Priming of Eosinophil by Granulocyte-Macrophage Colony Stimulating Factor is Mediated by Protein Kinase C βII Phosphorylated L-Plastin

Abstract

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein which we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. In addition, co-immunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with Protein Kinase C βII (PKCβII), GM-CSF receptor α chain, and two actin associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis (4-fluoroanilino)phtalimide or PKCβII specific siRNA blocked GM-CSF induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ2 integrin which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin which was abrogated by the above mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of ECP and EPX in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2–19) phosphorylated on Ser5 upregulated αMβ2 integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII -mediated L-plastin phosphorylation.

Introduction

The recruitment and activation of eosinophils in lung tissue is a hallmark of allergic inflammation. In asthma, eosinophils have been implicated in the characteristic pathophysiology of the disease in the airways, such as, mucus hypersecretion, desquamation of epithelium, and tissue remodeling (1–3). Therapy is still limited for asthma, but intervention at the key events that govern eosinophil recruitment and effector function may be one way forward (4). The observed increased recruitment of blood eosinophils into tissues in lung inflammation, e.g. allergy, is a multi-stage process that includes priming of blood eosinophils with cytokines, adhesion of eosinophils to, and transmigration across the endothelial cell layer, and finally migration towards a gradient of chemotactic factors in tissues (5–7). Among the gaps in our understanding of how eosinophils function is the mechanism by which the activated state of these cells is achieved. The transition of quiescent eosinophils to an activated state by an agonist-dependent induction of an eosinophil cell function, such as degranulation or chemotaxis, is preceded by priming (8, 9). Priming indicates transformation of eosinophils to a phenotype through exposure to an agent that does not fully activate the cell but renders eosinophils more susceptible to chemotaxis, degranulation and cytokine production. This state of heightened responsiveness by primed eosinophils typically allows the further activation of eosinophils at lower concentrations of agonists/cytokines that would typically not affect normal quiescent cells (10–12). Priming of eosinophils in vivo is an important phenomenon observed in patients with allergic inflammation and is likely caused by exposure to cytokines, primarily IL-3, IL-5 and GM-CSF, released after allergen exposure (13–15). Blood eosinophils from allergic patients exhibit increased chemotactic responsiveness to a range of chemotactic factors such as PAF or eotaxin (12, 16). Eosinophil priming “pre-activates” the eosinophils during the recruitment process allowing the cell to readily respond to the physiological gradients of chemotactic factors that direct them to tissue destinations where they are prepared to act and acquire further prolonged survival.

The role of GM-CSF in allergic inflammation is well established and there are numerous immunopathologic correlations between GM-CSF and eosinophil activation (17–19). In addition to its critical role in eosinophil survival and upregulation of adhesion molecules, GM-CSF initiates a priming state whereby eosinophils become responsive to chemotactic and degranulating factors. The mechanisms by which cytokines such as GM-CSF increase responsiveness to other stimuli are not completely understood and likely effect signaling cascades involving protein phosphorylation, integrin activation, and cytoskeleton reorganization (20–23). In this regard, opsonized zymosan particle-induced degranulation of primed eosinophils was shown to be mediated by tyrosine kinase activity in human eosinophils (21). Furthermore, GM-CSF-priming and the subsequent increased synthesis of inflammatory mediators in response to opsonized zymosan particles was shown to be dependent on the activity of integrin αMβ2 (24). Integrin αMβ2 is known to be critically involved in effector functions in granulocytes, such as, degranulation and release of reactive oxygen species (25). A common observation in other cells involving cytokine and integrin mediated activities is reorganization of the cytoskeleton (26). Thus, we have hypothesized that GM-CSF mediated eosinophil priming may involve the signal transduction pathway for cytoskeletal reorganization and a possible involvement of the actin bundling protein, L-plastin. We had previously identified for the first time the presence of L-plastin in eosinophils in a proteomic study of peripheral blood eosinophils which prompted this study (27). Plastins are actin binding proteins which cross-link actin filaments into tight bundles. Three isoforms sharing ~70% amino acid identity were characterized in mammals to have distinct expression patterns: plastin 1 (I-plastin) is expressed only in the renal and intestinal epithelium; plastin 2 (L-plastin) occurs predominantly in leukocytes; and plastin 3 (T-plastin) is broadly expressed (28). As with other plastins, L-plastin has two actin binding domains and an amino-terminal regulatory headpiece with two EF hand-type Ca²⁺ binding sites (29). Only the L-plastin isoform is phosphorylated (30) and phosphorylation on seryl residues was proposed to be required for targeting L-plastin to the actin cytoskeleton (29). Serine phosphorylation occurs in the regulatory Ca²⁺-binding headpiece domain and promotes F-actin binding and bundling leading to the stiffening of actin bundles (31). Additionally, phosphorylated L-plastin may also act as a scaffold for cytoskeleton-associated signaling complexes that contain paxilin, a cortactin interacting protein, and Syk tyrosine kinase (32, 33). Besides involvement in cytoskeleton reorganization, L-plastin phosphorylation has been implicated in integrin activation and induction of the adhesion-dependent respiratory burst by signaling to NADPH oxidase (34, 35). Phosphorylation of L-plastin involves complex mechanisms not yet fully understood and indicates to be dependent on cell and stimulus type. In polymorphonuclear leukocytes and peritoneal macrophages L-plastin is phosphorylated in response to cytokines (e.g., IL-1, TNFα), chemotactic factors (e.g., IL-8, fMLP), PKC activators (phorbol 12-myristate 13-acetate), lipopolysaccharide, and upon FcγRII ligation (reviewed in 36). Also, several protein kinases were implicated in upregulating L-plastin phosphorylation including PKA, PKC, and PI3K based on the sensitivity of L-plastin to kinase activators or inhibitors (37, 38).

In this report, we demonstrate a pivotal role for phosphorylated L-plastin in the regulation of GM-CSF dependent eosinophil priming. Using a proteomics approach, we identified GM-CSF- inducible phosphorylation of L-plastin and characterized the interaction of L-plastin with the α chain of the GM-CSF receptor (GMRα), PKCβII, and several cytoskeletal proteins. L-plastin phosphorylation and priming of eosinophils for chemotaxis and degranulation was sensitive to PKCβII inhibition indicating for the first time the role of this GM-CSF receptor-bound kinase in regulating L-plastin function. Moreover, the internalization of an L-plastin-derived phosphorylated peptide (residues 2–19) encompassing Ser5 also upregulated αMβ2 integrin expression and increased eosinophil chemotaxis independent of GM-CSF stimulation. Our results strongly suggested a critical role for phoshorylated actin bundling L-plastin in the crosstalk between signaling from the GM-CSF receptor and events associated with eosinophil priming, such as cytoskeleton rearrangement and integrin activation.

Materials and Methods

Reagents/materials

Recombinant, human GM-CSF, eotaxin, IL-5 and TNFα were purchased from Peprotech (Rocky Hill, NJ). Polyclonal antibodies against L-plastin, PKCβII, GM-CSF receptor β and GM-CSF receptor α, p-cofilin, p-paxilin, EPX and ECP were from Santa Cruz Biotechnology (Santa Cruz, CA). Two peptides representing the N-terminal region of L-plastin (residues 2–19), nonphosphorylated (ARGSVSDEEMMELREAFA) and phosphorylated on Ser5 (ARGpSVSDEEMMELREAFA) were synthesized in the UTMB Biomolecular Resources Facility (BRF) using a 433A peptide synthesizer (Applied Biosystems) employing Fmoc chemistry protocols as described by the manufacturer, and were purified by reversed-phase C18 HPLC to 99% purity and characterized by MALDI-TOF/MS. Polyclonal anti-phospho L-plastin antibody was prepared in the UTMB BRF by immunizing rabbits with synthetic peptide from the above described N-terminal region of L-plastin (residues 2–19) phosphorylated on Ser5 and purified by protein A and peptide affinity chromatography. Polyclonal anti-phospho-ERK antibodies and secondary HRP-conjugated anti-rabbit antibody were from Cell Signaling Technology (Danvers, MA). PKCβII siRNA and the appropriately scrambled control and transfecting reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE-conjugated anti-αMβ2 monoclonal antibody and isotype-specific control we also from Santa Cruz Biotechnology (Santa Cruz, CA). A cell permeable PKCβII inhibitor (I), 4,5-bis (4-fluoroanilino)-phtalimide, and ProteoExtract Protein Precipitation Kit (cat. 539180) were purchased from Calbiochem (San Diego, CA). In experiments involving PKC inhibition, all control cells were treated with solutions used for dissolving the PKC inhibitor (DMSO) and the final concentration of DMSO never exceeded 0.1% of the culture volume. The chemiluminescence reagent employed was purchased from Millipore Corporation (Bedford, MA).

Eosinophil isolation from peripheral human blood

Peripheral blood (120 ml) was drawn from healthy subjects as we previously reported (27) under a research protocol approved by the IRB committee at the University of Texas Medical Branch (IRB#04-371). Eosinophils were obtained by sedimentation in 4–6% dextran for 50 min at RT, followed by centrifugation in a Ficoll-Hypaque gradient as described previously (39). Following centrifugation at 500 × g, upper layers of plasma and mononuclear cells were removed and saved for further analysis. Erythrocytes were eliminated by hypotonic lysis, and then eosinophils were negatively selected using anti-CD16 immunomagnetic beads to remove neutrophils using the MACS system 9 (Miltenyi Biotec, Sunnyvale, CA). The final eosinophil purity was assayed by microscopic examination using a Wright-stained cytospin preparation. The purity of eosinophil preparations was ≥ 98%.

Human eosinophil cell culture

Although most experiments relating to eosinophil priming were performed on freshly isolated cells in the presence of 2% FBS, in certain experiments involving inhibition of PKCβII with siRNA, eosinophils were cultured for up to 48 h in the presence of 5% FBS (40). Briefly, eosinophils were suspended in RPMI 1640 medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 2–5% FBS (High Clone Laboratories, Inc., Logan, UT), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (GIBCO BRL/Life Technologies, Grand Island, NY). The effect of GM-CSF on eosinophils survival was tested by incubation of cells with GM-CSF at 1 ng/ml in the presence of 2% FBS. Cells were cultured at a density of 1 × 10⁶/ml in a humidified atmosphere containing 95% air and 5% CO₂. The cultures were maintained in 12-well sterile, flat bottom plates (Costar Corp., Cambridge, MA) previously coated with 1% human serum albumin.

Immunoprecipitation of protein complexes

Immunoprecipitation to establish the identity of proteins in complexes was conducted as we previously reported (39). Briefly, complexed proteins were crosslinked with dithiobis(succinimydyl)proprionate prior to affinity chromatography using antibody preparations bound to protein A-Sepharose. Bound protein complexes were eluted from the beads thrice with wash buffer containing 150 mM phenyl phosphate disodium salt. The pooled protein fractions were subsequently subjected to 1D SDS-PAGE and Western blot analysis.

2D gel electrophoresis

Two-dimensional gel electrophoresis was used to separate protein lysates and immunoprecipitates. After visualizing with Sypro Ruby fluorescent staining (BioRad Laboratories), UV-visible spots were excised and subjected to mass analysis after trypsin digestion as we previously reported (27). Mass spectra of peptide digests were obtained using a Model 4800 MALDI-TOF-TOF/MS (Applied Biosystems, Foster City, CA). Proteins were identified using the National Center for Biology Information (NCBI) protein database and Mascot software. Positive protein identifications were accepted for those with expectation scores of 1×10⁻³ or less (41).

Analysis of phosphorylated eosinophil proteins

Phosphoproteins from eosinophils stimulated with GM-CSF were enriched on a Qiagen PhosphoProtein Purification column using the manufacturer’s protocol. Briefly, eosinophil proteins were extracted by homogenization in lysis buffer containing 0.25% (w/v) CHAPS, protease/phosphatase inhibitors, and benzonase as described in the manufacturer’s phosphoprotein purification protocol (PhosphoProtein Purification Kit; Qiagen, Valencia, CA) for 30 min at 4°C and centrifuged at 10,000 × g at 4°C for 30 min to remove insoluble material. Total extracted eosinophil protein (200 μg from 4 × 10⁶ eosinophils) was diluted to a concentration of 0.1 mg/ml in lysis buffer (described above) and was applied to a lysis buffer-equilibrated PhosphoProtein purification column at RT. After washing the column with 6 ml of lysis buffer, the phosphoproteins were eluted with 2 ml of PhosphoProtein Elution Buffer. The yield of phosphorylated protein was determined by the Bradford assay and constituted 6–8% of the initial total protein load. The flow-through samples were passed through two additional Qiagen columns to ensure complete removal of phosphoproteins. The phosphoproteins were then concentrated by ultrafiltration using a 10-kDa cutoff Amicon filter (Millipore) and resolved on 1D SDS-PAGE followed by Western blotting.

Western blot analysis

For protein identification following gel electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MS). After transfer, membranes were blocked with 5% milk Tris-buffered saline (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.1% (v/v) Tween 20 for 1 h and then incubated with the appropriate antibody (1:10000 dilution) overnight at 4 C. Membranes were washed 4X in Tris-buffered saline with 0.1% (v/v) Tween 20 and then incubated with horseradish peroxidase-conjugated secondary antibody. After washing, immune complexes were detected by reaction with the enhanced-chemiluminescence assay (Millipore) according to the manufacturer’s protocol.

Densitometry

Densitometry analysis was performed on Western blots from experiments on time-course of protein expression and phosphorylation. Autoradiograms of the immunoblots were scanned using Adobe Photoshop (Adobe System, Inc., San Jose, CA). The blot’s contrast was adjusted for minimum background and the mean density for each band was analyzed using the ImageJ program (NIH, Bethesda, MD). The comparative data were calculated as “fold change” compared to control for each treatment time.

PKCβII siRNA transfection

Freshly isolated eosinophils were resuspended (10⁶/ml) in antibiotic-free siRNA transfection medium (sc-36868, Santa Cruz, CA). One ml aliquots of eosinophils in transfection medium were added to each well of a 12-well culture plate. An aliquot of 3.6 μl of PKCβII siRNA (sc-39170) was added to 40 μl of siRNA Transfection Medium (sc-36868, Santa Cruz, C) and kept at RT for 5 min. In a separate tube, 2.4 μl of siRNA Transfect ion Reagent (sc-29528) was mixed with 40 μl of siRNA Transfection Medium and kept at RT for 5 min. Subsequently, the contents from both tubes were mixed and incubated at RT for 20 min to form an siRNA-siRNA Transfection Reagent Complex. After 20 min of incubation 0.32 ml of siRNA Transfection Medium was added to each tube containing the siRNA-siRNA Transfection Reagent. Subsequently, cells were resuspended in 0.4 ml of resulting siRNA/siRNA Transfection reagent mixture and incubated for 8 h at 37°C. After incubation, cells were washed and resuspended in 1 ml of RPMI 1640 containing 5% of FBS followed by an additional incubation of 36 h. Transfection efficiency was evaluated by Western blotting for each experiment performed on eosinophil priming. Control siRNA (sc-370007) was a scrambled sequence that does not cause degradation of any known cellular mRNA.

Eosinophil chemotaxis assay

Chemotaxis of eosinophils was conducted in duplicate using 5 μm pore size, polycarbonate, polyvinylpyrrolidone-free membranes in Boyden chambers (42). Human eotaxin (R&D Systems) was diluted in RPMI 1640 (Invitrogen Life Technologies) with 2% FCS and placed in the lower wells (100 μl) at a 5–20 nM concentration. After incubation of the eosinophils with agonists or medium for 20 min at 37°C, the cells were washed twice. Aliquots of 100 μl of the cell suspension at 2.5 × 10⁵ cells/ml were placed in the upper chambers. The loaded chambers were incubated at 37°C in humidified air with 5% CO₂ for 1 h. Then the membrane was removed, subjected to fixation and stained for 3 min in a May-Grünwald solution. The cells that migrated and adhered to the lower surface of the membrane were counted by light microscopy and expressed as number of cells/10x field. In some experiments the cells were mixed the PKCβII inhibitor (I) before being incubated with GM-CSF and/or eotaxin.

Priming for degranulation

Degranulation of eosinophils was assessed by detection of extracellular ECP and EPX in the medium of eotaxin stimulated cells. Control and GM-CSF stimulated eosinophils (1 ng/ml for 1 h) were kept in 12-well culture plates at a density of 10⁶ cells per 1 ml of RPMI 1640 supplemented with 2% FBS and treated with increasing concentration of eotaxin (5, 10, 20 and 40 ng/ml) for 2 h. After stimulation, approximately 0.8 ml of culture medium was collected and centrifuged to separate remaining cells. Conditioned culture medium was subsequently mixed with 4 volumes of Proteoextract Precipitation reagent to isolate proteins. Resulting protein precipitates from equal volumes of medium were subsequently dissolved in SDS loading buffer, resolved on SDS and analyzed for expression of ECP and EPX by Western blot analysis using polyclonal rabbit antibody from Santa Cruz Biotechnology (Santa Cruz, CA).

Eosinophil viability

An Annexin V^PE Apoptosis Detection kit (BD Biosciences) was used to quantitatively determine eosinophils undergoing apoptosis by virtue of their ability to bind annexin V and exclude 7-aminoactinomycin (7-AAD). This assay detected viable cells (annexin V negative/7-AAD negative) cells undergoing early apoptosis (annexin V positive/7-AAD negative), and dead cells (annexin V positive/7-AAD positive). Eosinophils (2 × 10⁵) were stained with annexin A and 7-ADD according to the manufacturer’s instructions. Data were acquired on a FACScan instrument (BD Biosciences) and analyzed using CellQuest software (BD Biosciences); 10,000 events per sample were acquired.

Flow cytometry

Flow cytometric analysis of the expression of CD11b on the surface of eosinophils was performed after staining cells with PE-conjugated anti-αMβ2 monoclonal antibody (Santa Cruz, CA) according to the manufacturer’s protocol. For each population, a matched isotype control mAb was used to define the placement of the negative marker. Eosinophils were analyzed using a Becton Dickinson (San Jose, CA) FACSAria machine and at least 30,000 events were collected for each sample. The data were analyzed using CELLQuest software (Becton Dickinson).

Data analysis for eosinophil migration and viability

The results of eosinophil chemotaxis and viability measurements are expressed as means ± SD. To determine significant differences between the two groups, a two-tailed Student t test was performed using a Sigma-Plot software program (SPSS); p < 0.05 was considered significant.

Results

L-plastin phosphorylation in GM-CSF-stimulated eosinophils

We used two approaches to measure L-plastin phosphorylation in eosinophils stimulated with GM-CSF (1 ng/ml). First, total cell lysates from control and GM-CSF-stimulated cells were resolved by 2DE, followed by staining with the phosphosensitive fluorescent dye Pro-Q Diamond. A number of phosphoproteins indicated upregulation and especially prominent was a ~68 kDa phosphoprotein that was identified by MALDI-TOF-TOF/MS as L-plastin (Fig. 1). Subsequent staining of the same 2D gels with Sypro Ruby protein stain also indicated the L-plastin spots on both gels (results not shown). Multiple L-plastin spots shown in Fig. 1 likely were the result of many reported post-translational modifications, e.g. phosphorylation and acetylation (see Swiss-Prot accession #P13796). Our second approach used Western blot analysis first with antiphosphotyrosine Ab (clone 4G10) that showed no tyrosine phosphorylation of L-plastin, although some plastins were reported to have pTyr residues (Swiss-Prot above). Since previous reports using different cells indicated some significance to phosphorylation on Ser5 of L-plastin (34), we developed an Ab in rabbits against a synthetic phosphopeptide (residues 2–19) encompassing a putative phosphorylation site for L-plastin around Ser5. This antibody did not react with dephosphorylated L-plastin in control experiments involving treatment with phosphatase emphasizing its specificity for phosphorylated L-plastin (results not shown). Western blot analysis with this Ab did reveal trace constitutive phosphorylation of L-plastin in the control, non-stimulated eosinophils that was upregulated within 15 min of GM-CSF stimulation, reaching maximal phosphorylation within 60 min, and remaining elevated for the next 18 h (Fig. 2A and 2B). In addition, this Ab also revealed increased L-plastin phosphorylation in eosinophils stimulated with either IL-5 or TNFα, two cytokines known to prime eosinophil’s (Fig. 2C). Since L-plastin is an actin binding protein that stabilizes actin bundles, we reasoned that L-plastin may be contributing to eosinophil priming via F-actin interactions with possible cytoskeletal rearrangement.

Fig. 1.

2DE analysis of eosinophil whole cell lysates stained for phosphoproteins highlighted the identification of L-plastin by MALDI-TOF-TOF MS. A, Lysate samples (150 μg) from freshly isolated control eosinophils and cells stimulated with GM-CSF for 15 min (1 ng/ml) were focused on 18 cm IPG strips (pH 4–7) and subsequently resolved on 12% Tris-glycine polyacrylamide gels. Phosphorylated protein spots were visualized with the phosphosensitive fluorescent dye Pro-Q Diamond. Representative gel of 5 independent experiments on cells from 4 different donors is shown. B, The Mascot expectation scores for the L-plastin protein spots identified by MS analysis were highly significant as shown.

Fig. 2.

Western blot analysis of L-plastin phosphorylation on Ser5 in GM-CSF stimulated eosinophils. A, Time-course of L-plastin phosphorylation in GM-CSF-stimulated eosinophils. Western blot representative of 4 independent experiments showed increased phosphorylation of L-plastin occurring within 15 min of GM-CSF stimulation, and detectable 18 h later. Reblotting with anti-L-plastin as a control confirmed equal L-plastin content in the analyzed samples as shown. B, Densitometric quantification of the blots with anti-phospho-L-plastin used for detection of L-plastin phosphorylation in GM-CSF-stimulated eosinophils. The comparative data were calculated as “fold change” that is compared to control, nonstimulated cells for individual treatment time in each case and adjusted to expression of total L-plastin expression. Densitometry data shown are representative of four (3 h and 18 h stimulations) to seven (15 min and 1 h stimulations) independent experiments. The error bars represent the standard error of the mean and * represents p<0.05 as compared to non-stimulated cells. C, L-plastin phosphorylation was also inducible upon stimulation with other cytokines involved in eosionophil priming, such as IL-5 (5 ng/ml) and TNFα (10 ng/ml). Western blot is representative of 3 independent experiments.

Protein composition of L-plastin co-immunoprecipitates

The immediate phosphorylation of L-plastin in response to priming cytokines and a possible role in the rearrangement of cytoskeletal proteins led us to investigate the potential interaction of L-plastin complexed with other proteins. We thus performed a systematic analysis of proteins interacting with the L-plastin in eosinophils stimulated with GM-CSF (1 ng/ml, 15 min). Co-immunoprecipitation of eosinophil lysates using a commercial polyclonal anti-L-plastin Ab, followed by 2DE SDS-PAGE and MALDI-TOF-TOF/MS analysis, revealed binding to actin, cofilin, paxilin and PKC in the immunoprecipitates of L-plastin (results not shown). Subsequent verification by 1DE Western blot analysis (Fig. 3A) showed the co-precipitation of L-plastin with cofilin and paxilin as well as PKCβII and two subunits of the GM-CSF receptor–GMRα responsible for binding specificity and GMRβ responsible for intracellular signal propagation from GM-CSF and also IL-5, and IL-3. PKCβII was previously known to interact with GMRβ (43). These results were further verified by reverse co-immunoprecipitation that showed the presence of L-plastin in immunoprecipitates of GMRα, GMRβ and PKCβII kinase (Figs. 3B-3D). Importantly, as indicated in Fig. 3A, L-plastin’s interactions with cofilin and paxilin seemed to be constitutive and not modulated by GM-CSF treatment, whereas stimulation with GM-CSF clearly induced the upregulation of phosphorylated L-plastin with concomitantly upregulated and bound PKCβII, GMRα, and GMRβ. Western blot analysis of cell lysates from a time-course stimulation of eosinophils with GM-CSF over 18 h is given in Fig. 4. Phosphorylation of paxilin (Fig. 4A) indicated to parallel that of L-plastin phosphorylation (Fig. 2A) whereas cofilin was dephosphorylated after 15 min of GM-CSF stimulation (Fig. 4B).

Fig. 3.

Immunoprecipitation of L-plastin-associated proteins from eosinophils stimulated with GM-CSF (1 ng/ml for 15 min.). After GM-CSF stimulation, eosinophils were treated with a membrane-permeant cross-linker (DSP), followed by cell lysis. The lysates were then immunoprecipitated with annotated antibodies followed by Western blotting. A, Western blot analysis of L-plastin-coprecipitating proteins showed a GM-CSF-dependent interaction with PKCβII, GMRα and GM Rβ. Two additional proteins, paxilin and cofilin, coprecipitated with L-plastin in a GM-CSF-independent manner, suggesting their constitutive interaction with L-plastin. Reverse co-IP showing the presence of L-plastin in immmunoprecipitates of B, PKCβII, C, GMRα and D, GMRβ..

Fig. 4.

Western blots of GM-CSF-inducible phosphorylation of proteins interacting with the L-plastin in activated eosinophils. Freshly isolated control and GM-CSF-stimulated eosinophils were lysed and whole cell lysates were resolved on SDS-PAGE and Western blotted with annotated polyclonal antibodies. A, Western blot representative of 4 independent experiments shows increased phosphorylation of paxilin in stimulated eosinophils. Densitometry data are calculated as “fold change” compared to control, nonstimulated cells for individual treatment time in each and are representative of four independent experiments with reproducible findings. B, Western blot representative of 4 independent experiments shows decreased phosphorylation of cofilin in GM-CSF-stimulated eosinophils. Densitometry are representative of four independent experiments with reproducible findings. The error bars represent the standard error of the mean and * represents p<0.05 when compared to non-stimulated cells.

Inhibitory effect of PKCβII siRNA on GM-CSF-induced protein phosphorylation

To examine the role of PKCβII in GM-CSF-induced phosphorylation of L-plastin, we transfected eosinophils with a specific small interfering RNA (siRNA) designed to inhibit PKCβII expression. The observed inhibition of PKCβII protein expression led us to inquire further regarding the phosphorylation of the proteins that we found interacted with this kinase (Fig. 5A). Western blot analysis of eosinophils treated with siRNA confirmed suppressed PKCβII expression and showed no increase of L-plastin or paxilin phosphorylation in GM-CSF-stimulated cells. We also investigated the phosphorylation of cofilin, whose actin binding capacity is downregulated by phosphorylation at Ser3 in myocytes (44). We found prolonged dephosphorylation of cofilin upon GM-CSF stimulation (Fig. 4B); importantly, this dephosphorylation was suppressed in cells lacking PKCβII (Fig. 5A). Interestingly, siRNA inhibition of PKCβII had no significant effect on GM-CSF-inducible phosphorylation of Erk 1 and Erk2 kinases (Fig. 5A), indicating the relative specificity and nontoxicity of the siRNA used, and suggested the separation of Erk kinase activation from phosphorylation of PKCβII, L-plastin, paxilin, and cofilin. To further investigate whether L-plastin-interacting PKCβII is involved in the upregulation of αMβ2 expression, we examined the effect of PKCβII inhibition in response to GM-CSF stimulation (1 ng/ml for 90 min). Cells treated with PKCβII siRNA were unable to upregulate αMβ2 integrin upon GM-CSF stimulation, indicating a critical role for PKCβII in αMβ2 upregulation by GM-CSF (Fig. 5B).

Fig. 5.

Analysis of phosphoprotein expression in eosinophils treated with PKCβII siRNA. Eosinophils were transfected with siRNA against PKCβII for 36 h or with control scrambled siRNA for 36 h in the presence of 5% FBS, and stimulated with GM-CSF (1 ng/ml) for 15 min in the presence of 2% FBS. A, Western blot of PKCβII expression showed of significant inhibition of PKCβII and inhibition of GM-CSF-induced phosphorylation of L-plastin and paxilin. Dephosphorylation of cofilin, was also decreased in PKCβII siRNA-treated eosinophils. However, the same cells showed normal phosphorylation of Erk1 and Erk2 kinases. Western blot is representative of 5 independent experiments from 3 different donors. B, Results from flow cytometric analysis of αMβ2 expression using anti -CD11b-PE-conjugated mAb expressed as % change in expression as compared to control cells. GM-CSF stimulation (1 ng/ml for 90 min) showed significant upregulation of CD11b expression in control cells, and complete inhibition of integrin upregulation in PKCβII siRNA-treated eosinophils. Graph is representative of 3 independent experiments from 3 independent donors. The error bars represent the standard error of the mean.

Effect of PKCβII inhibitors on L-plastin phosphorylation and αMβ2 integrin expression

The availability of therapeutic inhibitors of PKCβ (45, 46) and the dependence of L-plastin phosphorylation on PKCβII suggested a possible pharmacological approach to inhibit eosinophil priming. We thus investigated the effects of PKCβII inhibition of L-plastin phosphorylation by treatment with the PKCβII inhibitor (I), a cell-permeable compound affecting PKC isoenzymes α, βI and βII with IC50 concentrations of 1.9, 3.8, and 1.0 μM, respectively (47). It was also reported to inhibit EGFR at lower concentrations (47). Pretreating eosinophils with PKCβII inhibitor (I) before GM-CSF stimulation completely inhibited the GM-CSF-inducible phosphorylation of L-plastin without any significant effect on its constitutive phosphorylation (Fig. 6A). The effect of inhibitor (I) on L-plastin phosphorylation was observed at all inhibitor concentrations tested, confirming the involvement of the PKCβII kinase in mediating GM-CSF-induced L-plastin phosphorylation. Inhibitor (I) did not have any significant effect on GM-CSF-induced phosphorylation of Erk1 and Erk2 kinases at concentrations specific for PKCβII which indicated selective specificity of inhibition consistent with the results obtained with the use of PKCβII siRNA (data not shown). Comparison of the surface expression of αMβ2 integrin showed upregulation of αMβ2 in eosinophils stimulated with GM-CSF and complete inhibition of αMβ2 upregulation upon treatment with inhibitor (I) (1 μM) (Fig. 6B). Also, inhibitor (I) showed no effect on the constitutive expression of αMβ2 expression at concentrations up to 10 μM. These results further confirmed the role of PKCβII in αMβ2 integrin upregulation obtained with the use specific siRNA.

Fig. 6.

Analysis of L-plastin phosphorylation and integrin expression in eosinophils treated with a PKCβ inhibitor. A, PKCβII inhibitor (I), was used to pretreat eosinophils for 30 min in three decremented concentrations affecting different isoenzymes of PKC (10 μM, 5 μM and 1 μM affecting PKCα, PKCβI and PKCβII, respectively); prior to GM-CSF stimulation for 15 min. Inhibitor I blocked GM-CSF-induced phosphorylation of L-plastin at doses of 10, 5, and 1 μM without a significant effect on constitutive phosphorylation of L-plastin. Western blot is representative of 5 independent experiments. B, Flow cytometric analysis of αMβ2 integrin expression on eosinophils stimulated with GM-CSF (1 ng/ml) for 90 min with and without the inhibitor of PKCβII (1 μM for 30 min prior to GM-CSF). Flow cytogram representative of 3 independent experiments confirms effect of PKCβII inhibition on GM-CSF stimulated upregulation of αMβ2 integrin observed in studies with the use of siRNA inhibitor.

Effect of PKCβII inhibition on priming for chemotaxis and eosinophil viability

We next examined the effect of PKCβII inhibition on GM-CSF-induced priming for chemotaxis toward eotaxin. Eosinophils were pretreated first with PKCβII inhibitor (I) at various concentrations followed by stimulation with GM-CSF (1 h, 1 ng/ml). Cell chemotaxis was then measured using Boyden chambers in which the lower chambers contained eotaxin at concentration of 5 and 20 nM. In preliminary studies we found that 5 nM eotaxin represented approximately 25% of the effective concentration for optimal chemotaxis. Fig. 7A shows that pretreatment of eosinophils with GM-CSF increased migration in response to a suboptimal concentration of eotaxin, indicating eosinophil priming. Cells pretreated with either PKCβII inhibitor (I) at concentrations preventing L-plastin phosphorylation (1 μM) completely abrogated the priming effect of GM-CSF. However, use of the inhibitor at concentration specific for PKCβII had no significant effect on chemotaxis when eotaxin was applied at higher doses (20 ng/ml) that elicited eosinophil migration independently upon GM-CSF stimulation, emphasizing the relatively high specificity of PKCβII inhibition on the eosinophil priming process. Taken together, these results indicated that the priming effect of GM-CSF on chemotaxis toward eotaxin was dependent on PKCβII expression and activity, and correlated with phosphorylation of L-plastin. Interestingly, prolongation of eosinophil survival was only partially affected by inhibitor (I) at PKCβII-specific concentrations capable of blocking L-plastin phosphorylation and integrin upregulation suggesting that either PKCβII inhibition alone is not able to completely block prosurvival effect of GM-CSF or that other cytokines released, perhaps by a paracrine mechanism and acting in a PKCβII-independent manner prolonged eosinophil survival (Fig. 7B). We previously showed that prolonged survival of GM-CSF-stimulated eosinophils was critically dependent on signals from both the GM-CSF receptor and the ICAM-1 adhesion molecule (48).

Fig. 7.

Effect of PKC inhibition on eosinophil chemotaxis and survival. A, Analysis of chemotaxis toward eotaxin in eosinophils primed with GM-CSF. Freshly isolated eosinophils were treated with PKCβII inhibitor at a concentration of 1 μM that was specific for PKCβII followed by stimulation with GM-CSF (1 ng/ml) for 1 h. Migration of control and primed eosinophils and effect of PKCβ inhibition was measured with a Boyden chamber using two different eotaxin concentrations (5 and 20 nM) in the lower chamber. Inhibition of PKCβII completely abrogated priming effect of GM-CSF on responsiveness to 5 nM concentration of eotaxin but had no significant effect on response to higher concentrations of eotaxin. Control cells were treated with diluent used for dissolving PKC inhibitor. The results are expressed as the mean of ± SEM from 3 independent experiments run in triplicates. B, Freshly isolated eosinophils were treated with PKCβII inhibitor (I) at two different concentrations (1 and 5 μM) followed by stimulation with GM-CSF (1 ng/ml) for 48 h. Comparison of viable eosinophils (7-ADD negative/annexin V negative) showed prolongation of eosinophil survival upon stimulation with GM-CSF and no significant effect of the PKCβ inhibition on viability of control, nonstimulated eosinophils. PKCβ inhibitor suppressed prolongation of eosinophil survival by GM-CSF at higher nonspecific for PKCβII concentration (5 μM); however, PKC inhibitor (I) at a concentration of 1 μM did not fully prevent GM-CSF-induced inhibition of apoptosis. The results are representative of 4 independent experiments.

Effect of PKCβII inhibition on priming for eosinophil degranulation

Since eotaxin is known to cause eosinophil degranulation when applied in higher concentrations, we tested whether preincubation with GM-CSF will elicit degranulation in response to lower doses of eotaxin. Degranulation of eosinophils was assessed by Western blot analysis of extracellular ECP and EPX and showed an increased level of EPX in the medium after stimulation with eotaxin at concentration of 20 and 40 ng/ml (Fig. 8A). Interestingly, ECP was detectable by our assay only at concentrations of 40 ng of eotaxin when used with nonprimed eosinophils. However, a 2 h pretreatment with GM-CSF significantly increased the degranulatory response to eotaxin and extracellular ECP was detectable after stimulation with eotaxin at concentrations as low as 5 ng/ml. Subsequently, we asked whether inhibition of PKCβII would affect priming for degranulation. We used eotaxin at concentration of 10 and 20 ng to test for the possible differential effect on priming and degranulation. Interestingly, siRNA induced inhibition of PKCβII completely blocked EPX and ECP release in response to both 10 and 20 ng/ml of eotaxin (Fig. 8B). These results complement our findings on the critical role of PKCβII in GM-CSF-induced upregulation of αMβ2 integrin and are in agreement with previous studies showing the dependence of eosinophil degranulation on αMβ2 integrin.

Fig. 8.

Effect of PKC inhibition on eosinophil priming for degranulation. A, Western blot of extracellular EPX and ECP was performed to test degranulation of eosinophils in response to eotaxin upon control and priming conditions. Eosinophils were primed with GM-CSF (1 ng/ml) for 1 h followed by stimulation with different concentrations of eotaxin for 2 h. Supernatant from cultured eosinophils was subsequently tested for released granule proteins by Western blotting. Western blots showed higher levels of ECP and EPX released from GM-CSF-treated eosinophils stimulated with eotaxin at concentration of 20 ng/ml suggesting their priming for degranulation. Representative blots from 3 independent experiment are showed. B, Effect of siRNA induced inhibition of PKCβII in eosinophil priming for degranulation. Eosinophil were transfected with specific siRNA to diminish expression of PKCβII or with control scrambled siRNA and subsequently cultured for 36 h to allow for efficient inhibition of targeted protein. Control and scrambled siRNA cells showed release of ECP and EPX in response to 10 ng/ml of eotaxin in cells primed with GM-CSF. Specific inhibition of PKCβII with siRNA completely blocked release of EPX and ECP in response to suboptimal (10 ng/ml) concentrations of eotaxin. Representative blots from 3 independent experiments on cells from two donors are shown.

Effect of p-L-plastin peptide (2–19) on αMβ2 expression and eosinophil sensitivity to eotaxin

Although GM-CSF-inducible phosphorylation of L-plastin correlated with the effects of PKCβII inhibition on eosinophil priming, we further questioned the role of L-plastin in these processes. Currently, there are no available inhibitors of L-plastin and our efforts to inhibit L-plastin expression with siRNA were not successful, likely due to the high abundance of the target protein and the short viability of eosinophils. To investigate the potential role of phosphorylated L-plastin in eosinophil priming, we studied the effect of an internalized synthetic peptides from the N-terminal region of L-plastin (residues 2–19) phosphorylated on Ser5, a site previously shown to be important in L-plastin involvement in adhesion (49). αMβ2 is one of the earliest integrins upregulated upon stimulation of eosinophils with GM-CSF and therefore suitable for evaluation in transfection experiments. For cellular internalization of the peptide we used the Chariot-mediated peptide transduction reagent (Active Motif, Carlsbad, CA) (50). Control experiments using fluorescein isothiocyanate-labeled control peptides demonstrated that nearly 100% of the eosinophils took up the peptide in the presence of the Chariot reagent without any appreciable toxic effects (data not shown). The phosphorylated L-plastin peptide rapidly upregulated the expression of αMβ2 (<30 min), with maximal expression at a peptide concentration of 50 μM. A control peptide in which Ser5 was not phosphorylated did not induce upregulation of αMβ2 (Fig. 9A). Neither peptide exerted any effect on αMβ2 expression in the absence of the Chariot reagent. The maximal expression of αMβ2 induced by the phosphorylated Ser5 peptide was almost equivalent to that stimulated by a priming dose of GM-CSF and was further slightly upregulated by additional stimulation with the cytokine. We also noted an inhibitory effect of the nonphosphorylated 2–19 peptide on GM-CSF-induced priming for chemotaxis (Fig. 9B); the mechanism of this inhibition is currently not known but may involve competition of the 2–19 peptide with nonphosphorylated L-plastin for PKCβII kinase. Interestingly, inhibition of PKCβII kinase activity had no inhibitory effect on the upregulation of αMβ2 by the phosphorylated Ser5 peptide, indicating that delivery of the phosphorylated L-plastin peptide alone is sufficient to cause upregulation of integrin expression in a manner independent of PKCβII activity.

Fig. 9.

Effect of L-plastin-derived peptides on expression of αMβ2 integrins and priming for chemotaxis. Peptide 2–19pS5 was phosphorylated on Ser5, while peptide 2–19 served as the nonphosphorylated control. A, Flow cytometric analysis of αMβ2 integrin expression in eosinophils upon internalization of phosphorylated and nonphosphorylated peptide 2–19. B, Priming for chemotaxis toward eotaxin upon internalization of L-plastin peptide 2–19. Treatment with phosphorylated 2–19pS5 peptide significantly increased eosinophil chemotactic responsiveness to suboptimal concentrations of eotaxin. There was no significant effect of the nonphosphorylated peptide 2–19 control on priming of eosinophil for chemotaxis. Experiments were repeated three times on eosinophils from two different donors. Control cells were treated with peptide diluents and transfection reagents without peptides.

We also investigated whether the phosphorylated L-plastin peptide affected eosinophil chemotaxis to eotaxin (Fig. 9B). Internalization of the phosphorylated L-plastin peptide significantly increased eosinophil chemotaxis toward suboptimal concentrations of eotaxin, whereas treatment with the non-phosphorylated L-plastin peptide did not. The observed eosinophil chemotaxis after treatment with the pSer5 peptide was comparable to that of eosinophils pretreated with GM-CSF. Taken together, these results underscore that Ser5-phosphorylated L-plastin is involved in GM-CSF induced priming of eosinophils for chemotaxis.

Discussion

In this study we have demonstrated a mechanism for eosinophil priming mediated by phosphorylation of the actin bundling protein, L-plastin. We found that L-plastin was critically involved in the upregulation of αMβ2 expression on GM-CSF-stimulated eosinophils and facilitated eosinophil migration and degranulation in response to eotaxin. We further found that the activation signal required phosphorylation of L-plastin by GM-CSF receptor-bound PKCβII since inhibition of PKCβII abrogates the effect of GM-CSF on eosinophil priming. Most importantly, we established that treatment of eosinophils with a synthetic peptide derived from the region of L-plastin containing phosphorylated Ser5 resulted in eosinophil priming in a manner independent of GM-CSF-activation. These results suggested that phosphorylated L-plastin was both essential and sufficient to propagate the upregulation of αMβ2 integrin and priming for chemotaxis and degranulation.

Our study provided several significant insights toward a better understanding of the signaling pathways that lead to eosinophil priming. First, we identified PKCβII as an upstream kinase necessary for GM-CSF-induced L-plastin phosphorylation and upregulation of αMβ2 integrins. Second, our results supported the hypothesis that L-plastin phosphorylation was an essential step for eosinophil αMβ2 integrin upregulation and activation. The role of L-plastin in these processes also pointed to the importance of actin bundling and cytoskeleton rearrangement in mediating priming of eosinophils.

Our findings that a PKCβII inhibitor blocked the upregulation of αMβ2 integrin were consistent with a proposed role for PKC in the induction of integrin-mediated cell functions such as adhesion or degranulation (51, 52). PKCα associates with β1 integrins and regulates their internalization (53), whereas PKCε has been implicated in the regulation of integrin-dependent cell spreading (54). The PKC isoforms α, βII, and βII activate integrin-mediated adhesion to ICAM-1 in a leukocyte model system (55). Moreover, the receptor for activated PKC (Rack1), a PKCβ-interacting protein that is believed to regulate its localization and substrate specificity, interacts with the membrane-proximal part of the integrin αMβ2 cytoplasmic tail in phorbol ester-activated leukocytes (56). Furthermore, a synthetic peptide corresponding to most of the cytoplasmic domain of the integrin αMβ2 chain was found to be phosphorylated by the active proteolytic fragments of PKCβ and PKCδ in vitro (57, 58). Two models employing PKCβ-deficient cells indirectly support our findings on the role of PKCβ in eosinophil priming. First, PKCβ−/− mast cells exhibited less degranulation activity than wild type cells in response to FcεRI stimulation (59). Second, stimulation of PKCβ−/− B lymphocytes with BCR or IL-4 failed to promote expression of the anti-apoptotic proteins BclxL and Bcl2 (60). Similarly, we found that eosinophils, which use BclxL for anti-apoptotic signaling did not respond to GM-CSF with prolonged survival upon inhibition of the PKCβ enzymes. We observed, however, that this effect was detectable when concentrations of PKC inhibitors were specific for PKCβI. Although PKCβI and PKCβII have 96% sequence identity, they exhibit homolog-specific subcellular localization with PKCβI localized mostly in the cytosol and membrane, whereas PKCβII is predominantly localized within the cytoskeleton (61). In eosinophils, cytoskeletal translocation of PKCβII was reported during stimulation with PAF, which was related to actin assembly, eosinophil shape change, adhesion, and superoxide anion generation (62). Thus, our findings support the role of PKCβII in the processes related to cytoskeleton reorganization and indicate that other PKC isoenzymes may also play a role in transducing anti-apoptotic signals from the GM-CSF receptor.

Although PKC was previously identified to be involved in αMβ2 upregulation, our results strongly support the participation of phosphorylated L-plastin in mediating PKCβII driven integrin upregulation. However, there is evidence of the involvement of L-plastin in αMβ2 integrin function (35). A variety of inflammatory mediators that activate αMβ2 integrins such as chemokines, cytokines, immune complexes, and phorbol 12-myristate 1-acetate, also induce L-plastin phosphorylation (63, 64). Since L-plastin remained phosphorylated after blocking αMβ2 integrins, this argued against L-plastin being dependent on integrin activation (65). On the other hand, we showed herein that the inhibition of αMβ2 integrin upregulation correlated with L-plastin phosphorylation. Furthermore, although the expression of αMβ2 and other β integrins in L-plastin −/− leukocytes was normal, adhesion-dependent signaling transduced by these adhesion molecules was severely impaired (66). L-plastin deficient leukocytes showed diminished phosphorylation of paxilin, a known downstream effector of αMβ2 integrin signaling, and were unable to mount a respiratory burst but, interestingly, showed normal migration to sites of infection (66). Similarly, we found that pharmacological inhibition of L-plastin phosphorylation had no significant effect on eosinophil migration toward higher concentrations of eotaxin consistent with phosphorylated L-plastin mediating processes related to priming.

The molecular involvement of L-plastin in priming processes is not yet fully elucidated but given that L-plastin represents an actin binding protein stabilizing actin bundles, it is likely that L-plastin exerts its action via activation-induced F-actin reorganization. It is also possible that the cortical F-actin localized below the plasma membrane functions as a physical barrier to exocytosis inhibiting the fusion of vesicles and cell membranes (67). For that reason, controlled disassembly of the actin network at the plasma membrane is a pre-requisite for exocytosis. Recent evidence suggests that the actin cytoskeleton restricts the lateral mobility of β2 integrins within the membrane of unactivated cells (68) and it was proposed that L-plastin plays a critical role in the cytoskeletal restriction of integrin diffusion in neutrophils (35). In this regard, phosphorylation of L-plastin, or as we have shown herein, the addition of a surrogate L-plastin phosphorylated peptide, may interfere with actin-L-plastin bundles resulting in cytoskeletal rearrangement and integrin diffusion. Consistent with the role of the amino-terminal region of L-plastin in regulating actin binding are findings that conformational changes in the L-plastin headpiece region inhibit actin bundling activity (49). However, it is also possible that the role of L-plastin in eosinophil priming may be more complex than only restriction of integrin diffusion. For example, phosphorylated L-plastin may affect the association of other actin-bound proteins or the activity of kinases or phosphatases that regulate cytoskeletal organization, resulting in an assembly of cytoskeletal structures that propagates integrin-mediated signaling.

The observed dependence of increased migration of eosinophils toward eotaxin with phosphorylation of L-plastin is not clear but can be explained by L-plastin effects on αMβ2 signaling. Although the intracellular mechanisms engaged by β2 integrins are mostly interpreted as mediated by adhesion, L-plastin deficiency revealed a role for β2 integrins in signaling clearly independent of adhesion (66). Another possible mechanism of integrin-mediated priming involves the possibility of αMβ2 integrin association with other receptors on the cell surface linking these receptors to the cytoskeleton and signal transduction pathways. Furthermore, protein tyrosine phosphorylation is a common feature following activation of the GM-CSF receptor, integrins, and chemokines and may represent a possible mechanism of priming responsiveness to chemoattractants by crosstalk with signaling elicited by activated GMR or αMβ2 inetgrins. Paxilin is one of the possible downstream targets of signaling pathways from integrin and the GM-CSF receptor. Paxilin phosphorylation was markedly diminished in L-plastin deficient and αMβ2 deficient leukocytes (66). Since we found paxilin in immunoprecipitates of L-plastin, and detected its phosphorylation in GM-CSF stimulated cells, paxilin may represent another testable crosstalk molecule linking signaling from GM-CSF and integrins to priming. Interestingly, Erk kinases which are utilized by both the GM-CSF and eotaxin receptors are unlikely targets of L-plastin, since we found no effect of PKCβII inhibition on the phosphorylation of Erk. These observations are consistent with findings utilizing L-plastin deficient leukocytes that showed overall intact phosphorylation of Erk kinases and supported a limited role of PKCβII and L-plastin in leukocyte activation. Another L-plastin-interacting molecule with the ability to promote changes in the configuration of the actin cytoskeleton is cofilin, a member of the cofilin/ADF family of small actin-binding proteins. In vitro, cofilin binds to both globular and filamentous actin and induces actin depolymerization (69). Moreover, binding of cofilin to F-actin leads to alterations in the F-actin filament twist which influences the interaction of other molecules with F-actin. The actin-binding capacity of cofilin is negatively regulated by the phosphorylation of cofilin at Ser3 (44) and we found early and prolonged dephosphorylation of cofilin upon GM-CSF stimulation. Thus, cofilin may be a possible candidate in a concept that links early signal transduction events with the functional role of the cytoskeleton upon eosinophil activation. A schematic representation of L-plastin’s association with actin components is shown in Fig. 10.

Fig. 10.

A schematic representation of L-plastin’s association with components of signaling pathways leading to priming of eosinophil for chemotaxis and degranulation. A, Nonactivated eosinophils are characterized by a low responsiveness to chemoattractants and degranulating stimuli which correlatse with low affinity of adhesion molecules (e.g. αMβ2) and eotaxin receptor (CCR3). The transition of eosinophils from a nonactivated to a primed phenotype is initiated by binding of a priming cytokine (e.g. GM-CSF) to its putative receptor resulting in the formation of signaling complexes and activation of PKCβII. Subsequently, activated PKCβII phosphorylates L-plastin promoting the disassembly of the actin network leading to integrin diffusion and αMβ2 and CCR3 activation. The surrogate L-plastin phosphorylated peptide mimics this process. B, Increased affinity of αMβ2 and CCR3 leads to enhanced eosinophil response to chemoattractants and degranulating stimuli resulting in primed phenotype

The specific reorganization of the actin cytoskeleton upon eosinophil activation could either enable the formation of coordinated structures composed of receptors and signaling molecules in order to reduce the threshold required for full activation, or prolong essential activating signals. It is significant that the inhibition of a single actin crosslinking protein can disrupt processes important for eosinophil effector function. Eosinophils are very rich in actin and the cytoskeleton is dynamic and very responsive to changes in the cell’s environment, a desirable property for a motile cell like the eosinophil migrating into an inflammatory site. Strikingly, the effect of L-plastin phosphorylation demonstrates that the precise arrangement of components in the cytoskeleton markedly influences the assembly of signal transduction cascades and subsequent effector functions. L-plastin with its capacity to promote such changes in the configuration of the actin cytoskeleton represents a likely candidate in a concept which links the functional role of the cytoskeleton with early signal transducing events upon stimulation of eosinophils with GM-CSF.

Finally, the suppression of inflammatory cells, such as eosinophils and T lymphocytes, through blockade of cell migration, cytokine production, degranulation, and survival is an obvious strategy of allergy research. Furhermore, since allergic reaction and asthma are typically characterized by preferential recruitment of eosinophils, it is reasonable to project that blocking priming will also affect IL-5/GM-CSF/IL-3 and eotaxin-driven eosinophil influx and activation. In addition, since L-plastin indicates at present to be leukocyte specific, at least among non-tumor cells, our study raises the question whether L-plastin may be a potential target for pharmacological therapy. Clearly further studies in this regard will be required to evaluate this potential. Of particular note, Wabnitz et al. (70) have shown that the phosphorylation of L-plastin on Ser5 in T lymphocytes by costimulation through TCR/CD3 plus CD2 or CD28 is an activation mechanism that can bring receptor molecules (e.g. CD25 and CD69) to the cell surface. The results of this report somewhat parallel our results described herein.

Abbreviations in this paper

αMβ2

CD11b/CD18 integrin

AAD

aminoactinomycin

BCR

B-cell receptor

PAF

platelet activating factor

ADF

actin depolymeryzing factor

DSP

dithio-bis(succinimydl)propionate

Co-IP

co-immunoprecipitation

ECP

eosinophil cationic protein

EPX

eosinophil peroxidase

GMRα

GM-CSF receptor α-chain

GMRβ

GM-CSF receptor β-chain

P-I3K

phosphatidylinositol 3-kinase

PKA

protein kinase A

PKCβII

protein kinase C βII

Syk

spleen tyrosine kinase

1DE

one-dimensional electrophoresis

2DE

two-dimensional electrophoresis

MS

mass spectrometry

RT

room temperature

F-actin

filamentous actin