Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Rα on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.

Asthma is a chronic obstructive disease of the airway that is characterized by recurrent episodes of shortness of breath, bronchial hyperreactivity, and inflammation marked by blood and pulmonary eosinophilia and increased levels of IL-5 and GM-CSF in the airway (1). The molecular mechanisms driving the pathogenesis of asthma are not clear and are likely to be complex. However, eosinophils are one cell type that has been implicated in promoting certain aspects of the pathophysiology occurring in asthma (1).

Previous studies have revealed that eosinophils are among the key effector cells responsible for the tissue destruction and inflammation in allergic airway diseases such as asthma (1). IL-5 and GM-CSF profoundly affect the differentiation, recruitment, and cytotoxic effector functions of the eosinophil (2). IL-5 and GM-CSF also modulate gene expression and suppress apoptosis in eosinophils (3).

One of the multiple signal transduction networks initiated by IL-5 family cytokines is the JAK/STAT pathway. Activation of this pathway by IL-5 family cytokines and other cytokines in various hemopoietic cell lines has indicated a diverse array of roles for the JAK/STAT pathway, including cell survival, differentiation, and proliferation (4). In general, reciprocal tyrosine phosphorylation of receptor-associated JAKs occurs upon cytokine binding to cytokine receptors. Subsequent tyrosine phosphorylation of the receptor by JAK creates binding sites for the Src homology 2 region of STAT proteins. Recruitment of the STAT proteins leads to their activation by phosphorylation by a JAK enzyme on a tyrosine residue at ∼aa 700, which allows for homo- or heterodimerization of STAT proteins. This activated STAT dimer then translocates into the nucleus, where it binds to consensus STAT-binding sequences and modulates transcription (5).

The JAK/STAT signal transduction pathway is vital for a number of functions, including the suppression of apoptosis in a variety of cell types. In eosinophils, IL-5- or GM-CSF-dependent survival is greatly attenuated when the enzymatic activity of Jak2 is inhibited (6, 7, 8). Furthermore, transcription appears necessary for the IL-5- or GM-CSF-mediated suppression of apoptosis in eosinophils, as assessed by using actinomycin D to block transcription (9). Because STAT proteins are transcription factors and are major substrates of JAKs, we therefore hypothesized that signal transduction through STAT pathway(s) occurs in human eosinophils treated with IL-5 or GM-CSF, resulting in the modulation of STAT-regulated gene expression. Furthermore, we hypothesize that eosinophils obtained from the airway following Ag challenge will be exposed to cytokines in vivo, and therefore will carry constitutively activated STAT proteins and express protein for STAT-dependent genes. To test these hypotheses, the current study examines the activation of STAT3, STAT5a, and STAT5b in blood eosinophils and airway eosinophils obtained from atopic patients 48 h after Ag challenge, and evaluates the expression of the STAT-dependent genes, the survival-associated kinase Pim-1, and the cell cycle regulator cyclin D3, in both cell populations. These studies suggest that STAT-dependent gene expression contributes to the increased IL-5- or GM-CSF-mediated viability of eosinophils and consequent eosinophilic inflammation often observed in asthma.

Percoll was purchased from Pharmacia (Piscataway, NJ), and anti-CD16-conjugated paramagnetic microbeads were obtained from Miltenyi Biotec (Auburn, CA). We acquired IL-5 from R&D Systems (Minneapolis, MN) and GM-CSF from PeproTech (Rocky Hill, NJ). Anti-STAT3 (06-596), anti-STAT5a (06-553), and anti-STAT5b (06-443) Abs were purchased from Upstate Biotechnology (Lake Placid, NY). We obtained an additional anti-STAT5b Ab (71-2500) from Zymed Laboratories (San Francisco, CA). From New England Biolabs (Beverly, MA), we obtained anti-phosphotyrosine STAT3 Ab, which detects tyrosine 705-phosphorylated STAT3 (9131S) and anti-phosphotyrosine 694 STAT5 (9351S). Anti-STAT3 (sc-7179), anti-STAT5a (sc-1081), and anti-STAT5b, which detects both STAT5a and b isoforms (sc-835); anti-Pim-1 (sc-13513), anti-cyclin D3 (sc-182) Abs; and HRP-conjugated goat anti-rabbit IgG and rabbit anti-mouse IgG were procured from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence substrate reagents LumiGlo and SuperSignal West were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD) and Pierce (Rockford, IL), respectively. For flow cytometric detection of the IL-5Rα subunit, a PE-conjugated mAb to IL-5Rα (CD125; BD Pharmingen, San Diego, CA) was used. The protease inhibitors MG-115 and Z-Asp-CH2-[(2,6,dichlorobenzoyl)oxy]methane were purchased from Calbiochem (San Diego, CA). β-glycerophosphate, sodium vanadate, additional protease inhibitors, and all other laboratory reagents, unless specified otherwise, were obtained from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Itasca, IL).

Subjects supplying blood for eosinophil purification ranged in age from 18 to 55 years. Healthy, nonallergic, nonasthmatic subjects were skin test negative, had no history of allergies or other medical conditions, and were taking no medications. Allergic subjects had a history of seasonal or perennial allergic rhinitis and were skin test positive (>3 mm by skin prick method) to at least one of the common allergens tested, including cat dander, ragweed, and dust mite allergens; they had no clinical history of asthma. These subjects’ only medications were antihistamines and/or Flonase, as needed. Allergic asthma patients were skin test positive. They were diagnosed clinically with asthma and were hyperresponsive on methacholine challenge. These subjects used only inhaled β2 agonists and had not been treated with oral or inhaled corticosteroids, long-acting β agonists, or leukotriene antagonist within 6 wk of participation.

Airway eosinophils were also acquired from 13 atopic patients using protocols described below. These subjects’ characteristics are described in Table I. Informed, written consent was obtained from all participants before inclusion in the study. All protocols used in the collection of human blood and airway eosinophils were approved by the University of Wisconsin Human Subjects Committee Internal Review Board.

Table I.

Segmental bronchoprovocation subject characteristics

SubjectaGenderAge (year)Meth PC20b (mg/ml)Ag PD20c (CBU)dFEV1 (% pred)eAg
29 17.9 224 88 CATf 
18 10.6 228 99 RWg 
30 4.8 10.7 94 HDMh 
19 1.9 51.9 120 CAT 
30 5.4 17.8 92 CAT 
21 1.2 5.6 82 HDM 
19 3.8 37 96 CAT 
20 0.3 1.4 87 RW 
20 1.3 8.2 107 HDM 
10 23 0.8 60.8 90 HDM 
11 20 1.9 33.7 116 CAT 
12 18 20 21 108 HDM 
13 19 3.5 118 105 HDM 
SubjectaGenderAge (year)Meth PC20b (mg/ml)Ag PD20c (CBU)dFEV1 (% pred)eAg
29 17.9 224 88 CATf 
18 10.6 228 99 RWg 
30 4.8 10.7 94 HDMh 
19 1.9 51.9 120 CAT 
30 5.4 17.8 92 CAT 
21 1.2 5.6 82 HDM 
19 3.8 37 96 CAT 
20 0.3 1.4 87 RW 
20 1.3 8.2 107 HDM 
10 23 0.8 60.8 90 HDM 
11 20 1.9 33.7 116 CAT 
12 18 20 21 108 HDM 
13 19 3.5 118 105 HDM 
a

Airway cosinophils used for these studies were acquired from 13 different patients.

b

Concentration of methacholine that caused a 20% fall in forced expiratory volume in 1 s (FEV1).

c

Provocative dose of Ag resulting in a 20% drop in FEV1.

d

Cumulative breath units.

e

Percentage of the predicted value.

f

Cat dander.

g

Ragweed Ag.

h

House dust mile.

Eosinophils were purified from the heparinized peripheral blood of volunteer human donors, with eosinophils composing 2–10% of the peripheral blood leukocytes. A granulocyte mixture was obtained from the leukocyte buffy coat after centrifugation through a Percoll monolayer (1.090 g/ml) and lysis of erythrocytes by hypotonic shock. The suspension was depleted of neutrophils by incubation with anti-CD16-conjugated paramagnetic microbeads.

Airway eosinophils were obtained from bronchoalveolar lavage (BAL)3 fluid 48 h following segmental bronchoprovocation of atopic human subjects with relevant Ag (Table I). Ag dose for segmental bronchoprovocation was defined, and segmental bronchoprovocations with Ag and BAL were performed, as previously described (10, 11, 13). Two different bronchopulmonary segments were identified for segmental bronchoprovocation with Ag. For each segment, a wedge position was achieved by the fiberoptic bronchoscope, and a baseline BAL was performed. Ag was instilled through the wedged bronchoscope. After 48 h, bronchoscopy was repeated and BAL was performed on each of the challenged segments. Airway eosinophils were purified by centrifugation of BAL cells through a Percoll bilayer (1.085/1.100 g/ml) and recovery of the cell population at the interface between the Percoll layers. The recovered blood and BAL eosinophils were resuspended in HBSS supplemented with 2% newborn calf serum. These cell preparations were at least 97% eosinophils with at least 95% viability, based on trypan blue staining and morphological examination of cytofuge preparations stained with Giemsa-based Diff-Quik stain (Baxter Scientific Products, McGaw Park, IL).

Freshly isolated eosinophils were preincubated at 37°C for 30 min; then stimulated with control buffer, IL-5, or GM-CSF at 100 pM (exceptions are noted in the figure legends); and incubated for various times, as indicated in each experiment. The cells were collected by centrifugation, and cell fractions were prepared.

Stimulated eosinophils were diluted in ice-cold buffer A (20 mM Tris, pH 7.4, 137 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM β-glycerophosphate, 10 mM NaF, and a mixture of protease inhibitors), pelleted by centrifugation, and lysed in buffer A plus detergents (1% Nonidet P-40, 0.25% deoxycholate, and 0.1% SDS) at a concentration of 2.5 × 107 cells/ml. Lysates were sonicated, and insoluble material was removed by centrifugation at 14,000 rpm for 10 min. The supernatants were assayed for total protein, diluted with electrophoresis sample buffer, and immunoblotted, as previously described (12).

For analysis of cell surface receptors, eosinophils were stained in whole blood (100 μl) and nonseparated BAL cell suspensions (105 cells per tube), as previously described (13). For analysis, 10,000 events were collected using a BD Biosciences (San Jose, CA) FACScan II flow cytometer, and data analyses were performed using CellQuest software (BD Biosciences). For analyses performed in whole blood and BAL fluid, all test samples contained a FITC- or PE-labeled anti-CD16 and anti-CD14 mixture, which allowed for electronic exclusion of any contaminating neutrophils and monocytes, respectively. Data are summarized as the median channel fluorescence of 104 events.

A protocol by van der Bruggen et al. (14) was used with modifications. Briefly, eosinophils were resuspended in 400 μl of a hypotonic buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM Na3VO4, 20 mM β-glycerophosphate, 0.5 mM DTT, 0.2 mM PMSF, and 10 μg/ml aprotinin. After incubation on ice for 15 min, Nonidet P-40 was added to a final concentration of 0.6% (v/v), and the mixture was vortexed for 10 s. The cytoplasmic fraction was collected after centrifugation at 4°C for 10 s at 7,200 × g. The pellet was resuspended in hypertonic buffer containing 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM Na3VO4, 0.2 mM PMSF, and 10 μg/ml aprotinin, and incubated on ice for 20 min. After centrifugation for 2 min at 13,500 × g, the soluble nuclear extract (supernatant) was collected. The fractions were stored at −80°C until used. Bicinchoninic acid assay (Pierce) was performed to determine the protein concentration.

Two DNA probes were used in these studies: the serum-inducible element (SIE) from the c-fos promoter, and the lactogenic hormone response region (LHRR) from the β-casein promoter. Complimentary oligonucleotides, including four base overhangs (5′-GATCT CATTT CCCGT AAATC TTGTCT-3′ and 5′-GATCA GACAA GATTT ACGGG AAATGA-3′ for the SIE probe, and 5′-GATCA GATTTC TAGGA ATTC AAATC-3′ and 5′-GATCG ATTTG AATTC CTAGA AATCT-3′ for the LHRR probe), were combined, heated to 95°C, and slowly cooled to room temperature. Duplexes were end labeled with [γ-32P]dATP (100 μCi) with T4 polynucleotide kinase (New England Biolabs).

An aliquot (5 μg of protein) of each nuclear extract was incubated on ice for 1 h with 10 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 5 mM DTT, 0.1 μg/μl herring sperm DNA, 0.1 μg/μl BSA, and, for the supershift experiments, 1 μg of the respective Ab. Radiolabeled probe (105 cpm) was added, and the mixture was incubated for 30 min at room temperature before electrophoresis on a 5% native polyacrylamide gel.

Previous reports have revealed that when the enzymatic activity of Jak2 is inhibited, eosinophil survival is attenuated even in the presence of IL-5 or GM-CSF (6, 7, 8). Given this background, together with the observation that STAT proteins are important substrates for JAKs, we tested the hypothesis that STAT proteins are involved in the signaling events controlling gene expression in eosinophils. We and others have observed that the transcription factors STAT1 (14, 15) and STAT5 (16, 17) are expressed and activated in human eosinophils; however, there are no reports of the expression of STAT3 nor which STAT5 isoform(s), STAT5a and/or STAT5b, is expressed in these cells. Recent studies have confirmed that the mRNA for all three isoforms is abundant in human peripheral blood eosinophils (18). Therefore, using immunoblotting analysis, we evaluated the expression of STAT3 (Fig. 1,A), STAT5a, or STAT5b (Fig. 1,B) protein in human eosinophils that had been treated with vehicle, IL-5, or GM-CSF for 15 min. STAT3 protein was detected in both vehicle-treated and IL-5- or GM-CSF-stimulated eosinophils. The mobility of the STAT3 protein band was slightly slower in the lanes from the IL-5- or GM-CSF-stimulated eosinophils (Fig. 1,A, lanes 2 and 3) than that from the lane from the vehicle-treated eosinophils (Fig. 1,A, lane 1), suggesting a protein modification such as phosphorylation. Fig. 1,B shows that both STAT5a (left panel) and STAT5b (right panel) proteins are expressed in eosinophils. Similar to the STAT3 immunoblot, the migration of STAT5b protein from the IL-5-stimulated eosinophils (Fig. 1,B, right panel, lane 2) shows retarded mobility compared with that from the vehicle treated (Fig. 1,B, right panel, lane 1), suggesting a protein modification similar to that seen with STAT3 (Fig. 1,B). In contrast, STAT5a showed no discernible shift in mobility following cytokine stimulation. The activation of STAT proteins occurs via JAK-dependent phosphorylation of a specific tyrosine residue that is necessary for STAT dimerization, nuclear translocation, and DNA binding (5). In addition, STAT proteins may be phosphorylated on serine or threonine residues in many model systems (19). Therefore, the alteration in mobility seen in STAT5b and STAT3 (Fig. 1, A and right panel of B) may reflect cytokine-stimulated changes in the phosphorylation status of STAT factors, but may or may not be related to the change in the DNA-binding activity.

FIGURE 1.

Expression of STAT3, STAT5a, and STAT5b in human eosinophils. Eosinophils were stimulated for 15 min with 100 pM IL-5 (lane 2), GM-CSF (lane 3), or control buffer PBS (lane 1). Protein (30 μg) from the resulting whole cell lysates was separated by SDS-PAGE and immunoblotted with A, anti-STAT3 Ab (Santa Cruz Biotechnology; sc-7179) (n = 10), or B, anti-STAT5a Ab (Upstate Biotechnology, 06-553; or Santa Cruz Biotechnology, sc-1081) (left panel) (n = 7) or anti-STAT5b Ab (Upstate Biotechnology; 06-443) (right panel) (n = 7). Blood donors for these experiments included nonallergic patients as well as allergic and allergic asthmatic patients.

FIGURE 1.

Expression of STAT3, STAT5a, and STAT5b in human eosinophils. Eosinophils were stimulated for 15 min with 100 pM IL-5 (lane 2), GM-CSF (lane 3), or control buffer PBS (lane 1). Protein (30 μg) from the resulting whole cell lysates was separated by SDS-PAGE and immunoblotted with A, anti-STAT3 Ab (Santa Cruz Biotechnology; sc-7179) (n = 10), or B, anti-STAT5a Ab (Upstate Biotechnology, 06-553; or Santa Cruz Biotechnology, sc-1081) (left panel) (n = 7) or anti-STAT5b Ab (Upstate Biotechnology; 06-443) (right panel) (n = 7). Blood donors for these experiments included nonallergic patients as well as allergic and allergic asthmatic patients.

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Phosphorylation at tyrosine 705 is a necessary prerequisite of activation of STAT3 (20). This phosphorylation allows the homo- or heterodimerization of STAT proteins via the phosphorylated tyrosine binding to the STAT partner’s Src homology 2 domain. Thus, the ability to detect phosphorylated tyrosine 705 STAT3 is a marker of STAT3 activation. In this present study, we tested whether IL-5 or GM-CSF could activate STAT3 by immunoblotting blood eosinophils (Fig. 2,A) with an Ab that detects STAT3 only when it is phosphorylated on tyrosine 705. Phosphotyrosine 705 STAT3 was detectable in the eosinophils stimulated in vitro with IL-5 or GM-CSF (Fig. 2,A, lanes 2 and 3), but not in unstimulated eosinophils (Fig. 2,A, lane 1). Equal protein loading and transfer were confirmed by stripping and reprobing the membrane with anti-STAT3, which detects both modified and unmodified STAT3 (Fig. 2 B).

FIGURE 2.

A, Phosphorylation status of STAT3 tyrosine 705 in peripheral blood eosinophils. Eosinophils from peripheral blood were stimulated with control buffer (lane 1), 300 pM IL-5 (lane 2), or 1 nM GM-CSF (lane 3) for 15 min and then lysed. Proteins (30 μg) from the lysates were subjected to electrophoresis, followed by immunoblotting with anti-phospho-STAT3 (New England Biolabs; 9131S), which detects STAT3 only when activated by phosphorylation at tyrosine 705. B, Membranes from A were stripped and reprobed with anti-STAT3 Ab (Santa Cruz Biotechnology; H-190, sc-7179) to demonstrate equivalent protein loading and transfer. These experiments were performed 13 times, using eosinophils from subjects without allergies as well as allergic and allergic asthmatic subjects. Similar results were observed with all patients. C, The tyrosine phosphorylation of STAT5a in eosinophils following IL-5-stimulation. Eosinophils from an allergic asthmatic blood donor were stimulated with 0 pM (lanes 1, 3 and 5) or 300 pM IL-5 (lanes 2, 4 and 6), and the resulting cell lysates were immunoprecipitated with anti-STAT5a (Santa Cruz Biotechnology; sc1081), anti-STAT5 (Santa Cruz Biotechnology; sc835), or rabbit IgG. The resulting immunoprecipitates were immunoblotted with anti-phosphotyrosine (upper panel) or anti-STAT5a (Santa Cruz Biotechnology; sc1081; lower panel) Abs. This experiment was conducted three times, with identical results, using eosinophils from one allergic nonasthmatic and two allergic asthmatic blood donors.

FIGURE 2.

A, Phosphorylation status of STAT3 tyrosine 705 in peripheral blood eosinophils. Eosinophils from peripheral blood were stimulated with control buffer (lane 1), 300 pM IL-5 (lane 2), or 1 nM GM-CSF (lane 3) for 15 min and then lysed. Proteins (30 μg) from the lysates were subjected to electrophoresis, followed by immunoblotting with anti-phospho-STAT3 (New England Biolabs; 9131S), which detects STAT3 only when activated by phosphorylation at tyrosine 705. B, Membranes from A were stripped and reprobed with anti-STAT3 Ab (Santa Cruz Biotechnology; H-190, sc-7179) to demonstrate equivalent protein loading and transfer. These experiments were performed 13 times, using eosinophils from subjects without allergies as well as allergic and allergic asthmatic subjects. Similar results were observed with all patients. C, The tyrosine phosphorylation of STAT5a in eosinophils following IL-5-stimulation. Eosinophils from an allergic asthmatic blood donor were stimulated with 0 pM (lanes 1, 3 and 5) or 300 pM IL-5 (lanes 2, 4 and 6), and the resulting cell lysates were immunoprecipitated with anti-STAT5a (Santa Cruz Biotechnology; sc1081), anti-STAT5 (Santa Cruz Biotechnology; sc835), or rabbit IgG. The resulting immunoprecipitates were immunoblotted with anti-phosphotyrosine (upper panel) or anti-STAT5a (Santa Cruz Biotechnology; sc1081; lower panel) Abs. This experiment was conducted three times, with identical results, using eosinophils from one allergic nonasthmatic and two allergic asthmatic blood donors.

Close modal

We have previously reported that IL-5 and GM-CSF treatment of human eosinophils results in phosphorylated tyrosine 694 STAT5, an indication of STAT5 activation (17). Whereas STAT5b demonstrates a mobility shift following IL-5 exposure, no apparent change in STAT5a mobility is discernible (Fig. 1,B). To determine whether STAT5a is tyrosine phosphorylated following exposure to IL-5, eosinophils were stimulated with IL-5 and then anti-STAT5a immunoprecipitates (Fig. 2,C, lanes 1 and 2) and anti-STAT5 immunoprecipitates (Fig. 2,C, lanes 3 and 4) were assessed for tyrosine phosphorylation and STAT5a content. These data demonstrate that STAT5a immunoprecipitates contain a tyrosine-phosphorylated protein following IL-5 stimulation, but not in the absence of cytokine (Fig. 2,C, upper panel, lane 2). The same cytokine-stimulated sample, immunoprecipitated with an Ab that binds both STAT5 isoforms, contains greater amounts of this tyrosine-phosphorylated protein (Fig. 2,C, upper panel, lane 4). Because the amount of STAT5a in all of the lanes is approximately equivalent (Fig. 2 C, lower panel), these data suggest that STAT5a is tyrosine phosphorylated following stimulation with IL-5. Furthermore, along with STAT5b, albeit to a lesser extent, STAT5a may participate in the regulation of STAT-dependent gene expression in eosinophils.

To determine whether STAT3, STAT5a, and/or STAT5b from IL-5- or GM-CSF-stimulated eosinophils have the ability to bind to DNA at STAT consensus sites, EMSAs were performed to assess such DNA binding. The SIE from the promoter of c-fos is a DNA sequence that binds tyrosine-phosphorylated STAT3 (21). As shown in Fig. 3,A, using a DNA probe that contains a variant high affinity SIE (21), we observed that the nuclear extracts from eosinophils treated with 300 pM IL-5 or 1 nM GM-CSF, but not from vehicle-treated eosinophils, contain protein that is able to bind the SIE DNA probe (Fig. 3,A). When anti-STAT3 Ab was included in the binding reaction, a supershift, or band of slower mobility, was observed (Fig. 3,A, lanes 4 and 7), supporting the concept that the protein/DNA complex contained STAT3. When preimmune rabbit IgG was included, there was no supershift, indicating the STAT3 supershift is specific (Fig. 3,A, lanes 5 and 8). Fig. 3 B shows an EMSA in which eosinophils were stimulated with 1 nM IL-5, resulting in a similar pattern of protein binding and supershifting.

FIGURE 3.

Ability of STAT3 and STAT5 protein from eosinophils to bind DNA. Eosinophils from an allergic asthmatic blood donor were stimulated with IL-5, GM-CSF, or vehicle control (C) for 15 min. Nuclear extracts (5 μg of protein) were incubated with a DNA probe containing STAT consensus binding sites. Complexes were resolved by electrophoresis on a native polyacrylamide gel. A, An EMSA was performed using the SIE DNA probe (see Materials and Methods) and, in lanes 4 and 7, the samples were incubated with anti-STAT3 (Upstate Biotechnology; 06-596) for supershift analysis. This experiment was repeated five times using eosinophils from nonallergic and allergic asthmatic blood donors, and similar results were observed. B, Results of an EMSA were performed similarly to that shown in A, except that eosinophils from an allergic nonasthmatic blood donor were stimulated with 0 nM (lane 1) or 1 nM IL-5 (lanes 2–4) and supershifted with anti-STAT3 (lane 3) or rabbit IgG (lane 4). This experiment was performed two separate times with similar results. C, An EMSA was performed using the LHRR DNA probe (see Materials and Methods), and the samples were incubated with either anti-STAT5a (Santa Cruz Biotechnology; sc-1081; lanes 3 and 7) or anti-STAT5b (Zymed Laboratories; 71-2500; lanes 4 and 8) or rabbit IgG (lanes 5 and 9) or buffer (lanes 1 and 10) for supershift analysis. This experiment was performed twice, and similar results were observed.

FIGURE 3.

Ability of STAT3 and STAT5 protein from eosinophils to bind DNA. Eosinophils from an allergic asthmatic blood donor were stimulated with IL-5, GM-CSF, or vehicle control (C) for 15 min. Nuclear extracts (5 μg of protein) were incubated with a DNA probe containing STAT consensus binding sites. Complexes were resolved by electrophoresis on a native polyacrylamide gel. A, An EMSA was performed using the SIE DNA probe (see Materials and Methods) and, in lanes 4 and 7, the samples were incubated with anti-STAT3 (Upstate Biotechnology; 06-596) for supershift analysis. This experiment was repeated five times using eosinophils from nonallergic and allergic asthmatic blood donors, and similar results were observed. B, Results of an EMSA were performed similarly to that shown in A, except that eosinophils from an allergic nonasthmatic blood donor were stimulated with 0 nM (lane 1) or 1 nM IL-5 (lanes 2–4) and supershifted with anti-STAT3 (lane 3) or rabbit IgG (lane 4). This experiment was performed two separate times with similar results. C, An EMSA was performed using the LHRR DNA probe (see Materials and Methods), and the samples were incubated with either anti-STAT5a (Santa Cruz Biotechnology; sc-1081; lanes 3 and 7) or anti-STAT5b (Zymed Laboratories; 71-2500; lanes 4 and 8) or rabbit IgG (lanes 5 and 9) or buffer (lanes 1 and 10) for supershift analysis. This experiment was performed twice, and similar results were observed.

Close modal

We, and others, have previously reported that STAT5 from IL-5- or GM-CSF-treated human eosinophils can bind DNA (16, 17), but it is unknown which STAT5 isoform, or whether both, bind to the STAT consensus DNA site. Therefore, we conducted EMSAs in which we included Abs specific for STAT5a or STATb that do not cross-react with the other isoforms and that are known to perform in Ab supershift experiments (22). Fig. 3 C (lanes 4 and 8) indicates that the majority of the protein/DNA complex is supershifted with anti-STAT5b, whereas there is a less marked supershift with anti-STAT5a (lanes 3 and 7), suggesting that STAT5b is a predominant member in the protein/DNA complex.

Because blood and airway eosinophils are characterized by multiple phenotypic differences and because levels of IL-5 and GM-CSF are increased in BAL fluid obtained 48 h after segmental bronchoprovocation with Ag (13, 18, 23, 24), we tested the idea that STAT3 and STAT5 are constitutively activated in airway eosinophils recovered from BAL fluid following segmental bronchoprovocation with Ag. In immunoblots detected with the anti-phosphotyrosine 705 STAT3 Ab, no phosphorylated STAT3 was detectable from untreated eosinophils isolated from BAL fluid (Fig. 4,A, lane 1), indicating that STAT3 is not constitutively activated in these eosinophils within the limits of detection by immunoblotting. Similar results were observed with STAT5 using an anti-phosphotyrosine 694 STAT5 Ab (Fig. 4,A, lane 3). Nonetheless, both STAT3 and STAT5 were still capable of becoming phosphorylated following IL-5 stimulation in vitro in some patients (Fig. 4,A, lanes 2 and 4; Table II). However, STAT3 and STAT5 activation in airway eosinophils following in vitro treatment with IL-5 was less pronounced than that seen in blood eosinophils in all patients (Table II) and undetectable in two of the four patients tested. Equal protein loading and transfer were confirmed by stripping and reprobing the membrane with anti-STAT3 or anti-STAT5 that detects both isoforms of STAT5 as a control for equivalent protein loading and transfer (Fig. 4, B and D). Given that cell surface expression of the IL-5Rα is decreased in BAL eosinophils (13) (Table II), these data suggest that airway eosinophils are less responsive to IL-5, a feature that may be mediated, at least in part, by decreased IL-5Rα expression.

FIGURE 4.

Phosphorylation status of STAT3 tyrosine 705 and STAT5 tyrosine 694 in airway eosinophils; the effect of IL-5 stimulation. A, Airway eosinophils recovered from BAL fluid of an allergic patient 48 h after segmental bronchoprovocation with Ag and were stimulated in vitro with 300 pM IL-5 (lanes 2 and 4) or vehicle (lanes 1 and 3) for 15 min. Cell lysate protein (30 μg) was immunoblotted with anti-phospho-STAT3, which detects STAT3 only when activated by phosphorylation at tyrosine 705 (lanes 1 and 2) or anti-phospho-STAT5, which detects STAT5 only when activated by phosphorylation at tyrosine 694 (lanes 3 and 4). B, Membranes from A were stripped and reprobed with anti-STAT3 Ab (H-190, sc-7179) in lanes 1 and 2 or anti-STAT5 Ab (C-17, sc-835) in lanes 3 and 4 to demonstrate equivalent protein loading and transfer. C and D, Blood eosinophils (lanes 1 and 2) and BAL eosinophils (lanes 3 and 4) from an allergic asthmatic patient (subject 12 in Tables I and II) were stimulated with 0 pM (lanes 1 and 3) or 300 pM IL-5 (lanes 2 and 4) for 15 min, and the resulting cell lysates were immunoblotted with anti-phosphotyrosine 694 STAT5 (C) or anti-STAT5 (D; Santa Cruz Biotechnology; sc-835). Similar experiments were performed four times with IL-5-stimulated STAT5 phosphorylation observed in BAL eosinophils from two of the donors and in blood eosinophils from all donors, as summarized in Table II.

FIGURE 4.

Phosphorylation status of STAT3 tyrosine 705 and STAT5 tyrosine 694 in airway eosinophils; the effect of IL-5 stimulation. A, Airway eosinophils recovered from BAL fluid of an allergic patient 48 h after segmental bronchoprovocation with Ag and were stimulated in vitro with 300 pM IL-5 (lanes 2 and 4) or vehicle (lanes 1 and 3) for 15 min. Cell lysate protein (30 μg) was immunoblotted with anti-phospho-STAT3, which detects STAT3 only when activated by phosphorylation at tyrosine 705 (lanes 1 and 2) or anti-phospho-STAT5, which detects STAT5 only when activated by phosphorylation at tyrosine 694 (lanes 3 and 4). B, Membranes from A were stripped and reprobed with anti-STAT3 Ab (H-190, sc-7179) in lanes 1 and 2 or anti-STAT5 Ab (C-17, sc-835) in lanes 3 and 4 to demonstrate equivalent protein loading and transfer. C and D, Blood eosinophils (lanes 1 and 2) and BAL eosinophils (lanes 3 and 4) from an allergic asthmatic patient (subject 12 in Tables I and II) were stimulated with 0 pM (lanes 1 and 3) or 300 pM IL-5 (lanes 2 and 4) for 15 min, and the resulting cell lysates were immunoblotted with anti-phosphotyrosine 694 STAT5 (C) or anti-STAT5 (D; Santa Cruz Biotechnology; sc-835). Similar experiments were performed four times with IL-5-stimulated STAT5 phosphorylation observed in BAL eosinophils from two of the donors and in blood eosinophils from all donors, as summarized in Table II.

Close modal
Table II.

Activation of STAT5 following IL-5 treatment of blood and BAL eosinophils

SubjectsaIL-5Rα (MCF)bIL-5-Stimulated STAT5 Phosphorylationc
BlooddBALeBlooddBALe
432 164 1.02 0.31 
292 80 0.26 < background 
12 372 32 0.73f 0.10f 
13 396 68 0.41 < background 
SubjectsaIL-5Rα (MCF)bIL-5-Stimulated STAT5 Phosphorylationc
BlooddBALeBlooddBALe
432 164 1.02 0.31 
292 80 0.26 < background 
12 372 32 0.73f 0.10f 
13 396 68 0.41 < background 
a

Characteristics of the subjects used for these studies can be found in Table I.

b

IL-5Rα was evaluated by flow cytometry for the eosinophils in whole blood or unpurified BAL fluid cells and reported as the median channel fluorescence (MCF) for 104 events.

c

STAT5 phosphorylation was analyzed, after stimulation of purified blood or BAL eosinophils with 300 pM IL-5 for 15 min, by immunoblotting with anti-phosphotyrosine 694 STAT5. Immunoblot bands were quantified by densitometry, background was subtracted, and the values were normalized to the signal from the anti-STAT5 immunoblot of the same membrane. These are arbitrary units.

d

Results are reported for the analysis of peripheral blood eosinophils acquired 48 h after segmental bronchoprovocation with Ag.

e

Results are reported for the analysis of airway eosinophils acquired from BAL fluid 48 h after segmental bronchoprovocation with Ag.

f

The immunoblots from which these values were derived are pictured in Fig. 4C, lanes 2 and 4. The data are normalized to the signal of the same lanes reprobed with anti-STAT5 antisera (Fig. 4 D, lanes 2 and 4).

The promoter of Pim-1 has STAT-binding consensus sequences (25, 26) and is considered a STAT target gene (25, 26, 27, 28). Furthermore, Pim-1 transcripts were among the most highly induced genes in IL-5- or GM-CSF-treated human eosinophils, as evaluated by DNA microarray analysis (18, 29). This induction of Pim-1 mRNA was also observed with RT-PCR (29) (results not shown). We therefore evaluated the kinetics of Pim-1 protein expression following IL-5 treatment. Fig. 5,A demonstrates that Pim-1 protein is detectable in eosinophils following stimulation with IL-5, and that Pim-1 protein expression is evident as early as 2 h posttreatment (lanes 3, 5, 7, and 9). Similar results were observed with GM-CSF stimulation (results not shown). In contrast, airway eosinophils and eosinophils derived from the peripheral blood 48 h after segmental bronchoprovocation with Ag expressed detectable levels of Pim-1 protein in the absence of in vitro cytokine stimulation (Fig. 5,B) in eight of the nine tested subjects, all of whom were allergic asthmatic patients (Table I, subjects 3–11). Pim-1 expression in BAL eosinophils was greater than in blood eosinophils in all patients showing detectable expression (Fig. 5 C) and was variable from patient to patient. These studies suggest that Pim-1 expression in blood and airway eosinophils is a marker of activation following in vivo exposure of eosinophils to the inflammatory cytokines that accompany Ag challenge.

FIGURE 5.

Protein expression of the STAT-dependent target Pim-1. A, Blood eosinophils were stimulated with 0 pM (lanes 1, 2, 4, 6 and 8) or 100 pM IL-5 (lanes 3, 5, 7 and 9) for the times indicated. Eosinophils were lysed, and 50 μg of lysate protein was immunoblotted with anti-Pim-1 Ab (Santa Cruz Biotechnology; sc-13513). This experiment was performed seven times, and similar results were observed using eosinophils obtained from allergic asthmatic and nonasthmatic patients. B, Pim-1 protein expression in BAL (lane 1) or blood (Bld) (lane 2) eosinophils collected 48 h after segmental bronchoprovocation with Ag from an allergic asthmatic patient. Data shown are from eosinophils of subject 3 in Table I. C, Summary of the relative expression of Pim-1 protein, as determined by immunoblotting and densitometry analysis, for eight subjects for whom there was detectable Pim-1 protein in both blood and BAL eosinophils acquired 48 h after segmental bronchoprovocation with Ag. One additional patient (patient 6 in Table I) had no detectable Pim-1 protein in either blood or BAL eosinophils. The expression of Pim-1 protein was significantly greater (n = 9, p = 0.0015) in BAL eosinophils. The characteristics of these subjects are listed in Table I.

FIGURE 5.

Protein expression of the STAT-dependent target Pim-1. A, Blood eosinophils were stimulated with 0 pM (lanes 1, 2, 4, 6 and 8) or 100 pM IL-5 (lanes 3, 5, 7 and 9) for the times indicated. Eosinophils were lysed, and 50 μg of lysate protein was immunoblotted with anti-Pim-1 Ab (Santa Cruz Biotechnology; sc-13513). This experiment was performed seven times, and similar results were observed using eosinophils obtained from allergic asthmatic and nonasthmatic patients. B, Pim-1 protein expression in BAL (lane 1) or blood (Bld) (lane 2) eosinophils collected 48 h after segmental bronchoprovocation with Ag from an allergic asthmatic patient. Data shown are from eosinophils of subject 3 in Table I. C, Summary of the relative expression of Pim-1 protein, as determined by immunoblotting and densitometry analysis, for eight subjects for whom there was detectable Pim-1 protein in both blood and BAL eosinophils acquired 48 h after segmental bronchoprovocation with Ag. One additional patient (patient 6 in Table I) had no detectable Pim-1 protein in either blood or BAL eosinophils. The expression of Pim-1 protein was significantly greater (n = 9, p = 0.0015) in BAL eosinophils. The characteristics of these subjects are listed in Table I.

Close modal

Cyclin D3 mRNA was also up-regulated in IL-5- or GM-CSF-treated eosinophils, as indicated by a DNA microarray analysis of eosinophils (18), which is consistent with the observation that the promoter region of cyclin D3 contains STAT consensus sequences, as well as SP1, activating protein 1, and activating enhancer binding protein 2 sites (30), and that cyclin D3 expression is up-regulated via the activation of STAT3 (31). Therefore, given that IL-5 and GM-CSF activate STAT3 and STAT5, we sought to determine whether IL-5 or GM-CSF affects cyclin D3 protein levels in eosinophils. Eosinophils from peripheral blood of allergic or nonallergic patients were treated with IL-5 for 0–9 h. Then lysates were resolved by electrophoresis and immunoblotted with anti-cyclin D3 Ab (Fig. 6,A). Unstimulated blood eosinophils express cyclin D3 protein (Fig. 6,A, lane 1), but this expression decreases with time of incubation in the absence of added cytokine (Fig. 6,A, lanes 2, 4, and 6). This decrease in cyclin D3 protein is attenuated in eosinophils treated with IL-5 (Fig. 6 A, lanes 3, 5, and 7). Similar findings were observed following treatment of eosinophils with 100 pM GM-CSF (data not shown).

FIGURE 6.

Expression of the STAT-dependent target cyclin D3. A, Blood eosinophils were stimulated with 0 pM (lanes 1, 2, 4 and 6) or 100 pM IL-5 (lanes 3, 5, and 7) for the times indicated, and cell lysates were prepared. Lysate protein (30 μg) was subjected to SDS-PAGE, followed by immunoblotting analysis using an anti-cyclin D3 Ab (n = 9, including nonallergic patients and allergic patients with and without asthma). B, Blood eosinophils were incubated with 0 pM (lane 1) or 100 pM IL-5 for 16 h in the presence (lanes 4 and 5) or absence (lanes 2 and 3) of the caspase inhibitor Z-Asp-CH2- (lanes 2–7) [(2,6-dichlorobenzoyl)oxy]methane (100 μM) or the proteasome inhibitor MG-115 (50 μM) (lanes 6 and 7). Cell lysates were prepared and immunoblotted with anti-cyclin D3. The experiment shown is representative of three experiments using the eosinophils of allergic donors, two of whom had asthma.

FIGURE 6.

Expression of the STAT-dependent target cyclin D3. A, Blood eosinophils were stimulated with 0 pM (lanes 1, 2, 4 and 6) or 100 pM IL-5 (lanes 3, 5, and 7) for the times indicated, and cell lysates were prepared. Lysate protein (30 μg) was subjected to SDS-PAGE, followed by immunoblotting analysis using an anti-cyclin D3 Ab (n = 9, including nonallergic patients and allergic patients with and without asthma). B, Blood eosinophils were incubated with 0 pM (lane 1) or 100 pM IL-5 for 16 h in the presence (lanes 4 and 5) or absence (lanes 2 and 3) of the caspase inhibitor Z-Asp-CH2- (lanes 2–7) [(2,6-dichlorobenzoyl)oxy]methane (100 μM) or the proteasome inhibitor MG-115 (50 μM) (lanes 6 and 7). Cell lysates were prepared and immunoblotted with anti-cyclin D3. The experiment shown is representative of three experiments using the eosinophils of allergic donors, two of whom had asthma.

Close modal

The decrease in cyclin D3 expression seen in the absence of in vitro cytokine treatment is most likely the result of proteolytic degradation, as has been reported in other cell systems (32). A series of experiments were conducted to determine whether inhibition of selected caspases or ubiquitin-mediated proteolysis could attenuate the degradation of cyclin D3 seen in the absence of cytokine stimulation. As seen in Fig. 6,B, cyclin D3 is abundantly expressed in blood eosinophils (Fig. 6,B, lane 1) and decreased following incubation for 16 h in the absence of IL-5 (Fig. 6,B, lane 2). In eosinophils incubated in the presence of IL-5 (Fig. 6,B, lane 3), the mass of cyclin D3 is comparable to that seen in lane 1. Inclusion of a caspase inhibitor, known to effectively increase eosinophil survival in the absence of added cytokine (100 μM Z-Asp-CH2-[(2,6-dichlorobenzoyl)oxy]methane) (33), did not attenuate the decrease in cyclin D3 protein levels (Fig. 6,B, lane 4), whereas an inhibitor of the ubiquitin proteosome system, MG115 (50 μM), prevented the loss of immunoreactive cyclin D3 (Fig. 6 B, lane 6). These data suggest that levels of cyclin D3 protein in eosinophils reflect a balance between the rate of biosynthesis, regulated in part by STAT-dependent transcriptional processes, and the rate of ubiquitin-mediated degradation.

The JAK/STAT signal transduction pathway is important in regulating many functions in hemopoietic cells, including the suppression of apoptosis. In eosinophils, inhibition of Jak2 attenuates cell survival even in the presence of IL-5 or GM-CSF (6, 7, 8). Because gene transcription is necessary for the IL-5- or GM-CSF-mediated suppression of apoptosis in eosinophils (9), and because STAT proteins are transcriptional regulators, the objective of these studies was to test whether STAT-regulated gene expression is modulated in human eosinophils treated with IL-5 or GM-CSF. In this study, we found that IL-5 and GM-CSF stimulate STAT3- and STAT5-associated pathways in human eosinophils, and that protein expression of the STAT-regulated and/or survival-associated genes Pim-1 and cyclin D3 is enhanced.

Previous studies have revealed that IL-5 can activate STAT3 in COS cells transfected with the IL-5R (34), but until the present studies it has been unclear whether this process occurs in the context of human cells that naturally express the IL-5R. In this regard, only a few types of human cells express functional IL-5R, including eosinophils, basophils, and a small subset of B lymphocytes (35, 36). In this study, we show that: 1) eosinophils express STAT3, STAT5a, and STAT5b protein; 2) IL-5 and GM-CSF treatment of human eosinophils results in the tyrosine phosphorylation of STAT3 on a site that is critical for dimer formation and entry into the nucleus; and 3) STAT3 and STAT5 from IL-5- or GM-CSF-stimulated eosinophils bind STAT consensus DNA sequences. In addition, we observed that STAT5b was the principal factor from IL-5- or GM-CSF-stimulated eosinophils that bound to a DNA element with a STAT5 consensus sequence. These observations are consistent with the findings that STAT3 and STAT5a/b are important for various hematological functions. Interestingly, our observation that STAT5b is responsible for much of the binding to STAT5 consensus sites suggests that, while STATa and b do have redundant roles, STAT5b may be more important to STAT5-driven transcription, and thus function, in mature human eosinophils.

We had hypothesized that, because of the high concentration of IL-5 and GM-CSF in the human airway following segmental bronchoprovocation with Ag (23, 24), STAT3 and STAT5 would be constitutively phosphorylated in airway eosinophils obtained after segmental bronchoprovocation with Ag. However, freshly isolated airway eosinophils, procured via segmental bronchoprovocation with Ag and lavage, were not detectably phosphorylated on tyrosine 705 of STAT3 or tyrosine 694 of STAT5, suggesting that these STAT proteins are not constitutively activated within the limits of detection by immunoblotting. Nonetheless, these findings complement the report by Sampath et al. (37), who observed that although STAT1 was constitutively activated in airway epithelial cells from endobronchial biopsies from asthmatic human subjects, STAT3 was not apparently constitutively activated.

Another issue relevant to the present studies pertains to the observation that following in vitro stimulation of BAL eosinophils with IL-5, the airway eosinophils from only 50% of the donors responded with detectable STAT3 or STAT5 phosphorylation, which in all subjects was markedly lower in BAL eosinophils than in their circulating counterparts. This reduced responsiveness to IL-5 may be related to the recent finding that the α subunit of the IL-5R is down-regulated in eosinophils derived from human airways following segmental bronchoprovocation with Ag (13) (Table II). These data suggest that the lack of responsiveness of the JAK/STAT pathways to in vitro IL-5 exposure in airway eosinophils may be related to the modulation of the receptor expression in vivo.

We and others have observed that treatment of eosinophils with IL-5 or GM-CSF results in increased mRNA expression of the STAT-dependent gene Pim-1 (18, 29). The expression of Pim-1 has been reported to be STAT3 dependent (28) as well as STAT5 dependent (27, 38). However, because mRNA levels may not reflect protein expression, we examined Pim-1 expression using immunoblotting, and we demonstrated that Pim-1 protein is induced by IL-5 or GM-CSF. We consistently observed induction of Pim-1 in eosinophils isolated from peripheral blood following in vitro stimulation with IL-5 or GM-CSF. We also detected the presence of Pim-1 protein in freshly isolated airway and blood eosinophils obtained 48 h postsegmental bronchoprovocation with Ag, suggesting that the high concentration of IL-5 and GM-CSF present in the blood and airway following Ag challenge may contribute to Pim-1 induction in vivo. However, it is important to note that there was considerable variability in the levels of Pim-1 in blood and BAL eosinophils among the patients tested; therefore, this biological endpoint reflects the functional heterogeneity of human eosinophils and immune responses, in general.

Pim-1 may be critical to eosinophil function in asthma, as suggested by its role in preventing apoptosis in other cytokine-dependent hemopoietic cells (28, 39, 40, 41). One of the important effects of IL-5 and GM-CSF on eosinophils is the suppression of apoptosis, thus allowing the persistence of tissue eosinophilia at sites of inflammation, and thus potentially causing more inflammation and damage to the tissues. The mechanism(s) behind the antiapoptotic effects of Pim-1 observed in the various reports is unknown, but included among the Pim-1 substrates are the transcriptional coactivator p100 (42); the cell cycle phosphatase Cdc25A (43); the transcriptional repressor heterochromatin protein 1 γ (44); a protein of unknown function, PAP-1 (45); the phosphatase PTP-U2S (46); the transcription factor NF-ATc1 (47); and a suppressor of the JAK/STAT signaling pathway, i.e., suppressor of cytokine signaling-1 (SOCS-1) (48). In the latter study, the phosphorylation of SOCS-1 by Pim kinases stabilizes SOCS-1 protein, thus down-regulating the activity of the JAK/STAT signaling pathway. Interestingly, we previously reported that IL-5 and GM-CSF treatment of human eosinophils induces the mRNA for cytokine-inducible SH2-containing protein 1, which is another member of the SOCS family (17). The role of Pim-1 in eosinophils is undefined at this time, and demands further exploration.

In contrast to the induction of Pim-1 in peripheral blood eosinophils, cyclin D3 protein was expressed strongly in freshly isolated peripheral blood eosinophils (Fig. 6,A) and airway eosinophils (n = 3; data not shown) from all patients examined. In the blood eosinophils, cyclin D3 expression decreased with the time of incubation in untreated eosinophils, but this decrease was attenuated if the eosinophils were treated with IL-5 or GM-CSF or with MG115, an inhibitor of the ubiquitin proteosome (Fig. 6 B). Cyclin D proteins have generally been linked to the regulation of the cell cycle, i.e., members of the cyclin D family are required for cells to progress from G1 to S phase (49, 50, 51, 52). The presence and apparent regulation of cyclin D3 protein levels by IL-5 and GM-CSF are surprising because eosinophils are terminally differentiated cells and do not proceed through the cell cycle. It is possible that the presence and modulation of cyclin D3 protein in mature eosinophils are a molecular vestige remaining from earlier stages of differentiation and proliferation. Alternatively, cyclin D3 may have a novel function in addition to its function in the regulation of the cell cycle.

To assess whether IL-5-induced cyclin D3 expression is associated with classical cell cycle events, we evaluated eosinophils for the expression of other proteins involved in cell cycle progression through G1, and we assessed whether IL-5 or GM-CSF could effect an increase, decrease, or modification of these proteins. Previous studies in our laboratories had shown that mRNA for cyclin-dependent kinase 2 (CDK2), Rb p110, and p21, a member of the cyclin-dependent kinase inhibitor protein (CIP) family. are expressed at the mRNA level in peripheral blood eosinophils, but no message for CDK4 was detected (18). However, when assessed for protein expression by immunoblotting, these molecules were undetectable or expressed at very low levels, and no alteration in protein expression or mobility or mRNA abundance was observed following cytokine stimulation (18) (data not shown). Therefore, these observations do not support a role for cyclin D3 in early or residual cell cycle events in human blood eosinophils.

Interestingly, other investigators have also found cyclin D3 protein expression in differentiating and differentiated cell types (53, 54, 55), suggesting that cyclin D3 has additional role(s) in cell function besides cell cycle progression. Although other putative functions of cyclin D3 are presently unknown, it is possible that cyclin D3 is also associated with maintaining cell survival. In this regard, Boonen et al. (56) have reported the intriguing finding that exogenous cyclin D3 protein expression in actively proliferating leukemic T cells inhibits apoptosis triggered by PMA and TCR activation. Based on this observation that there seems to be a link between cell cycling and cell viability with respect to cyclin D3 expression, one could speculate that cyclin D3 perhaps could play a role in the suppression of apoptosis in nonreplicating cells such as terminally differentiated blood eosinophils.

In summary, we have shown that treatment of human eosinophils with IL-5 or GM-CSF results in the phosphorylation and activation of STAT3, STAT5a, and STAT5b; the induction of the STAT-target Pim-1 protein; and the regulation of cyclin D3 protein. These events are likely to regulate eosinophil viability in the airway and influence other aspects of eosinophil biology relevant to airway function in asthma. The regulation of gene expression is clearly a critical process through which the proinflammatory effector function of eosinophils is modulated by IL-5 family cytokines. As additional STAT target genes are identified, possibly including cell surface receptors, cytokines/chemokines, and enzymes critical for the elaboration of other proinflammatory mediators, additional molecular mechanisms that drive the differentiation, accumulation, and activation of eosinophils may be elucidated.

We are grateful to Julie Sedgwick, Heather Gerbyshak, Kristin Jansen, and Anne Brooks for preparation of blood eosinophils. We thank the Pulmonary Research Group, including Nizar Jarjour, Mary Jo Jackson, and Erin Billmeyer, for subject screening, bronchoscopies, and segmental bronchoprovocation; Sarah Panzer and Rebecca Lawniczak for processing BAL samples; and Keith Meyer and Richard Cornwell for assisting with bronchoscopies. We also appreciate the assistance of Gregory Wiepz and E. A. Becky Kelly in the preparation and critical review of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was sponsored by National Institutes of Health Grants HL56396, including the administrative supplement to HL56396 for microarray applications and analyses, and MO1RR03186.

3

Abbreviations used in this paper: BAL, bronchoalveolar lavage; CDK, cyclin-dependent kinase; LHRR, lactogenic hormone response region; SIE, serum-inducible element; SOCS, suppressor of cytokine signaling.

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