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 Cε, and glycogen synthase kinase 3 α and β. 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 2α are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-β in eosinophils. TGF-β directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-β 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 phospho-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-β and that this could be modulated by a Th2 microenvironment in vivo.

The eosinophil is an essential component of the immune system and plays an important role in Th2-mediated allergic and parasitic diseases (1). Cytokines and chemokines are potent activators of eosinophils. However, there are significant differences between cytokines and chemokines in regard to their activation of eosinophils (2). Cytokines with growth factor-like activity such as IL-3, IL-5, and GM-CSF regulate differentiation, activation, and survival of eosinophils. In the absence of these growth factors, eosinophil differentiation and survival are severely impaired (3). In contrast, chemokines like CCL11 (eotaxin) and RANTES primarily regulate eosinophil chemotaxis, homing, and activation. They have very little effect on differentiation, and especially on survival (4, 5). The signaling mechanism underlying these differences between chemokines and growth factors is largely unknown.

The receptors for IL-3, IL-5, and GM-CSF are heterodimers; each cytokine has its specific α receptor but they share a common β receptor subunit (βc) (6). CCRs are Gi-coupled 7-transmembrane heptahelical receptors (4). The signaling mechanism of activation of eosinophils by some of the cytokines and chemokines (e.g., IL-5 and CCL11) has previously been investigated. However, most of these studies have been performed as a part of separate and independent investigations.

We have performed an Ab-based phosphoprotein screen of eosinophil activation by IL-5 and CCL11—the most prominent representatives of growth factors and CC chemokines for eosinophils. This analysis led to the identification of a number of novel signaling molecules that are differentially activated by IL-5 and CCL11. Based upon these novel findings, we undertook a systematic analysis of signaling determinants of eosinophil survival.

Abs used were: rabbit anti-pThr451-PKR (Cell Signaling), rabbit anti-pSer51-eukaryotic initiation factor 2α (EIF2α3; Epitomics), rabbit anti-PKR, mouse anti-p65, goat anti-β-actin, goat anti-pSer433/435-SMAD2/3 (Santa Cruz Biotechnology), mouse monoclonal anti-TGFβ (R&D Systems), and IgG isotype controls (DakoCytomation). Pharmaceutical inhibitors and inert controls for PKR were obtained from Calbiochem; PKR smart-pool small interfering RNA (siRNA) and nontargeting (NT)-siRNA were obtained from Dharmacon, whereas FITC-labeled control siRNA was 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 chemokines were obtained from PeproTech. RPMI 1640 with l-glutamine was obtained from Cellgro; FCS was obtained from HyClone; and Hetastarch was obtained from Hospira. MACS separation reagents and the Vario MACS magnet were obtained from Miltenyi Biotec.

The procedure was conducted with sterile solutions in a hood. A minimum of 60 ml of whole blood was obtained from healthy to mildly allergic donors. Whole blood was combined with 2.5 ml of 100 mM EDTA and 7.5 ml of 6% hetastarch in 0.9% NaCl. The RBC were allowed to sediment over a period of 45 min and the buffy coat was collected every 5–10 min. The buffy coat was washed several times with cold HBSS plus 4 mM HEPES (pH 7.3) before resuspending in 30 ml of room temperature HBSS plus 4 mM HEPES and underlying 15 ml of Percoll (1.089 g/ml) followed by centrifugation at 400 × g for 35 min at room temperature with minimum acceleration/deceleration. The monocyte/lymphocyte and granulocyte fractions were washed one time in 50 ml of HBSS plus 4 mM HEPES plus 4 mM EDTA before hypotonic lysis of residual RBCs with ice-cold sterile double-distilled water (12 ml). After 50 s of lysis on ice, cold 10× HBSS buffer (1.5 ml) and FCS (1 ml) were added and brought to 50 ml in cold HBSS plus 4 mM EDTA. Cell fractions were washed three times in cold HBSS plus 4 mM HEPES/2% FCS. Granulocytes were labeled with anti-human CD16 microbeads for positive selection of neutrophils as per manufacturer’s (Miltenyi Biotec) protocol. Granulocytes were run down the appropriate size column with HBSS plus 2% FCS in the presence of a magnetic field (Vario MACS), and the eluent (50 ml) containing eosinophils was collected via a 21-gauge flow resistor. Labeled neutrophils were collected by taking the column out of the magnetic field and collecting 50 ml of eluent. Before stimulation or culture, cells were resuspended in RPMI 1640/10% FCS containing antibiotics and mycotics and allowed to equilibrate to 37°C in HSA (3%) coated tubes/wells.

Purified eosinophils were prepared as above and stimulated with either medium, CCL11 (10−8 M) for 3 min, and IL-5 (10−10 M) for 3 and 5 min. This time point was previously shown to have the highest activation of upstream tyrosine kinases and downstream MAPKs (7, 8, 9). Cell lysates were sent to Kinexus Bioinformatics for a Western blot-based phosphosite screen. The results are compared with trace amounts of known proteins relative to various phosphospecific proteins in each sample via densitometry readings.

Leukocytes were prepared as above, stimulated or cultured as indicated, and fixed in 2% paraformaldehyde in PBS (pH 7.4) at room temperature for 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 −80°C until use. Cells were blocked in 10% goat serum in PBS plus 0.05% saponin at room temperature for 1 h. Primary Abs and isotype controls were applied at 1–2 μg/ml overnight at 4°C in 5% goat serum. Slides were washed three times for 10 min each in PBS plus 0.05% saponin before applying the FITC- or tetramethylrhodamine isothiocyanate-conjugated secondary Ab at 1:200 at the same time as 4′,6′-diamidino-2-phenylindole (DAPI; 0.1 nM) in 5% goat serum at room temperature for 1 h. Slides were washed as above, checked for background (IgG control), and cover slides are mounted. Slides are visualized with an epifluorescence microscope (Nikon Eclipse 2000) equipped with a Cool-Snap CCD camera and Metamorph image analysis software (Molecular Devices). Exposure times are chosen in a manner that background IgG staining is negligible and only signal above background is analyzed. Morphometric data was obtained by viewing a minimum of five high-powered fields per slide containing between 30 and 50 cells/field. In most cases, mean fluorescent intensity or integrated fluorescent intensity (normalized to cell number) was analyzed. Means are expressed with error bars representative of the SEM.

SDS-PAGE was performed on 8–10% gels with 20 μg of protein per sample. Protein was transferred onto polyvinylidene difluoride (PVDF) membranes and blocked in 5% BSA before immunoblotting with primary Abs overnight at 4°C in 1% BSA. Blots were washed three times in TBST 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 ECL reagent (ECL from Amersham) per the manufacturer’s instructions before exposure to film.

A total of 40 μg of protein was precleared for nonspecific binding by incubation with protein A/G agarose beads for 2 h and then incubated overnight with 2 μg of an anti-PKR (Santa Cruz Biotechnology) before immunoprecipitating with protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h. Immunoprecipitates were washed three times in 1 ml of 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 magnesium acetate, 2 mM MnCl2, 10% glycerol with protease inhibitors added fresh: 1 mM PMSF, 1 μg/ml apropeptin, leupeptin, and peptin, and 1 mM Na3VO4). The kinase reaction was conducted in 25 μl of the above buffer with 10 μCi [γ-32P]ATP and 40 μM nonradioactive ATP per reaction for 30 min at 30°C followed by addition of an SDS protein loading dye with 2-ME and boiling for 5 min before loading supernatants onto a polyacrylamide gel and transferring to PVDF as above. PVDF membranes were then exposed to a phosphor screen for 24 h and read on a phosphor imager (Typhoon).

Purified eosinophils were cultured at 1 × 106 cells/ml in RPMI 1640 plus 10% FCS in the presence or absence of a pharmacological inhibitor of protein kinase R (PKR; an imidazole-oxindole compound from Calbiochem) or its inert analog. Preliminary dose-response experiments with 100, 200, and 400 nM concentrations of the inhibitor demonstrated the best response with the 200 nM dose. IL-5 (10−10 M; PeproTech) was used as a positive control. siRNA was used at a 2 μg/ml concentration. TGF-β was used at 4 × 10−10 M, and anti-TGF-β and mouse IgG1 isotype control were used at 2 μg/ml. A total of 200,000 cells were taken from each culture at the indicated time point and resuspended in 20 μl of 0.25 mg/ml propidium iodide in PBS. Cells were put onto slides and a minimum of five fields (×20 magnification) per sample were viewed to obtain an average viability for each sample. Experiments were done in triplicate.

Female C57BL/6 mice from The Jackson Laboratory were acquired and housed under pathogen-free conditions at the Biological Resource Center (National Jewish Medical and Research Center, Denver, CO). Mice were used under an experimental protocol approved by the institutional animal care and use committee. Animals were maintained on an OVA-free diet. Mice were immunized at 8 wk of age via s.c. injections of 50 μg of OVA in alum. Animals were given an identical s.c. booster 2 wk after the first immunization. Two weeks following the booster, animals were challenged 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 sacrificed and bone marrow was isolated and fixed as above for intracellular immunofluorescent staining.

Data are presented as mean ± SEM of multiple experiments as indicated in the text. Statistical analyses were performed with the Student t test, the Mann-Whitney U test, the Spearman’s correlation coefficient test, and the Fisher’s exact probability test. A value of p < 0.05 was considered significant.

Eosinophils were purified to 97–99% purity from the peripheral blood of mildly allergic patients by a combination of Percoll gradient centrifugation and negative selection using anti-human CD16-coated magnetic beads as described previously (2, 8). Eosinophils were stimulated with CCL11 for 3 min and with IL-5 for 5 min. The foregoing time points have previously been shown to be optimal for tyrosine kinase and MAPK activation (7, 8, 9). The cell lysate from the stimulated eosinophils from two donors was sent out to Kinexus for phosphosite screening. Eosinophils from two additional donors were processed in-house for confirmation of the Kinexus screen. These samples were Western blotted using a similar set of phosphospecific Abs. The Kinexus data are reported in densitometric units. The trace quantity of the known proteins from each screen type is compared against all relevant samples. The corrected data standardizes the trace quantity (actual trace quantity times coefficient) for all samples. The in-house Western blot and its densitometric analyses confirmed the overall direction of change (positive or negative) in target protein phosphorylation following IL-5 and CCL11 stimulation as reported by the Kinexus screen.

We examined 44 different phosphosites covering 36 signaling molecules. Of the 44 phosphosites studied, the phosphorylation (or lack thereof) of 38 phosphosites was concordant in all four samples. Data for six molecules were discordant among the samples (p < 0.00005, Fisher’s exact probability test). IL-5 and CCL11 increased phosphorylation of 9 sites, decreased phosphorylation of 2 sites, and differentially regulated phosphorylation of 4 sites (Fig. 1). Phosphorylation of 29 sites was unchanged by either activator (data not shown).

FIGURE 1.

Differential phosphorylation of signaling molecules in IL 5- and CCL11-stimulated eosinophils. Purified human peripheral blood eosinophils were stimulated with IL-5 (10−10 M) for 5 min, CCL11 (10−7 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 as a bar graph. Right panel, Western blot. ∗, p < 0.05 compared with the medium control (Mann-Whitney U test, n = 4).

FIGURE 1.

Differential phosphorylation of signaling molecules in IL 5- and CCL11-stimulated eosinophils. Purified human peripheral blood eosinophils were stimulated with IL-5 (10−10 M) for 5 min, CCL11 (10−7 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 as a bar graph. Right panel, Western blot. ∗, p < 0.05 compared with the medium control (Mann-Whitney U test, n = 4).

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The signaling molecules that were phosphorylated by both IL-5 and CCL11 include the members of the MAPK cascade: MEK1/2 and ERK1/2 (Fig. 1,A, left panel), p38 and its substrate mitogen and stress activated kinase (MSK)1/2 (Fig. 1 A, right panel). IL-5 consistently induced higher levels of phosphorylation of ERK1/2. One of the physiological substrates of activated ERK1/2 is ribosomal S6 kinase (RSK)1 (10). Both IL-5 and CCL11 induced phosphorylation of RSK1, thus confirming biological relevance of activation of the ERK pathway. The role of the MAPK signaling pathway in eosinophil function has previously been studied. They are involved in eosinophil differentiation, survival, and degranulation (2, 7, 11, 12).

Both activators inhibited the phosphorylation of S6 kinase and protein kinase Cε (Fig. 1,B, left panel). In addition, they increased the inhibitory phosphorylation of Ser21 and Ser9 of glycogen synthase kinase 3 (GSK3) α and β, respectively (Fig. 1,B, middle panel). Phosphorylation of the foregoing residues, which is likely mediated by Akt, inhibits the kinase activity of GSK3 (13, 14, 15). In agreement with our finding, a recent report demonstrated that GSK3-β serine phosphorylation was important for IL5-induced eosinophil survival (16). IL-5 increased phosphorylation of Tyr216 of GSK3β (Fig. 1 B, right panel). The significance of tyrosine phosphorylation of GSK3 is currently unknown.

The phosphorylation of STAT1 and STAT3 was increased by IL-5 but not by CCL11 (Fig. 1 C). Previously, the phosphorylation of STATS 1, 3, and 5 in IL5-stimulated eosinophils has been described (9, 17, 18, 19). Furthermore, we and others have reported the phosphorylation of Jak1 and Jak2 by IL-5 but not by chemokines (2, 9, 12, 17, 20). Both Jaks and STATs, especially STAT5, are important for IL5-induecd eosinophil differentiation and survival.

Eosinophils show a high level of basal phosphorylation of PKR, a dsRNA-dependent protein kinase (Fig. 1 D). IL-5 induced a dramatic inhibition of PKR phosphorylation. CCL11 did not have any effect on PKR phosphorylation.

Phosphorylation of the following signaling molecules was not affected by either cytokines: adducin α and γ, c-Jun, cyclin-dependent kinase 1, CREB, JNK, MKK3, protein kinase B, protein kinase C-α, -β, and -δ, retinoblastoma protein, and SMAD1. The 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.

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. 2 A). Immunofluorescence 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.

FIGURE 2.

Phospho-PKR in eosinophils. A, Immunofluorescent staining of pPKR in a mixture of leukocytes. PBLs were stained with a rabbit phospho-PKR (Thr451) Ab (green) and the nuclei were counterstained with DAPI and pseudocolored red: 1, monocyte; 2, lymphocyte; 3, neutrophil; and 4, eosinophil. B, Immunofluorescent staining of eosinophils for phospho-PKR and the effect of IL-5. Purified eosinophils were immunostained with the rabbit phospho-PKR (Thr451) Ab (green) with and without stimulation with IL-5 10−10 M for 5 or 30 min. Nuclei were stained with DAPI and pseudocolored with red. Left panel, Representative immunofluorescent images from one of four independent experiments. Bottom panel, Total PKR immunostaining under similar experimental conditions. Right panel, Quantitative analyses of mean fluorescence intensity (MFI) of PKR staining of 200 eosinophils from four different donors. Data are normalized to medium control (Cntrl) which ranged from 600 to 1455 of fluorescence intensity units depending upon the eosinophil donor. C, An immune-complex kinase assay for PKR. PKR was immunoprecipitated (anti-PKR) from eosinophils that were treated with medium (−) or IL-5 (+) for 30 min. In the control experiment, rabbit IgG was used for immunoprecipitation. The immunoprecipitates were assayed for autophosphorylation [32P]PKR and autoradiographed. Bottom panel, The Ig H chain (IgH) reflecting the loading of the immunoprecipitates. D, The effect of GM-CSF on eosinophil PKR phosphorylation. Eosinophils were stimulated with GM-CSF for the indicated time period and Western blotted with an anti-pPKR Ab. The membrane was reprobed with the anti-PKR Ab. E, Immunofluorescent staining of eosinophils for the NF-κB p65 subunit and phospho-EIF2α and the effect of IL-5. Purified eosinophils were processed as above and immunostained using a mouse mAb against p65 and a rabbit anti-phospho-EIF2α. Representative images from one of three independent experiments are shown. Right panels in B and E, Immunostaining with control Abs (green) and nuclear staining (red) with DAPI. Rabbit IgG is a control for the anti-pPKR and anti-pEIF2α Abs, mIgG2a is a control for the anti-PKR Ab, and mIgG1 is a control for the anti-NFκB p65 Ab.

FIGURE 2.

Phospho-PKR in eosinophils. A, Immunofluorescent staining of pPKR in a mixture of leukocytes. PBLs were stained with a rabbit phospho-PKR (Thr451) Ab (green) and the nuclei were counterstained with DAPI and pseudocolored red: 1, monocyte; 2, lymphocyte; 3, neutrophil; and 4, eosinophil. B, Immunofluorescent staining of eosinophils for phospho-PKR and the effect of IL-5. Purified eosinophils were immunostained with the rabbit phospho-PKR (Thr451) Ab (green) with and without stimulation with IL-5 10−10 M for 5 or 30 min. Nuclei were stained with DAPI and pseudocolored with red. Left panel, Representative immunofluorescent images from one of four independent experiments. Bottom panel, Total PKR immunostaining under similar experimental conditions. Right panel, Quantitative analyses of mean fluorescence intensity (MFI) of PKR staining of 200 eosinophils from four different donors. Data are normalized to medium control (Cntrl) which ranged from 600 to 1455 of fluorescence intensity units depending upon the eosinophil donor. C, An immune-complex kinase assay for PKR. PKR was immunoprecipitated (anti-PKR) from eosinophils that were treated with medium (−) or IL-5 (+) for 30 min. In the control experiment, rabbit IgG was used for immunoprecipitation. The immunoprecipitates were assayed for autophosphorylation [32P]PKR and autoradiographed. Bottom panel, The Ig H chain (IgH) reflecting the loading of the immunoprecipitates. D, The effect of GM-CSF on eosinophil PKR phosphorylation. Eosinophils were stimulated with GM-CSF for the indicated time period and Western blotted with an anti-pPKR Ab. The membrane was reprobed with the anti-PKR Ab. E, Immunofluorescent staining of eosinophils for the NF-κB p65 subunit and phospho-EIF2α and the effect of IL-5. Purified eosinophils were processed as above and immunostained using a mouse mAb against p65 and a rabbit anti-phospho-EIF2α. Representative images from one of three independent experiments are shown. Right panels in B and E, Immunostaining with control Abs (green) and nuclear staining (red) with DAPI. Rabbit IgG is a control for the anti-pPKR and anti-pEIF2α Abs, mIgG2a is a control for the anti-PKR Ab, and mIgG1 is a control for the anti-NFκB p65 Ab.

Close modal

The incubation of eosinophils with IL-5 resulted in near-complete dephosphorylation of PKR rapidly within 5 min (Fig. 2,B, upper panels). There were no changes in the level of PKR staining during this time period (Fig. 2,B, left lower panel). The IFS results were confirmed by an immune complex kinase assay for PKR (Fig. 2,C). PKR signals through two antagonistic pathways: the survival pathway through the activation of NF-κB and the apoptotic pathway through phosphorylation of EIF2α (22, 23). Phosphorylation of the EIF2α regulatory site Ser51 inhibits protein synthesis and drives the apoptotic cell death. We observed very little translocation of the p65 subunit of NF-κB into the nucleus under basal condition. IL-5 stimulation significantly increased p65 (green) translocation into the nucleus (red), turning them yellow upon overlay of the images (Fig. 2,E). Thus, increased basal phospho-PKR does not result in increased NF-κB activation in eosinophils. Next, we examined phosphorylation of EIF2α under basal conditions. Phosphorylation of EIF2α regulatory site Ser51 closely paralleled activation of PKR in eosinophils. There was significant cytosolic immunofluorescence (green) of Ser51-phosphorylated EIF2α under the basal condition (Fig. 2 E). Stimulation of cells with IL-5 induced near complete dephosphorylation of EIF2α after 30 min.

It has previously been reported that IL-3 stimulation abrogates PKR signaling (24). It was therefore of interest to see whether GM-CSF (whose receptor shares the common β-chain with the IL-3 and IL-5 receptors) could have the same effects on PKR. Treatment with GM-CSF (10−10 M) inhibited PKR phosphorylation in eosinophils (Fig. 2 D), which was similar to that observed with IL-5. Together, these findings implicate the common β-chain in cytokine-induced down-regulation of PKR signaling.

To determine whether increased PKR activation contributes to eosinophil survival, we used a pharmacological inhibitor of PKR. Incubation with a PKR inhibitor (an imidazole-oxindole compound, C13H8N4OS obtained from Calbiochem) but not its inert oxindole control (C15H8Cl3NO2) improved cell survival in eosinophils (Fig. 3,A). We also examined the effect of PKR knockdown by siRNA in eosinophils. We have previously studied transfection of eosinophils with oligos and plasmids by various methods involving cationic lipids and electroporation. The success rate of these methods is low and highly variable because of extreme adhesiveness of eosinophils following treatment with the transfecting agents resulting in cell loss and also because of increased cell death. While studying these methods we observed that control cells (eosinophils treated with siRNA alone without transfecting agents) spontaneously internalized large quantities of siRNA (Fig. 3, B and D). Treatment of purified eosinophils with siRNA for PKR significantly reduced the expression of the PKR protein (Fig. 3,C, left panel). Concomitantly, and in accord with the PKR inhibitor data, survival of eosinophils was significantly prolonged in PKR knockdown but not in control eosinophils (Fig. 3,C, right panel). We speculated that the spontaneous internalization of siRNA (observed in 85% of eosinophils) was an active cellular process and was dependent on endocytosis. All endocytic processes are dependent upon the activity of dynamin, a GTPase, which facilitates budding of endocytic vesicles from the cell membrane. To address this question, we pretreated cells with NT or dynamin II siRNA for 24 h before incubation with a fluorescent NT siRNA for 24 h. Next, we fixed the cells and immunostained for dynamin II (red) and stained nuclei (blue) with DAPI (Fig. 3 D). Preincubation of cells with dynamin siRNA, but not NT siRNA, caused markedly decreased dynamin II expression. The internalization of the fluorescently labeled siRNA correlated positively with the level of dynamin II expression. Together, these data suggest that the spontaneous uptake of siRNA is an active endocytotic process mediated by dynamin II.

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 survival. The PKR inhibitor prolonged eosinophil survival as compared with the diluent control at all time points (n = 4; ∗, p < 0.05). B, Spontaneous 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 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 = 3). D, Spontaneous uptake of siRNA is a dynamin II-dependent endocytotic process. Eosinophils were cultured with 2 μg/ml NT or dynamin siRNA for 24 h followed by incubation for an additional 24 h with 2 μg/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.

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 survival. The PKR inhibitor prolonged eosinophil survival as compared with the diluent control at all time points (n = 4; ∗, p < 0.05). B, Spontaneous 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 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 = 3). D, Spontaneous uptake of siRNA is a dynamin II-dependent endocytotic process. Eosinophils were cultured with 2 μg/ml NT or dynamin siRNA for 24 h followed by incubation for an additional 24 h with 2 μg/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.

Close modal

Because we demonstrated that PKR is important for eosinophil survival, we wondered whether apoptosis-promoting factors could increase PKR activation in eosinophils. The phosphorylation of PKR was studied by IFS and immunoblotting. Indeed, we discovered that TGF-β does activate PKR and its downstream substrate EIF2α in eosinophils (Fig. 4,A). We next wondered whether the down-regulation of PKR activation by IL-5 shown earlier could be rescued by TGF-β. To this goal, the cells were first treated with IL-5 for 30 min and then treated with TGF-β, a proapoptotic agent for 30 min. Indeed, TGF-β could restore PKR (Fig. 4,B) and EIF2α (Fig. 4 C) phosphorylation to baseline levels after being down-regulated by IL-5. These data further explain the mechanism by which TGF-β induces apoptosis in eosinophils in addition to those investigated previously (9, 25).

FIGURE 4.

TGF-β regulation of PKR in eosinophils. A, TGF-β enhances PKR/EIF2α phosphorylation in eosinophils. Eosinophils were treated with or without TGF-β for 5–60 min and then immunostained for pPKR (Thr451), pEIF2 α (Ser51), or PKR (green). Nuclei were stained with DAPI and pseudocolored red. Basal phosphorylation of PKR in eosinophils was further enhanced by the treatment with TGF-β. Left panel, Images from the 30-min time point. Right panel, Quantitative analyses of normalized mean fluorescence intensity (MFI) from four (EIF2α) and five (PKR) independent experiments. ∗ and #, p < 0.05 for pPKR- and pEIF2α-stained samples with respect to medium controls; ∧, p < 0.05 for 5 min vs 30 min TGF-β treatment. B and C, The effect of IL-5 treatment first followed by TGF-β stimulation on pPKR and pEIF2α expression by eosinophils. Purified eosinophils were treated with IL-5 (10−10 M) for 30 min, washed two times, followed by stimulation with TGF-β (10−10 M) for 30 min (for pPKR) or 60 min (for pEIF2α). Phosphorylation of PKR (Thr451) was detected by immunoblotting (B). The membrane was reprobed with an anti-PKR Ab. The phosphorylation of EIF2α (Ser51) was detected by immunofluorescent staining followed by morphometric quantitative analyses (C, n = 3). $, p < 0.05 for IL-5 vs IL-5 + TGF-β 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 pSMAD+ eosinophils and eosinophils with nuclear and cytosolic only (cyto) pSMAD is shown in the bottom panel (n = 3). E, The effect of a neutralizing anti-TGF-β Ab on PKR and SMAD phosphorylation. Purified eosinophils were cultured in the presence of a mouse monoclonal anti-TGF-β (αTGFβ) or a control mIgG1 Ab (2 μg/ml) for 72 h. In parallel, we also cultured another aliquot of eosinophils with IL-5 as a positive control (mIgG1+IL5). 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 experiments. Basal MFI in nonstimulated (mIgG1-treated) cells was normalized to 100%. (∗ and ∧, p < 0.005 vs isotype control for pPKR and pSMAD2/3, respectively).

FIGURE 4.

TGF-β regulation of PKR in eosinophils. A, TGF-β enhances PKR/EIF2α phosphorylation in eosinophils. Eosinophils were treated with or without TGF-β for 5–60 min and then immunostained for pPKR (Thr451), pEIF2 α (Ser51), or PKR (green). Nuclei were stained with DAPI and pseudocolored red. Basal phosphorylation of PKR in eosinophils was further enhanced by the treatment with TGF-β. Left panel, Images from the 30-min time point. Right panel, Quantitative analyses of normalized mean fluorescence intensity (MFI) from four (EIF2α) and five (PKR) independent experiments. ∗ and #, p < 0.05 for pPKR- and pEIF2α-stained samples with respect to medium controls; ∧, p < 0.05 for 5 min vs 30 min TGF-β treatment. B and C, The effect of IL-5 treatment first followed by TGF-β stimulation on pPKR and pEIF2α expression by eosinophils. Purified eosinophils were treated with IL-5 (10−10 M) for 30 min, washed two times, followed by stimulation with TGF-β (10−10 M) for 30 min (for pPKR) or 60 min (for pEIF2α). Phosphorylation of PKR (Thr451) was detected by immunoblotting (B). The membrane was reprobed with an anti-PKR Ab. The phosphorylation of EIF2α (Ser51) was detected by immunofluorescent staining followed by morphometric quantitative analyses (C, n = 3). $, p < 0.05 for IL-5 vs IL-5 + TGF-β 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 pSMAD+ eosinophils and eosinophils with nuclear and cytosolic only (cyto) pSMAD is shown in the bottom panel (n = 3). E, The effect of a neutralizing anti-TGF-β Ab on PKR and SMAD phosphorylation. Purified eosinophils were cultured in the presence of a mouse monoclonal anti-TGF-β (αTGFβ) or a control mIgG1 Ab (2 μg/ml) for 72 h. In parallel, we also cultured another aliquot of eosinophils with IL-5 as a positive control (mIgG1+IL5). 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 experiments. Basal MFI in nonstimulated (mIgG1-treated) cells was normalized to 100%. (∗ and ∧, p < 0.005 vs isotype control for pPKR and pSMAD2/3, respectively).

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We have previously shown that autocrine secretion of TGF-β from eosinophils contributes to their short life (9, 25). It was therefore of interest whether we could detect baseline phospho-SMAD 2/3 signaling in eosinophils. Indeed, we found baseline p-SMAD 2/3 in both the cytosol and nucleus of eosinophils (Fig. 4,D, upper panel). We analyzed 200 randomly selected and immunofluorescently stained eosinophils per slide from three donors (minimum of five high-powered fields per slide with ∼40 cells/field). Phospho-SMAD 2/3 was detected by IFS in 84 ± 3% of eosinophils under basal conditions; of these, 67 ± 5% had nuclear phospho-SMAD 2/3 (Fig. 4,D, lower panel). We subsequently showed that both this baseline TGF-β signaling through phospho-SMAD2/3 and phospho-PKR could be inhibited by treatment with a mAb to TGF-β 1/2 but not by the mIgG1 isotype control Ab (Fig. 4 E). We also observed that pretreatment with the pharmacological inhibitor of PKR had no effect on baseline or TGFβ-induced pSMAD 2/3 levels suggesting that PKR is downstream of SMAD2/3 in this cascade (data not shown). These results suggest that autocrine TGF-β stimulates PKR and subsequent inhibition of EIF2α contributes to shortened life spans of eosinophils.

It is becoming apparent that PKR plays a role in regulating important signaling pathways of the immune system including NF-κB, STAT 1 and 3, and may signal via MAPKs (26, 27, 28, 29). It has also been suggested that PKR may have a regulatory role in Th2-mediated diseases, and that CD8+ T cells are hyperproliferative in PKR knockout mice (30). For this reason, we sought to study PKR behavior in eosinophils in the setting of a Th2-mediated experimental model of asthma. To this goal, we examined phospho-PKR staining in eosinophils (major basic protein-positive cells) from bone marrow obtained from OVA-sensitized and -challenged mice and control mice. We observed a significant number of phospho-PKR-positive eosinophils in the bone marrow from control animals (Fig. 5). In contrast, <10% of eosinophils were positive for phospho-PKR in the bone marrow from OVA-sensitized mice. This would suggest that a Th2 microenvironment is sufficient to silence baseline PKR and likely increase 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 = 30 eosinophils per bone marrow slide from a total of two mice per group). C, Quantitative analysis of double positive (MBP + pPKR) eosinophils from A.

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 = 30 eosinophils per bone marrow slide from a total of two mice per group). C, Quantitative analysis of double positive (MBP + pPKR) eosinophils from A.

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This article presents the results of a broad phosphoprotein screen of signaling molecules that are activated by IL-5 and CCL11 in eosinophils. The phosphoscreen covered a total of 44 phosphosites encompassing 36 signaling molecules. IL-5 and CCL11 modified phosphorylation of 15 phosphosites. Twenty-nine phosphosites were unchanged by either activator. This is not surprising because we have measured the phosphorylation state only at two time points: 3 and 5 min after stimulation. Many signaling molecules are likely to be phosphorylated at later time points. Also pertinent to this issue is the fact that not all signaling molecules require a change in the phosphorylation status for signal transduction.

Our study has identified a number of new signaling molecules that were previously not known to be involved in IL-5 and CCL11 signaling. These newly identified signaling molecules include MSK1/2, RSK, GSK3 α and β, and PKR. Of the foregoing, only the phosphorylation of RSK could have been predicted based upon the previous knowledge of ERK1/2 activation by IL-5 and CCL11 (11, 21). MSK1/2 is a new family of signaling molecule which functions downstream of ERK1/2 and p38 (31, 32). MSK1/2 phosphorylates NF-κB, which results in increased transcriptional activity. Its function in eosinophils is unknown. GSK3 α and β are important regulatory molecules that function at the crossroad of many signaling pathways. They are basally active and function as an inhibitor of many signaling pathways such as the Akt-mammalian target of rapamycin pathway (33). Phosphorylation of GSK3α and GSK3β at serine 21 and 9, respectively, inhibits their regulatory function. GSK3 phosphorylation and inhibition have recently been shown to be important for IL5-mediated eosinophil survival (16).

One of the key findings of this study is that the stress-response kinase PKR is activated under basal conditions in peripheral blood eosinophils. PKR is a serine/threonine kinase initially discovered to regulate antiviral responses through activation by dsRNA and IFN-α/IFN-β and subsequent phosphorylation of the EIF2α regulatory site (34). Upon binding dsRNA, PKR catalytic domains dimerize and autophosphorylate Thr446, facilitating deactivation of EIF2α by phosphorylation of the regulatory Ser51 residue leading to the inhibition of protein synthesis and hence viral replication (23, 35, 36). Alternatively, under acute stress conditions, PKR activates NF-κB and antagonizes the apoptotic pathway (22, 37). PKR has also been implicated in regulating the cell cycle and it has been shown that a loss-of-function PKR mutant can lead to tumorigenesis (38). In addition, a variety of extracellular stimuli appear to signal through PKR such as IFN-α and -β, LPS (39), dsRNA (34), TNF-α (40), and platelet-derived growth factor (41); there is also recent evidence that regulatory RNAs may signal via PKR (42). Thus, PKR appears to mediate a myriad of signals from regular cell growth to innate inflammation, bacterial infection, and viral replication.

Because the eosinophil is a short-lived granulocyte, we wondered whether basal PKR activation was seminal to their short survival. In this regard, it is of interest that the neutrophil, which is another short-lived granulocyte, also showed high basal levels of PKR phosphorylation. This is in contrast to long-lived lymphocytes and monocytes, which manifest minimal PKR phosphorylation. Basal phosphorylation of PKR in eosinophils was rapidly down-regulated by their survival-promoting factor IL-5. Both a pharmacological inhibitor of PKR and PKR siRNA prolonged eosinophil survival, suggesting that this signaling molecule indeed contributes to eosinophil apoptosis. In agreement with the basal phosphorylation of PKR, we observed increased EIF2α phosphorylation in eosinophils. We have previously reported that TGF-β is a strong inducer of apoptosis in eosinophils and that autocrine TGF-β may in part be responsible for the low survival rate of eosinophils (9, 25). However, the mechanism by which TGF-β induces apoptosis in this context is only moderately understood (9).

Due to PKR’s link to apoptosis (43, 44), we investigated whether TGF-β could activate PKR. We not only discovered that TGF-β activates PKR but also found TGF-β signaling in nonstimulated eosinophils in the form of nuclear phospho-SMAD 2/3. Both the basal phosphorylation of PKR and SMAD2/3 could be abrogated by a neutralizing anti-TGF-β Ab. This line of evidence is highly suggestive of a TGF-β autocrine mechanism of PKR activation in eosinophils.

One of the more prominent challenges that remain unresolved regarding the PKR/EIF2α pathway is deciphering the mechanisms of its down-regulation. It has previously been reported that IL-3 inhibits PKR activation in a murine growth factor-dependent cell line (24). Here, we show that stimulation of human eosinophils with IL-5 or GM-CSF has the ability to down-regulate PKR and reinitiate EIF2α. Because IL-3, IL-5, and GM-CSF receptors all share the common β-chain, these findings could implicate their common β-chain receptor subunit in cytokine-induced down-regulation of PKR- and EIF2α-mediated protein translation inhibition.

In light of our findings that show PKR is deactivated by IL-5, as well as others’ findings that PKR possesses negative regulatory functions relevant to T cell-mediated diseases (30, 45), we sought to investigate whether the IL-5 inhibition of PKR phosphorylation occurs in vivo in mice that are sensitized under Th2-immunizing conditions. We decided to study eosinophils from the bone marrow as opposed to the lung because of easily identifiable morphological features and the lack of the background tissue-derived noise. It has been previously demonstrated that bone marrow actively participates in Th2 inflammation through increased production of eosinophils (46, 47, 48). When we costained for phospho-PKR and major basic protein, we found that eosinophils isolated from bone marrow with a Th2 milieu did not show activated PKR in contrast to sham-immunized mice bone marrow eosinophils that had a high degree of basal phospho-PKR ex vivo. It is known that eosinophils isolated from a Th2 microenvironment (e.g., asthmatic lung) have belated apoptosis and increased survival (49). Our observations suggest that down-regulation of phospho-PKR is an early event in eosinophilic conditions such as allergy and parasitic infections.

The authors have no financial conflict of interest.

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

1

This work was supported by National Institutes of Health Grants R01 AI50179, AI059719, and AI68088.

3

Abbreviations used in this paper: EIF2α, eukaryotic initiation factor 2α; DAPI, 4′,6′-diamidino-2-phenylindole; PVDF, polyvinylidene difluoride; IFS, immunofluorescent staining; GSK3, glycogen synthase kinase 3; PKR, protein kinase R; NT, nontargeting; siRNA, small interfering RNA; MSK, mitogen and stress activated kinase; RSK, ribosomal S6 kinase.

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