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The Journal of Immunology, 2001, 166: 4658-4663.
Copyright © 2001 by The American Association of Immunologists

Granulocyte Macrophage-Colony-Stimulating Factor mRNA Is Stabilized in Airway Eosinophils and Peripheral Blood Eosinophils Activated by TNF-{alpha} Plus Fibronectin1

Stéphane Esnault and James S. Malter2

Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies have shown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controlling cytokine production remain obscure. We compared GM-CSF mRNA decay in eosinophils from bronchoalveolar lavage (BALEos) after allergen challenge or from PBEos. BALEos showed prolonged survival in vitro (60% at 4 days) and expressed GM-CSF mRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfection studies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin and TNF-{alpha} increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seen in BALEos. These data suggest that TNF-{alpha} plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSF production at a posttranscriptional level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inflammatory stage of asthma and other allergic diseases is characterized by pulmonary eosinophilia (1). Eosinophil numbers are increased in the airways and pulmonary tissues following experimental or environmental allergen challenge (2, 3). After recruitment from the peripheral blood, pulmonary eosinophils show markedly enhanced survival, which further contributes to their accumulation. In vitro, eosinophils from bronchoalveolar lavage (BALEos)3 survived longer than control eosinophils from peripheral blood (PBEos) (4). Naive cells exposed to IL-5, IL-3, or GM-CSF were similarly resistant to apoptosis, suggesting that these cytokines likely enhance eosinophil survival in vivo (5, 6, 7). GM-CSF levels were increased in BAL from active asthmatics (8) as well as allergen-challenged atopic subjects (9, 10, 11). It is likely that multiple sources including activated T lymphocytes, macrophages, pulmonary fibroblasts, and eosinophils themselves produce GM-CSF (12, 13).

The molecular mechanism responsible for GM-CSF elaboration by activated eosinophils remains unclear. In resting T cells or eosinophils, GM-CSF mRNA is rapidly degraded, preventing mRNA accumulation and GM-CSF protein production. T lymphocytes activated with phorbol ester (14) or anti-CD3 and anti-CD28 Abs (15) stabilized GM-CSF mRNA, accounting for the majority of mRNA accumulation and GM-CSF protein production. We have recently demonstrated that GM-CSF mRNA was very unstable in resting PBEos but stabilized following ionomycin activation (16, 17). Recently, {beta}7 integrin ligation with plate-bound fibronectin or anti-{beta}7 Abs increased PBEos survival in a GM-CSF-dependant manner (18). Similarly, TNF-{alpha} has been shown to positively influence in vitro eosinophil survival (19) although the mechanism for this effect remains unclear. Both TNF-{alpha} and fibronectin were increased in BAL from asthmatic or allergen-challenged subjects (8, 10, 20, 21), suggesting that these agonists might contribute to intrapulmonary eosinophil survival by stimulating GM-CSF production. Here we show that PBEos treated in vitro with TNF-{alpha} plus fibronectin secreted GM-CSF and displayed enhanced survival. Cytokine production was preceded by GM-CSF mRNA stabilization. Eosinophils obtained from BAL of allergen-challenged subjects also showed prolonged GM-CSF mRNA half-life. These data suggest that exposure to TNF-{alpha} plus fibronectin during or after PBEos migration into pulmonary tissues and air spaces stabilized GM-CSF mRNA accounting for GM-CSF production and increased eosinophil survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects and eosinophil preparation

Each subject for BAL underwent a medical history and physical examination after obtaining of informed consent. Histamine challenge was performed to determine nonspecific bronchial responsiveness as previously described (22). All subjects had a history of allergic rhinitis with normal lung function. Peripheral blood was obtained by venipuncture from patients with allergic rhinitis or asthma. All informed consent was acquired according to a protocol approved by the University of Wisconsin Human Subjects Committee.

At least 1 month before bronchoscopy, a graded nebulized challenge of Ag was performed in each subject to determine the Ag dose that provoked a 20% fall in forced expiratory volume in 1 s (Ag 20% provocative dose (PD20)). The Ag PD20 was calculated from a cumulative dose-response curve as described previously (22). Bronchoscopy and segmental bronchoprovocation were performed as described previously (3, 23). Briefly, one bronchopulmonary segment was identified, and the fiberoptic bronchoscope was wedged into the segment. Segmental bronchoprovocation was performed by injecting the Ag dose (10% of the calculated Ag PD20) diluted in 10 ml of 0.9% NaCl followed by 5 ml of air to clear the bronchoscope channel. A second bronchoscopy was done 48 h later and BAL was performed. For BAL, three or six 40-ml aliquots of warm (37°C) 0.9% NaCl was used in each segment. The fluid was sequentially recovered by gentle hand suction.

From peripheral blood or BAL, eosinophils were purified using a negative immunomagnetic procedure as previously described (24). Briefly, heparinized whole blood or BAL was centrifuged (700 x g, 20 min) over a Percoll density gradient (density 1.090 g/ml; Pharmacia Biotech, Piscataway, NJ) to separate mononuclear cells from granulocytes. After removal of the mononuclear cell band, RBC were lysed by twice incubating (for 30 s) in sterile, deionized water. The remaining white blood cells were incubated with anti-CD16-coated microbeads (100 ml per 108 cells) for 40 min and then passed through steel mesh columns that had been previously washed with 2% newborn calf serum. The cells in the eluent were stained (Diff Quik; Baxter, Miami, FL), and 400 cells were examined microscopically. The cells were used only if >98% were eosinophils. The few contaminating cells were either neutrophils or mononuclear cells. After isolation, PBEos were maintained in RPMI 1640 medium, 10% FCS, and 50 µg/ml gentamicin (all obtained from Life Technologies, Grand Island, NY) at 37°C in a 5% CO2 environment. Patient characteristics and experiments performed with each donor are presented in Table IGo.


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  		Table I. Subjects used for GM-CSF mRNA expression (E), viability (V) or mRNA decays (D)1

 
Reagents and eosinophil activation

Recombinant human TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). Human cellular fibronectin was purchased from Sigma (St. Louis, MO). Human plasma fibronectin-coated 96-well tissue culture plates were bought from Becton Dickinson (Bedford, MA). PBEos were activated with TNF-{alpha} (10 ng/ml) in 96-well tissue culture plates (Becton Dickinson, Meylan, France). For fibronectin experiments, eosinophils were cultured in fibronectin-coated plates with medium supplemented with 20 µg/ml of soluble cellular fibronectin. Eosinophils are activated for 5 h at 1 x 106 cells/ml.

RT-PCR

RT-PCR for GM-CSF was performed as previously described (16). Briefly, after 5 h of activation, 1 x 106 eosinophils/ml were pelleted and lysed in TRIreagent (Molecular Research Center, Cincinnati, OH), and total RNA was isolated as described by the manufacturer. RT-PCR was performed using the manufacturer’s protocol (Promega, Madison, WI). Primers for {beta}-actin mRNA were complementary to nucleotides 227–246 (5'-TCACCAACTGGGACGACATG-3') and 429–410 (5'-AGGCTGTGCTATCCCTGTAC-3'), whereas those for GM-CSF mRNA corresponded to nucleotides 241–260 (5'-CAGGGCCTGCGGGGCAGCCT-3') and 438–421 (5'-GTCTCACTCCTGGACTGG-3'). Thirty-two or 34 cycles, respectively, were performed for {beta}-actin or GM-CSF. Because the signals obtained in an ethidium bromide gel were generally weak for the GM-CSF PCR products, Southern blotting was performed using a radioactively labeled GM-CSF cDNA probe as previously described (16).

Eosinophil survival

Eosinophils (1 x 106 cells/ml) were cultured in 96-well tissue culture plates. PBEos viability was assessed by trypan blue exclusion on a hemocytometer (4). The percent PBEos survival was determined by the following equation: % survival = (no. of viable cells at 96 h)/(no. of viable cells at 0 h) x 100. Where used, neutralizing anti-GM-CSF or anti-IL-5 (5 µg/ml; R&D Systems) were added at the initiation of culture.

Plasmid constructions

cDNA coding for human GM-CSF was obtained from the American Type Culture Collection (Manassas, VA). The plasmid for in vitro, wild-type GM-CSF mRNA synthesis has been described previously (25) and contained a complete 5'UTR, coding and 3'UTR. In addition, the in vitro transcript was capped at the 5' end and terminated with a 90 base, polyadenylate tail. After production, mRNAs were phenol/chloroform-extracted and precipitated at -20°C. The quality and the quantity of synthesized mRNAs were verified by agarose gel electrophoresis and by absorbance at 260 nm.

mRNA transfection

Particle-mediated gene transfer (PMGT) of expression vectors or in vitro transcribed mRNAs into cultured cells was performed using the Accell Gene-Gun (Powderject, Madison, WI), as previously described (25, 26). Briefly, mRNAs in aqueous solution were precipitated at -20°C for 1 h with 1 volume of 2-propanol and 0.10 volume of 5 M ammonium acetate onto 1 µm gold beads at a concentration of 5 µg of mRNA/mg of gold beads. Eighty to 95% of the input mRNA was typically loaded onto the beads. Successive transfections of 2 x 106 cells were pooled and washed twice in culture medium to remove any extracellular mRNAs. The transfected PBEos were placed in culture at 1 x 107 cells/ml.

Northern blotting

At indicated times, cells were pelleted and lysed in TRIreagent (Molecular Research Center), and total RNA was quantitatively isolated and analyzed by Northern blotting with a radioactively labeled cDNA GM-CSF or actin probe as described previously (25). GM-CSF mRNA signals were normalized to those for actin mRNA to accommodate any differences in the extraction, gel loading, and transfer of total RNA. After stringent washing at 50°C for 5 min with 0.1x SSC, 0.1% SDS, the blots were quantitated by phosphorimaging (model 445SI; Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GM-CSF mRNA steady-state level in BALEos and PBEos activated with TNF-{alpha} plus fibronectin

In active asthmatics, eosinophils expressed GM-CSF mRNA at sites of allergic pulmonary inflammation (12). However, these studies used relatively insensitive in situ hybridization. We reassessed these conclusions by RT-PCR analysis immediately after purification of airway eosinophils obtained 48 h after segmental allergen challenge of consenting, atopic subjects. As shown in Fig. 1Go, GM-CSF mRNA was a consistent finding in BALEos from donor to donor, which was usually absent or barely detected in PBEos from allergic donors with no current symptoms.



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  		FIGURE 1. GM-CSF mRNA expression in BALEos or PBEos. GM-CSF and {beta}-actin RT-PCR were performed with total RNA extracted from BALEos 48 h after allergen challenge, resting PBEos or those activated with fibronectin (Fn), TNF-{alpha} (TNF), or both for 5 h. PBEos 1 and 2 were isolated from control allergic donors. Top, Southern blot using a 32P-labeled GM-CSF cDNA probe; bottom, ethidium bromide-stained agarose gel of actin RT-PCR. C+ (GM-CSF mRNA in vitro synthesized) and C- (no cDNA) refer to positive and negative control RT-PCR for GM-CSF. For PBEos, data are representative of nine experiments with eight different donors (Table IGo).

 
To establish whether extracellular mediators stimulated GM-CSF mRNA accumulation in BALEos, we exposed resting PBEos from nonsymptomatic allergic donors to different cytokines and/or ligands for {alpha}4, {beta}1, and {beta}7 integrins. As shown in Fig. 1Go for PBEos1, fibronectin increased GM-CSF mRNA levels compared with resting controls. However, this response was variable and only observed in 50% of the studied donors. As eosinophils are exposed to a variety of chemokines and cytokines before and during migration into, and residence in the airways, we stimulated PBEos with fibronectin plus TNF-{alpha}. As shown in Fig. 1Go (PBEos 2), resting eosinophils contained little GM-CSF mRNA, which was typically unaffected by TNF-{alpha} alone. However, the combination of TNF-{alpha} plus fibronectin dramatically and reproducibly induced GM-CSF mRNA accumulation. These data show that GM-CSF mRNA levels can be significantly enhanced by cell surface TNF-{alpha} and integrin-mediated signaling.

BALEos and TNF-{alpha} plus fibronectin-mediated PBEos survival

If TNF-{alpha} and fibronectin modulated airway eosinophil apoptosis through a GM-CSF-dependent mechanism, we reasoned similar responses should be observed in identically treated PBEos cultures. Thus, we compared the survival of BALEos to PBEos treated with TNF-{alpha}, fibronectin, or both. As seen previously (4), BALEos were highly resistant to apoptosis ({approx}60% survival at 4 days in vitro) (Fig. 2Go), whereas only 5% of unstimulated PBEos survived 4 days in culture. TNF-{alpha} or fibronectin alone increased survival 3-fold, but the combination was approximately additive with >30% survival at 4 days (Fig. 2Go). Interestingly, fibronectin-mediated survival could be entirely inhibited by anti-GM-CSF Ab demonstrating dependence on extracellular GM-CSF. However, the TNF-{alpha} response was GM-CSF independent. Anti-GM-CSF Ab also blocked the survival advantage induced by TNF-{alpha} plus fibronectin (Fig. 2Go). These data suggest that TNF-{alpha} signaling may be modulated by costimulation of PBEos with fibronectin. These data are consistent with our RT-PCR results showing substantial and reproducibly increased GM-CSF mRNA in PBEos stimulated with fibronectin, fibronectin plus TNF-{alpha}, but not TNF-{alpha} alone.



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  		FIGURE 2. BALEos survival is prolonged (A) as well as PBEos activated in vitro by TNF-{alpha}, fibronectin (Fn), or TNF-{alpha} plus fibronectin (B). Cell viability was determined after 4 days by trypan blue exclusion. Neutralizing anti-GM-CSF mAb was added at the beginning of the culture. Each value represents the mean of three (SD) or two cultures (no SD) from the same donor representative of three different donors for BALEos and six donors for PBEos.

 
As the up-regulation of GM-CSF mRNA and protein secretion appeared necessary for enhanced survival of PBEos, we hypothesized in vitro BALEos survival would be similarly dependent on GM-CSF. The viability of BALEos obtained 48 h after segmental allergen challenge was accessed after addition of anti-GM-CSF, anti-IL-5, or irrelevant Ab. As shown in Fig. 3Go, the majority of BALEos were dead at 6 days in the presence of neutralizing anti-GM-CSF, whereas irrelevant Ab or anti-IL-5 had no effect. These data show that GM-CSF rather than IL-5 is critical for long-term BALEos survival in vitro. In addition, BALEos become GM-CSF secretors during migration into the lung or after residence there.



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  		FIGURE 3. PBALEos survival is inhibited with a neutralizing anti-GM-CSF. Cell viability was determined after 6 days by trypan blue exclusion. Neutralizing anti-GM-CSF or anti-IL-5 mAb was added at the beginning of the culture. Each value represents the mean of three (SD) cultures from the same donor representative of two different donors.

 
Stabilization of GM-CSF mRNA in BALEos and TNF-{alpha} plus fibronectin-activated PBEos

GM-CSF production is regulated at transcriptional and posttranscriptional levels (14, 15). Recently, we have shown that GM-CSF mRNA was stabilized in eosinophils after ionomycin activation (17). Therefore, we transfected BALEos, PBEos stimulated with TNF-{alpha} plus fibronectin, or control PBEos with GM-CSF mRNA by PMGT and measured its decay. GM-CSF mRNA was extremely unstable in resting PBEos with half-life (t1/2) of 11 min (Fig. 4Go) but was nearly 2.5-fold more stable (t1/2 = 26 min) in BALEos. GM-CSF mRNA in PBEos activated with fibronectin plus TNF-{alpha} decayed at a rate nearly identical with that seen in BALEos. The stability of GM-CSF mRNA in BALEos or activated PBEos was 35% greater than we previously observed for PBEos activated with ionophore alone (17). Finally, activation with TNF-{alpha} alone had no effect on GM-CSF mRNA stability, whereas fibronectin alone had an intermediate effect (Fig. 5Go). We were consistently unable to detect GM-CSF mRNA by Northern blot in the absence of PMGT or transfection with naked gold beads (data not shown). Thus, GM-CSF mRNA detected in these experiments must reflect exogenous message. These data show that increased GM-CSF mRNA stability occurs in BALEos. A similar phenotype can be induced by TNF-{alpha} plus fibronectin activation of PBEos, suggesting that these mediators may be responsible for GM-CSF up-regulation in vivo.



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  		FIGURE 4. GM-CSF mRNA is stabilized in BALEos and in PBEos activated by TNF-{alpha} plus fibronectin. Resting PBEos (control), BALEos, or TNF-{alpha} + fibronectin-activated PBEos were transfected with GM-CSF mRNA. A, At the indicated time points, equal numbers of cells were harvested, and total RNA was quantitatively isolated and Northern blotted with 32P-labeled GM-CSF or {beta}-actin cDNA probes. Signals were visualized using a PhosphorImager. B, Radioactive signals were quantified using a PhosphorImager and normalized to {beta}-actin mRNA and plotted vs time. Each point is the mean SD of three experiments with three different donors or two for the control.

 


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  		FIGURE 5. Fibronectin-activated PBEos partially stabilized GM-CSF mRNA. PBEos activated by TNF-{alpha} or fibronectin for 5 h were transfected with GM-CSF mRNA. At indicated time points, equal numbers of cells were harvested, and total RNA was isolated and Northern blotted as for Fig. 3Go. Radioactive signals were quantified and normalized to {beta}-actin mRNA and plotted vs time. Each point is the mean of two experiments with two different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GM-CSF is a critical cytokine implicated in eosinophil differentiation, function, migration, and survival (7, 27). Besides responding to cytokine produced by activated inflammatory or resident pulmonary cells, eosinophils themselves produce GM-CSF (12, 13, 28). The mechanisms that control the elaboration of GM-CSF by activated eosinophils remain largely unknown. We report herein that GM-CSF mRNA levels are increased in BALEos or PBEos activated with a combination of TNF-{alpha} plus fibronectin. Stabilization of GM-CSF mRNA accounted for increased steady-state levels and preceded secretion of GM-CSF.

GM-CSF secretion by PBEos can be induced by IFN-{gamma} (28), LPS (29), anti-CD40 mAb (30), anti-CD9 mAb (31), anti-CD32 mAb (32), or fibronectin (33). Because fibronectin concentrations are significantly increased and strongly correlated with the eosinophil content of BAL fluid 48 h after Ag challenge (21), we asked whether fibronectin could affect GM-CSF mRNA accumulation. However, in our hands fibronectin had inconsistent effects suggesting differences in donor responsiveness or the need for additional and possibly additive agonists. TNF-{alpha} was chosen as a potential cofactor as it was abundant in BAL fluid from patients with symptomatic asthma (8) or after segmental allergen challenge (10). Combined with IL-4, TNF-{alpha} enhanced eosinophil adhesion to human pulmonary microvascular endothelial cell monolayers (34). Finally, Levi-Schaffer et al. (19) demonstrated that TNF-{alpha} in the context of mast cell lysates increased eosinophil survival by inducing autocrine GM-CSF production. Here, PBEos activated with fibronectin and TNF-{alpha} showed increased GM-CSF mRNA accumulation (Fig. 1Go). Interestingly, TNF-{alpha} alone had little effect on GM-CSF mRNA levels (Fig. 1Go) but was able to prolong eosinophil survival through a GM-CSF-independent pathway (Fig. 2Go). These data are supported by Tsukahara et al. (35) who recently reported that TNF-{alpha} inhibited eosinophil apoptosis independently of GM-CSF via p38 mitogen-activated protein kinase activation. Thus, TNF-{alpha} may regulate airway eosinophil survival either independently of GM-CSF through p38-mediated signaling or in association with other activators such as fibronectin in a GM-CSF-dependent fashion. As we have shown that GM-CSF rapidly activates the JAK2/STAT5 pathway in PBEos (36), anti-apoptotic signals may be up-regulated through this cascade.

Fibronectin signaling might occur through several possible integrins. PBEos express cell surface {alpha}4{beta}7, {alpha}4{beta}1, and {alpha}d{beta}2 (37, 38, 39). Recently, Meerschaert demonstrated that PBEos survival was markedly enhanced after {alpha}4{beta}7 ligation with anti-{beta}7 mAbs (18). Survival was blocked by anti-GM-CSF Abs suggesting that integrin engagement triggered GM-CSF release. The production, accumulation, or stability of GM-CSF mRNA was not assessed in these studies but based on our data was unlikely significantly altered.

In previous reports, we have demonstrated that a relatively modest 2- or 3-fold prolongation of GM-CSF mRNA half-life time increased GM-CSF protein production by 15- to 20-fold (16, 25) with corresponding increased PBEos in vitro survival (17). Thus, we hypothesized that TNF-{alpha} plus fibronectin could be a physiologic equivalent to ionomycin causing GM-CSF elaboration by blocking GM-CSF mRNA decay. Of note, {beta}2 integrin signaling in leukocytes stabilized urokinase plasminogen activator receptor mRNA (40). Thus, there is a precedent for integrin signaling affecting mRNA decay. In addition, urokinase plasminogen activator receptor mRNA contains, as does GM-CSF, multiple adenosine uridine (AUUUA) motifs in the 3' untranslated region that are required for regulated decay. Also consistent with our observations are prior data demonstrating IL-1 and TNF-{alpha} regulated, respectively, GM-CSF, gro{alpha}, {beta}, {gamma}, IL-8, and IL-1 mRNA stability (41, 42, 43). RANTES production by activated pulmonary epithelium was preceded by stabilization of its mRNA (44). Thus posttranscriptional regulation may control the production of multiple cytokines and chemokines by activated cells in allergic diseases.

The degree of GM-CSF mRNA stabilization after activation was correlated with survival. Thus mRNA stabilization likely accounts for a majority of the observed increase in GM-CSF mRNA content and subsequent cytokine secretion in eosinophils. Similar data has been shown for mitogen-activated T lymphocytes (15) where transcriptional up-regulation of IL-2, GM-CSF, IFN-{gamma}, and TNF-{alpha} was very small. As BALEos also displayed nuclease-resistant GM-CSF mRNA, we propose that eosinophils during migration or after residence in the lung block normal GM-CSF mRNA decay. The mechanism responsible for this phenotype could be general down-regulation of mRNA decay, although this is unlikely. Rather, we favor a specific effect on GM-CSF mediated by mRNA binding proteins. Overexpression of HuR, an AUUUA-specific RNA binding protein, stabilized c-fos mRNA in transfected NIH 3T3 cells (45) and p21 mRNA in human colorectal carcinoma RKO cells (46). Heterogeneous nuclear ribonucleoproteins (hnRNP) C and L stabilized amyloid protein precursor (47) and vascular endothelial growth factor (48) mRNAs, respectively. Consistent with this hypothesis, we have shown that ionophore up-regulated the activity of multiple AUUUA specific binding proteins in an eosinophil cell line (AML14.3D10) concomitant with GM-CSF mRNA stabilization and accumulation (49). We are currently looking for mRNA binding proteins in eosinophils involved in GM-CSF mRNA posttranscriptional regulation.

The accumulation of GM-CSF mRNA in BALEos (Fig. 1Go) and requirement for GM-CSF to support long-term survival in vitro suggest that GM-CSF plays an important functional role in initiating and/or maintaining pulmonary eosinophilia in vivo. This is supported by several studies showing elevations of GM-CSF in BAL fluid after segmental challenge correlated with eosinophil content elevation during asthma (9, 11) or inhibition of eosinophil survival after treatment of BAL fluid with neutralizing anti-GM-CSF Ab (50). In this last study, anti-IL-5 Ab had no effect on eosinophil survival. Altogether these findings raise the importance for considering not only IL-5 but also GM-CSF in clinical studies targeting lower eosinophilia and late asthmatic response.


   Acknowledgments
 
We thank the other members of the Specialized Center of Research-asthma group, particularly Julie B. Sedgwick for providing PBEos and Nizar N. Jarjour and E.A.B. Kelly for BALEos.


   Footnotes
 
1 This work was supported by the National Institutes of Health (Project 5 of Specialized Center of Research-asthma-P50HL56396, to J.S.M.). Back

2 Address correspondence and reprint requests to Dr. James S. Malter, Department of Pathology and Laboratory Medicine, K4/812-CSC, University of Wisconsin Hospital and Clinic, 600 Highland Avenue, Madison, WI 53792. Back

3 Abbreviations used in this paper: BALEos, eosinophils from bronchoalveolar lavage; PBEos, eosinophils from peripheral blood; PMGT, particle-mediated gene transfer; PD20, 20% provocative dose; AUUUA, adenosine/uridine. Back

Received for publication November 7, 2000. Accepted for publication January 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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