Cyclin-dependent kinase 5 regulates degranulation in human eosinophils
Introduction
The secretion of cationic proteins from eosinophil crystalloid granules is thought to be an important contributing factor to bronchial epithelial damage associated with allergic asthma.12 Indeed, studies using a transgenic mouse model co-expressing interleukin-5 and eotaxin-2 showed that eosinophil degranulation is a crucial component of allergic asthma.2 Eosinophil numbers, as well as their degranulation and release of eosinophil granule products in the airways, broadly correlate with the severity of asthma in human patients.34 The mechanisms underlying this secretory process are not fully understood.
Evidence from a range of secretory cells, including neuronal cells, suggests that exocytosis involves docking of intracellular secretory granules to the inner leaflet of the plasma membrane before fusion and secretion of their contents via SNAREs [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors].5 The SNARE hypothesis proposes a requirement for specific interactions between proteins located on the membranes of transported intracellular vesicular compartments (R-SNAREs, formerly known as v-SNAREs) and those on the target membrane (Q-SNAREs, formerly t-SNAREs) before membrane fusion.5
Eosinophils express several members of the SNARE family, including VAMP-2, -7 and -8, as well as syntaxin-4 and SNAP-23.68 We previously demonstrated that regulated release of stored mediators from the crystalloid granules and secretory vesicles of eosinophils depends on these SNARE proteins.6 Additionally, the upstream sequence of intracellular events leading to SNARE-mediated membrane fusion depends on other proteins capable of interacting with R- and Q-SNAREs, either directly or indirectly. Among other factors, an indirect regulator of SNARE function has been found to be required for insulin secretion from pancreatic β cells: cyclin-dependent kinase 5 (Cdk5), a proline-directed serine-threonine kinase.910
Cyclin-dependent kinases (Cdk) are cell cycle control proteins activated by cyclins.11 The Cdk family has more than 20 members, with distinct functions and interactions with effector proteins. Unlike other family members, however, Cdk5 is neither involved in cell-cycle regulation nor controlled by cell-cycle-associated cyclins.12 Although Cdk5 is ubiquitously expressed in mammalian cells, activation of Cdk5 is regulated by non-cyclin subunits, p35 and p39, found mainly in post-mitotic neurons.13 However, recent studies have associated Cdk5 activation with various functions in non-neuronal cells,14 including the control of the interaction of another family of proteins, Sec-1/Munc-18, with Q-SNAREs on the plasma membrane.910
The Sec-1/Munc-18, or SM, proteins are central and indispensable factors in intracellular vesicle trafficking and membrane fusion in a wide range of species and cell types.1519 In mammals, seven SM proteins have been identified. However, only three isoforms, Munc18a, -b, and -c (also known as Munc18-1, -2 and -3) are involved in exocytosis at the plasma membrane.20 While Munc18a is primarily expressed in neurons and neuroendocrine cells, Munc18b and Munc18c are more widely expressed.21 In addition, Munc18a and Munc18b bind to syntaxins 1–3. However, only Munc18c binds to syntaxin-4 with high affinity.22 In resting neurons, Munc18 maintains syntaxin-4 in a closed conformation, so preventing interaction with R-SNAREs.23 Following cellular activation, however, Cdk5 phosphorylates Munc18 to remove this inhibitory effect on Q-SNAREs and promote interaction with R-SNAREs, leading to exocytosis.24
In inflammatory cells, Cdk5 activity has been identified in monocytes and neutrophils14 and was shown to be associated with the differentiation of the HL-60 cell line to monocytes.25 In contrast, a recent study ascribed a role to Cdk5 in mediator release from GTP-γ-S-stimulated, permeabilized neutrophils.26 We therefore hypothesized that Cdk5 and its activators, p35 and p39, are expressed and actively involved in the release of granule products from human peripheral blood eosinophils through phosphorylation of Munc18c.
Materials and methods
Materials and antibodies
Anti-human Cdk5 (C8, DC17 and J3), anti-phospho Cdk5 (pCdk5, ser159), anti-p35 (C19), anti-p39 (C20 and N16) and anti-Unc18-c (Munc18c) antibodies were obtained from Santa Cruz Biotechnology Inc., (Dallas, TX). RPMI and chemicals were from Sigma (Mississauga, ON, Canada). Eotaxin/CCL11, platelet-activating factor (PAF), interleukin-3, interleukin-5, and granulocyte–macrophage colony stimulating factor were from R&D Systems (Minneapolis, MN). Omnia kinase assay kit was obtained from Invitrogen (Burlington, ON, Canada).
Eosinophil isolation
Blood samples (100 ml) were collected from both atopic and non-atopic donors according to the protocol approved by the University of Alberta Health Sciences Research Ethics Committee. Eosinophil purification from healthy donors with eosinophil counts ranging from 1% to 5% was achieved by negative selection using an AutoMACS system (Miltenyi Biotec, Gladbach, Germany) as described previously.27
Reverse transcription-polymerase chain reaction
Total RNA was extracted from eosinophils using an RNeasy Mini kit (Qiagen, Mississauga, ON, Canada), as described by the manufacturer. RT-PCR for Munc18c detection was carried out using the SuperScript RT-PCR system (Invitrogen) with intron-spanning primers 5′-TGATTCGTAA CTGGAGCCAC-3 (forward) and 5′-TTTCTGGCGAGCACTCACA-3′ (reverse).28 Product amplicon (235 bp) was gel-purified and sequenced in both directions to confirm its identity as well as primer specificity.
HL-60 clone 15 cell culture and differentiation into an eosinophilic phenotype
HL-60 clone 15 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to recommended protocols (using sterile RPMI-1640 supplemented with 25 mm HEPES, 20% heat-inactivated fetal bovine serum, 2 mm l-glutamine and 100 U/ml penicillin and streptomycin). Cells were maintained at 37° in a humidified incubator with 5% CO2 and passaged no more than 10 times for experiments. For differentiation into an eosinophil-like phenotype, HL-60 clone 15 cells were treated with 500 μm sodium butyrate for 5 days with media replenishment, containing fresh sodium butyrate at 500 μm, at day 2–3.2930
Western blot analyses
We used whole cell lysates or proteins immunoprecipitated using Sepharose A/G-conjugated antibodies for Western blot assays. Antibody reactivity was detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence.
Measurement of eosinophil peroxidase release
The release of eosinophil peroxidase (EPX) by eosinophils treated with secretory IgA beads or PMA was measured as described previously.31
Determination of Cdk5 phosphorylation using flow cytometry
Levels of phosphorylated Cdk5 (pCdk5) in eosinophils following activation were determined using a modification of the BD Phosflow protocol (BD Biosciences, Mississauga, ON, Canada). Briefly, eosinophils were activated as described previously,31 and then fixed for 15 min on ice, by adding an equal volume of 5% paraformaldehyde. After pelleting, cells were permeabilized by washing three times with 1 ml of BD Phosflow Perm/Wash Buffer 1 and centrifuging for 10 min at 300 g. Cells were then resuspended at 1 × 10 cells/ml in permeabilization buffer, and 100 μl (1 × 10 cells) was distributed to each flow cytometry tube for staining. Primary antibody (anti-pCdk5) or rabbit IgG (control) was added to a final concentration of 2 μg/ml and incubated for 30 min at room temperature. After three rounds of washing with permeabilization buffer, secondary fluorochrome-conjugated (Alexa-488) mouse anti-rabbit antibody was added, followed by incubation at room temperature for 30 min. Flow cytometric events were acquired using FACsCalibur (BD Biosciences).
Detection of phosphorylated Munc18c
Total phosphorylated protein was extracted from eosinophil lysates using the PhosphoProtein Purification Kit (Qiagen) according to manufacturers’ instructions. Briefly, 1 × 10 cells/ml were suspended in colour-free RPMI supplemented with 0·5% BSA. Cells were then incubated for 15 min at 37° in an Eppendorf tube previously coated with 1% human serum albumin to prevent eosinophil adhesion and activation. Following the addition of secretory-IgA-coupled beads, cells were activated for 15 min at 37°. The reaction was stopped by rapid chilling on ice and pelletting by centrifugation at 300 g for 10 min. The pellet was resuspended in 5 ml PhosphoProtein Lysis Buffer containing CHAPS, protease inhibitors and Benzonase (Qiagen), followed by a 30-min incubation at 4°. The protein concentration of the lysates was measured and adjusted to 0·1 mg/ml before using the PhosphoProtein purification column (Qiagen).
Small interfering RNA-mediated knockdown of Cdk5
A pool of small interfering RNA (siRNA; SMARTPOOL) targeting human Cdk5 (M-003239-01) and the non-targeting control (D-001210-01) were obtained from Dharmacon (Lafayette, CO) and transfected into eosinophils using RNAiFect transfection reagents (Qiagen). Following siRNA treatment, the cells were cultured for 24 hr at 37° in medium to which 10 pg granulocyte–macrophage colony stimulating factor per 1 × 10 cells had been added to preserve eosinophil viability. Transfection efficiency was measured by flow cytometry using fluorescently labelled siRNA control (siGLO; Thermo Scientific, Ottawa, ON, Canada). Western blotting for Cdk5 with mouse monoclonal anti-Cdk5 antibodies (DC17 and J3) was used to confirm Cdk5 knockdown. Secretion assays to determine the effect of Cdk5 knockdown on secretory IgA-induced EPX secretion were performed after 48 hr of culture, with silencing or control RNA.
Statistical analyses
Multiple comparisons of treatment and control groups were made using analysis of variance followed by Dunnett's post hoc analysis (comparison to control) or Tukey's method (pairwise comparisons) as executed with the S-PLUS programming language (Insightful Corporation, Seattle, WA). For dose–response analysis, two-way analysis of variance with Bonferroni post hoc test was selected (prism software; GraphPad, La Jolla, CA). For the analysis of the EPX release data in the siRNA experiments, Student's t-test was used. A P value < 0·05 was considered statistically significant.
Materials and antibodies
Anti-human Cdk5 (C8, DC17 and J3), anti-phospho Cdk5 (pCdk5, ser159), anti-p35 (C19), anti-p39 (C20 and N16) and anti-Unc18-c (Munc18c) antibodies were obtained from Santa Cruz Biotechnology Inc., (Dallas, TX). RPMI and chemicals were from Sigma (Mississauga, ON, Canada). Eotaxin/CCL11, platelet-activating factor (PAF), interleukin-3, interleukin-5, and granulocyte–macrophage colony stimulating factor were from R&D Systems (Minneapolis, MN). Omnia kinase assay kit was obtained from Invitrogen (Burlington, ON, Canada).
Eosinophil isolation
Blood samples (100 ml) were collected from both atopic and non-atopic donors according to the protocol approved by the University of Alberta Health Sciences Research Ethics Committee. Eosinophil purification from healthy donors with eosinophil counts ranging from 1% to 5% was achieved by negative selection using an AutoMACS system (Miltenyi Biotec, Gladbach, Germany) as described previously.27
Reverse transcription-polymerase chain reaction
Total RNA was extracted from eosinophils using an RNeasy Mini kit (Qiagen, Mississauga, ON, Canada), as described by the manufacturer. RT-PCR for Munc18c detection was carried out using the SuperScript RT-PCR system (Invitrogen) with intron-spanning primers 5′-TGATTCGTAA CTGGAGCCAC-3 (forward) and 5′-TTTCTGGCGAGCACTCACA-3′ (reverse).28 Product amplicon (235 bp) was gel-purified and sequenced in both directions to confirm its identity as well as primer specificity.
HL-60 clone 15 cell culture and differentiation into an eosinophilic phenotype
HL-60 clone 15 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to recommended protocols (using sterile RPMI-1640 supplemented with 25 mm HEPES, 20% heat-inactivated fetal bovine serum, 2 mm l-glutamine and 100 U/ml penicillin and streptomycin). Cells were maintained at 37° in a humidified incubator with 5% CO2 and passaged no more than 10 times for experiments. For differentiation into an eosinophil-like phenotype, HL-60 clone 15 cells were treated with 500 μm sodium butyrate for 5 days with media replenishment, containing fresh sodium butyrate at 500 μm, at day 2–3.2930
Western blot analyses
We used whole cell lysates or proteins immunoprecipitated using Sepharose A/G-conjugated antibodies for Western blot assays. Antibody reactivity was detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence.
Measurement of eosinophil peroxidase release
The release of eosinophil peroxidase (EPX) by eosinophils treated with secretory IgA beads or PMA was measured as described previously.31
Determination of Cdk5 phosphorylation using flow cytometry
Levels of phosphorylated Cdk5 (pCdk5) in eosinophils following activation were determined using a modification of the BD Phosflow protocol (BD Biosciences, Mississauga, ON, Canada). Briefly, eosinophils were activated as described previously,31 and then fixed for 15 min on ice, by adding an equal volume of 5% paraformaldehyde. After pelleting, cells were permeabilized by washing three times with 1 ml of BD Phosflow Perm/Wash Buffer 1 and centrifuging for 10 min at 300 g. Cells were then resuspended at 1 × 10 cells/ml in permeabilization buffer, and 100 μl (1 × 10 cells) was distributed to each flow cytometry tube for staining. Primary antibody (anti-pCdk5) or rabbit IgG (control) was added to a final concentration of 2 μg/ml and incubated for 30 min at room temperature. After three rounds of washing with permeabilization buffer, secondary fluorochrome-conjugated (Alexa-488) mouse anti-rabbit antibody was added, followed by incubation at room temperature for 30 min. Flow cytometric events were acquired using FACsCalibur (BD Biosciences).
Detection of phosphorylated Munc18c
Total phosphorylated protein was extracted from eosinophil lysates using the PhosphoProtein Purification Kit (Qiagen) according to manufacturers’ instructions. Briefly, 1 × 10 cells/ml were suspended in colour-free RPMI supplemented with 0·5% BSA. Cells were then incubated for 15 min at 37° in an Eppendorf tube previously coated with 1% human serum albumin to prevent eosinophil adhesion and activation. Following the addition of secretory-IgA-coupled beads, cells were activated for 15 min at 37°. The reaction was stopped by rapid chilling on ice and pelletting by centrifugation at 300 g for 10 min. The pellet was resuspended in 5 ml PhosphoProtein Lysis Buffer containing CHAPS, protease inhibitors and Benzonase (Qiagen), followed by a 30-min incubation at 4°. The protein concentration of the lysates was measured and adjusted to 0·1 mg/ml before using the PhosphoProtein purification column (Qiagen).
Small interfering RNA-mediated knockdown of Cdk5
A pool of small interfering RNA (siRNA; SMARTPOOL) targeting human Cdk5 (M-003239-01) and the non-targeting control (D-001210-01) were obtained from Dharmacon (Lafayette, CO) and transfected into eosinophils using RNAiFect transfection reagents (Qiagen). Following siRNA treatment, the cells were cultured for 24 hr at 37° in medium to which 10 pg granulocyte–macrophage colony stimulating factor per 1 × 10 cells had been added to preserve eosinophil viability. Transfection efficiency was measured by flow cytometry using fluorescently labelled siRNA control (siGLO; Thermo Scientific, Ottawa, ON, Canada). Western blotting for Cdk5 with mouse monoclonal anti-Cdk5 antibodies (DC17 and J3) was used to confirm Cdk5 knockdown. Secretion assays to determine the effect of Cdk5 knockdown on secretory IgA-induced EPX secretion were performed after 48 hr of culture, with silencing or control RNA.
Statistical analyses
Multiple comparisons of treatment and control groups were made using analysis of variance followed by Dunnett's post hoc analysis (comparison to control) or Tukey's method (pairwise comparisons) as executed with the S-PLUS programming language (Insightful Corporation, Seattle, WA). For dose–response analysis, two-way analysis of variance with Bonferroni post hoc test was selected (prism software; GraphPad, La Jolla, CA). For the analysis of the EPX release data in the siRNA experiments, Student's t-test was used. A P value < 0·05 was considered statistically significant.
Results
Human eosinophils express functionally active Cdk5
We confirmed the expression of Cdk5 in human eosinophils and eosinophil-differentiated HL-60 clone 15 cells (HL-60c15) by Western blot analysis, using a specific monoclonal antibody (Fig.(Fig.1a).1a). Human eosinophils and neutrophils expressed less Cdk5 than eosinophil-differentiated HL-60c15 cells or mouse brain lysate, based on relatively similar amounts loaded (indicated by the β-actin loading controls). In addition, human eosinophils express Munc18c, p35 and p39, although HL-60c15 cells appeared to express only Munc18c and p35.
Cyclin-dependent kinase 5 (Cdk5) is expressed and phosphorylated upon activation in human eosinophils. (a) Cdk5, Munc18c, p35, and p39 expression in whole cell lysates of human eosinophils and eosinophil-differentiated HL-60 clone 15 cells was confirmed using specific monoclonal antibodies on whole cell lysates (20 μg protein/lane). (b) Immunoprecipitation with Cdk5-specific polyclonal antibody followed by immunoblotting with anti-p39 demonstrates Cdk5 association with p39 in human eosinophils. (c) Confirmation of Cdk5 interaction with p35 and p39 by immunoprecipitation. Cell lysates were immunoprecipitated with p35 or p39, followed by immunoblotting with mouse monoclonal anti-Cdk5 antibody. Each figure is representative of three to five experiments.
To identify interactions with proposed effector molecules p35 and p39, Cdk5 was immunoprecipitated from human eosinophil and neutrophil lysates using a rabbit polyclonal anti-Cdk5 antibody. Using anti-p39 for immunoblotting, we demonstrated a physical association of p39 with Cdk5 (Fig.(Fig.1b).1b). Similarly, probing of immunoprecipitates obtained using p35 or p39, with mouse monoclonal anti-Cdk5 confirmed co-immunoprecipitation of these activators with Cdk5 (Fig.(Fig.1c1c).
In its inactive state, the catalytic site on Cdk proteins is obstructed by a loop structure, the T loop.32 This site is opened after activation and simultaneous phosphorylation of a threonine or serine residue (depending on the Cdk family member) on the T loop in position 159 of the protein;33 Cdk5 has a serine in position 159. To investigate whether Cdk5 was activated in eosinophils, we used flow cytometry to determine phosphorylation on the serine in position 159 of Cdk5, using an antibody specific for this phosphorylated form. Our data showed a rapid serine-159 phosphorylation of Cdk5 following activation with calcium ionophore A23187 (5 μm), platelet-activating factor (PAF, 1 μm) or eotaxin/CCL11 (10 ng/ml, Fig.Fig.2a2a–c).
Cyclin-dependent kinase 5 (Cdk5) is activated by physiological stimulation in human eosinophils. Flow cytometric analysis using anti-phospho-Cdk5 antibody (anti-pCdk5) showed phosphorylation of Cdk5 induced by (a) calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187, 5 μm), (b) platelet-activating factor (PAF; 1 μm), or (c) eotaxin/CCL11 (10 ng/ml), resulting in phosphorylation of Cdk5 on the serine residue. Representative flow cytometry histograms out of three individual experiments are shown for each condition. (d) Following activation with eotaxin/CCL11 (10 ng/ml) or PAF (1 μm) for 10 min, Cdk5 was immunoprecipitated from cell lysates and assayed in a fluorescence-based Omnia Kinase assay kit containing a Cdk5-specific substrate. The plot shows the kinetic analysis of Cdk5 kinase activity of PAF-activated (open circles), eotaxin/CCL11-treated (closed triangles) and unstimulated (closed circles) cells, measured as fluorescence units. The positive control (inverted open triangles) consisted of recombinant Cdk5 supplied by the manufacturer. Representative data shown from three separate experiments.
To confirm activation of Cdk5, we used an Omnia Kinase assay, employing a substrate specific for Cdk5. Kinase activity was determined in eosinophils activated for 10 min by eotaxin/CCL11 (10 ng/ml) or PAF (1 μm) by subjecting lysates to Cdk5 immunoprecipitation with an anti-Cdk5 antibody (Fig.(Fig.2d).2d). The positive control consisted of recombinant Cdk5 supplied by the manufacturer, which was spiked into the buffer and then treated in the same manner as the protein extracts. Our results showed that activation of eosinophils by eotaxin/CCL11 or PAF is accompanied by significant Cdk5 kinase activity (P < 0·001).
Human eosinophils express Munc18c that is bound to Cdk5 during stimulation
Cdk5 functions in exocytosis by phosphorylating SM proteins bound to SNARE proteins on the plasma membrane, which in turn makes Q-SNAREs accessible to R-SNAREs. While Munc18a and Munc18b bind to syntaxins 1–3, Munc18c binds only to syntaxin-4. As we had previously identified syntaxin-4 as an essential component in eosinophil exocytosis,8 we probed for the expression of Munc18c using RT-PCR and Western blotting. Expression of both mRNA and protein for Munc18c was detected in human peripheral blood eosinophils (Figs(Figs11 and and3a).3a). Munc18c was detected by Western blot in immunoprecipitation experiments using an anti-Cdk5 antibody with protein extracts from HL-60 cells differentiated to an eosinophilic phenotype that were activated with PMA (Fig.(Fig.3b),3b), suggesting a direct interaction between Cdk5 and Munc18c. Furthermore, the association of Munc18c with Cdk5 appears to be activation-dependent, as longer PMA activation of eosinophils resulted in increased Munc18c protein in Cdk5 immunoprecipitates.
Munc18c is expressed in eosinophils and phosphorylated following activation. (a) Munc18 is expressed in eosinophils at mRNA level. Using specific intron-spanning primers, we amplified a fragment of Munc18c from human eosinophils. The identity of the amplicon was confirmed by sequencing. Mouse embryo 3T3-L1 cells and neutrophils were used as positive controls. (b) Top, Western blot to detect Munc18c in cyclin-dependent kinase 5 (Cdk5) immunoprecipitates of HL-60 cells differentiated to an eosinophilic phenotype, and activated with PMA for 0 (unstimulated), 5, 10 or 15 min; immunoprecipitation using an isotype control, instead of the anti-Cdk antibody, is also shown (IP isotype); fluorescence of protein bands was measured for 5, 10 and 15 min (middle) and ratios were calculated against total immunoprecipitated Cdk5; bottom, a Western blot of the same samples using anti-Cdk5 antibody as a loading control.
Cdk5 regulates eosinophil degranulation using pharmacological inhibition
Eosinophil degranulation, indicated by the release of EPX occurs by exocytosis in response to secretagogue stimulation. To understand the role of Cdk5 in receptor-mediated degranulation, we tested the effect of chemical inhibitors of Cdk5 on EPX secretion. However, as there are no existing Cdk5-specific inhibitors, we used roscovitine and AT7519, which are broad inhibitors of cyclin-dependent kinases, including Cdk5. We used human peripheral blood eosinophils as well as eosinophil-differentiated HL-60 clone 15 cells, which release EPX in response to phorbol ester (PMA) stimulation. We used PMA as a secretagogue for HL-60c15 cells because physiological stimuli (PAF, eotaxin/CCL11, and secretory IgA) failed to evoke significant EPX release from HL-60c15 cells, and PMA was a potent inducer of EPX release from both eosinophils and HL-60c15 cells (data not shown). Preliminary cytotoxicity assays demonstrated that roscovitine or AT7519 treatment (10–30 μm) for 30 min affected neither mitochondrial membrane potential nor surface binding of annexin V/propidium iodide uptake (by flow cytometry) on eosinophils or HL-60c15 cells (with viability > 95%), suggesting that cell viability was not affected during the short 30-min incubation period with inhibitors (data not shown).
Using HL-60c15 cells, a 30-min pre-incubation of eosinophils with roscovitine (30 μm) led to a significant reduction in EPX release after stimulation with PMA, compared with untreated (control) cells (Fig.(Fig.4a).4a). We confirmed such roscovitine-sensitive EPX release using freshly isolated human peripheral blood eosinophils stimulated with either PMA (Fig.(Fig.4b)4b) or the physiological stimulus, secretory IgA (Fig.(Fig.4c).4c). To further establish a role for Cdk in eosinophil degranulation, we used another Cdk-specific inhibitor, AT7519 (Fig.(Fig.4d4d),34 and found similar inhibition of degranulation in eosinophils at a lower dose (10 μm). These findings suggest that Cdk contributes to exocytosis in eosinophils.
Cyclin-dependent kinase 5 (Cdk5) regulates agonist-induced eosinophil peroxidase (EPX) release from human eosinophils. HL-60c15 cells and human peripheral blood eosinophils were subjected to agonist stimulation with PMA or secretory IgA (sIgA). (a) Treatment with the Cdk inhibitor roscovitine (30 μm) resulted in decreased EPX release when differentiated HL-60 cells were stimulated with PMA. Human eosinophils stimulated with either PMA (b) or sIgA-coated beads (c) showed similar results. (d) AT7519 (10 and 30 μm), another Cdk-specific inhibitor, also inhibited PMA-induced EPX release. (e) Transfection of eosinophils with a pool of Cdk5 small interfering RNA (siRNA) resulted in knockdown of Cdk5 protein expression. Briefly, eosinophils were cultured for 48 hr in the presence of granulocyte–macrophage colony stimulating factor, without treatment (lanes 1 and 2), or after being treated with transfection reagent without siRNA (lane 3) or with 20 μm (lane 4) and 40 μm (lane 5) of Cdk5 siRNA pool. Lane 6 corresponds to cells transfected with a control RISK-free non-targeting siRNA (Dharmacon). A representative result of three successful transfections is shown. (f) EPX release was reduced in cells treated with Cdk5 siRNA, compared with those treated with a non-targeting siRNA pool (siControl). Data were analysed by two-way analysis of variance with Bonferroni post hoc testing (a, c and d), comparing values to matching control (n = 7); one-way analysis of variance (b) with Tukey's post hoc testing, comparing to control (n = 3); and Student's t-test (f, n = 5,). *P < 0·05, **P < 0·01, ***P < 0·001.
However, these two inhibitors are broadly specific for several Cdk isoforms, and do not target Cdk5 activity alone. We sought to determine the specific role of Cdk5 in exocytosis by inhibiting the expression of Cdk5 using Cdk5-specific siRNA. We obtained a transfection efficiency of approximately 50% in peripheral blood eosinophils in five donor eosinophil samples, identified after 24 hr of incubation with fluorescence-labelled siRNA. In Cdk5 siRNA-treated samples, we observed reduced levels of Cdk5 expression, as indicated by the ratios of Cdk5/β-actin band intensities (Fig.(Fig.4e).4e). Treatment with Cdk siRNA resulted in a significant reduction of EPX release following secretory IgA stimulation of eosinophils (Fig.(Fig.4f,4f, P < 0·05).
Human eosinophils express functionally active Cdk5
We confirmed the expression of Cdk5 in human eosinophils and eosinophil-differentiated HL-60 clone 15 cells (HL-60c15) by Western blot analysis, using a specific monoclonal antibody (Fig.(Fig.1a).1a). Human eosinophils and neutrophils expressed less Cdk5 than eosinophil-differentiated HL-60c15 cells or mouse brain lysate, based on relatively similar amounts loaded (indicated by the β-actin loading controls). In addition, human eosinophils express Munc18c, p35 and p39, although HL-60c15 cells appeared to express only Munc18c and p35.
Cyclin-dependent kinase 5 (Cdk5) is expressed and phosphorylated upon activation in human eosinophils. (a) Cdk5, Munc18c, p35, and p39 expression in whole cell lysates of human eosinophils and eosinophil-differentiated HL-60 clone 15 cells was confirmed using specific monoclonal antibodies on whole cell lysates (20 μg protein/lane). (b) Immunoprecipitation with Cdk5-specific polyclonal antibody followed by immunoblotting with anti-p39 demonstrates Cdk5 association with p39 in human eosinophils. (c) Confirmation of Cdk5 interaction with p35 and p39 by immunoprecipitation. Cell lysates were immunoprecipitated with p35 or p39, followed by immunoblotting with mouse monoclonal anti-Cdk5 antibody. Each figure is representative of three to five experiments.
To identify interactions with proposed effector molecules p35 and p39, Cdk5 was immunoprecipitated from human eosinophil and neutrophil lysates using a rabbit polyclonal anti-Cdk5 antibody. Using anti-p39 for immunoblotting, we demonstrated a physical association of p39 with Cdk5 (Fig.(Fig.1b).1b). Similarly, probing of immunoprecipitates obtained using p35 or p39, with mouse monoclonal anti-Cdk5 confirmed co-immunoprecipitation of these activators with Cdk5 (Fig.(Fig.1c1c).
In its inactive state, the catalytic site on Cdk proteins is obstructed by a loop structure, the T loop.32 This site is opened after activation and simultaneous phosphorylation of a threonine or serine residue (depending on the Cdk family member) on the T loop in position 159 of the protein;33 Cdk5 has a serine in position 159. To investigate whether Cdk5 was activated in eosinophils, we used flow cytometry to determine phosphorylation on the serine in position 159 of Cdk5, using an antibody specific for this phosphorylated form. Our data showed a rapid serine-159 phosphorylation of Cdk5 following activation with calcium ionophore A23187 (5 μm), platelet-activating factor (PAF, 1 μm) or eotaxin/CCL11 (10 ng/ml, Fig.Fig.2a2a–c).
Cyclin-dependent kinase 5 (Cdk5) is activated by physiological stimulation in human eosinophils. Flow cytometric analysis using anti-phospho-Cdk5 antibody (anti-pCdk5) showed phosphorylation of Cdk5 induced by (a) calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187, 5 μm), (b) platelet-activating factor (PAF; 1 μm), or (c) eotaxin/CCL11 (10 ng/ml), resulting in phosphorylation of Cdk5 on the serine residue. Representative flow cytometry histograms out of three individual experiments are shown for each condition. (d) Following activation with eotaxin/CCL11 (10 ng/ml) or PAF (1 μm) for 10 min, Cdk5 was immunoprecipitated from cell lysates and assayed in a fluorescence-based Omnia Kinase assay kit containing a Cdk5-specific substrate. The plot shows the kinetic analysis of Cdk5 kinase activity of PAF-activated (open circles), eotaxin/CCL11-treated (closed triangles) and unstimulated (closed circles) cells, measured as fluorescence units. The positive control (inverted open triangles) consisted of recombinant Cdk5 supplied by the manufacturer. Representative data shown from three separate experiments.
To confirm activation of Cdk5, we used an Omnia Kinase assay, employing a substrate specific for Cdk5. Kinase activity was determined in eosinophils activated for 10 min by eotaxin/CCL11 (10 ng/ml) or PAF (1 μm) by subjecting lysates to Cdk5 immunoprecipitation with an anti-Cdk5 antibody (Fig.(Fig.2d).2d). The positive control consisted of recombinant Cdk5 supplied by the manufacturer, which was spiked into the buffer and then treated in the same manner as the protein extracts. Our results showed that activation of eosinophils by eotaxin/CCL11 or PAF is accompanied by significant Cdk5 kinase activity (P < 0·001).
Human eosinophils express Munc18c that is bound to Cdk5 during stimulation
Cdk5 functions in exocytosis by phosphorylating SM proteins bound to SNARE proteins on the plasma membrane, which in turn makes Q-SNAREs accessible to R-SNAREs. While Munc18a and Munc18b bind to syntaxins 1–3, Munc18c binds only to syntaxin-4. As we had previously identified syntaxin-4 as an essential component in eosinophil exocytosis,8 we probed for the expression of Munc18c using RT-PCR and Western blotting. Expression of both mRNA and protein for Munc18c was detected in human peripheral blood eosinophils (Figs(Figs11 and and3a).3a). Munc18c was detected by Western blot in immunoprecipitation experiments using an anti-Cdk5 antibody with protein extracts from HL-60 cells differentiated to an eosinophilic phenotype that were activated with PMA (Fig.(Fig.3b),3b), suggesting a direct interaction between Cdk5 and Munc18c. Furthermore, the association of Munc18c with Cdk5 appears to be activation-dependent, as longer PMA activation of eosinophils resulted in increased Munc18c protein in Cdk5 immunoprecipitates.
Munc18c is expressed in eosinophils and phosphorylated following activation. (a) Munc18 is expressed in eosinophils at mRNA level. Using specific intron-spanning primers, we amplified a fragment of Munc18c from human eosinophils. The identity of the amplicon was confirmed by sequencing. Mouse embryo 3T3-L1 cells and neutrophils were used as positive controls. (b) Top, Western blot to detect Munc18c in cyclin-dependent kinase 5 (Cdk5) immunoprecipitates of HL-60 cells differentiated to an eosinophilic phenotype, and activated with PMA for 0 (unstimulated), 5, 10 or 15 min; immunoprecipitation using an isotype control, instead of the anti-Cdk antibody, is also shown (IP isotype); fluorescence of protein bands was measured for 5, 10 and 15 min (middle) and ratios were calculated against total immunoprecipitated Cdk5; bottom, a Western blot of the same samples using anti-Cdk5 antibody as a loading control.
Cdk5 regulates eosinophil degranulation using pharmacological inhibition
Eosinophil degranulation, indicated by the release of EPX occurs by exocytosis in response to secretagogue stimulation. To understand the role of Cdk5 in receptor-mediated degranulation, we tested the effect of chemical inhibitors of Cdk5 on EPX secretion. However, as there are no existing Cdk5-specific inhibitors, we used roscovitine and AT7519, which are broad inhibitors of cyclin-dependent kinases, including Cdk5. We used human peripheral blood eosinophils as well as eosinophil-differentiated HL-60 clone 15 cells, which release EPX in response to phorbol ester (PMA) stimulation. We used PMA as a secretagogue for HL-60c15 cells because physiological stimuli (PAF, eotaxin/CCL11, and secretory IgA) failed to evoke significant EPX release from HL-60c15 cells, and PMA was a potent inducer of EPX release from both eosinophils and HL-60c15 cells (data not shown). Preliminary cytotoxicity assays demonstrated that roscovitine or AT7519 treatment (10–30 μm) for 30 min affected neither mitochondrial membrane potential nor surface binding of annexin V/propidium iodide uptake (by flow cytometry) on eosinophils or HL-60c15 cells (with viability > 95%), suggesting that cell viability was not affected during the short 30-min incubation period with inhibitors (data not shown).
Using HL-60c15 cells, a 30-min pre-incubation of eosinophils with roscovitine (30 μm) led to a significant reduction in EPX release after stimulation with PMA, compared with untreated (control) cells (Fig.(Fig.4a).4a). We confirmed such roscovitine-sensitive EPX release using freshly isolated human peripheral blood eosinophils stimulated with either PMA (Fig.(Fig.4b)4b) or the physiological stimulus, secretory IgA (Fig.(Fig.4c).4c). To further establish a role for Cdk in eosinophil degranulation, we used another Cdk-specific inhibitor, AT7519 (Fig.(Fig.4d4d),34 and found similar inhibition of degranulation in eosinophils at a lower dose (10 μm). These findings suggest that Cdk contributes to exocytosis in eosinophils.
Cyclin-dependent kinase 5 (Cdk5) regulates agonist-induced eosinophil peroxidase (EPX) release from human eosinophils. HL-60c15 cells and human peripheral blood eosinophils were subjected to agonist stimulation with PMA or secretory IgA (sIgA). (a) Treatment with the Cdk inhibitor roscovitine (30 μm) resulted in decreased EPX release when differentiated HL-60 cells were stimulated with PMA. Human eosinophils stimulated with either PMA (b) or sIgA-coated beads (c) showed similar results. (d) AT7519 (10 and 30 μm), another Cdk-specific inhibitor, also inhibited PMA-induced EPX release. (e) Transfection of eosinophils with a pool of Cdk5 small interfering RNA (siRNA) resulted in knockdown of Cdk5 protein expression. Briefly, eosinophils were cultured for 48 hr in the presence of granulocyte–macrophage colony stimulating factor, without treatment (lanes 1 and 2), or after being treated with transfection reagent without siRNA (lane 3) or with 20 μm (lane 4) and 40 μm (lane 5) of Cdk5 siRNA pool. Lane 6 corresponds to cells transfected with a control RISK-free non-targeting siRNA (Dharmacon). A representative result of three successful transfections is shown. (f) EPX release was reduced in cells treated with Cdk5 siRNA, compared with those treated with a non-targeting siRNA pool (siControl). Data were analysed by two-way analysis of variance with Bonferroni post hoc testing (a, c and d), comparing values to matching control (n = 7); one-way analysis of variance (b) with Tukey's post hoc testing, comparing to control (n = 3); and Student's t-test (f, n = 5,). *P < 0·05, **P < 0·01, ***P < 0·001.
However, these two inhibitors are broadly specific for several Cdk isoforms, and do not target Cdk5 activity alone. We sought to determine the specific role of Cdk5 in exocytosis by inhibiting the expression of Cdk5 using Cdk5-specific siRNA. We obtained a transfection efficiency of approximately 50% in peripheral blood eosinophils in five donor eosinophil samples, identified after 24 hr of incubation with fluorescence-labelled siRNA. In Cdk5 siRNA-treated samples, we observed reduced levels of Cdk5 expression, as indicated by the ratios of Cdk5/β-actin band intensities (Fig.(Fig.4e).4e). Treatment with Cdk siRNA resulted in a significant reduction of EPX release following secretory IgA stimulation of eosinophils (Fig.(Fig.4f,4f, P < 0·05).
Discussion
Our study shows for the first time that Cdk5 contributes to human eosinophil degranulation. Although association of p35 with Cdk5 is sufficient to activate its kinase activity,35in vitro studies showed this association would result in an extremely low catalytic rate.36 Full activation and physiological function of Cdk5 require phosphorylation of the serine residue on the T loop (Ser-159)36 by the more potent activator p25, product of calpain-mediated cleavage of p35.37 We demonstrated not only the association of Cdk5 in eosinophils with its effector molecules, p35 and p39, but also the specific phosphorylation of Cdk5 on Ser-159 following activation. The functional importance of this observation in eosinophil exocytosis was further confirmed by the increase in kinase activity of Cdk5 in cells activated with the secretagogues, eotaxin/CCL11 and PAF. An increase in Cdk5 kinase activity following activation has previously been identified as a strong marker of Cdk5-mediated secretory events in neuronal cells.38
A major target of the kinase activity of Cdk5 is Munc18c, which in turn opens syntaxin-4 following cell activation to interact with R-SNAREs on granules.22 We detected the expression of Munc18c, the syntaxin-interacting protein known to maintain membrane-bound syntaxin-4 in a closed conformation in resting cells, in human eosinophils. We have previously shown that the interaction of the Q-SNARE syntaxin-4 on the plasma membrane with the R-SNAREs VAMP-7, on the large crystalloid granules, or VAMP-2, on small secretory vesicles, is crucial for membrane fusion and exocytosis in human eosinophils.68 We have now shown that Munc18c is not only present on the plasma membrane but also in enriched crystalloid granule fractions, and that Munc18c interacts with Cdk5 during cell activation. Hence, in human eosinophils, degranulation involves phosphorylation of Cdk5, which binds Munc18c on the plasma membrane, permitting the interaction of VAMP-2 or VAMP-7 with syntaxin-4, and leading to membrane fusion and mediator release.
We confirmed our model of Cdk5-Munc18c-SNARE-dependent exocytosis in human eosinophils by using pharmacological inhibitors. Our observation, based principally on the ability of roscovitine, AT7519 and Cdk5 siRNA to inhibit human blood eosinophil exocytosis, established a role for Cdk5 in exocytosis of EPX in eosinophils. Roscovitine has been shown to induce eosinophil apoptosis by inhibiting Cdk1, -2, -5, -7 and -9.3940 However, these studies indicated an absence of any significant apoptosis within the first 4 hr of incubation of human eosinophils with roscovitine. In the present work, we incubated Cdk inhibitors roscovitine and AT7519 with eosinophils for no more than 30 min, well before the apoptosis-inducing effects of these drugs. We found that the viability of eosinophils was not affected after 30 min of incubation with these inhibitors, and determined that eosinophil degranulation was significantly inhibited in the presence of roscovitine and AT7519.
Our attempts at knocking down Cdk5 expression using siRNA yielded diminished, but not abolished, expression of Cdk5 in transfected cells. This, we believe, resulted in a partial, but significant, decrease in EPX release in transfected cells. It is known that eosinophils are difficult to transfect successfully.4142 showing low transfection efficiency and probe degradation.43 Our transfection efficiency reached 50% in human peripheral blood eosinophils, suggesting that we were able to partially knock down Cdk5 expression, and that this led to significantly reduced EPX release.
We propose that Cdk5 is a critical element in the complex intracellular events regulating exocytosis of vesicular and granule mediator release, whether from eosinophils, pancreatic cells14 or permeabilized neutrophils.26 In our proposed model, stimulation of eosinophils leads to Ca influx, activation of calpain and cleavage of p35 into p25, with the subsequent phosphorylation of Cdk5; pCdk5 in turn phosphorylates Munc18c, releasing it from its binding to syntaxin-4, making the latter available for vesicle docking via VAMP-2 or VAMP-7 interactions. Hence, inhibition of Cdk5-dependent secretion may be a potential target for preventing the local release of mediators during chronic inflammation in diseases including allergy and atopic asthma. Interestingly, roscovitine is currently undergoing clinical trials for the treatment of cervical cancer targeting Cdk5.44 Our studies point to the possibility of extending this therapeutic strategy by applying Cdk5 inhibitors to the treatment of allergic airway disease.
Abstract
Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.
Acknowledgments
During the execution of the study, RM was an Alberta Heritage Medical Senior Investigator and supported by CIHR grant MOP-13441. RM and PL were supported by a CIHR grant MOP-89748. DJA was an Alberta Heritage Clinical Investigator.
Glossary
Abbreviations:
| Cdk | cyclin-dependent kinase |
| EPX | eosinophil peroxidase |
| Munc18 | syntaxin binding protein 3 (mammalian uncoordinated-18c protein homolog) |
| PAF | platelet-activating factor |
| siRNA | small interfering RNA |
| SM | Sec-1/Munc-18 |
| SNARE | soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor |
References
- 1. [PubMed]
- 2. [PubMed]
- 3. [PubMed]
- 4. [PubMed]
- 5. [PubMed]
- 6. [PubMed]
- 7. [PubMed]
- 8. [PubMed]
- 9. [PubMed]
- 10. [PubMed]
- 11. [PubMed]
- 12. [PubMed]
- 13. [PubMed]
- 14. [PubMed]
- 15.
- 16.
- 17.
- 18. [PubMed]
- 19. [PubMed]
- 20. [PubMed]
- 21.
- 22. [PubMed]
- 23. [PubMed]
- 24. [PubMed]
- 25. [PubMed]
- 26. [PubMed]
- 27. [PubMed]
- 28. [PubMed]
- 29.
- 30. [PubMed]
- 31. [PubMed]
- 32. [PubMed]
- 33. [PubMed]
- 34. [PubMed]
- 35. [PubMed]
- 36.
- 37. [PubMed]
- 38. [PubMed]
- 39. [PubMed]
- 40. [PubMed]
- 41. [PubMed]
- 42. [PubMed]
- 43. [PubMed]
- 44. [PubMed]