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Published ahead of print on November 20, 2003, doi:10.1165/rcmb.2003-0234OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 736-743, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0234OC

Expression of Interleukin-5– and Granulocyte Macrophage–Colony-Stimulating Factor–Responsive Genes in Blood and Airway Eosinophils

Mary E. Bates, Lin Ying Liu, Stephane Esnault, Barbara A. Stout, Ekokobe Fonkem, Vanderlene Kung, Julie B. Sedgwick, Elizabeth A. B. Kelly, Douglas M. Bates, James S. Malter, William W. Busse and Paul J. Bertics

Departments of Biomolecular Chemistry, Medicine, Pathology, and Laboratory Medicine and Statistics, University of Wisconsin–Madison, Madison, Wisconsin

Address correspondence to: Paul J. Bertics, Ph.D., Dept. of Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. E-mail: pbertics{at}facstaff.wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage–colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor {alpha} subunit, lymphotoxin ß, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5– and GM-CSF–responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor {alpha} subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin ß and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

Abbreviations: bronchoalveolar lavage, BAL • biliary glycoprotein a, BGPa • interleukin, IL • granulocyte macrophage–colony-stimulating factor, GM-CSF • lymphotoxin, LT • Model-Based Expression Index, MBEI • median channel fluorescence, MCF • segmental bronchoprovocation with antigen, SBP-Ag • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The eosinophil is a granulocytic leukocyte that is produced in the bone marrow and trafficks to the tissues, most markedly those with a mucosal interface with the environment (1). Eosinophils are increased in number in allergic disease, suggesting a role for these cells in the pathogenesis of asthma and allergies (2). They contribute to tissue injury and inflammation through a number of mechanisms including the elaboration of enzymatic and bioactive proteins, the release of proinflammatory lipid mediators, and the production of reactive oxygen metabolites (1).

Whereas eosinophil accumulation in the tissues is promoted by chemoattractants, notably the CC chemokine eotaxin (3), the primary eosinophil-active cytokines are interleukin (IL)-5, IL-3, and granulocyte macrophage–colony-stimulating factor (GM-CSF). These IL-5-family cytokines have distinct and overlapping effects to increase the proliferation and differentiation of eosinophilic precursor cells in the bone marrow, release eosinophils into the circulation, and recruit them to sites of inflammation through effects on adherence, endothelial transmigration, and motility (4). The IL-5 family cytokines also promote the accumulation of eosinophils by reducing the rate at which they are cleared from the tissues through suppression of apoptotic cell death (5). Furthermore, IL-5 family cytokines enhance eosinophil proinflammatory effector functions either directly or indirectly by enhancing the responsiveness of the eosinophil to secondary stimulation by other factors in a process known as priming (1, 6, 7). In many respects, the enhanced responsiveness seen in IL-5–primed blood eosinophils is recapitulated in vivo by eosinophils recruited to the airway 48 h after segmental bronchoprovocation with antigen (SBP-Ag). For example, compared with cells in the circulation, airway eosinophils have heightened release of toxic oxygen species, enhanced ex vivo survival, and a greater expression of activation markers (8). The contribution of IL-5 to these features in vivo is not well understood, however.

The responsiveness of the eosinophil to IL-5 family cytokines is mediated through the activation of numerous intracellular signal transduction networks (reviewed in Ref. 5), many of which can impinge on mRNA expression through effects on both transcriptional and post-transcriptional processes (9–11). It is likely, therefore, that the phenotypic properties of the eosinophil in the airway are, in part, affected through the regulation of eosinophil gene expression by the IL-5 family cytokines, because localized expression of IL-5 family cytokines is a feature of allergic inflammation (12). In the current study, we sought to identify proteins regulated at the level of gene expression by IL-5 family cytokines in human blood eosinophils through microarray analysis. We then examined the expression of selected gene products in blood eosinophils to determine if there is a subsequent modulation of protein expression following ex vivo stimulation with IL-5 family cytokines. In addition, the expression of several IL-5–responsive gene products localized to the cell surface was evaluated in blood and airway eosinophils, given that cell surface proteins are critical to the responsiveness of the eosinophil to the various matrix proteins, cells, and mediators in the inflammatory microenvironment of the airway in asthma. These studies identify the cell surface molecules IL-3 receptor {alpha} subunit and lymphotoxin (LT)ß and the signaling molecules Pim-1 and cyclin D3 as being increased in expression in blood eosinophils following ex vivo stimulation with IL-5. Furthermore, four IL-5– and GM-CSF–responsive genes are increased in expression in airway eosinophils relative to their circulating counterparts; the {alpha} subunit of the IL-3 receptor, CD25, CD44, and CD66e.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Materials
Reagents for immunoblotting included anti-LTß mAb B27B2 and B2C2, a generous gift from Dr. Jeff Browning of Biogen (Cambridge, MA), horseradish peroxidase–conjugated anti-mouse IgG anti-Rabbit IgG, anti–Pim-1, and anti–cyclin D3 purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce Biotechnology Co. (Rockford, IL). Directly conjugated mAb for flow cytometry included PE-conjugated antibodies to IL-3 receptor {alpha} (IL-3R{alpha}, CDx123; BD Pharmingen, San Diego, CA), CD24 (Immunotech, Marseille, France), and FITC-conjugated mAb to CD25 (from Caltag Laboratories, Burlingame, CA), CD44 (Becton Dickinson Immunocytometry systems, San Jose, CA), CD66b (Immunotech, Prague, Czech Republic), and CD66e (CLB, Amsterdam, Netherlands). HuGeneFL microarrays were purchased from Affymetrix Inc. (Santa Clara, CA). These oligonucleotide microarrays permit the detection of ~ 5,000 human full-length sequences. Recombinant human IL-5 and GM-CSF were purchased from Peprotech, Inc. (Rocky Hill, NJ) and from R&D Systems Inc. (Minneapolis, MN).

SBP-Ag
Bronchoalveolar lavage (BAL) fluid was obtained from allergic subjects 48 h after airway allergen challenge, and BAL eosinophils were isolated for further analysis as described below. SBP protocols were approved by the University of Wisconsin–Madison Health Sciences Human Subjects Committee, and informed consent was obtained from the subjects before participation. Allergen dose for SBP was defined, and bronchoscopy was conducted, as recently described (13).

Eosinophil Purification
For immunoblotting experiments, BAL eosinophils were purified by a Percoll gradient. BAL cells (50 x 106/5 ml) were layered over a 1.085/1.100 g/ml isotonic Percoll bilayer and centrifuged for 20 min at 700 x g. The BAL eosinophils were collected from the 1.085/1.100 g/ml interface. Peripheral blood eosinophils were isolated by negative selection using immunomagnetic beads. Briefly, venous peripheral blood was centrifuged over Percoll (1.090 g/ml) to exclude mononuclear cells, erythrocytes were removed by hypotonic lysis, and then neutrophils were depleted with anti-CD16 immunomagnetic beads (MACS system; Miltenyi, San Jose, CA) (14). Both purified BAL and blood eosinophils were > 97% viable and > 98% pure.

Cell Culture
Purified eosinophils were suspended in RPMI with 1% fetal bovine serum with or without added cytokine (100 pM IL-5 or GM-CSF). The cell suspensions were incubated for 4–24 h at 37°C in a humidified incubator with 5% CO2. Viability was > 94% following cell stimulation. Eosinophils were then examined for expression of selected proteins by immunoblotting or flow cytometry or for RNA expression by microarray analysis.

RNA Preparation and Sample Labeling
After incubation with cytokines, eosinophils were lysed using TRI reagent (Molecular Research Center Inc., Cincinnati, OH) and total RNA was isolated as described by the manufacturer. For each cell culture condition of the blood eosinophils, the total RNAs from 5–7 eosinophil donors were pooled, enabling us to acquire adequate amounts of eosinophil RNA for microarray analysis from patients without pronounced eosinophilia. Five micrograms of total RNA was reverse-transcribed to cDNA and the cDNA was used to synthesize biotin-labeled complementary RNA using ENZO BioArray High Yield RNA Transcript Kit (ENZO Diagnostics, Farmingdale, NY).

Hybridization
Hybridization of the biotin-labeled complementary RNA to Affymetrix HuGeneFL arrays was performed as described (15) (Affymetrix Expression Analysis Technical Manual available from Affymetrix, Santa Clara, CA). The fluorescence signal was amplified by the addition of streptavidin and PE-labeled anti-streptavidin antibodies, and the hybridization intensity was determined by laser scanning.

Microarray Data Analysis
Pixel intensity levels were acquired from the digitized images of six oligonucleotide microarrays. These images were acquired from the labeled nucleotide preparations representing the two independent eosinophil pools from a total of 12 patients. Each patient's eosinophils were treated with control buffer, IL-5, and GM-CSF before RNA purification and RNA samples from like-treated eosinophils were pooled. The scanned microarray images were first analyzed by GeneChip software (Affymetrix) and sample values were normalized according to the total signal intensity on each chip. To quantitate the abundance of each transcript, the Model-Based Expression Index (MBEI) was calculated by dChip software (16). The determination of whether a transcript was increased or decreased by the various cytokine treatments was yielded by GeneChip software.

Flow Cytometry
For analysis of cell surface receptors, eosinophils were stained in whole blood (100 µl) and nonpurified BAL cell suspensions (105 cells/tube) as previously described (13). In some experiments, eosinophils were stained following in vitro incubation of purified eosinophil preparations with exogenous IL-5. For analysis, 10,000 events were collected using a Becton Dickinson FACScan II flow cytometer, and data analyses were performed using CellQuest software (Becton Dickinson). For analyses performed in whole blood and BAL fluid, all test samples contained an FITC- or PE-labeled anti-CD16 and anti-CD14 cocktail, which allowed for electronic exclusion of any contaminating neutrophils and monocytes, respectively. Data are summarized as the median channel fluorescence (MCF) of 104 events. The statistical significance of receptor expression was determined by a Student's t test applied to the MCF (17).

Immunoblotting
After stimulation with IL-5 or GM-CSF, eosinophil suspensions were diluted with Buffer A (25 mM HEPES pH 7.4, 137 mM NaCl, 1 mM EDTA, 10 mM NaF, 20 mM ß glycerophosphate, 1 mM sodium vanadate, and 1:100 [vol/vol] dilution of Sigma Mammalian Protease Inhibitor cocktail; Sigma Chemical Co., St. Louis, MO), pelleted by centrifugation, and lysed in Buffer B (Buffer A with 1% Triton-X-100, 0.1% SDS, and 0.25% deoxycholic acid). Equal mass of eosinophil lysate protein was loaded into the wells of an SDS-polyacrylamide gel and immunoblotting was conducted as previously described (18). Images were scanned and quantified using densitometry and NIH Image software. The statistical significance of differences between various samples was determined by the Student's t test for paired samples applied to the density of the digitized immunoblot image. In some cases, the densitometry data were log-transformed before statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Global Gene Expression in Human Eosinophils Stimulated with IL-5 or GM-CSF
Using oligonucleotide microarrays, the absolute expression levels of eosinophil transcripts were determined following stimulation with control buffer, 100 pM IL-5, or GM-CSF for 4 h (see Table E1 in the online supplement for expression data of ~ 5,000 human genes in peripheral blood eosinophils stimulated with or without IL-5 or GM-CSF). Approximately one-third of the 7,000 transcripts detectable by the microarrays were present (as defined by GeneChip and dChip softwares) in the eosinophil samples. An overall view of the relative expression of all the transcripts, as it is affected by cytokine treatment, is presented in the panels of Figure 1. In the two upper panels of Figure 1, the scatterplot demonstrates that a number of transcripts were more abundantly expressed in the IL-5–treated samples than in the control-treated sample (points falling above the diagonal trend lines). Likewise, a smaller number of transcripts were decreased in abundance following IL-5 treatment (points falling below the diagonal trend lines). A similar distribution of transcripts were increased or decreased following treatment with GM-CSF (see center panels in Figure 1). Therefore, the upper four panels of Figure 1 suggest that treatment of blood eosinophils for 4 h with either IL-5 or GM-CSF induces the increased expression of many transcripts and, for a smaller number of transcripts, a modest reduction in expression.



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  		Figure 1. Scatterplot of mRNA abundance for each transcript present in eosinophils. To visualize the effects of IL-5 and GM-CSF on global gene expression in eosinophils, dChip software was used to calculate the MBEI for each transcript encoded by the Affymetrix HuGeneFL genechips. The expression data were excluded for those transcripts that were absent in all the eosinophil samples. The log10 MBEIs for the remaining transcripts were plotted pairwise for the eosinophil treatment samples from donor pool 1 on the left panels and donor pool 2 on the right panels. On each panel, the trend lines for x = y, 2x = y, 3x = y, x = 2y, and x = 3y on a linear scale are plotted as a visual aid for comparison. All panels of Figure 1 are plotted on identical axes, although the numerical scale is included only on alternate panels.

 
Gene Expression Profiles Stimulated by IL-5 Relative to GM-CSF
The two bottom panels of Figure 1 show the expression of transcripts in the IL-5–treated blood eosinophil samples relative to the GM-CSF–treated eosinophil samples and reveal that there is little variation between the samples treated by the two cytokines. Most transcripts fall very close to the diagonal trend lines plotting x = y. To determine if there are discrete transcriptional targets of the IL-5 and GM-CSF receptors, genes that were recognized by GeneChip software as being altered by IL-5 treatment relative to the expression in GM-CSF–treated blood eosinophils in both patient sample pools were identified. This analysis identified no transcripts that were increased by treatment with 100 pM IL-5 relative to 100 pM GM-CSF in both eosinophil pools. However, 94 transcripts were more abundantly expressed in GM-CSF–treated eosinophils than in IL-5–treated eosinophils in both sample pools. Of these 94 transcripts, 65 transcripts were among those also identified as being induced by both IL-5 and GM-CSF relative to control-treated eosinophils. Therefore, the argument can be made that the greater mRNA expression induced by GM-CSF relative to IL-5 with respect to these 65 transcripts reflects effects on the magnitude of the response rather than the nature of the response. For the remaining transcripts in this group, i.e., transcripts that were induced by GM-CSF relative to IL-5 but not by IL-5 relative to control, the magnitude of the induction by GM-CSF relative to IL-5 was quite modest. The average expression index in the GM-CSF–treated eosinophils was only 1.0 to 1.6 times the expression index in the IL-5–treated eosinophil, a marginal level of induction. Therefore, these analyses have provided no compelling evidence for the existence of discrete transcriptional targets of GM-CSF that are not also stimulated by IL-5.

Genes Increased in Abundance Following Treatment with IL-5 or GM-CSF Relative to Control
From the absolute expression data, comparisons were made by GeneChip software of the abundance of each transcript in the various eosinophil treatments. This analysis identified 254 genes that were increased by both IL-5 and GM-CSF stimulation in both pools of eosinophil donors. Table 1 summarizes the expression data on the most highly induced of these 254 genes, selected for presentation because they showed an average increase in the MBEI of at least 3-fold following cytokine treatment.


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  		TABLE 1. mRNA expression of genes highly induced by both IL-5 and GM-CSF*

 
Several of the transcripts identified in Table 1 to be IL-5–responsive had been reported in previous studies to be induced by IL-5 or GM-CSF in eosinophils (1, 19–21), thereby validating microarray analysis of pooled eosinophil RNA samples as a useful approach in examining the regulation of eosinophil gene expression. We next sought to determine if the protein products of selected IL-5– and GM-CSF–responsive genes were more highly expressed in blood eosinophils following in vitro incubation with IL-5 or GM-CSF. As summarized in Figure 2, the protein expression of CD69 (Figure 2A, included as a positive control), the {alpha} subunit of the IL-3 receptor (Figure 2B), LT-ß (Figure 2C), Pim1 (Figure 2D), and cyclin D3 (Figure 2E) were significantly increased in expression following IL-5 or GM-CSF (data not shown) treatment. However, the expression of p27-IEX-1 protein (Figure 2F), a protein related to regulation of apoptosis in some model systems (22), was affected to a modest degree in a smaller proportion of the eosinophil preparations examined. Therefore, several, but not all, of the IL-5–responsive genes show enhanced protein expression following exposure to IL-5 or GM-CSF, suggesting the importance of other post-transcriptional modes of regulation in the control of eosinophil phenotypic properties.



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  		Figure 2. Protein expression of several IL-5–responsive genes in purified blood eosinophils following treatment with control buffer or IL-5. In each panel, each line represents data from an independent experiment. Statistical significance between control-treated and IL-5–treated eosinophils, as determined by the Student's t test, is given at the top of each panel. (A) CD69 was determined by flow cytometry after 24 h stimulation with 300 pM IL-5 and quantified as the MCF. (B) IL-3R{alpha} was determined by flow cytometry after 24 h stimulation with 300 pM IL-5 and quantified as the MCF. (C) LT-ß was evaluated by immunoblotting and densitometry analysis after 4 h of treatment with 0 or 100 pM IL-5 and quantified as the optical density (in arbitrary units). (D) Pim-1 was evaluated by immunoblotting and densitometry analysis after 4 h of treatment with 0 or 100 pM IL-5 and quantified as the optical density (in arbitrary units). (E) Cyclin D3 was evaluated by immunoblotting and densitometry analysis after 4 h of treatment with 0 or 100 pM IL-5 and quantified as the optical density (in arbitrary units). (F) p27-IEX-1 expression was evaluated by immunoblotting and densitometry analysis after 4 h of treatment with 0 or 100 pM IL-5 and quantified as the optical density (in arbitrary units).

 
Protein Expression of IL-5– and GM-CSF–Responsive Genes in Blood and BAL Eosinophils
It is noteworthy that several of the transcripts summarized in Table 1 encode cell surface proteins. Because of the importance of plasma membrane proteins in the regulation of cellular functions, the protein expression was analyzed, in blood and BAL eosinophils, of several of these transcriptionally-regulated membrane proteins, given that BAL eosinophils acquired 48 h after SBP-Ag are exposed in vivo to greater concentrations of IL-5 family cytokines (23). Other studies had previously evaluated the protein expression of CD69, CD32, CSF2RB, and CCR1 in BAL eosinophils (24–27), so these gene products were not considered further in this study. In examining cell surface expression of other transcripts identified by microarray analysis, we observed markedly upregulated cell surface protein expression in BAL eosinophils for four molecules. These included IL-3R{alpha} (Figure 3A), IL-2R{alpha}/CD25 (Figure 3B), and CD44, a product of the Epican gene (Figure 3C). In addition, a protein product of the BGPa gene, an epitope labeled by an antibody to CD66e, was markedly upregulated in airway eosinophils (Figure 3D). Furthermore, it was noted that expression of IL-3R{alpha}, CD44, and CD66e was significantly higher on blood eosinophils 48 h after SBP-Ag than it was on the blood eosinophils taken from the same patients before Ag challenge (Figures 3A, 3C, and 3D). However, the increase in expression of these molecules in post-Ag blood relative to pre-Ag blood was modest when compared with the magnitude of the increased expression in airway eosinophils (Table 2). Nonetheless, taken together, these data suggest that four IL-5–responsive genes encoding cell surface proteins are increased in expression in blood and/or airway eosinophils following antigen challenge of allergic patients.



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  		Figure 3. Protein expression of IL-5–responsive genes for blood and BAL eosinophils: The cell surface expression for IL3R{alpha} (A), CD25 (B), CD44 (C), CD66e (D), and CD24 (E) was determined by flow cytometry for peripheral blood eosinophils prior to SBP-Ag and blood and BAL eosinophils 48 h after SBP-Ag. Each line represents data on a separate patient. Plotted values are the MCF for 104 events. The statistical significance for comparisons between the three populations of cells is given at the top of each panel. LTß was determined by immunoblotting for peripheral blood eosinophils and BAL eosinophils (F). A representative immunoblot (n = 3) is shown of the expression in BAL (lane 1) and peripheral blood (lane 2) eosinophils 48 h after SBP-Ag as well as in the blood eosinophils from a non–Ag-challenged donor after incubation for 4 h with 0 pM (lane 3) or 100 pM (lane 4) IL-5.

 

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  		TABLE 2. Expression of cell surface markers on blood and BAL eosinophils as determined by flow cytometry*

 
In contrast, two additional protein products of IL-5–responsive genes were examined and were not found to be discernibly upregulated in BAL eosinophils relative to their expression in peripheral blood eosinophils. One of these is CD24, a GPI-anchored member of the immunoglobulin family of adhesion molecules (28). CD24 was expressed in blood eosinophils but significantly decreased in BAL eosinophils following SBP-Ag (Figure 3E and Table 2).

A second transcript whose protein product was not discernibly increased in abundance in BAL eosinophils is LTß, a membrane-associated cytokine of the tumor necrosis factor (TNF) family (29). The expression of LTß was evaluated on blood and BAL eosinophils by flow cytometry using two separate anti-LTß mAb (30). No difference in the cell surface labeling of eosinophils was observed relative to labeling by isotype control antibodies, suggesting that little or no LTß is expressed on the plasma membrane of eosinophils (n = 3, data not shown). Whereas immunoblotting studies showed that expression of LTß protein was increased in blood eosinophils following incubation with IL-5 (Figure 2F) or GM-CSF (data not shown), densitometry analysis of anti-LTß immunoblots revealed no consistent differences in the expression levels of LTß in blood versus BAL eosinophils (P = 0.89, n = 3). These data suggest that LTß is an IL-5– and GM-CSF–responsive gene, that its protein product is expressed in blood and BAL eosinophils at approximately equal levels but that the protein is not discernibly expressed on the cell surface of eosinophils as they were evaluated in these biological samples.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Members of the IL-5 family are principal eosinophil-active cytokines and modulate multiple eosinophil biological activities. In this regard, the goal of the present study was to test the concept that IL-5 family cytokines exert extensive control over the expression of several proteins in eosinophils and thereby affect the biological properties of the eosinophil. Accordingly, we used oligonucleotide microarrays to evaluate the gene expression profiles of eosinophils following stimulation with IL-5 or GM-CSF for 4 h (Figure 1), and we observed that ~ 9% of the ~ 3,000 transcripts detected as present in the eosinophils by the microarrays were altered in their expression level by the cytokine treatments in both pools of eosinophils (264 transcripts).

Among the transcripts for which the abundance of the mRNA was increased by both cytokines, 54 genes exhibited average MBEI that was at least 3-fold greater in cytokine-treated eosinophils than in control-treated cells (Table 1). These highly induced genes included several signaling molecules and transcription factors, as well as multiple enzymes, a few proinflammatory mediators, several transcripts encoding proteins of unknown function, and other proteins that contribute to metabolic and homeostatic processes in the cell.

To affect the phenotype of the eosinophil, a transcript must be productively translated into protein. We assessed the level of protein expression of several gene products of IL-5– and GM-CSF–responsive genes, and observed that the {alpha} subunit of the IL-3R, the TNF family cytokine, LTß, the apoptosis-associated protein kinase, Pim-1, and the cell cycle regulatory protein cyclin D3 all showed greater protein expression in blood eosinophils treated with IL-5 (Figures 2B–2E) or GM-CSF (data not shown; 31). However, the apoptosis-related protein p27-IEX-1 was not consistently upregulated in expression after IL-5 treatment for 4 h (Figure 2F) or 24 h (data not shown).

Cell surface proteins that were identified as being highly induced at the mRNA level by both IL-5 and GM-CSF included several that may be important effectors of eosinophil responsiveness to the cells, tissues, and soluble factors present in the airway. Therefore we hypothesized that, after migration from the circulation to the lumen of the airway following SBP-Ag, human eosinophils would show altered phenotypic characteristics consistent with stimulation with IL-5 family cytokines. Although it is clear that other factors during this transmigration process can affect the phenotypic characteristics of the eosinophil (e.g., adherence to extracellular matrix proteins and endothelium, soluble factors elaborated by cells of the airway, and proteins localized to the inflammatory microenvironment), these experiments were initiated to identify gene products that may be altered by exposure in vivo to IL-5 family cytokines.

The IL-3R{alpha} subunit binds IL-3, and its protein expression was markedly higher in airway eosinophils (Figure 3A). The expression of GM-CSFR{alpha} in BAL eosinophils was reported in an earlier study from our laboratories (24) that also noted that the protein expression of IL-5R{alpha} and the common ß receptor subunit were reduced in airway eosinophils following SBP-Ag relative to their expression in blood eosinophils. Furthermore, in the study by Liu and coworkers, the authors observed that BAL eosinophils were less responsive to IL-5 than blood eosinophils with regard to their ability to elaborate the cytotoxic granule protein eosinophil-derived neurotoxin (24). Taken together, these data show that the cell surface receptors for IL-3 and GM-CSF are more highly expressed in BAL eosinophils than in blood eosinophils, and although IL-3, IL-5, and GM-CSF may be present in the airway of patients with asthma (32–34), the activation of airway eosinophils may be more directly regulated by IL-3 and GM-CSF than IL-5 because the abundance of their receptor subunits on eosinophils appears to be greater. The complex and diverse processes regulating the cytokine receptor subunit expression, including transcriptional control and downregulation by internalization/proteolytic degradation are therefore important determinants of eosinophil responsiveness to IL-5 family cytokines in the airway.

Microarray analysis revealed that mRNA for the immunoglobulin supergene family molecule BGPa (CD66) was highly induced by IL-5 and GM-CSF (21) (Table 1). Interestingly, an earlier report identified greater CD66 protein expression in eosinophils acquired from patients infected with helminthic parasites (35), which is another pathologic state associated with high circulating and localized levels of IL-5 family cytokines. Whereas the mRNA for the CD66 is rapidly and markedly induced by cytokine treatment, the cell surface expression was not significantly affected by the cytokine treatments as studied by Mawhorter and colleagues (35), perhaps because the immunochemicals chosen for the study were reactive with multiple isoforms of CD66. In agreement with those findings, we detected no difference in the cell surface expression of CD66b on blood versus BAL eosinophils (n = 3, data not shown). However, the epitope for CD66e was expressed on blood eosinophils, increased by in vitro treatment with both IL-5 and GM-CSF (not shown), increased on blood eosinophils acquired 48 h following SBP-Ag, and markedly increased on airway eosinophils relative to their circulating counterparts (Figure 3D). The various isoforms of CD66 represent alternatively spliced variants of a single gene in humans (36), and these molecules are considered to be activation markers for myeloid cells including neutrophils (37). They are believed to be important for endothelial transmigration, cell aggregation, and responsiveness to selected microbial proteins (37). Therefore, these findings suggest that CD66e is a previously unidentified marker of eosinophil activation or inflammatory capacity.

The increased expression of CD25 (the IL-2 receptor {alpha} subunit), and CD44 mRNA and protein in eosinophils has been previously reported in response to in vitro exposure to cytokines or in blood eosinophils under various circumstances of eosinophilic inflammation (20, 21, 35, 38, 39). Our microarray analysis of IL-5– and GM-CSF–treated blood eosinophils also showed increased mRNA expression of these genes (Table 1). Cell surface expression of CD25 (Figure 3B) and CD44 (Figure 3C) was markedly increased in BAL eosinophils relative to their circulating counterparts acquired at the same time. Furthermore, the protein expression for CD44 was marginally but significantly higher in blood eosinophils acquired 48 h after SBP-Ag than before Ag exposure (Figure 3C, Table 2). Therefore, these cell surface proteins also appear to be sensitive markers of in vivo activation of eosinophils.

In contrast, CD24, for which the mRNA was highly induced by IL-5 and GM-CSF (Table 1) (21), exhibited significantly decreased expression in BAL eosinophils relative to the expression in blood eosinophils (Figure 3E, Table 2). CD24 is a widely expressed membrane protein and may participate in eosinophil recruitment and adherence through interaction with P-Selectin (28). Our data suggest, however, that CD24 may be shed or internalized before eosinophils reach the lumen of the airway. To our knowledge, this report is the first demonstration of LTß expression in human eosinophils (Figure 2C, Figure 3F). LTß is a TNF family cytokine that exerts its biological effects by interacting with the LTß receptor expressed on a wide variety of cells. Current understanding of the function of LTß suggests that this cytokine is noncovalently associated with another TNF family cytokine, LT{alpha} (also designated TNF-ß), on the cell surface as a heterotrimer called membrane lymphotoxin (29). To date, no cell surface expression of LTß in the absence of LT{alpha} has been reported in any model system. Notably, LT{alpha} mRNA was not detected in any of the eosinophil samples, and no previous reports of its production by eosinophils have appeared. It is possible that exposure of eosinophils to a second stimulus may induce expression of LT{alpha} and mobilize intracellular stores of LTß to the cell surface. However, given that membrane lymphotoxin was not detected by flow cytometry on blood or BAL eosinophils (not shown), there is currently insufficient evidence to suggest a role for LTß in eosinophil biological function in allergic inflammation and asthma.

Several previous reports in a variety of cell backgrounds have attributed distinct signaling capabilities to the {alpha} subunits of the IL-5 and GM-CSF receptors (40, 41). It was surprising, therefore, that in this analysis we found no distinct transcriptional targets for IL-5 relative to those for GM-CSF. No transcripts were more highly induced by IL-5 relative to the expression in GM-CSF–treated eosinophils in both sample pools. Although a number of transcripts were more abundantly expressed in GM-CSF–treated eosinophils than in IL-5–treated eosinophils, all of these transcripts were induced by a very modest degree (1.6-fold or less) or were among those genes increased by both IL-5 and GM-CSF relative to control-stimulated eosinophils. This analysis did not reveal a distinct pattern of transcriptional responses stimulated by IL-5 versus GM-CSF, but it is possible that there are discrete transcriptional targets that are manifested in only a subpopulation of patients, or with different kinetics than those employed in this study. Within these limitations, however, this study suggests that the differences in gene expression induced by these two cytokines in human eosinophils appear to be in the magnitude of the responsiveness. These data have provided no compelling evidence for the existence of distinct gene expression profiles induced by IL-5 and GM-CSF in human blood eosinophils.

In conclusion, we have provided expression data for ~ 5,000 genes in human eosinophils (see Table E1 in the online supplement) and evidence for the regulation by IL-5 and GM-CSF of the mRNA abundance of numerous transcripts. Among the transcripts most profoundly affected by these cytokines are several cell surface proteins, proinflammatory mediators, enzymes, signaling molecules, and proteins of unknown function (Table 1). We have shown for the first time that the protein products of a subset of these IL-5–responsive genes are expressed more abundantly in blood eosinophils following IL-5 treatment (Figure 2), and that several membrane receptors that are IL-5–responsive genes are increased in expression on the cell surface of BAL eosinophils relative to their circulating counterparts (CD25, CD66e, IL-3R{alpha}, CD44), suggesting a role for these IL-5– and GM-CSF–responsive genes in the regulation of eosinophil function in the airway.


   Acknowledgments
 
The authors are grateful to Dr. Mats Johansson, Dr. Deane Mosher, and Dr. Gregory Wiepz for helpful discussion. They thank Heather Gerbyshak, Kristin Jansen, and Sarah Panzer for technical assistance in eosinophil and BAL cell preparation, Dr. Nizar Jarjour and Dr. Keith Myers for BAL cells, and the University of Wisconsin Gene Expression Center for technical assistance with microarray hybridization, scanning, and analysis.


   Footnotes
 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form June 17, 2003

Received in final form November 14, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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