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A Phosphosite Screen Identifies Autocrine TGF- β-Driven Activation of Protein Kinase R as a Survival-Limiting Factor for Eosinophils This information is current as of September 27, 2021. Nicholas Goplen, Magdalena M. Gorska, Susan J. Stafford, Sadee Rozario, Lei Guo, Qiaoling Liang and Rafeul Alam J Immunol 2008; 180:4256-4264; ; doi: 10.4049/jimmunol.180.6.4256 http://www.jimmunol.org/content/180/6/4256 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Phosphosite Screen Identiﬁes Autocrine TGF-␤-Driven Activation of Protein Kinase R as a Survival-Limiting Factor for Eosinophils1

Nicholas Goplen,* Magdalena M. Gorska,*† Susan J. Stafford,‡ Sadee Rozario,* Lei Guo,* Qiaoling Liang,* and Rafeul Alam2*†

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase C␧, and glycogen Downloaded from synthase kinase 3 ␣ and ␤. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2␣ are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-␤ in eosinophils. TGF-␤ directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-␤ Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phos- http://www.jimmunol.org/ pho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-␤ and that this could be modulated by a Th2 microenvironment in vivo. The Journal of Immunology, 2008, 180: 4256–4264.

he eosinophil is an essential component of the immune sys- these studies have been performed as a part of separate and inde- tem and plays an important role in Th2-mediated allergic pendent investigations. and parasitic diseases (1). Cytokines and chemokines are We have performed an Ab-based phosphoprotein screen of eo- T by guest on September 27, 2021 potent activators of eosinophils. However, there are significant differ- sinophil activation by IL-5 and CCL11—the most prominent rep- ences between cytokines and chemokines in regard to their activation resentatives of growth factors and CC chemokines for eosinophils. of eosinophils (2). Cytokines with growth factor-like activity such as This analysis led to the identification of a number of novel signal- IL-3, IL-5, and GM-CSF regulate differentiation, activation, and sur- ing molecules that are differentially activated by IL-5 and CCL11. vival of eosinophils. In the absence of these growth factors, eosinophil Based upon these novel findings, we undertook a systematic anal- differentiation and survival are severely impaired (3). In contrast, che- ysis of signaling determinants of eosinophil survival. mokines like CCL11 (eotaxin) and RANTES primarily regulate eosinophil chemotaxis, homing, and activation. They have very little Materials and Methods effect on differentiation, and especially on survival (4, 5). The signal- Reagents ing mechanism underlying these differences between chemokines and Abs used were: rabbit anti-pThr451-PKR (Cell Signaling), rabbit anti- growth factors is largely unknown. pSer51-eukaryotic initiation factor 2␣ (EIF2␣3; Epitomics), rabbit anti- The receptors for IL-3, IL-5, and GM-CSF are heterodimers; PKR, mouse anti-p65, goat anti-␤-actin, goat anti-pSer433/435-SMAD2/3 each cytokine has its specific ␣ receptor but they share a common (Santa Cruz Biotechnology), mouse monoclonal anti-TGF␤ (R&D Sys- ␤ ␤ tems), and IgG isotype controls (DakoCytomation). Pharmaceutical inhib- receptor subunit ( c) (6). CCRs are Gi-coupled 7-transmembrane heptahelical receptors (4). The signaling mechanism of activation itors and inert controls for PKR were obtained from Calbiochem; PKR smart-pool small interfering RNA (siRNA) and nontargeting (NT)-siRNA of eosinophils by some of the cytokines and chemokines (e.g., IL-5 were obtained from Dharmacon, whereas FITC-labeled control siRNA was and CCL11) has previously been investigated. However, most of obtained from Santa Cruz Biotechnology. Propidium iodide, OVA grade V, HBSS, HEPES, EDTA, and HSA were obtained from Sigma-Aldrich; Imject Alum was obtained from Pierce; and all recombinant cytokines and † *National Jewish Medical and Research Center, Denver, CO 80206; University of chemokines were obtained from PeproTech. RPMI 1640 with L-glutamine ‡ Colorado at Denver Health Sciences Center, Denver, CO 80206; and University of was obtained from Cellgro; FCS was obtained from HyClone; and Texas Medical Branch, Galveston, TX 77555 Hetastarch was obtained from Hospira. MACS separation reagents and the Received for publication July 5, 2007. Accepted for publication January 9, 2008. Vario MACS magnet were obtained from Miltenyi Biotec. 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. 3 Abbreviations used in this paper: EIF2␣, eukaryotic initiation factor 2␣; DAPI, Ј Ј 1 4 ,6 -diamidino-2-phenylindole; PVDF, polyvinylidene difluoride; IFS, immunoflu- This work was supported by National Institutes of Health Grants R01 AI50179, orescent staining; GSK3, glycogen synthase kinase 3; PKR, protein kinase R; NT, AI059719, and AI68088. nontargeting; siRNA, small interfering RNA; MSK, mitogen and stress activated 2 Address correspondence and reprint requests to Dr. Rafeul Alam, National Jewish kinase; RSK, ribosomal S6 kinase. Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 4257

Leukocyte isolation radio-immunoprecipitation assay buffer with protease inhibitors and a final time in 1 ml of kinase buffer (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM The procedure was conducted with sterile solutions in a hood. A minimum magnesium acetate, 2 mM MnCl2, 10% glycerol with protease inhibitors of 60 ml of whole blood was obtained from healthy to mildly allergic added fresh: 1 mM PMSF, 1 ␮g/ml apropeptin, leupeptin, and peptin, and 1 donors. Whole blood was combined with 2.5 ml of 100 mM EDTA and 7.5 ␮ mM Na3VO4). The kinase reaction was conducted in 25 l of the above buffer ml of 6% hetastarch in 0.9% NaCl. The RBC were allowed to sediment with 10 ␮Ci [␥-32P]ATP and 40 ␮M nonradioactive ATP per reaction for 30 over a period of 45 min and the buffy coat was collected every 5–10 min. min at 30°C followed by addition of an SDS protein loading dye with 2-ME The buffy coat was washed several times with cold HBSS plus 4 mM and boiling for 5 min before loading supernatants onto a polyacrylamide gel HEPES (pH 7.3) before resuspending in 30 ml of room temperature HBSS and transferring to PVDF as above. PVDF membranes were then exposed to plus 4 mM HEPES and underlying 15 ml of Percoll (1.089 g/ml) followed a phosphor screen for 24 h and read on a phosphor imager (Typhoon). by centrifugation at 400 ϫ g for 35 min at room temperature with minimum acceleration/deceleration. The monocyte/lymphocyte and granulo- Survival assay cyte fractions were washed one time in 50 ml of HBSS plus 4 mM HEPES 6 plus 4 mM EDTA before hypotonic lysis of residual RBCs with ice-cold Purified eosinophils were cultured at 1 ϫ 10 cells/ml in RPMI 1640 plus sterile double-distilled water (12 ml). After 50 s of lysis on ice, cold 10ϫ 10% FCS in the presence or absence of a pharmacological inhibitor of HBSS buffer (1.5 ml) and FCS (1 ml) were added and brought to 50 ml in protein kinase R (PKR; an imidazole-oxindole compound from Calbio- cold HBSS plus 4 mM EDTA. Cell fractions were washed three times in chem) or its inert analog. Preliminary dose-response experiments with 100, cold HBSS plus 4 mM HEPES/2% FCS. Granulocytes were labeled with 200, and 400 nM concentrations of the inhibitor demonstrated the best Ϫ10 anti-human CD16 microbeads for positive selection of neutrophils as per response with the 200 nM dose. IL-5 (10 M; PeproTech) was used as a manufacturer’s (Miltenyi Biotec) protocol. Granulocytes were run down positive control. siRNA was used at a 2 ␮g/ml concentration. TGF-␤ was Ϫ10 the appropriate size column with HBSS plus 2% FCS in the presence of a used at 4 ϫ 10 M, and anti-TGF-␤ and mouse IgG1 isotype control magnetic field (Vario MACS), and the eluent (50 ml) containing eosino- were used at 2 ␮g/ml. A total of 200,000 cells were taken from each culture at the indicated time point and resuspended in 20 ␮l of 0.25 mg/ml pro- phils was collected via a 21-gauge flow resistor. Labeled neutrophils were Downloaded from collected by taking the column out of the magnetic field and collecting 50 pidium iodide in PBS. Cells were put onto slides and a minimum of five ml of eluent. Before stimulation or culture, cells were resuspended in fields (ϫ20 magnification) per sample were viewed to obtain an average RPMI 1640/10% FCS containing antibiotics and mycotics and allowed to viability for each sample. Experiments were done in triplicate. equilibrate to 37°C in HSA (3%) coated tubes/wells. Animals and immunization protocol Phosphosite screen Female C57BL/6 mice from The Jackson Laboratory were acquired and Purified eosinophils were prepared as above and stimulated with either housed under pathogen-free conditions at the Biological Resource Center http://www.jimmunol.org/ medium, CCL11 (10Ϫ8 M) for 3 min, and IL-5 (10Ϫ10 M) for 3 and 5 min. (National Jewish Medical and Research Center, Denver, CO). Mice were This time point was previously shown to have the highest activation of used under an experimental protocol approved by the institutional animal upstream tyrosine kinases and downstream MAPKs (7–9). Cell lysates care and use committee. Animals were maintained on an OVA-free diet. were sent to Kinexus Bioinformatics for a Western blot-based phosphosite Mice were immunized at 8 wk of age via s.c. injections of 50 ␮gofOVA screen. The results are compared with trace amounts of known proteins in alum. Animals were given an identical s.c. booster 2 wk after the first relative to various phosphospecific proteins in each sample via densitom- immunization. Two weeks following the booster, animals were challenged etry readings. with a 1% aerosol of OVA in 0.9% saline for 30 min for 3 consecutive days. Three days following the final aerosol challenge, animals were sac- Immunofluorescent staining rificed and bone marrow was isolated and fixed as above for intracellular immunofluorescent staining. Leukocytes were prepared as above, stimulated or cultured as indicated, by guest on September 27, 2021 and fixed in 2% paraformaldehyde in PBS (pH 7.4) at room temperature for Statistical analyses 45 min. Cells were washed four times in PBS and 200,000 cells were placed on a slide and either immediately stained or frozen at Ϫ80°C until Data are presented as mean Ϯ SEM of multiple experiments as indicated use. Cells were blocked in 10% goat serum in PBS plus 0.05% saponin at in the text. Statistical analyses were performed with the Student t test, the room temperature for 1 h. Primary Abs and isotype controls were applied Mann-Whitney U test, the Spearman’s correlation coefficient test, and the at 1–2 ␮g/ml overnight at 4°C in 5% goat serum. Slides were washed three Fisher’s exact probability test. A value of p Ͻ 0.05 was considered times for 10 min each in PBS plus 0.05% saponin before applying the significant. FITC- or tetramethylrhodamine isothiocyanate-conjugated secondary Ab at 1:200 at the same time as 4Ј,6Ј-diamidino-2-phenylindole (DAPI; 0.1 nM) Results in 5% goat serum at room temperature for 1 h. Slides were washed as Phosphospecific Ab-based signaling screen above, checked for background (IgG control), and cover slides are mounted. Slides are visualized with an epifluorescence microscope (Nikon Eosinophils were purified to 97–99% purity from the peripheral Eclipse 2000) equipped with a Cool-Snap CCD camera and Metamorph blood of mildly allergic patients by a combination of Percoll gra- image analysis software (Molecular Devices). Exposure times are chosen dient centrifugation and negative selection using anti-human in a manner that background IgG staining is negligible and only signal above background is analyzed. Morphometric data was obtained by view- CD16-coated magnetic beads as described previously (2, 8). Eo- ing a minimum of five high-powered fields per slide containing between 30 sinophils were stimulated with CCL11 for 3 min and with IL-5 for and 50 cells/field. In most cases, mean fluorescent intensity or integrated 5 min. The foregoing time points have previously been shown to fluorescent intensity (normalized to cell number) was analyzed. Means are be optimal for tyrosine kinase and MAPK activation (7–9). The expressed with error bars representative of the SEM. cell lysate from the stimulated eosinophils from two donors was Immunoblotting sent out to Kinexus for phosphosite screening. Eosinophils from SDS-PAGE was performed on 8–10% gels with 20 ␮g of protein per two additional donors were processed in-house for confirmation of sample. Protein was transferred onto polyvinylidene difluoride (PVDF) the Kinexus screen. These samples were Western blotted using a membranes and blocked in 5% BSA before immunoblotting with primary similar set of phosphospecific Abs. The Kinexus data are reported Abs overnight at 4°C in 1% BSA. Blots were washed three times in TBST in densitometric units. The trace quantity of the known proteins before application of the HRP-conjugated secondary Ab in 1% BSA for 1 h at room temperature. Blots were washed as above and developed with the from each screen type is compared against all relevant samples. ECL reagent (ECL from Amersham) per the manufacturer’s instructions The corrected data standardizes the trace quantity (actual trace before exposure to film. quantity times coefficient) for all samples. The in-house Western Immunoprecipitation and in vitro kinase assay blot and its densitometric analyses confirmed the overall direction of change (positive or negative) in target protein phosphorylation A total of 40 ␮g of protein was precleared for nonspecific binding by following IL-5 and CCL11 stimulation as reported by the Kinexus incubation with protein A/G agarose beads for 2 h and then incubated overnight with 2 ␮g of an anti-PKR (Santa Cruz Biotechnology) before screen. immunoprecipitating with protein A/G agarose beads (Santa Cruz Biotech- We examined 44 different phosphosites covering 36 signaling nology) for 2 h. Immunoprecipitates were washed three times in 1 ml of molecules. Of the 44 phosphosites studied, the phosphorylation (or 4258 PKR REGULATION OF EOSINOPHIL SURVIVAL Downloaded from http://www.jimmunol.org/

FIGURE 1. Differential phosphorylation of signaling molecules in IL 5- and CCL11-stimulated eosinophils. Purified human peripheral blood eosinophils were stimulated with IL-5 (10Ϫ10 M) for 5 min, CCL11 (10Ϫ7 M) for 3 min, or medium control for 5 min and then lysed and analyzed for the expression of phosphoproteins. Samples from two donors were analyzed by the “Kinetworks Phosphosite Screen” (Kinexus). Eosinophil lysate from another two donors was studied by Western blotting with phosphospecific Abs in our laboratory. Densitometric data from all four donors were pooled and analyzed statistically. A, Signaling molecules phosphorylated by both IL-5 and CCL11. B, Signaling molecule inhibited by both IL-5 and CCL11. Left panel, Inhibition of activating phosphorylation. Middle and right panel, Induction of inhibitory phosphorylation. C, Differential phosphorylation by IL-5 and CCL11. Phosphorylation induced by IL-5 but not CCL11. D, Phosphorylation inhibited by IL-5 but not by CCL11. Left panel, Densitometric data presented by guest on September 27, 2021 .(p Ͻ 0.05 compared with the medium control (Mann-Whitney U test, n ϭ 4 ,ء .as a bar graph. Right panel, Western blot lack thereof) of 38 phosphosites was concordant in all four sam- mediated by Akt, inhibits the kinase activity of GSK3 (13–15). In ples. Data for six molecules were discordant among the samples agreement with our finding, a recent report demonstrated that ( p Ͻ 0.00005, Fisher’s exact probability test). IL-5 and CCL11 GSK3-␤ serine phosphorylation was important for IL5-induced increased phosphorylation of 9 sites, decreased phosphorylation of eosinophil survival (16). IL-5 increased phosphorylation of Tyr216 2 sites, and differentially regulated phosphorylation of 4 sites (Fig. of GSK3␤ (Fig. 1B, right panel). The significance of tyrosine 1). Phosphorylation of 29 sites was unchanged by either activator phosphorylation of GSK3 is currently unknown. (data not shown). Activation increased by IL-5 but not by CCL11 Phosphorylation increased by both IL-5 and CCL11 The phosphorylation of STAT1 and STAT3 was increased by IL-5 The signaling molecules that were phosphorylated by both IL-5 and but not by CCL11 (Fig. 1C). Previously, the phosphorylation of CCL11 include the members of the MAPK cascade: MEK1/2 and STATS 1, 3, and 5 in IL5-stimulated eosinophils has been de- ERK1/2 (Fig. 1A, left panel), p38 and its substrate mitogen and stress scribed (9, 17–19). Furthermore, we and others have reported the activated kinase (MSK)1/2 (Fig. 1A, right panel). IL-5 consistently phosphorylation of Jak1 and Jak2 by IL-5 but not by chemokines induced higher levels of phosphorylation of ERK1/2. One of the phys- (2, 9, 12, 17, 20). Both Jaks and STATs, especially STAT5, are iological substrates of activated ERK1/2 is ribosomal S6 kinase important for IL5-induecd eosinophil differentiation and survival. (RSK)1 (10). Both IL-5 and CCL11 induced phosphorylation of RSK1, thus confirming biological relevance of activation of the ERK Kinases inhibited by IL-5 but not by CCL11 pathway. The role of the MAPK signaling pathway in eosinophil Eosinophils show a high level of basal phosphorylation of PKR, a function has previously been studied. They are involved in eosinophil dsRNA-dependent protein kinase (Fig. 1D). IL-5 induced a dra- differentiation, survival, and degranulation (2, 7, 11, 12). matic inhibition of PKR phosphorylation. CCL11 did not have any effect on PKR phosphorylation. Activation inhibited by both IL-5 and CCL11 Both activators inhibited the phosphorylation of S6 kinase and Signaling molecules not affected by either cytokines protein kinase C␧ (Fig. 1B, left panel). In addition, they increased Phosphorylation of the following signaling molecules was not af- the inhibitory phosphorylation of Ser21 and Ser9 of glycogen syn- fected by either cytokines: adducin ␣ and ␥, c-Jun, cyclin-depen- thase kinase 3 (GSK3) ␣ and ␤, respectively (Fig. 1B, middle dent kinase 1, CREB, JNK, MKK3, protein kinase B, protein ki- panel). Phosphorylation of the foregoing residues, which is likely nase C-␣,-␤, and -␦, retinoblastoma protein, and SMAD1. The The Journal of Immunology 4259

lack of phosphorylation of JNK and c-Jun is in agreement with previously published reports (21). The lack of phosphorylation of other signaling molecules could be due to inadequate length of stimulation with the activators, use of an insensitive Ab, or may reflect a true absence of activation. Phosphorylation of PKR, its downstream effectors, and the effect of PKR on eosinophil survival One of the molecules that showed the most dramatic differential response to IL-5 and CCL11 was PKR. Another startling finding in this regard was the high level of basal phosphorylation of this molecule in eosinophils. Remarkably, increased basal phosphorylation was most prominent in short-lived leukocytes (e.g., eosinophils and neutrophils) as compared with long-lived leukocytes such as lymphocytes and monocytes (Fig. 2A). Immunofluores- cence staining (IFS) for phospho-PKR confirmed the results of Western blotting. For these reasons, we examined the PKR pathway and its biological relevance in-depth.

The incubation of eosinophils with IL-5 resulted in near-com- Downloaded from plete dephosphorylation of PKR rapidly within 5 min (Fig. 2B, upper panels). There were no changes in the level of PKR staining during this time period (Fig. 2B, left lower panel). The IFS results were conﬁrmed by an immune complex kinase assay for PKR (Fig. 2C). PKR signals through two antagonistic pathways: the survival

pathway through the activation of NF-␬B and the apoptotic path- http://www.jimmunol.org/ way through phosphorylation of EIF2␣ (22, 23). Phosphorylation of the EIF2␣ regulatory site Ser51 inhibits protein synthesis and drives the apoptotic cell death. We observed very little translocation of the p65 subunit of NF-␬B into the nucleus under basal condition. IL-5 stimulation significantly increased p65 (green) FIGURE 2. Phospho-PKR in eosinophils. A, Immunofluorescent stain- translocation into the nucleus (red), turning them yellow upon ing of pPKR in a mixture of leukocytes. PBLs were stained with a rabbit overlay of the images (Fig. 2E). Thus, increased basal phospho- phospho-PKR (Thr451) Ab (green) and the nuclei were counterstained with PKR does not result in increased NF-␬B activation in eosinophils. DAPI and pseudocolored red: 1, monocyte; 2, lymphocyte; 3, neutrophil; Next, we examined phosphorylation of EIF2␣ under basal condi- and 4, eosinophil. B, Immunofluorescent staining of eosinophils for phos- by guest on September 27, 2021 tions. Phosphorylation of EIF2␣ regulatory site Ser51 closely par- pho-PKR and the effect of IL-5. Purified eosinophils were immunostained alleled activation of PKR in eosinophils. There was significant with the rabbit phospho-PKR (Thr451) Ab (green) with and without stim- Ϫ cytosolic immunofluorescence (green) of Ser51-phosphorylated ulation with IL-5 10 10 M for 5 or 30 min. Nuclei were stained with DAPI EIF2␣ under the basal condition (Fig. 2E). Stimulation of cells and pseudocolored with red. Left panel, Representative immunofluorescent with IL-5 induced near complete dephosphorylation of EIF2␣ after images from one of four independent experiments. Bottom panel, Total PKR immunostaining under similar experimental conditions. Right panel, 30 min. Quantitative analyses of mean fluorescence intensity (MFI) of PKR stain- It has previously been reported that IL-3 stimulation abrogates ing of 200 eosinophils from four different donors. Data are normalized to PKR signaling (24). It was therefore of interest to see whether medium control (Cntrl) which ranged from 600 to 1455 of fluorescence GM-CSF (whose receptor shares the common ␤-chain with the intensity units depending upon the eosinophil donor. C, An immune-com- IL-3 and IL-5 receptors) could have the same effects on PKR. plex kinase assay for PKR. PKR was immunoprecipitated (anti-PKR) from Ϫ Treatment with GM-CSF (10 10 M) inhibited PKR phosphoryla- eosinophils that were treated with medium (Ϫ) or IL-5 (ϩ) for 30 min. In tion in eosinophils (Fig. 2D), which was similar to that observed the control experiment, rabbit IgG was used for immunoprecipitation. The with IL-5. Together, these findings implicate the common ␤-chain immunoprecipitates were assayed for autophosphorylation [32P]PKR and in cytokine-induced down-regulation of PKR signaling. autoradiographed. Bottom panel, The Ig H chain (IgH) reflecting the loading of the immunoprecipitates. D, The effect of GM-CSF on eosinophil PKR is important for eosinophil survival PKR phosphorylation. Eosinophils were stimulated with GM-CSF for the indicated time period and Western blotted with an anti-pPKR Ab. The To determine whether increased PKR activation contributes to eo- membrane was reprobed with the anti-PKR Ab. E, Immunofluorescent sinophil survival, we used a pharmacological inhibitor of PKR. staining of eosinophils for the NF-␬B p65 subunit and phospho-EIF2␣ and Incubation with a PKR inhibitor (an imidazole-oxindole com- the effect of IL-5. Purified eosinophils were processed as above and im- pound, C13H8N4OS obtained from Calbiochem) but not its inert munostained using a mouse mAb against p65 and a rabbit anti-phospho- ␣ oxindole control (C15H8Cl3NO2) improved cell survival in eosin- EIF2 . Representative images from one of three independent experiments ophils (Fig. 3A). We also examined the effect of PKR knockdown are shown. Right panels in B and E, Immunostaining with control Abs by siRNA in eosinophils. We have previously studied transfection (green) and nuclear staining (red) with DAPI. Rabbit IgG is a control for of eosinophils with oligos and plasmids by various methods the anti-pPKR and anti-pEIF2␣ Abs, mIgG2a is a control for the anti-PKR ␬ involving cationic lipids and electroporation. The success rate of Ab, and mIgG1 is a control for the anti-NF B p65 Ab. these methods is low and highly variable because of extreme ad- hesiveness of eosinophils following treatment with the transfecting agents resulting in cell loss and also because of increased cell spontaneously internalized large quantities of siRNA (Fig. 3, B and death. While studying these methods we observed that control cells D). Treatment of purified eosinophils with siRNA for PKR signif- (eosinophils treated with siRNA alone without transfecting agents) icantly reduced the expression of the PKR protein (Fig. 3C, left 4260 PKR REGULATION OF EOSINOPHIL SURVIVAL Downloaded from http://www.jimmunol.org/

FIGURE 3. The effect of PKR on eosinophil survival. A, Purified eosinophils were incubated with an imidazole-oxindole PKR inhibitor (PKR inhibitor) and its inert control (Inhibitor Cntrl) (both obtained from Calbiochem) for various periods of time. Live cells were counted after staining with propidium iodide and excluding the propidium-positive cells. Vehicle control (0.2% DMSO) and IL-5 were used as negative and positive controls, respectively, for p Ͻ 0.05). B, Spontaneous ,ء ;survival. The PKR inhibitor prolonged eosinophil survival as compared with the diluent control at all time points (n ϭ 4 uptake of labeled siRNA by eosinophils (red autofluorescence). A mixed leukocyte population was incubated with a FITC-labeled NT siRNA (green) for 24 h. The cells were then fixed and nuclei (blue) were stained with DAPI followed by analysis by epifluorescence microscopy. Eosinophils were identified by their autofluorescence in the TRITC (red) channel and by their nuclear morphology. The yellow color indicates the presence of FITC-labeled siRNA (green) superimposed on the red autofluorescence of eosinophils. C, PKR knockdown by siRNA (left panel). Purified eosinophils were incubated with the

PKR siRNA or a control NT siRNA for 60 h and then the cells were lysed and Western blotted for PKR. The membrane was reprobed for actin to show by guest on September 27, 2021 equal protein loading. The effect of PKR knockdown on eosinophil survival (right panel). Purified eosinophils were cultured with the PKR siRNA or NT control siRNA and the cell viability was assessed at 60 h. Eosinophils cultured in the presence of siRNA showed significantly increased survival (n ϭ 3). D, Spontaneous uptake of siRNA is a dynamin II-dependent endocytotic process. Eosinophils were cultured with 2 ␮g/ml NT or dynamin siRNA for 24 h followed by incubation for an additional 24 h with 2 ␮g/ml FITC-labeled NT-siRNA (green). Cells were fixed and stained with an anti-dynamin II Ab (red) and DAPI (blue). The fluorescence of immunostaining was presented as integrated fluorescent intensity (IFI) normalized to the cell number. Left upper panel, The effect of dynamin siRNA (Dyn SiRNA) on dynamin expression. Right panel, Immunofluorescent images of the FITC-labeled NT-siRNA uptake by dynamin-sufficient (NT Si) and dynamin-deficient (Dyn Si) eosinophils. Right lower panel, A correlation between the expression level of dynamin and the uptake of FITC-NT siRNA. panel). Concomitantly, and in accord with the PKR inhibitor data, increase PKR activation in eosinophils. The phosphorylation of survival of eosinophils was significantly prolonged in PKR knock- PKR was studied by IFS and immunoblotting. Indeed, we discov- down but not in control eosinophils (Fig. 3C, right panel). We ered that TGF-␤ does activate PKR and its downstream substrate speculated that the spontaneous internalization of siRNA (ob- EIF2␣ in eosinophils (Fig. 4A). We next wondered whether the served in 85% of eosinophils) was an active cellular process and down-regulation of PKR activation by IL-5 shown earlier could be was dependent on endocytosis. All endocytic processes are depen- rescued by TGF-␤. To this goal, the cells were first treated with dent upon the activity of dynamin, a GTPase, which facilitates IL-5 for 30 min and then treated with TGF-␤, a proapoptotic agent budding of endocytic vesicles from the cell membrane. To address for 30 min. Indeed, TGF-␤ could restore PKR (Fig. 4B) and EIF2␣ this question, we pretreated cells with NT or dynamin II siRNA for (Fig. 4C) phosphorylation to baseline levels after being down-reg- 24 h before incubation with a fluorescent NT siRNA for 24 h. ulated by IL-5. These data further explain the mechanism by which Next, we fixed the cells and immunostained for dynamin II (red) TGF-␤ induces apoptosis in eosinophils in addition to those in- and stained nuclei (blue) with DAPI (Fig. 3D). Preincubation of vestigated previously (9, 25). cells with dynamin siRNA, but not NT siRNA, caused markedly decreased dynamin II expression. The internalization of the fluo- ␤ rescently labeled siRNA correlated positively with the level of PKR activation by TGF- is an autocrine effect in eosinophils dynamin II expression. Together, these data suggest that the spon- We have previously shown that autocrine secretion of TGF-␤ from taneous uptake of siRNA is an active endocytotic process mediated eosinophils contributes to their short life (9, 25). It was therefore by dynamin II. of interest whether we could detect baseline phospho-SMAD 2/3 signaling in eosinophils. Indeed, we found baseline p-SMAD 2/3 ␤ TGF- promotes phosphorylation of PKR in eosinophils in both the cytosol and nucleus of eosinophils (Fig. 4D, upper Because we demonstrated that PKR is important for eosinophil panel). We analyzed 200 randomly selected and immunofluores- survival, we wondered whether apoptosis-promoting factors could cently stained eosinophils per slide from three donors (minimum The Journal of Immunology 4261 Downloaded from http://www.jimmunol.org/

FIGURE 4. TGF-␤ regulation of PKR in eosinophils. A, TGF-␤ enhances PKR/EIF2␣ phosphorylation in eosinophils. Eosinophils were treated with or without TGF-␤ for 5–60 min and then immunostained for pPKR (Thr451), pEIF2 ␣ (Ser51), or PKR (green). Nuclei were stained with DAPI and by guest on September 27, 2021 pseudocolored red. Basal phosphorylation of PKR in eosinophils was further enhanced by the treatment with TGF-␤. Left panel, Images from the 30-min time point. Right panel, Quantitative analyses of normalized mean fluorescence intensity (MFI) from four (EIF2␣) and five (PKR) independent experiments. ,and #, p Ͻ 0.05 for pPKR- and pEIF2␣-stained samples with respect to medium controls; ∧, p Ͻ 0.05 for 5 min vs 30 min TGF-␤ treatment. B and C ء The effect of IL-5 treatment first followed by TGF-␤ stimulation on pPKR and pEIF2␣ expression by eosinophils. Purified eosinophils were treated with IL-5 (10Ϫ10 M) for 30 min, washed two times, followed by stimulation with TGF-␤ (10Ϫ10 M) for 30 min (for pPKR) or 60 min (for pEIF2␣). Phosphorylation of PKR (Thr451) was detected by immunoblotting (B). The membrane was reprobed with an anti-PKR Ab. The phosphorylation of EIF2␣ (Ser51) was detected by immunofluorescent staining followed by morphometric quantitative analyses (C, n ϭ 3). $, p Ͻ 0.05 for IL-5 vs IL-5 ϩ TGF-␤ 60 min. D, Basal phosphorylation of SMAD2/3 (Ser433/435) in eosinophils. Purified eosinophils were immunostained with phospho-SMAD2/3 and the nuclei were stained with DAPI. Phosphorylated SMAD2/3 stained the cytosol green and the nucleus yellow because of green overlaid with red. A representative image from three independent experiments is shown (upper panel). A quantitative analysis of total pSMADϩ eosinophils and eosinophils with nuclear and cytosolic only (cyto) pSMAD is shown in the bottom panel (n ϭ 3). E, The effect of a neutralizing anti-TGF-␤ Ab on PKR and SMAD phosphorylation. Purified eosinophils were cultured in the presence of a mouse monoclonal anti-TGF-␤ (␣TGF␤) or a control mIgG1 Ab (2 ␮g/ml) for 72 h. In parallel, we also cultured another aliquot of eosinophils with IL-5 as a positive control (mIgG1ϩIL5). The cells were then stained with anti-pPKR (upper panel), anti-pSMAD2/3 (middle panel), or anti-PKR Abs. Right panel, A quantitative analysis of mean fluorescent intensity (MFI) from three independent ,and ∧, p Ͻ 0.005 vs isotype control for pPKR and pSMAD2/3 ء) .experiments. Basal MFI in nonstimulated (mIgG1-treated) cells was normalized to 100% respectively).

of five high-powered fields per slide with ϳ40 cells/field). Phos- PKR activation is rarely observed in bone marrow-derived pho-SMAD 2/3 was detected by IFS in 84 Ϯ 3% of eosinophils eosinophils from OVA-immunized mice under basal conditions; of these, 67 Ϯ 5% had nuclear phospho- SMAD 2/3 (Fig. 4D, lower panel). We subsequently showed that It is becoming apparent that PKR plays a role in regulating im- both this baseline TGF-␤ signaling through phospho-SMAD2/3 portant signaling pathways of the immune system including NF- ␬ and phospho-PKR could be inhibited by treatment with a mAb to B, STAT 1 and 3, and may signal via MAPKs (26–29). It has also TGF-␤ 1/2 but not by the mIgG1 isotype control Ab (Fig. 4E). We been suggested that PKR may have a regulatory role in Th2-me- ϩ also observed that pretreatment with the pharmacological inhibitor diated diseases, and that CD8 T cells are hyperproliferative in of PKR had no effect on baseline or TGF␤-induced pSMAD 2/3 PKR knockout mice (30). For this reason, we sought to study PKR levels suggesting that PKR is downstream of SMAD2/3 in this behavior in eosinophils in the setting of a Th2-mediated experi- cascade (data not shown). These results suggest that autocrine mental model of asthma. To this goal, we examined phospho-PKR TGF-␤ stimulates PKR and subsequent inhibition of EIF2␣ con- staining in eosinophils (major basic protein-positive cells) from tributes to shortened life spans of eosinophils. bone marrow obtained from OVA-sensitized and -challenged mice 4262 PKR REGULATION OF EOSINOPHIL SURVIVAL

FIGURE 5. PKR activation is down-regulated in a Th2 environment. A and B, Bone marrow cells were isolated from mice immunized with either sham treatment (saline) or OVA. After fixing, cells were immunostained with an anti-major basic protein (MBP) Ab (stained green) to mark eosinophils and an anti-pPKR (stained red in A) or an anti-PKR Ab (stained red in B). Nuclei were stained with DAPI (blue). Phosphorylated PKR was found in 85% of the bone marrow-derived eosinophils from the two sham-treated mice. Six percent of eosinophils from two OVA-immunized mice showed activation of PKR (n ϭ 30 eosinophils per bone marrow slide from a total of two mice per group). C, Quantita- tive analysis of double positive (MBP ϩ pPKR) eosinophils from A. Downloaded from http://www.jimmunol.org/ and control mice. We observed a significant number of phospho- One of the key findings of this study is that the stress-response PKR-positive eosinophils in the bone marrow from control ani- kinase PKR is activated under basal conditions in peripheral blood mals (Fig. 5). In contrast, Ͻ10% of eosinophils were positive for eosinophils. PKR is a serine/threonine kinase initially discovered phospho-PKR in the bone marrow from OVA-sensitized mice. to regulate antiviral responses through activation by dsRNA and This would suggest that a Th2 microenvironment is sufficient to IFN-␣/IFN-␤ and subsequent phosphorylation of the EIF2␣ regu- silence baseline PKR and likely increase eosinophil survival. latory site (34). Upon binding dsRNA, PKR catalytic domains dimerize and autophosphorylate Thr446, facilitating deactivation of by guest on September 27, 2021 Discussion EIF2␣ by phosphorylation of the regulatory Ser51 residue leading This article presents the results of a broad phosphoprotein screen to the inhibition of protein synthesis and hence viral replication of signaling molecules that are activated by IL-5 and CCL11 in (23, 35, 36). Alternatively, under acute stress conditions, PKR ac- eosinophils. The phosphoscreen covered a total of 44 phosphosites tivates NF-␬B and antagonizes the apoptotic pathway (22, 37). encompassing 36 signaling molecules. IL-5 and CCL11 modified PKR has also been implicated in regulating the cell cycle and it has phosphorylation of 15 phosphosites. Twenty-nine phosphosites been shown that a loss-of-function PKR mutant can lead to tumor- were unchanged by either activator. This is not surprising because igenesis (38). In addition, a variety of extracellular stimuli appear we have measured the phosphorylation state only at two time to signal through PKR such as IFN-␣ and -␤, LPS (39), dsRNA points: 3 and 5 min after stimulation. Many signaling molecules (34), TNF-␣ (40), and platelet-derived growth factor (41); there is are likely to be phosphorylated at later time points. Also pertinent also recent evidence that regulatory RNAs may signal via PKR to this issue is the fact that not all signaling molecules require a (42). Thus, PKR appears to mediate a myriad of signals from reg- change in the phosphorylation status for signal transduction. ular cell growth to innate inflammation, bacterial infection, and Our study has identified a number of new signaling molecules viral replication. that were previously not known to be involved in IL-5 and CCL11 Because the eosinophil is a short-lived granulocyte, we won- signaling. These newly identified signaling molecules include dered whether basal PKR activation was seminal to their short MSK1/2, RSK, GSK3 ␣ and ␤, and PKR. Of the foregoing, only the phosphorylation of RSK could have been predicted based upon survival. In this regard, it is of interest that the neutrophil, which the previous knowledge of ERK1/2 activation by IL-5 and CCL11 is another short-lived granulocyte, also showed high basal levels of (11, 21). MSK1/2 is a new family of signaling molecule which PKR phosphorylation. This is in contrast to long-lived lympho- functions downstream of ERK1/2 and p38 (31, 32). MSK1/2 phos- cytes and monocytes, which manifest minimal PKR phosphoryla- phorylates NF-␬B, which results in increased transcriptional action. Basal phosphorylation of PKR in eosinophils was rapidly tivity. Its function in eosinophils is unknown. GSK3 ␣ and ␤ are down-regulated by their survival-promoting factor IL-5. Both a important regulatory molecules that function at the crossroad of pharmacological inhibitor of PKR and PKR siRNA prolonged eo- many signaling pathways. They are basally active and function as sinophil survival, suggesting that this signaling molecule indeed an inhibitor of many signaling pathways such as the Akt-mamma- contributes to eosinophil apoptosis. In agreement with the basal lian target of rapamycin pathway (33). Phosphorylation of GSK3␣ phosphorylation of PKR, we observed increased EIF2␣ phosphor- and GSK3␤ at serine 21 and 9, respectively, inhibits their regula- ylation in eosinophils. We have previously reported that TGF-␤ is tory function. GSK3 phosphorylation and inhibition have recently a strong inducer of apoptosis in eosinophils and that autocrine been shown to be important for IL5-mediated eosinophil survival TGF-␤ may in part be responsible for the low survival rate of (16). eosinophils (9, 25). However, the mechanism by which TGF-␤ The Journal of Immunology 4263 induces apoptosis in this context is only moderately understood ways leading to the prolongation of eosinophil survival. J. Exp. Med. 186: (9). 561–568. 8. Pazdrak, K., D. Schreiber, P. Forsythe, L. Justement, and R. Alam. 1995. The Due to PKR’s link to apoptosis (43, 44), we investigated intracellular signal transduction mechanism of interleukin 5 in eosinophils: the whether TGF-␤ could activate PKR. We not only discovered that involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associ- TGF-␤ activates PKR but also found TGF-␤ signaling in non- ated protein kinase pathway. J. Exp. Med. 181: 1827–1834. 9. Pazdrak, K., S. Stafford, and R. Alam. 1995. The activation of the Jak-STAT 1 stimulated eosinophils in the form of nuclear phospho-SMAD 2/3. signaling pathway by IL-5 in eosinophils. J. Immunol. 155: 397–402. Both the basal phosphorylation of PKR and SMAD2/3 could be 10. Smith, J. A., C. E. 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