of January 19, 2016.This information is current as Plus Fibronectinαby TNF-

and Peripheral Blood Eosinophils Activated mRNA Is Stabilized in Airway EosinophilsMacrophage-Colony-Stimulating Factor Granulocyte

Stéphane Esnault and James S. Malter

http://www.jimmunol.org/content/166/7/4658doi: 10.4049/jimmunol.166.7.4658

2001; 166:4658-4663; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/7/4658.full#ref-list-1

, 27 of which you can access for free at: cites 50 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on January 19, 2016

http://ww

w.jim

munol.org/

Dow

nloaded from

Granulocyte Macrophage-Colony-Stimulating Factor mRNA IsStabilized in Airway Eosinophils and Peripheral BloodEosinophils Activated by TNF-a Plus Fibronectin1

Stephane Esnault and James S. Malter2

Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies haveshown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controllingcytokine 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-CSFmRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfectionstudies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin andTNF-a increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seenin BALEos. These data suggest that TNF-a plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSFproduction at a posttranscriptional level. The Journal of Immunology,2001, 166: 4658–4663.

T he inflammatory stage of asthma and other allergic dis-eases is characterized by pulmonary eosinophilia (1). Eo-sinophil numbers are increased in the airways and pul-

monary tissues following experimental or environmental allergenchallenge (2, 3). After recruitment from the peripheral blood, pul-monary eosinophils show markedly enhanced survival, which fur-ther contributes to their accumulation. In vitro, eosinophils frombronchoalveolar lavage (BALEos)3 survived longer than controleosinophils from peripheral blood (PBEos) (4). Naive cells ex-posed to IL-5, IL-3, or GM-CSF were similarly resistant to apo-ptosis, suggesting that these cytokines likely enhance eosinophilsurvival in vivo (5–7). GM-CSF levels were increased in BALfrom active asthmatics (8) as well as allergen-challenged atopicsubjects (9–11). It is likely that multiple sources including acti-vated T lymphocytes, macrophages, pulmonary fibroblasts, and eo-sinophils themselves produce GM-CSF (12, 13).

The molecular mechanism responsible for GM-CSF elaborationby activated eosinophils remains unclear. In resting T cells or eo-sinophils, GM-CSF mRNA is rapidly degraded, preventing mRNAaccumulation and GM-CSF protein production. T lymphocytes ac-tivated with phorbol ester (14) or anti-CD3 and anti-CD28 Abs(15) stabilized GM-CSF mRNA, accounting for the majority ofmRNA accumulation and GM-CSF protein production. We haverecently demonstrated that GM-CSF mRNA was very unstable in

resting PBEos but stabilized following ionomycin activation (16,17). Recently,b7 integrin ligation with plate-bound fibronectin oranti-b7 Abs increased PBEos survival in a GM-CSF-dependantmanner (18). Similarly, TNF-a has been shown to positively in-fluence in vitro eosinophil survival (19) although the mechanismfor this effect remains unclear. Both TNF-a and fibronectin wereincreased in BAL from asthmatic or allergen-challenged subjects(8, 10, 20, 21), suggesting that these agonists might contribute tointrapulmonary eosinophil survival by stimulating GM-CSF pro-duction. Here we show that PBEos treated in vitro with TNF-aplus fibronectin secreted GM-CSF and displayed enhanced sur-vival. Cytokine production was preceded by GM-CSF mRNA sta-bilization. Eosinophils obtained from BAL of allergen-challengedsubjects also showed prolonged GM-CSF mRNA half-life. Thesedata suggest that exposure to TNF-a plus fibronectin during orafter PBEos migration into pulmonary tissues and air spaces sta-bilized GM-CSF mRNA accounting for GM-CSF production andincreased eosinophil survival.

Materials and MethodsSubjects and eosinophil preparation

Each subject for BALunderwent a medical history and physical examinationafter obtaining of informed consent. Histamine challenge was performed todetermine nonspecific bronchial responsiveness as previously described (22).All subjects had a history of allergic rhinitis with normal lung function. Pe-ripheral blood was obtained by venipuncture from patients with allergic rhinitisor asthma. All informed consent was acquired according to a protocol ap-proved by the University of Wisconsin Human Subjects Committee.

At least 1 month before bronchoscopy, a graded nebulized challenge ofAg was performed in each subject to determine the Ag dose that provokeda 20% fall in forced expiratory volume in 1 s (Ag 20% provocative dose(PD20)). The Ag PD20 was calculated from a cumulative dose-responsecurve as described previously (22). Bronchoscopy and segmental broncho-provocation were performed as described previously (3, 23). Briefly, onebronchopulmonary segment was identified, and the fiberoptic broncho-scope was wedged into the segment. Segmental bronchoprovocation wasperformed by injecting the Ag dose (10% of the calculated Ag PD20) di-luted in 10 ml of 0.9% NaCl followed by 5 ml of air to clear the bron-choscope channel. A second bronchoscopy was done 48 h later and BALwas 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 bygentle hand suction.

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

Received for publication November 7, 2000. Accepted for publication January19, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the National Institutes of Health (Project 5 of Special-ized Center of Research-asthma-P50HL56396, to J.S.M.).2 Address correspondence and reprint requests to Dr. James S. Malter, Department ofPathology and Laboratory Medicine, K4/812-CSC, University of Wisconsin Hospitaland Clinic, 600 Highland Avenue, Madison, WI 53792. E-mail address:jsmalter@facstaff.wisc.edu3 Abbreviations used in this paper: BALEos, eosinophils from bronchoalveolar la-vage; PBEos, eosinophils from peripheral blood; PMGT, particle-mediated genetransfer; PD20, 20% provocative dose; AUUUA, adenosine/uridine.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

From peripheralblood or BAL, eosinophils were purified using a negativeimmunomagnetic procedure as previously described (24). Briefly, heparinizedwhole blood or BAL was centrifuged (7003 g, 20 min) over a Percoll densitygradient (density 1.090 g/ml; Pharmacia Biotech, Piscataway, NJ) to separatemononuclear cells from granulocytes. After removal of the mononuclear cellband, RBC were lysed by twice incubating (for 30 s) in sterile, deionizedwater. The remaining white blood cells were incubated with anti-CD16-coatedmicrobeads (100 ml per 108 cells) for 40 min and then passed through steelmesh columns that had been previously washed with 2% newborn calf serum.The cells in the eluent were stained (Diff Quik; Baxter, Miami, FL), and 400cells were examined microscopically. The cells were used only if.98% wereeosinophils. The few contaminating cells were either neutrophils or mononu-clear cells. After isolation, PBEos were maintained in RPMI 1640 medium,10% FCS, and 50mg/ml gentamicin (all obtained from Life Technologies,Grand Island, NY) at 37°C in a 5% CO2 environment. Patient characteristicsand experiments performed with each donor are presented in Table I.

Reagents and eosinophil activation

Recombinant human TNF-a was purchased from R&D Systems (Minne-apolis, MN). Human cellular fibronectin was purchased from Sigma (St.Louis, MO). Human plasma fibronectin-coated 96-well tissue culture plateswere bought from Becton Dickinson (Bedford, MA). PBEos were activatedwith TNF-a (10 ng/ml) in 96-well tissue culture plates (Becton Dickinson,Meylan, France). For fibronectin experiments, eosinophils were cultured infibronectin-coated plates with medium supplemented with 20mg/ml ofsoluble cellular fibronectin. Eosinophils are activated for 5 h at 13 106

cells/ml.

RT-PCR

RT-PCR for GM-CSF was performed as previously described (16). Briefly,after 5 h of activation, 13 106 eosinophils/ml were pelleted and lysed inTRIreagent (Molecular Research Center, Cincinnati, OH), and total RNAwas isolated as described by the manufacturer. RT-PCR was performedusing the manufacturer’s protocol (Promega, Madison, WI). Primers forb-actin mRNA were complementary to nucleotides 227–246 (59-TCACCAACTGGGACGACATG-39) and 429–410 (59-AGGCTGTGCTATCCCTGTAC-39), whereas those for GM-CSF mRNA corresponded to nu-cleotides 241–260 (59-CAGGGCCTGCGGGGCAGCCT-39) and 438–421(59-GTCTCACTCCTGGACTGG-39). Thirty-two or 34 cycles, respec-tively, were performed forb-actin or GM-CSF. Because the signals ob-tained in an ethidium bromide gel were generally weak for the GM-CSFPCR products, Southern blotting was performed using a radioactively la-beled GM-CSF cDNA probe as previously described (16).

Eosinophil survival

Eosinophils (13 106 cells/ml) were cultured in 96-well tissue cultureplates. PBEos viability was assessed by trypan blue exclusion on a hemo-cytometer (4). The percent PBEos survival was determined by the follow-ing equation: % survival5 (no. of viable cells at 96 h)/(no. of viable cellsat 0 h) 3 100. Where used, neutralizing anti-GM-CSF or anti-IL-5 (5mg/ml; R&D Systems) were added at the initiation of culture.

Plasmid constructions

cDNA coding for human GM-CSF was obtained from the American TypeCulture Collection (Manassas, VA). The plasmid for in vitro, wild-typeGM-CSF mRNA synthesis has been described previously (25) and con-tained a complete 59UTR, coding and 39UTR. In addition, the in vitrotranscript was capped at the 59end and terminated with a 90 base, poly-adenylate tail. After production, mRNAs were phenol/chloroform-ex-tracted and precipitated at220°C. The quality and the quantity of synthe-sized mRNAs were verified by agarose gel electrophoresis and byabsorbance at 260 nm.

mRNA transfection

Particle-mediated gene transfer (PMGT) of expression vectors or in vitrotranscribed mRNAs into cultured cells was performed using the AccellGene-Gun (Powderject, Madison, WI), as previously described (25, 26).Briefly, mRNAs in aqueous solution were precipitated at220°C for 1 hwith 1 volume of 2-propanol and 0.10 volume of 5 M ammonium acetateonto 1 mm gold beads at a concentration of 5mg of mRNA/mg of goldbeads. Eighty to 95% of the input mRNA was typically loaded onto thebeads. Successive transfections of 23 106 cells were pooled and washedtwice in culture medium to remove any extracellular mRNAs. The trans-fected PBEos were placed in culture at 13 107 cells/ml.

Northern blotting

At indicated times, cells were pelleted and lysed in TRIreagent (MolecularResearch Center), and total RNA was quantitatively isolated and analyzedby Northern blotting with a radioactively labeled cDNA GM-CSF or actinprobe as described previously (25). GM-CSF mRNA signals were normal-ized to those for actin mRNA to accommodate any differences in the ex-traction, gel loading, and transfer of total RNA. After stringent washing at50°C for 5 min with 0.13SSC, 0.1% SDS, the blots were quantitated byphosphorimaging (model 445SI; Molecular Dynamics, Sunnyvale, CA).

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

Identification Category Ag Symptoms Experiments

PGEos 1; P S AR RW, HDM No E2; M D AA RW, Cat, dog, No E3; N M AA RW, Cat, dog, HRM No D4; M G AA Rat No E; V5; ML J AR RW, HDM, pollen No E; V6; D B AR RW, HDM, cat No E7; K M AA HDM, cat Moderate D8; M D AA RW, cat, dog, Moderate D9; D K AR RW No E10; J V AA Car, horse No E11; G C AA Dog No E; V12; M G AA Rat No E; V13; J S AA RW, HDM, cat No V; D14; J VE AA RW, pollen, cat Moderate V; D15; M B AR HDM, pollen, animals Moderate D16; M M AR Cat Yes D17; N T AR Grass, HDM, cat No V

BALEos 1; S H AA RW, HDM Yes D2; B S AR RW, HDM, pollen Yes D3; M R AA Cat Yes D4; B D AA Cat Yes D5; J H AA RW No E; V6; C P AR RW Yes E; D7; S G AA HDM Yes E; V8; S K AA HDM Yes V

a Definition of abbreviations: AR, allergic rhinitis; AA, allergic asthmatic; RW, ragweed; HDM, house dust mite.

4659The Journal of Immunology

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

ResultsGM-CSF mRNA steady-state level in BALEos and PBEosactivated with TNF-a plus fibronectin

In active asthmatics, eosinophils expressed GM-CSF mRNA atsites of allergic pulmonary inflammation (12). However, thesestudies used relatively insensitive in situ hybridization. We reas-sessed these conclusions by RT-PCR analysis immediately afterpurification of airway eosinophils obtained 48 h after segmentalallergen challenge of consenting, atopic subjects. As shown in Fig.1, GM-CSF mRNA was a consistent finding in BALEos from do-nor to donor, which was usually absent or barely detected inPBEos from allergic donors with no current symptoms.

To establish whether extracellular mediators stimulated GM-CSF mRNA accumulation in BALEos, we exposed resting PBEosfrom nonsymptomatic allergic donors to different cytokines and/orligands for a4, b1, and b7 integrins. As shown in Fig. 1 forPBEos1, fibronectin increased GM-CSF mRNA levels comparedwith resting controls. However, this response was variable andonly observed in 50% of the studied donors. As eosinophils areexposed to a variety of chemokines and cytokines before and dur-ing migration into, and residence in the airways, we stimulatedPBEos with fibronectin plus TNF-a. As shown in Fig. 1 (PBEos2), resting eosinophils contained little GM-CSF mRNA, whichwas typically unaffected by TNF-a alone. However, the combina-tion of TNF-a plus fibronectin dramatically and reproducibly in-duced GM-CSF mRNA accumulation. These data show that GM-CSF mRNA levels can be significantly enhanced by cell surfaceTNF-a and integrin-mediated signaling.

BALEos and TNF-a plus fibronectin-mediated PBEos survival

If TNF-a and fibronectin modulated airway eosinophil apoptosisthrough a GM-CSF-dependent mechanism, we reasoned similarresponses should be observed in identically treated PBEos cul-tures. Thus, we compared the survival of BALEos to PBEostreated with TNF-a, fibronectin, or both. As seen previously (4),BALEos were highly resistant to apoptosis ('60% survival at 4days in vitro) (Fig. 2), whereas only 5% of unstimulated PBEossurvived 4 days in culture. TNF-a or fibronectin alone increasedsurvival 3-fold, but the combination was approximately additivewith .30% survival at 4 days (Fig. 2). Interestingly, fibronectin-mediated survival could be entirely inhibited by anti-GM-CSF Abdemonstrating dependence on extracellular GM-CSF. However,the TNF-a response was GM-CSF independent. Anti-GM-CSF Abalso blocked the survival advantage induced by TNF-a plus fi-bronectin (Fig. 2). These data suggest that TNF-a signaling may

be modulated by costimulation of PBEos with fibronectin. Thesedata are consistent with our RT-PCR results showing substantialand reproducibly increased GM-CSF mRNA in PBEos stimulatedwith fibronectin, fibronectin plus TNF-a, but not TNF-a alone.

As the up-regulation of GM-CSF mRNA and protein secretionappeared necessary for enhanced survival of PBEos, we hypothe-sized in vitro BALEos survival would be similarly dependent onGM-CSF. The viability of BALEos obtained 48 h after segmentalallergen challenge was accessed after addition of anti-GM-CSF,anti-IL-5, or irrelevant Ab. As shown in Fig. 3, the majority ofBALEos were dead at 6 days in the presence of neutralizing anti-GM-CSF, whereas irrelevant Ab or anti-IL-5 had no effect. Thesedata show that GM-CSF rather than IL-5 is critical for long-termBALEos survival in vitro. In addition, BALEos become GM-CSFsecretors during migration into the lung or after residence there.

FIGURE 1. GM-CSF mRNA expression in BALEos or PBEos. GM-CSF andb-actin RT-PCR were performed with total RNA extracted fromBALEos 48 h after allergen challenge, resting PBEos or those activatedwith fibronectin (Fn), TNF-a (TNF), or both for 5 h. PBEos 1 and 2 wereisolated from control allergic donors.Top, Southern blot using a32P-la-beled GM-CSF cDNA probe;bottom, ethidium bromide-stained agarosegel of actin RT-PCR. C1(GM-CSF mRNA in vitro synthesized) and C2(no cDNA) refer to positive and negative control RT-PCR for GM-CSF.For PBEos, data are representative of nine experiments with eight differentdonors (Table I).

FIGURE 2. BALEos survival isprolonged (A) as well as PBEos activatedin vitro by TNF-a, fibronectin (Fn), or TNF-a plus fibronectin (B). Cellviability was determined after 4 days by trypan blue exclusion. Neutralizinganti-GM-CSF mAb was added at the beginning of the culture. Each valuerepresents the mean of three (SD) or two cultures (no SD) from the same donorrepresentative of three different donors for BALEos and six donors for PBEos.

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 ofthe culture. Each value represents the mean of three (SD) cultures from thesame donor representative of two different donors.

4660 GM-CSF mRNA STABILIZATION IN AIRWAY EOSINOPHILS

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

Stabilization of GM-CSF mRNA in BALEos and TNF-a plusfibronectin-activated PBEos

GM-CSF production is regulated at transcriptional and posttran-scriptional levels (14, 15). Recently, wehave shown that GM-CSFmRNA was stabilized in eosinophils after ionomycin activation (17).Therefore, we transfected BALEos, PBEos stimulated with TNF-aplus fibronectin, or control PBEos with GM-CSF mRNA by PMGTand measured its decay. GM-CSF mRNA was extremely unstable inresting PBEos with half-life (t1/2) of 11 min (Fig. 4) but was nearly2.5-fold more stable (t1/2 5 26 min) in BALEos. GM-CSF mRNA inPBEos activated with fibronectin plus TNF-a decayed at a rate nearlyidentical with that seen in BALEos. The stability of GM-CSF mRNAin BALEos or activated PBEos was 35% greater than we previouslyobserved for PBEos activated with ionophore alone (17). Finally,activation with TNF-a alone had no effect on GM-CSF mRNAstability, whereas fibronectin alone had an intermediate effect (Fig. 5).We were consistently unable to detect GM-CSF mRNA by Northernblot in the absence of PMGT or transfection with naked gold beads(data not shown). Thus, GM-CSF mRNA detected in these experi-ments must reflect exogenous message. These data show that in-creased GM-CSF mRNA stability occurs in BALEos. A similarphenotype can be induced by TNF-a plus fibronectin activation of

PBEos, suggesting that these mediators may be responsible for GM-CSF up-regulation in vivo.

DiscussionGM-CSF is a critical cytokine implicated in eosinophil differenti-ation, function, migration, and survival (7, 27). Besides respondingto cytokine produced by activated inflammatory or resident pul-monary cells, eosinophils themselves produce GM-CSF (12, 13,28). The mechanisms that control the elaboration of GM-CSF byactivated eosinophils remain largely unknown. We report hereinthat GM-CSF mRNA levels are increased in BALEos or PBEosactivated with a combination of TNF-a plus fibronectin. Stabili-zation of GM-CSF mRNA accounted for increased steady-statelevels and preceded secretion of GM-CSF.

GM-CSF secretion by PBEos can be induced by IFN-g (28),LPS (29), anti-CD40 mAb (30), anti-CD9 mAb (31), anti-CD32mAb (32), or fibronectin (33). Because fibronectin concentrationsare significantly increased and strongly correlated with the eosin-ophil content of BAL fluid 48 h after Ag challenge (21), we askedwhether fibronectin could affect GM-CSF mRNA accumulation.However, in our hands fibronectin had inconsistent effects sug-gesting differences in donor responsiveness or the need for addi-tional and possibly additive agonists. TNF-a was chosen as a po-tential cofactor as it was abundant in BAL fluid from patients withsymptomatic asthma (8) or after segmental allergen challenge (10).Combined with IL-4, TNF-a enhanced eosinophil adhesion to hu-man pulmonary microvascular endothelial cell monolayers (34).Finally, Levi-Schaffer et al. (19) demonstrated that TNF-a in thecontext of mast cell lysates increased eosinophil survival by in-ducing autocrine GM-CSF production. Here, PBEos activated withfibronectin and TNF-a showed increased GM-CSF mRNA accu-mulation (Fig. 1). Interestingly, TNF-a alone had little effect onGM-CSF mRNA levels (Fig. 1) but was able to prolong eosinophilsurvival through a GM-CSF-independent pathway (Fig. 2). Thesedata are supported by Tsukahara et al. (35) who recently reportedthat TNF-a inhibited eosinophil apoptosis independently of GM-CSF via p38 mitogen-activated protein kinase activation. Thus,

FIGURE 4. GM-CSF mRNA is stabilized in BALEos and in PBEosactivated by TNF-a plus fibronectin. Resting PBEos (control), BALEos, orTNF-a 1 fibronectin-activated PBEos were transfected with GM-CSFmRNA. A, At the indicated time points, equal numbers of cells were har-vested, and total RNA was quantitatively isolated and Northern blottedwith 32P-labeled GM-CSF orb-actin cDNA probes. Signals were visual-ized using a PhosphorImager.B, Radioactive signals were quantified usinga PhosphorImager and normalized tob-actin mRNA and plotted vs time.Each point is the mean SD of three experiments with three different donorsor two for the control.

FIGURE 5. Fibronectin-activatedPBEos partially stabilized GM-CSFmRNA. PBEos activated by TNF-a or fibronectin for 5 h were transfectedwith GM-CSF mRNA. At indicated time points, equal numbers of cells wereharvested, and total RNA was isolated and Northern blotted as for Fig. 3.Radioactive signals were quantified and normalized tob-actin mRNA andplotted vs time. Each point is the mean of two experiments with two differentdonors.

4661The Journal of Immunology

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

TNF-a may regulate airway eosinophil survival either indepen-dently of GM-CSF through p38-mediated signaling or in associa-tion with other activators such as fibronectin in a GM-CSF-depen-dent fashion. As we have shown that GM-CSF rapidly activates theJAK2/STAT5 pathway in PBEos (36), anti-apoptotic signals maybe up-regulated through this cascade.

Fibronectin signaling might occur through several possible in-tegrins. PBEos express cell surfacea4b7, a4b1, andadb2 (37–39).Recently,Meerschaert demonstrated that PBEos survival was mark-edly enhanced aftera4b7 ligation with anti-b7 mAbs (18). Survivalwas blocked by anti-GM-CSF Abs suggesting that integrin engage-ment triggered GM-CSF release. The production, accumulation, orstability of GM-CSF mRNA was not assessed in these studies butbased on our data was unlikely significantly altered.

In previous reports, we have demonstrated that a relatively mod-est 2- or 3-fold prolongation of GM-CSF mRNA half-life timeincreased GM-CSF protein production by 15- to 20-fold (16, 25)with corresponding increased PBEos in vitro survival (17). Thus,we hypothesized that TNF-a plus fibronectin could be a physio-logic equivalent to ionomycin causing GM-CSF elaboration byblocking GM-CSF mRNA decay. Of note,b2 integrin signaling inleukocytes stabilized urokinase plasminogen activator receptormRNA (40). Thus, there is a precedent for integrin signaling af-fecting mRNA decay. In addition, urokinase plasminogen activatorreceptor mRNA contains, as does GM-CSF, multiple adenosineuridine (AUUUA) motifs in the 39 untranslated region that arerequired for regulated decay. Also consistent with our observationsare prior data demonstrating IL-1 and TNF-a regulated, respec-tively, GM-CSF, groa, b, g, IL-8, and IL-1 mRNA stability (41–43). RANTES production by activated pulmonary epithelium waspreceded by stabilization of its mRNA (44). Thus posttranscrip-tional regulation may control the production of multiple cytokinesand chemokines by activated cells in allergic diseases.

The degree of GM-CSF mRNA stabilization after activation wascorrelated with survival. Thus mRNA stabilization likely accountsfor a majority of the observed increase in GM-CSF mRNA contentand subsequent cytokine secretion in eosinophils. Similar data hasbeen shown for mitogen-activated T lymphocytes (15) where tran-scriptional up-regulation of IL-2, GM-CSF, IFN-g, and TNF-awas very small. As BALEos also displayed nuclease-resistant GM-CSF mRNA, we propose that eosinophils during migration or afterresidence in the lung block normal GM-CSF mRNA decay. Themechanism responsible for this phenotype could be general down-regulation of mRNA decay, although this is unlikely. Rather, wefavor a specific effect onGM-CSF mediated by mRNA bindingproteins. Overexpression of HuR, an AUUUA-specific RNA bindingprotein, stabilized c-fosmRNA in transfected NIH 3T3 cells (45) andp21 mRNA in human colorectal carcinoma RKO cells (46). Hetero-geneous nuclear ribonucleoproteins (hnRNP) C and L stabilizedamyloid protein precursor (47) and vascular endothelial growth factor(48) mRNAs, respectively. Consistent with this hypothesis, we haveshown that ionophore up-regulated the activity of multiple AUUUAspecific 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 eosin-ophils involved in GM-CSF mRNA posttranscriptional regulation.

The accumulation of GM-CSF mRNA in BALEos (Fig. 1) andrequirement for GM-CSF to support long-term survival in vitrosuggest that GM-CSF plays an important functional role in initi-ating and/or maintaining pulmonary eosinophilia in vivo. This issupported by several studies showing elevations of GM-CSF inBAL fluid after segmental challenge correlated with eosinophilcontent elevation during asthma (9, 11) or inhibition of eosinophilsurvival after treatment of BAL fluid with neutralizing anti-GM-

CSF Ab (50). In this last study, anti-IL-5 Ab had no effect oneosinophil survival. Altogether these findings raise the importancefor considering not only IL-5 but also GM-CSF in clinical studiestargeting lower eosinophilia and late asthmatic response.

AcknowledgmentsWe thank the other members of the Specialized Center of Research-asthmagroup, particularly Julie B. Sedgwick for providing PBEos and NizarN. Jarjour and E.A.B. Kelly for BALEos.

References1. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barneon, N. Ghavanian, I. Enander,

P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F. B. Michel. 1990.Eosinophilic inflammation in asthma.N. Engl. J. Med. 323:1033.

2. Aalbers, R., H. F. Kauffman, B. Vrugt, G. H. Koeter, and J. G. de Monchy. 1993.Allergen-induced recruitment of inflammatory cells in lavages 3 and 24 h afterchallenge in allergic asthmatic lungs.Chest 103:1178.

3. Jarjour, N. N., W. J. Calhoun, E. A. B. Kelly, G. J. Gleich, L. B. Schwartz, andW. W. Busse. 1997. The immediate and late allergic response to segmental bron-chopulmonary provocation in asthma.Am. J. Respir. Crit. Care Med. 155:1515.

4. Segwick, J. B., W. J. Calhoun, R. F. Vrtis, M. E. Bates, P. K. McAllister, andW. W. Busse. 1992. Comparison of airway and blood eosinophil function after invivo antigen challenge.J. Immunol. 149:3710.

5. Yamaguchi, Y., T. Suda, S. Ohta, K. Tominaga, Y. Miura, and T. Kasahara. 1991.Analysis of the survival of mature human eosinophils: interleukin-5 preventsapoptosis in mature human eosinophils.Blood 78:2542.

6. Rothenberg, M. E., W. F. Owen, Jr., D. S. Silberstein, J. Woods, R. J. Soberman,K. F. Austen, and R. L. Stevens. 1988. Human eosinophils have prolonged sur-vival, enhanced functional properties, and become hypodense when exposed tohuman interleukin 3.J. Clin. Invest. 81:1986.

7. Owen, W. F., M. E. Rothenberg, D. S. Silberstein, J. C. Gasson, R. L. Stevens,K. F. Austen, and R. J. Soberman. 1987. Regulation of human eosinophil via-bility, density, and function by granulocyte/macrophage colony-stimulating fac-tor in the presence of 3T3 fibroblasts.J. Exp. Med. 166:129.

8. Broide, D. H., M. Lotz, A. J. Cuomo, D. A. Coburn, E. C. Federman, andS. I. Wasserman. 1992. Cytokines in symptomatic asthma airways.J. Allergy Clin. Immunol. 89:958.

9. Woolley, K. L., E. Adelroth, M. J. Woolley, R. Ellis, M. Jordana, and P. M.O’Byrne. 1995. Effects of allergen challenge on eosinophils, eosinophil cationicprotein, and granulocyte-macrophage colony-stimulating factor in mild asthma.Am. J. Respir. Crit. Care Med. 151:1915.

10. Virchow, J.-C., C. Walker, D. Hafner, C. Kortsik, P. Werner, H. Matthys, andC. Kroegel. 1995. T cells and cytokines in bronchoalveolar lavage fluid aftersegmental allergen provocation in atopic asthma.Am. J. Respir. Crit. Care Med.151:960.

11. Shaver, J. R., J. G. Zangrilli, S.-K. Cho, R. A. Cirelli, M. Pollice, A. T. Hastie,J. E. Fish, and S. P. Peters. 1997. Kinetics of the development and recovery of thelung from IgE-mediated inflammation.Am. J. Respir. Crit. Care Med. 155:442.

12. Broide, D. H., and G. S. Firestein. 1991. Endobronchial allergen challenge inasthma: demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization.J. Clin. Invest. 88:1048.

13. Kita, H., T. Ohnishi, Y. Okubo, D. Weiler, J. S. Abrams, and G. J. Gleich. 1991.Granulocyte/macrophage colony-stimulating factor and interleukin 3 release fromhuman peripheral blood eosinophils and neutrophils.J. Exp. Med. 174:745.

14. Bickel, M., R. B. Cohen, and D. H. Pluznik. 1990. Post-transcriptional regulationof granulocyte-macrophage colony-stimulating factor synthesis in murine T cells.J. Immunol. 145:840.

15. Lindsten, T., C. H. June, J. A. Ledbetter, G. Stella, and C. B. Thompson. 1989.Regulation of lymphokine messenger RNA stability by a surface-mediated T cellactivation pathway.Science 244:339.

16. Esnault, S., J. A. Jarzembowski, and J. S. Malter. 1998. Stabilization of granu-locyte-macrophage colony-stimulating factor RNA in a human eosinophil-likecell line requires the AUUUA motifs.Proc. Assoc. Am. Physicians 110:575.

17. Esnault, S., and J. S. Malter. 1999. Primary peripheral blood eosinophils rapidlydegrade transfected granulocyte-macrophage colony-stimulating factor mRNA.J. Immunol. 163:5228.

18. Meerschaert, J., R. F. Vrtis, Y. Shikama, J. B. Sedgwick, W. W. Busse, andD. F. Mosher. 1999. Engagement ofa4b7 integrins by monoclonal antibodies orligands enhances survival of human eosinophils in vitro.J. Immunol. 163:6217.

19. Levi-Schaffer, F., V. Temkin, V. Malamud, S. Feld, and Y. Zilberman. 1998.Mast cells enhance eosinophil survival in vitro: role of TNF-a and granulocyte-macrophage colony-stimulating factor.J. Immunol. 160:5554.

20. Mattoli, S., V. L. Mattoso, M. Soloperto, L. Allegra, and A. Fasoli. 1991. Cellularand biochemical characteristics of bronchoalveolar lavage fluid in symptomaticnonallergic asthma.J. Allergy Clin. Immunol. 87:794.

21. Meerschaert, J., E. A. B. Kelly, D. F. Mosher, W. W. Busse, and N. N. Jarjour.1999. Segmental antigen challenge increases fibronectin in bronchoalveolar la-vage fluid.Am. J. Respir. Crit. Care Med. 159:619.

22. Chai, H., R. Farr, and L. A. Froehlich. 1975. Standardization of bronchial inha-lation challenge procedures.J. Allergy Clin. Immunol. 56:323.

23. Calhoun, W. J., N. N. Jarjour, G. J. Gleich, C. A. Stevens, and W. W. Busse.1993. Increased airway inflammation with segmental versus aerosol antigen chal-lenge.Am. Rev. Respir. Dis. 147:1465.

4662 GM-CSF mRNA STABILIZATION IN AIRWAY EOSINOPHILS

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

24. Hansel, T. T., I. J. M. de Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser,and C. Walker. 1991. An improved immunomagnetic procedure for the isolationof highly purified human blood eosinophils.J. Immunol. Methods 145:105.

25. Rajagopalan, L. E., and J. S. Malter. 1996. Turnover and translation of in vitrosynthesized messenger RNAs in transfected, normal cells.J. Biol. Chem. 271:19871.

26. Burkholder, J. K., J. Decker, and N.-S. Yang. 1993. Rapid transgene expressionin lymphocyte and macrophage primary cultures after particle bombardment-mediated gene transfer.J. Immunol. Methods 165:149.

27. Sedgwick, J. B., S. F. Quan, W. J. Calhoun, and W. W. Busse. 1995. Effect ofinterleukin-5 and granulocyte-macrophage colony stimulating factor on in vitroeosinophil function: comparison with airway eosinophils.J. Allergy Clin. Immu-nol. 96:375.

28. Moqdel, R., Q. Hamid, S. Ying, J. Barkans, A. Hartnell, A. Tsicopoulos,A. J. Wardlaw, and A. B. Kay. 1991. Expression of mRNA and immunoreactivityfor the granulocyte/macrophage colony-stimulating factor in activated human eo-sinophils.J. Exp. Med. 174:749.

29. Takanashi, S., R. Nonaka, Z. Xing, P. O’Byrne, J. Dolovich, and M. Jordana.1994. Interleukin 10 inhibits lipopolysaccharide-induced survival and cytokineproduction by human peripheral blood eosinophils.J. Exp. Med. 180:711.

30. Ohkawara, Y., K. G. Lim, Z. Xing, M. Glibetic, K. Nakano, J. Dolovich,K. Croitoru, P. F. Weller, and M. Jordana. 1996. CD40 expression by humanperipheral blood eosinophils.J. Clin. Invest. 97:1761.

31. Kim, J.-T., G. J. Gleich, and H. Kita. 1997. Roles of CD9 molecules in survivaland activation of human eosinophils.J. Immunol. 159:926.

32. Kim, J.-T., A. W. Schimming, and H. Kita. 1999. Ligation of FcgRII (CD32)pivotally regulates survival of human eosinophils.J. Immunol. 162:4253.

33. Anwar, A. R. F., R. Moqdel, G. M. Walsh, A. B. Kay, and A. J. Wardlaw. 1993.Adhesion to fibronectin prolongs eosinophil survival.J. Exp. Med. 177:839.

34. Yamamoto, H., J. B. Sedgwick, and W. W. Busse. 1998. Differential regulationof eosinophil adhesion and transmigration by pulmonary microvascular endothe-lial cells. J. Immunol. 161:971.

35. Tsukahara, K., A. Nakao, M. Hiraguri, S. Miike, M. Mamura, Y. Saito, andI. Iwamoto. 1999. Tumor necrosis factor-a mediates antiapoptotic signals par-tially via p38 MAPKinase activation in human eosinophils.Int. Arch. AllergyImmunol. 120(Suppl. 1):54.

36. Bhattacharya, S., B. Stout, M. E. Bates, P. J. Bertics, and J. S. Malter. 2001.Granulocyte macrophage colony-stimulating factor and interleukin-5 activateSTAT5 and induce CSI mRNA in human peripheral blood eosinophils.Am. J.Respir. Cell. Mol. Biol. 24:1.

37. Walsh, G. M., F. A. Symon, A. I. Lazarovits, and A. J. Wardlaw. 1996. Integrina4b7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 andfibronectin.Immunology 89:112.

38. Carlos, T. M., and J. M. Harlan. 1994. Leukocyte-endothelial adhesion molecule.Blood 84:2068.

39. Grayson, M. H., M. Van der Vieren, S. A. Sterbinsky, W. M. Gallatin,P. A. Hoffman, D. E. Staunton, and B. S. Bochner. 1998.aD/b2 integrin is ex-pressed on human eosinophils and functions as an alternative ligand for vascularcell adhesion molecule 1 (VCAM-1).J. Exp. Med. 188:2187.

40. Wang, G. J., M. Collinge, F. Blasi, R. Pardi, and J. R. Bender. 1998. Posttran-scriptional regulation of urokinase plasminogen activator receptor messengerRNA by leukocyte integrin engagement.Proc. Natl. Acad. Sci. USA 95:6296.

41. Bagby, G. C., G. Shaw, M. C. Heinrich, S. Hefeneider, M. A. Brown,T. G. DeLoughery, G. M. Segal, and L. Band. 1990. Interleukin-1 stimulationstabilizes GM-CSF mRNA in human vascular endothelial cells: preliminary stud-ies on the role of the 39AU rich motif. Prog. Clin. Biol. Res. 352:233.

42. Stoeckle, M. Y. 1991. Post-transcriptional regulation of groa, b, g, and IL-8mRNAs by IL-1b. Nucleic Acids Res. 19:917.

43. Gorospe, M. S. Kumar, and C. Baglioni. 1993. Tumor necrosis factor increasesstability of interleukin-1 mRNA by activating protein kinase C.J. Biol. Chem.268:6214.

44. Koga, T., E.Sardina, R. M. Tidwell, M. Pelletier, D. C. Look, and M. J. Holtzman.1999. Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization.Proc. Natl. Acad. Sci. USA 96:5680.

45. Peng, S.-Y., C. Y. Chen, N. Xu, and A. B. Shyu. 1998. RNA stabilization by theAU-rich element binding protein, HuR, an ELAV protein.EMBO J. 17:3461.

46. Wang, W., H. Furneaux, H. Cheng, M. C. Caldwell, D. Hutter, Y. Liu,N. Holbrook, and M. Gorospe. 2000. HuR regulates p21 mRNA stabilization byUV light. Mol. Cell. Biol. 20:760.

47. Rajagopalan, L. E., C. J. Westmark, J. A. Jarzembowski, and J. S. Malter. 1998.hnRNP C increases amyloid precursor protein (APP) production by stabilizingAPP mRNA.Nucleic Acids Res. 26:3418.

48. Shih, S. C., and K. P. Claffey. 1999. Regulation of human vascular endothelialgrowth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleo-protein L.J. Biol. Chem. 274:1359.

49. Ruth, J. H., S. Esnault, J. A. Jarzembowski, and J. S. Malter. 1999. Calciumionophore upregulation of AUUUA-specific binding protein activity is contem-poraneous with granulocyte macrophage colony-stimulating factor messengerRNA stabilization in AML14.3D10 cells.Am. J. Respir. Cell Mol. Biol. 21:621.

50. Park, C. S., Y. S. Choi, S. Y. Ki, S. H. Moon, S. W. Jeong, S.-T. Uh, andY. H. Kim. 1998. Granulocyte macrophage colony-stimulating factor is the maincytokine enhancing survival of eosinophils in asthmatic airways.Eur. Respir. J.12:872.

4663The Journal of Immunology

by guest on January 19, 2016http://w

ww

.jimm

unol.org/D

ownloaded from