. 4Go and 5Go), the failure to detect by the standard FACS technique (Figs. 2Go and 3Go) suggested that the density of Fc{epsilon}RI expressed on the surface of the eosinophil is extremely low. Basophils are known to express a high density of Fc{epsilon}RI (31) and blood monocytes from patients with allergy also express Fc{epsilon}RI (32). Therefore, we compared the expression levels of Fc{epsilon}RI by eosinophils to other leukocytes. To this end, total leukocytes were sensitized with biotinylated cIgE and visualized by PE-conjugated streptavidin and flow cytometry. The individual cell populations were electronically gated on their unique green autofluorescence and side scatter characteristics. This strategy avoided unanticipated cell activation during the isolation procedures, avoided inadvertent subset selection, and enabled simultaneous analyses of different cell populations from the same sample. As shown in Fig. 6Go, A and B, green fluorescence/side scatter dot-plot of leukocytes incubated with PE-streptavidin alone without sensitization with cIgE revealed four distinct cell populations, including lymphocytes plus basophils, monocytes, neutrophils, and eosinophils. When cells were sensitized with biotinylated cIgE and stained with PE-streptavidin, we found that cIgE binds to all the cell populations to various degrees (Fig. 6GoC). Notably, basophils were intensely stained, forming a unique population distinct from the lymphocyte cluster in PE fluorescence reading (Fig. 6GoC) and even in the green fluorescence reading (Fig. 6GoD). The distribution of cell clusters in green fluorescence/side scatter dot-plot did not change otherwise (Fig. 6GoD). Interestingly, when leukocytes were pretreated with anti-Fc{epsilon}RI mAb, the binding of cIgE to the basophil cluster was completely inhibited (Fig. 6EGo). In contrast, the inhibitory effects of anti-Fc{epsilon}RI mAb on cIgE binding to eosinophil and neutrophil clusters were not apparent in the dot-plot.



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  		FIGURE 6. FACS analyses of whole blood for cIgE binding to leukocytes. Buffy coat leukocytes were treated with lactic acid buffer to remove in vivo bound IgE molecules. Cells were preincubated without (A–D) or with (E and F) anti-Fc{epsilon}RI mAb (15-1). Cells were then passively sensitized with (C–F) or without (A and B) 25 µg/ml biotinylated cIgE, and stained with PE-conjugated streptavidin. Fluorescence analysis used a FACScan flow cytometer. Two-dimensional dot-plot analysis of the green fluorescence intensity and side scatter of leukocytes showed four distinct populations (B), including lymphocytes (Ly), monocytes (Mo), neutrophils (Ne), and eosinophils (Eo). Basophils (Ba) fell within the population of lymphocytes. However, when stained with biotinylated cIgE and PE-conjugated streptavidin (D), basophils (Ba) formed one unique population in green fluorescence/side scatter dot-plot analysis (C) due to their extremely strong PE fluorescence. The identity of the individual cell populations was confirmed as described in Materials and Methods.

 
The quantitative analyses of cIgE binding and the inhibitory effects of anti-Fc{epsilon}RI mAb (15-1) are shown in Fig. 7Go. Binding of cIgE to leukocytes was observed in the following order: basophils (5062 ± 221) >> neutrophils (346 ± 23) > eosinophils (124 ± 15) {cong} monocytes (90 ± 9) (means ± SEM of {Delta}MFI, n = 27). Pretreatment of cells with anti-Fc{epsilon}RI mAb completely inhibited the cIgE binding to basophils (99.0 ± 0.1% of inhibition, n = 27), suggesting that Fc{epsilon}RI plays a major role in cIgE binding to basophils. The binding of cIgE to monocytes was inhibited moderately but significantly by anti-Fc{epsilon}RI mAb in 24 of 27 patients (45.0 ± 5.5% of inhibition, p < 0.01), suggesting roles for Fc{epsilon}RI and for other binding site(s) in cIgE binding to monocytes. In contrast, in neutrophils anti-Fc{epsilon}RI mAb minimally affected the binding of cIgE (5.1 ± 5.3% of inhibition), and the inhibitory effects of anti-Fc{epsilon}RI mAb varied considerably among individuals as evidenced by a wide range of {Delta}(W/O-With) values. The effects of anti-Fc{epsilon}RI mAb on eosinophils were between those on monocytes and neutrophils, namely significant (p < 0.01) but small and variable inhibitory effects (18.2 ± 3.8% of inhibition). The binding of cIgE to leukocytes due to Fc{epsilon}RI was observed in the following order: basophils (5015 ± 223) >> monocytes (40 ± 12) > eosinophils (25 ± 7) > neutrophils (13 ± 13) (means ± SEM of the {Delta}MFI with or without anti-Fc{epsilon}RI mAb). Thus, although the expression of Fc{epsilon}RI by eosinophils is detectable, the level is strikingly less than that by basophils and less than that by monocytes.



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  		FIGURE 7. Binding of cIgE to leukocyte populations and the inhibitory effects of anti-Fc{epsilon}RI mAb (15-1). Buffy coat leukocytes were treated with lactic acid buffer, preincubated with or without Fc{epsilon}RI mAb, and sensitized with cIgE. The bound cIgE was visualized by PE-conjugated streptavidin and analyzed by a FACScan flow cytometer. Individual cell populations were electronically gated, based on their unique green fluorescence and side scatter characteristics, as shown in the dot plots of Fig. 6Go, B and D. PE fluorescence intensity was determined on 50,000 total cells from each sample using logarithmic amplification, which was converted to the linear equivalent by Becton Dickinson Lysis II software. Data are presented as the differences in the mean fluorescence intensity ({Delta}MFI) between test sample (e.g., biotinylated cIgE plus PE-conjugated streptavidin) and control (PE-conjugated streptavidin alone). Each dot represents an individual subject, and the statistical differences with or without preincubation with anti-Fc{epsilon}RI mAb are shown. {Delta}(W/O-With) was calculated by subtracting ({Delta}MFI value without anti-Fc{epsilon}RI mAb pretreatment) from ({Delta}MFI value with anti-Fc{epsilon}RI mAb pretreatment) for each individual subject. Open bars and horizontal bars show the 25th to 75th percentile range and median of the data points, respectively.

 
Roles of IgE and IgG receptors for degranulation of basophils and eosinophils

In the next series of experiments, we examined the functional significance of IgE receptors for mediator release by eosinophils. The engagement of IgE receptors on basophils and mast cells by ligation of bound IgE with anti-IgE Ab is commonly used to trigger various effector functions of these cell types, including degranulation and production of lipid mediators and cytokines (reviewed in Refs. 33 and 34). Therefore, we investigated whether anti-IgE Ab induces degranulation of eosinophils and compared the responses of basophils and eosinophils using cells isolated from the same donors. When basophils were stimulated with 1 µg/ml anti-IgE Ab in solution, they released a large amount of histamine (Fig. 8GoA, 85.7 ± 4% of total histamine, n = 6). The effect of 10 µg/ml anti-IgE Ab in solution was weaker, but substantial amounts of histamine were released (51.3 ± 10.5% of total, n = 6). In contrast, 1 µg/ml anti-IgE Ab in solution failed to induce EDN release from eosinophils (Fig. 8GoB), and higher concentrations of anti-IgE Ab (5~50 µg/ml) did not induce EDN release from eosinophils (data not shown). Furthermore, immobilized anti-IgE Ab induced histamine release from basophils but not EDN release from eosinophils. In contrast, immobilized anti-IgG Ab induced both basophil histamine release and eosinophil EDN release.



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  		FIGURE 8. Degranulation of basophils and eosinophils induced by anti-IgE or anti-IgG Ab. Isolated eosinophils or basophil preparations were stimulated with 1 µg/ml F(ab')2 fragments of goat anti-human IgE (soluble stimulus), or F(ab')2 fragments of goat anti-human IgE, goat anti-human IgG, or goat IgG (control) immobilized onto 96-well tissue culture plates at 50 µg/ml (immobilized stimuli). Cells were incubated for 60 min (basophil preparations) or 180 min (eosinophils) at 37°C and 5% CO2. To quantitate basophil degranulation, the concentration of histamine in the sample supernatants was measured by histamine EIA kit. To quantitate eosinophil degranulation, the concentration of EDN in the sample supernatants was measured by RIA. Total cellular histamine and EDN contents were measured using supernatants from cells lysed with 0.5% Nonidet P-40 detergent. Each dot represents the result from an individual subject.

 
Production of LTC4 and superoxide anion by eosinophils

The experiments described above suggest that a degranulation response is provoked by anti-IgE Ab in basophils, but not in eosinophils. Perhaps the amounts of IgE mounted on eosinophils are too small to trigger sufficient signals for cellular function when cross-linked by anti-IgE Ab. Furthermore, if the affinity of the IgE binding sites is low, the sites may be partially occupied and partially unoccupied in freshly isolated eosinophils (see Figs. 2Go and 3Go). Therefore, to characterize completely the eosinophil responses to Ig, we stimulated eosinophils with Ig itself or anti-Ig Ab immobilized onto tissue culture plates and examined the production of an eicosanoid, LTC4. As shown in Fig. 9Go, ligation of unoccupied IgG receptors by immobilized IgG provoked LTC4 production in a concentration-dependent manner. In contrast, ligation of unoccupied IgE receptors (or binding sites) with immobilized IgE failed to induce LTC4 production. Similarly, ligation of cell-bound IgG by anti-IgG Ab stimulated eosinophil LTC4 production; however, anti-IgE Ab did not induce significant production of LTC4. Furthermore, as shown in Fig. 10GoA, eosinophils produced superoxide anion in a time-dependent manner when stimulated with immobilized IgG; immobilized IgE stimulated only minimal superoxide production. In addition, anti-IgG Ab, but not anti-IgE Ab, induced eosinophil superoxide production (Fig. 10GoB). Altogether, these findings suggest that eosinophil degranulation and production of LTC4 and superoxide anion are only minimally induced by IgE-dependent stimuli, such as IgE or anti-IgE Ab, whereas these functions are induced vigorously by IgG-dependent stimuli.



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  		FIGURE 9. LTC4 production by eosinophils stimulated with IgE- or IgG-dependent stimuli. Isolated eosinophils were stimulated with serial dilutions of human IgE or human IgG immobilized onto 96-well tissue culture plates, or with F(ab')2 fragments of goat anti-human IgE, goat anti-human IgG, or goat IgG (control) immobilized onto tissue culture plates at 50 µg/ml. Cells were incubated for 1 h at 37°C and 5% CO2, and concentrations of LTC4 in the sample supernatants were measured by LTC4 ELISA kit. Data show means ± SEM from three experiments using cells from three patients with hay fever.

 


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  		FIGURE 10. Superoxide anion production by eosinophils stimulated with IgE- or IgG-dependent stimuli. Isolated eosinophils were stimulated with human IgE or human IgG immobilized onto 96-well tissue culture plates at 10, 30, or 100 µg/ml, or with F(ab')2 fragments of goat anti-human IgE, goat anti-human IgG or goat IgG (control) immobilized onto tissue culture plates at 50 µg/ml. The kinetics of superoxide production were examined by reduction of cytochrome c using a microplate autoreader. Between absorbance measurements, the plate was incubated at 37°C. Data are presented as means ± SEM from six experiments using cells from six different patients with hay fever.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Eosinophils and their inflammatory products play important roles in the pathophysiology of bronchial asthma and other allergic disorders (reviewed in 1). Allergen-specific IgE is one of the major components in allergic diseases in humans, and the ligation of specific IgE bound to Fc{epsilon}RI on mast cells and basophils triggers production and release of inflammatory mediators from these cells (reviewed in Refs. 33 and 34). Because eosinophils from hypereosinophilic patients may express Fc{epsilon}RI (13), it has been suggested that Fc{epsilon}RI plays an important role in mediator release by eosinophils in allergic disease (35). However, we have made four major observations suggesting that this is unlikely. First, by standard FACS analysis, eosinophils from patients with hay fever during the peak of the season express no or minimal levels of Fc{epsilon}RI or Fc{epsilon}RII on their surfaces (Figs. 2Go and 3Go); however, these eosinophils do express eosinophil activation markers and Fc{gamma}RII. Second, eosinophils are able to bind IgE (Fig. 3Go); however, in contrast to basophils, the contribution of Fc{epsilon}RI toward eosinophil IgE binding is minor (Fig. 7Go). Third, IgE-dependent stimuli fail to provoke the release of inflammatory mediators by eosinophils (Figs. 8Go, 9Go, and 10Go). Fourth, in contrast, eosinophils from the same donors express Fc{gamma}RII (Fig. 2Go) and bind IgG (Fig. 3Go), and their effector functions are triggered by IgG-dependent stimuli (Figs. 8Go, 9Go, and 10Go). Therefore, eosinophils from patients with ragweed hay fever express negligible Fc{epsilon}RI or Fc{epsilon}RII. Unlike mast cells and basophils, factors other than IgE, such as IgG and cytokines, likely play roles in activation of eosinophils in allergic diseases.

The expression of IgE receptors by eosinophils has been a controversial issue. Murine eosinophils do not express Fc{epsilon}RI or Fc{epsilon}RII (36). Although human eosinophils were considered to express only the low affinity IgE receptor (Fc{epsilon}RII) (12, 37, 38, 39), a more recent study by the same investigators demonstrated that eosinophils from hypereosinophilic patients express Fc{epsilon}RI (13). This latter report triggered clinical studies to examine eosinophil expression of Fc{epsilon}RI. For example, the transcription of mRNA for {alpha}-, ß-, and/or {gamma}-chains of Fc{epsilon}RI in eosinophils was detected during allergen-induced late phase cutaneous reactions in patients with allergy (16). In addition, local allergen provocation induced expression of Fc{epsilon}RI protein by eosinophils infiltrating into the airways (14) and skin (15, 16). However, other investigators showed that the expression of Fc{epsilon}RI protein was not detected on the surface of the eosinophil (40, 41). In addition, we showed that eosinophils from patients with hay fever express very low numbers of Fc{epsilon}RI on their surface, which can be detected by the biotin-streptavidin technique but not by the standard FACS technique. Although the reasons for the discrepancies in our observations and some of the previous studies are unknown, we speculate that synthesis of receptor proteins and surface expression of the receptor proteins are regulated under different mechanisms. In other words, the detection of receptor proteins by immunohistochemistry may not necessarily represent the expression of the proteins on cell surface. For example, human T cells contain proteins for low affinity IgG receptor (Fc{gamma}RII, CD32) within the cytoplasm, but these receptors are not expressed on the cell surface (18). A similar scenario is known for Fc{gamma}RIII (CD16) in eosinophils (42). In addition, the earlier studies often used eosinophils from patients with hematologic or immunologic disorders, including idiopathic hypereosinophilic syndrome and lymphomas. Perhaps disease heterogeneity accounts for the receptor expression heterogeneity.

The strikingly different responses to IgE between basophils and eosinophils are apparent in this study. From the MFI of FACS analysis, ~50 times more cIgE was bound to basophils than to eosinophils (Fig. 7Go). Furthermore, >99% of cIgE binding to basophils was inhibited by anti-Fc{epsilon}RI mAb pretreatment, suggesting that Fc{epsilon}RI is the predominant IgE receptor in basophils. In contrast, anti-Fc{epsilon}RI mAb only partially (18%) inhibited cIgE binding to eosinophils. Thus, we estimate that eosinophils from these ragweed hay fever patients express ~0.5% of the Fc{epsilon}RI compared with basophils. In addition, the basophil degranulation response was triggered by ligation of Fc{epsilon}RI, either by anti-IgE Ab or by immobilized IgE; these same stimuli failed to induce degranulation or release of mediators from eosinophils. Interestingly, treatment of patients with atopy with humanized anti-IgE Ab resulted in ~96% decrease in the expression of Fc{epsilon}RI and ~40% decrease in anti-IgE-induced histamine release response by basophils (30). Because eosinophils possess only ~0.5% of the Fc{epsilon}RI possessed by basophils, this paucity of Fc{epsilon}RI expression by eosinophils may explain the heterogeneous responses of eosinophils and basophils to Fc{epsilon}RI ligation. Perhaps eosinophils lose or basophils acquire their capacity to express high levels of Fc{epsilon}RI during their differentiation from common progenitors (43).

Then, the question remains as to which receptors or binding sites contribute to the binding of myeloma IgE (Figs. 2Go and 3Go) and cIgE (Figs. 4 through 7GoGoGoGo) by eosinophils. The results of our experiments and a review of the literature provide some insights. As shown in Figs. 2Go and 3Go, none to minimal expression of Fc{epsilon}RI or Fc{epsilon}RII (CD23) was detectable using 15-1 or 9P25 mAb, respectively, and the standard FACS technique. These findings are also consistent with previous reports, demonstrating negligible binding of anti-Fc{epsilon}RI mAb (clones 22E7 and 15-1) (17) and a panel of mAb against CD23 (44, 45) to eosinophils. Therefore, the expression levels of high affinity IgE receptor (Fc{epsilon}RI) and low affinity IgE receptor (Fc{epsilon}RII) on eosinophils are likely too low to play major roles in IgE binding. Furthermore, the minimal or lack of binding of IgE on eosinophils in vivo (Figs. 2Go and 3Go) suggests that IgE binding to eosinophils is of low affinity. Besides these IgE Fc receptors, an IgE-binding molecule belonging to an S-type lectin family, called Mac-2/{epsilon}BP, can also bind IgE through the carbohydrate recognition domain (46). Mac-2/{epsilon}BP is expressed by various cell types, including neutrophils and eosinophils (47, 48). Indeed, Ab against Mac-2/{epsilon}BP strongly inhibited IgE binding and IgE-dependent activation of human neutrophils (47). Therefore, by analogy to neutrophils, Mac-2/{epsilon}BP is a good candidate molecule for IgE-binding sites on eosinophils. Our findings, showing large amounts of IgE binding to both eosinophils and neutrophils independent of Fc{epsilon}RI (Fig. 7Go), are consistent with this speculation. Alternatively, IgE may bind nonspecifically through its carbohydrate moiety to lectin-like binding sites on the surface of the eosinophil.

Two other questions remain. Because previous histological studies (14, 15, 16) could not differentiate between the proteins within the cells and those on the cell surface, the first question involves the surface expression of Fc{epsilon}R by tissue eosinophils. As shown in Fig. 5GoB, the levels of Fc{epsilon}RI expression by eosinophils show a positive correlation with serum IgE levels, suggesting that the expression of Fc{epsilon}RI by eosinophils is regulated by IgE-dependent mechanisms similar to those in mast cells (29) and basophils (30). In addition, IL-4 enhances the expression of mRNA and protein for the {alpha}-chain of Fc{epsilon}RI (40). It is well known that IL-4 is expressed in the local tissues of patients with allergy (49, 50); a more recent report suggests that IgE may be produced by B cells in the airway tissues in patients with allergy (51). Therefore, the tissue microenvironment may be optimal for Fc{epsilon}RI expression, suggesting that the surface expression of Fc{epsilon}RI by tissue eosinophils may be higher than the surface expression in peripheral blood eosinophils. On the other hand, even in the optimal tissue environment, the ability of eosinophils to express Fc{epsilon}RI on their surfaces may be limited. For example, eosinophils obtained from bronchoalveolar lavage fluids from patients with allergy do not express detectable levels of Fc{epsilon}RI on their surfaces (41). Eosinophils cultured for up to 11 days with myeloma IgE or IL-4, conditions known to up-regulate Fc{epsilon}RI on basophils, failed to induce any detectable surface Fc{epsilon}RI (41). Furthermore, Terada et al. (40) found that after culture with IL-4, eosinophil production of Fc{epsilon}RI {alpha}-subunit protein was increased, but expression of Fc{epsilon}RI was not detectable. In preliminary studies, we were not able to detect Fc{epsilon}RI by standard FACS analysis on eosinophils cultured for 3 days with human myeloma IgE in the presence of various cytokines, such as IL-5, IL-3, IL-4, TNF-{alpha} and fibroblast supernatants (data not shown).

The second question concerns the interaction of IgE with other eosinophil agonists. We found that anti-IgE Ab or immobilized IgE by themselves failed to provoke eosinophil mediator release (Figs. 8 through 10GoGoGo). However, a previous report also suggests that IgE mediates eosinophil killing of schistosomula of Schistosoma mansoni when cells are stimulated with lipid mediators, such as platelet-activating factor or LTB4, without the apparent expression of Fc{epsilon}R (52). Therefore, IgE may collaborate with other eosinophil agonists to provoke some functions of the cells, such as cellular cytotoxicity. In preliminary studies, IgE did not enhance EDN release from eosinophils stimulated with platelet-activating factor or IL-5 (H. Kita and M. Muraki, unpublished observation). Furthermore, it is also possible that Fc{epsilon}RI or IgE may be involved in other functions than secretory process of eosinophils, such as Ag presentation, as proposed in other cell types (53).

Remaining questions aside, our study clearly shows that the surface expression of Fc{epsilon}RI by blood eosinophils from patients with hay fever is minimal and that, in contrast to IgG, IgE does not mediate release of inflammatory mediators from these eosinophils. Therefore, previous observations regarding the expression of Fc{epsilon}RI and Fc{epsilon}RII by blood eosinophils from patients with eosinophilia may not be directly applicable to patients with allergy. Although our study may be limited because only patients with hay fever were studied, our observations raise questions about the current conception that eosinophil IgE receptors play a role in the pathophysiology of allergic diseases. The observations in IgE knockout mice, which demonstrated eosinophilic inflammation and increased bronchial hyperreactivity in the absence of IgE (54), may in part be applicable to humans; i.e., factors other than IgE may act as trigger(s) for mediator release by eosinophils in allergic inflammation.


   Acknowledgments
 
We thank Dr. J.-P. Kinet for the kind gift of anti-Fc{epsilon}RI mAb (15-1) and biotinylated cIgE; Mr. James E. Tarara, Ms. Teresa J. Halsey, and Ms. Holly B. Lamb for their excellent technical assistance in flow cytometry; Ms. Cheryl Adolphson for editorial assistance; Ms. Linda Arneson for secretarial assistance; and Ms. Judith Blomgren and Ms. Kay Bachman for recruiting and obtaining blood samples from patients.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health, AI 34577 and AI 34486, and by Mayo Foundation. Back

2 Address correspondence and reprint requests to Dr. Hirohito Kita, Department of Immunology, Mayo Clinic, Rochester, MN 55905. E-mail address: Back

3 Abbreviations used in this paper: mIgG, mouse IgG1; cIgE, chimeric mouse/human IgE; EDN, eosinophil-derived neurotoxin; HSA, human serum albumin; LT, leukotriene; PAB, PBS containing 0.1% NaN3 and 1% BSA; {Delta}MFC, differences in the mean fluorescence channel; EIA, enzyme immunoassay. Back

Received for publication November 10, 1998. Accepted for publication February 10, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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