Ionomycin causes activation of p38 and p42/44 mitogen-activated protein kinases in human neutrophils

David J. Elzi1, A. Jason Bjornsen2, Todd MacKenzie2, Travis H. Wyman2, and Christopher C. Silliman1,2,3

1 Bonfils Blood Center, Denver 80230; and Departments of 2 Pediatrics and 3 Surgery, University of Colorado School of Medicine, Denver, Colorado 80262


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+ concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+ in the activation of p38 and p42/44 MAP kinases, we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+ that was due to both a release of cytosolic Ca2+ stores and Ca2+ influx. Ionomycin also activated (2 µM) and primed (20-200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2-2 µM. SB-203580, a p38 MAP kinase antagonist, inhibited ionomycin-induced activation of the oxidase (68 ± 8%, P < 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely, PD-98059, an inhibitor of MAP/extracellular signal-related kinase 1, had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+ completely inhibited ionomycin activation of p38 MAP kinase, whereas chelation of extracellular Ca2+ abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+ for MAP kinase activation in PMNs.

cytosolic calcium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLYMORPHONUCLEAR NEUTROPHILS (PMNs) comprise an essential component of host defense against microbial invaders (50, 56). PMNs circulate freely in the blood stream and exert their major microbicidal functions in the tissues. To perform these microbicidal functions, PMNs migrate to areas of infection by adhesion to the vascular endothelium, diapedesis through the endothelial monolayer, chemotaxis to the site of infection, phagocytosis of microbial invaders, and release of cytotoxic metabolites into the phagolysozome, resulting in killing of the microorganisms (2, 18, 22). Priming and activation of PMNs are distinct events important for normal PMN function. Priming results in both conformational changes of the beta 2-integrin adhesion molecules, e.g., CD11b/CD18, which increases their avidity for endothelial ligands, including intracellular adhesion molecule-1, and increases the surface expression of CD11b/CD18, which is important for chemotaxis (2, 22). PMN activation results in the release of maximal amounts of reactive oxygen metabolites and proteases for optimal antimicrobial function. The respiratory burst in PMNs describes the generation of superoxide anions and other toxic oxygen metabolites by the NADPH oxidase, whereby oxygen molecules are reduced to superoxide anions through the electron transfer from NADPH (11). Receptor-linked agents, such as platelet-activating factor (PAF) and leukotriene B4, can prime the NADPH oxidase, while chemoattractants such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8, and C5a can prime or activate the respiratory burst depending on their concentration (17, 31, 51, 55, 57).

Ca2+ ionophores are small hydrophobic molecules that intercalate into the lipid bilayer of the cell membrane and increase Ca2+ permeability. These increases in cytosolic Ca2+ concentration mediate many cellular functions, including priming and activation of the PMN oxidase (36). Ionomycin also modulates other PMN functions, including adhesion to endothelial cells, as well as eliciting changes in tyrosine protein phosphorylation (4, 17, 28). Indeed, changes in cytosolic Ca2+ play an integral role in receptor-mediated signaling in PMNs, and these events are the result of either the release of Ca2+ from intracellular stores (calciosomes) or influx of extracellular Ca2+, or both (3). Receptor-linked priming and activating agents, e.g., PAF and FMLP, cause rapid rises in cytosolic Ca2+ levels, and these changes in cytosolic Ca2+ are necessary for chemotaxis, protein tyrosine phosphorylation, and in the case of FMLP, oxidase assembly (14, 15, 20, 26, 37, 45). In addition, extracellular Ca2+ is also required for maximal activity of PMN priming agents, including PAF (15).

Protein phosphorylation is a prerequisite for many PMN signal transduction pathways, including those that are receptor linked. Many PMN agonists signal through a complex series of protein kinase cascades that include activation of mitogen-activated protein (MAP) kinases (6, 14, 16). p38 and p42/44 MAP kinases are members of distinct, parallel serine/threonine kinase pathways in PMNs that relay signals from a cell surface receptor to changes in cellular functions including transcription, chemotaxis, and phagocytosis (24, 32, 39, 47). Both PAF and FMLP cause activation of p38 and p42/44 MAP kinases in PMNs, and these MAP kinase pathways have been implicated in regulating multiple PMN functions, including oxidase activity and chemotaxis (24, 32, 39, 40, 47). It is important to note that activation of the p38 and p42/44 MAP kinases requires dual phosphorylation of discrete tyrosine and threonine residues (Thr-180/Tyr-182 and Thr-202/Tyr-204, respectively) (7, 27).

Changes in cytosolic Ca2+ concentration have been implicated in regulation of protein phosphorylation in PMNs, especially for receptor-linked priming and activation of the PMN oxidase by PAF or FMLP (14, 45). However, newer data have implied that intracellular Ca2+ is not sufficient or necessary for p38 MAP kinase phosphorylation or activity (32). Because ionomycin directly causes changes in cytosolic Ca2+ concentration, affects multiple PMN functions, and activates PMN signal transduction in a receptor-independent manner, we investigated ionomycin-induced changes in both p38 and p42/44 MAP kinase activity. We hypothesize that ionomycin activates MAP kinases directly through Ca2+ influx, and this Ca2+ influx and MAP kinase activity are important for ionomycin-induced priming and activation of the PMN oxidase.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. Cell isolation reagents, electrophoresis materials, ammonium persulfate, cytochrome c, superoxide dismutase, PAF, FMLP, phorbol 12-myristate 13-acetate (PMA), ionomycin, EGTA, leupeptin, phenylmethylsulfonyl fluoride (PMSF), p-nitrophenylphosphate, and sodium orthovanadate were obtained from Sigma Chemical (St. Louis, MO). PD-98059 and SB-203580 were obtained from Calbiochem (La Jolla, CA). Acrylamide, bisacrylamide, and N,N,N',N'-tetramethylenediamine were obtained from Bio-Rad (Hercules, CA). Dual phosphospecific antibodies to the p42/44 and p38 MAP kinases (Thr-202/Tyr-204), p38 (activating transcription factor-2 or ATF-2) and p42/44 (Elk-1) MAP kinase activity assay kits, and a horseradish peroxidase-linked goat anti-rabbit secondary antibody were obtained by Cell Signaling Technology (Beverly, MA). An antibody against phosphotyrosine and an enhanced chemiluminescence detection system were purchased from Zymed Laboratories (San Francisco, CA) and Amersham (Chicago, IL), respectively. Indo 1-acetoxymethyl ester (indo 1-AM), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and thapsigargin were obtained from Molecular Probes (Eugene, OR).

Neutrophil isolation. Whole blood was drawn from healthy human volunteers by venipuncture after obtaining informed consent via a protocol approved by the Colorado Multiple Institution Review Board. PMNs were isolated by dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic lysis as previously described (3). Cells were resuspended to a concentration of 2.5 × 107 cells/ml in Krebs-Ringer phosphate buffer with 2% dextrose and used immediately for all subsequent manipulations.

Superoxide anion measurement and cytosolic surements. The maximal rate of superoxide anion generation was measured by monitoring the superoxide dismutase inhibitable reduction of cytochrome c at 550 nm in a Molecular Devices microplate reader (Menlo Park, CA) as previously described (49). For measuring activation and priming of the PMN oxidase by ionomycin, PMNs were primed in wells for 5 min with 20 nM-20 µM ionomycin and subsequently activated with 1 µM FMLP. For MAP kinase inhibitor studies, PMNs were incubated for 30 min in the dark at 37°C with 0.1-10 µM PD-98059, an inhibitor of MAP/extracellular signal-related kinase (ERK) 1 (MEK1) (IC50 = 2 µM), the upstream kinase that phosphorylates p42/44 MAP kinase, and SB-203580, a p38 MAP kinase inhibitor (IC50 = 0.2-1.0 µM) that binds the p38 MAP kinase ATP pocket, or DMSO vehicle (9, 13, 52). In selected experiments, PMNs were loaded with 50 µM BAPTA, a chelator of cytosolic Ca2+ that is loaded intracellularly, or with DMSO vehicle for 30 min in the dark at 37°C with shaking. This concentration of BAPTA completely abrogated the intracellular Ca2+ flux, while lower BAPTA concentrations (1-25 µM) did not completely inhibit the cytosolic Ca2+ rise to either PAF (40 nM) or FMLP (1 µM) (results not shown). Moreover, in selected experiments, cells were pretreated in the plate for 2 min with 5 mM EGTA, a chelator of extracellular Ca2+. PMN cytosolic Ca2+ levels were determined by indo 1 loading of PMNs and analysis in a Perkin-Elmer LS50B spectrofluorimeter over real time (Perkin-Elmer, Norwalk, CT) as previously described employing the Grynkiewicz equation (15, 23).

MAP kinase immunoblotting. PMNs were pretreated in selected experiments with PD-98059, SB-203580, genistein (100 µM), a general tyrosine kinase inhibitor (54), BAPTA (50 µM), or DMSO control at 37°C as described above. The dose of genistein was determined by both examining inhibition of the FMLP-activated respiratory burst and tyrosine phosphorylation over a range of concentrations from 1-100 µM, compared with the inactive analog daidzein. Moreover, at the 100-µM concentration, genistein does not affect many Ser/Thr protein kinases (1). PMNs (1.25 × 106, 5 µg of protein) were stimulated with 2 nM-2 µM ionomycin, 2 µM PAF, 1 µM FMLP, or DMSO control for 30-300 s and immediately placed in Laemmli digestion buffer with freshly prepared protease inhibitors: 10 µg/ml leupeptin, 2 mM sodium orthovanadate, 10 mM p-nitrophenylphosphate, and 1 mM PMSF (34). It is important to note that the exact numbers of cell equivalents were added to each well. Samples were boiled for 15 min, and the proteins were separated by 10% SDS-PAGE overnight. Gels were subsequently immunoblotted onto nitrocellulose membranes using Towbin Tris-glycine transfer buffer at 4°C (53). Blots were blocked in 5% skim milk diluted in Tris-buffered saline with 0.1% Tween 20 (Tris buffer), incubated with either dual-phosphorylated p42/44 or p38 MAP kinase antibodies overnight, washed three times with Tris buffer, incubated with a horseradish peroxidase-linked goat anti-rabbit secondary antibody, and subsequently developed on X-ray film using an enhanced chemiluminescence detection system. Densitometry was performed using Scion Image version 4.0.2 downloaded from the National Institutes of Health website, and the densitometry values are expressed in arbitrary units.

MAP kinase activity assays. PMNs were isolated and stimulated for 60 s with differing concentrations (20 nM-2 µM) of ionomycin. The PMNs were lysed in a buffer (lysis buffer) containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerol phosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin. The lysate was sonicated four times for 5 s at 4°C, centrifuged at 15,000 g for 10 min at 4°C, and the supernatant was transferred to a fresh tube. The amount of protein was measured by a modified Lowry protein assay, and 200 µg of lysate were incubated with a 1:50 dilution of p38 or p42/44 MAP kinase antibody overnight with gentle rocking at 4°C. Protein A-Sepharose beads (20 µl) were added to the mixture and incubated for 3 h at 4°C. The mixture was centrifuged at 15,000 g for 30 s at 4°C, and the pellet was washed twice with lysis buffer followed by two washes with 25 mM Tris (pH 7.5), 5 mM beta -glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2 (kinase buffer). The pellet was resuspended in kinase buffer supplemented with 200 mM ATP and 2 mg ATF-2 fusion protein, and the mixture was incubated for 30 min at 30°C. The reaction was terminated with the addition of Laemmli sample buffer and boiled for 5 min. The proteins were separated by gel electrophoresis, transferred to nitrocellulose, and probed with a phospho-ATF (Thr-71) antibody or a phospho-Elk-1 (Ser-383) antibody. The bands were visualized by an enhanced chemiluminescence detection system with subsequent exposure to X-ray film.

Statistical analysis. The mean, SD, and SE of the mean were calculated by standard techniques. Paired analysis of variance, followed by Tukey's post hoc analysis for multiple comparisons, were employed with statistical significance determined at P < 0.05.


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Ionomycin-induced cytosolic Ca2+ flux. To determine the contributions of intracellular and extracellular Ca2+ in the ionomycin-induced cytosolic Ca2+ flux, indo 1-loaded PMNs were pretreated for 2 min with 5 mM EGTA, a chelator of extracellular Ca2+, or for 30 min in the dark with 50 µM BAPTA, a chelator of cytosolic Ca2+, and cytosolic Ca2+ levels were determined with addition of 20 nM ionomycin (Fig. 1). PMNs stimulated with 20 nM ionomycin showed a rapid cytosolic Ca2+ response comparable to other priming agents, such as PAF (15). PMNs pretreated with 5 mM EGTA for 2 min and subsequently stimulated with 20 nM ionomycin showed diminished Ca2+ flux compared with ionomycin alone (670 ± 90 vs. 1,890 ± 60 nM Ca2+). Moreover, PMNs pretreated with 50 µM BAPTA and subsequently stimulated with 20 nM ionomycin showed total inhibition of cytosolic Ca2+ flux. Because extracellular chelation of Ca2+ with EGTA only partially inhibited the ionomycin-induced Ca2+ transient, it appears that ionomycin directly caused the release of Ca2+ from calciosomes, the intracellular storage compartment of PMNs. Similar results were obtained with higher doses of ionomycin (200 nM - 2 µM), and the 2-nM concentration elicited a small but reproducible increase in cytosolic Ca2+ concentration (200 ± 45 nM).


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Fig. 1.   Measurement of ionomycin-induced changes in cytosolic Ca2+ concentration. Measurement of cytosolic Ca2+ concentration was determined by employing indo 1-loaded polymorphonuclear neutrophils (PMNs). Cytosolic Ca2+ levels in PMNs primed with 20 nM ionomycin were compared with pretreatment with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 30 min) to chelate cytosolic Ca2+ or 5 mM EGTA (2 min) to chelate extracellular Ca2+. Ionomycin was added 20 s after the Ca2+ measurements were started. Representative of 4 identical experiments.

Ionomycin activation and priming of the PMN oxidase. To determine whether ionomycin primed or activated the PMN oxidase in a dose-dependent manner, the maximal rates of superoxide production were measured over a range of ionomycin concentrations (2 nM-20 µM). Both activation of the oxidase and priming of the FMLP-activated respiratory burst were examined (Table 1). Ionomycin significantly activated the NADPH oxidase at 2 µM (7.5 ± 0.9 nmol O<UP><SUB>2</SUB><SUP>−</SUP></UP>/3.75 × 105 PMNs per min) compared with DMSO-treated controls (0.1 ± 0.02 nmol O<UP><SUB>2</SUB><SUP>−</SUP></UP>/3.75 × 105 PMNs per min) or lesser concentrations of ionomycin (2 - 200 nM; n = 9; P < 0.01). Treatment with 20 µM ionomycin did not result in any superoxide generation and actually caused an increased percentage of PMNs that could not extrude trypan blue (12 ± 3 vs. 3 ± 1%, respectively). Additionally, 5 min of pretreatment of PMNs with 20 or 200 nM ionomycin, compared with DMSO-pretreated controls, primed the FMLP-activated respiratory burst (4.3 ± 0.5 and 9.8 ± 0.9 vs. 0.5 ± 0.1 nmol O<UP><SUB>2</SUB><SUP>−</SUP></UP>/3.75 × 105 PMNs per min, P < 0.05). Lower concentrations of ionomycin (0.2-2 nM) did not prime the oxidase. Thus these experiments confirmed the ability of ionomycin to prime (20 - 200 nM) or activate (2 µM) the PMN oxidase in a dose-dependent fashion.

                              
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Table 1.   Priming and activation of the PMN oxidase by ionomycin

Dual phosphorylation and activation of the p38 and p42/44 MAP kinases in PMNs stimulated with ionomycin. Because dual phosphorylation of discrete threonine and tyrosine residues is required for p38 and p42/44 MAP kinase activation, activation of p38 and p42/44 MAP kinases by ionomycin was investigated by Western blotting whole cell extracts with antibodies to dually phosphorylated (Thr-180/Tyr-182) p38 and (Thr-202/Tyr-204) p42/44 MAP kinases (7). PMNs were stimulated with 2 nM - 2 µM ionomycin for 1 min, and whole cell lysates were subjected to SDS-PAGE and immunoblotting. In addition, PMNs were stimulated with 2 µM PAF or 1 µM FMLP as positive controls or with DMSO vehicle control. Samples were taken concurrently from the same aliquot of PMNs for p38 and p42/44 MAP kinase dual phosphorylation to examine differential phosphorylation patterns. Ionomycin stimulation of PMNs for 1 min showed dual phosphorylation of p38 MAP kinase at 20 nM - 2 µM with the 2 nM dose demonstrating minimal dual phosphorylation of the p38 MAP kinase (Fig. 2A). This dual phosphorylation of p38 MAP kinase was similar to the PAF- and FMLP-positive controls, and no dual phosphorylation was detected in the DMSO-treated PMNs. The identical PMN lysates were also subjected to immunoblotting with an antibody against dual-phosphorylated (Thr-202, Tyr-204) p42/44 MAP kinase. Ionomycin elicited dual phosphorylation of p42/44 MAP kinases at concentrations of 200 nM and 2 µM, similar to the PAF- and FMLP-positive controls (Fig. 2B). Analogous to the DMSO control, incubation with ionomycin at concentrations of 2-20 nM did not cause dual phosphorylation of p42/44 MAP kinases. Ionomycin stimulation of PMNs resulted in dual phosphorylation of p38 MAP kinase at concentrations (20 nM - 2 µM) that either primed or activated the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase was elicited at slightly higher doses of ionomycin (200 nM - 2 µM). As controls, identical aliquots from the same PMNs employed above as well as stripped blots from the above experiments were probed with a monoclonal antibody to p38 and p42/44 MAP kinases. No differences in the amounts of immunoreactivity were visualized among any of the wells (results not shown).


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Fig. 2.   Dual phosphorylation of p38 and p42/44 mitogen-activated protein (MAP) kinases in PMNs stimulated with ionomycin. A: isolated PMNs were incubated with 2 nM-2 µM ionomycin (ION), 2 µM platelet-activating factor (PAF), 1 µM N-formyl-methionyl-leucyl-phenylalanine (FMLP), or DMSO vehicle for 1 min. Dual phosphorylation of p38 MAP kinase was measured by SDS-PAGE and immunoblotted with an antibody (Thr-180/Tyr-182) to p38 MAP kinase as described in MATERIALS AND METHODS. B: lysates of the identical PMNs stimulated in A were immunoblotted with an antibody to dual-phosphorylated (Thr-202/Tyr-204) p42/44 MAP kinase. Representative of 4 separate experiments. MW, molecular weight; C, control.

Although dual phosphorylation of both p38 and p42/44 MAP kinases is required for activation of these kinases, the activity of these kinases was determined by assaying phosphorylation of known endogenous substrates of these enzymes. As shown in Fig. 3A, 2 µM ionomycin caused phosphorylation of ATF-2, a known substrate of p38 MAP kinase. In addition, both 20 and 200 nM doses of ionomycin also elicited phosphorylation of ATF-2, demonstrating that ionomycin concentrations that caused dual phosphorylation of p38 MAP kinase also caused activation of this enzyme (32, 39). Similarly, ionomycin concentrations that caused dual phosphorylation of p42/44 MAP kinases (2 µM but not 20 nM) also rapidly phosphorylated Elk-1, a recognized substrate of the p42/44 MAP kinases (Fig. 3B) (47). Thus dual phosphorylation of p38 and p42/44 MAP kinases is equivalent to activation as determined by phosphorylation of well-described substrates of these kinases.


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Fig. 3.   p38 and p42/44 MAP kinase induced phosphorylation of activating transcription factor (ATF)-2 and Elk-1. PMNs were incubated with ionomycin (20 nM or 2 µM), FMLP (1 µM), or DMSO vehicle control for 1 min and lysed. Whole cell lysates were immunoprecipitated with antibodies to dual-phosphorylated p38 (Thr-180/Tyr-182) (A) or p42/44 (Thr-202/Tyr-204) (B) overnight. An in vitro kinase assay was performed to assay for p38 or p42/44 MAP kinase activity and was completed with the addition of ATP and purified ATF-2 or Elk-1 proteins. The proteins were separated by 10% PAGE, transferred to nitrocellulose, and probed with an antibody to phospho-ATF-2 (A) or an antibody to phospho-Elk-1 (B). In the p38 MAP kinase activity assays (A), PMNs were pretreated with DMSO or 1 µM SB-203580 (SB) and then stimulated for 1 min with 2 µM ionomycin or 1 µM FMLP (positive control). In the p42/44 MAP kinase activity assays (B), the PMNs were stimulated with DMSO, 20 nM and 2 µM ionomycin, or 1 µM FMLP. Representative of 3 identical experiments.

Because ionomycin caused rapid changes in cytosolic Ca2+ concentration, we investigated whether dual phosphorylation of MAP kinases occurred concurrently over the same time interval with the cytosolic Ca2+ transient (Fig. 4). PMNs were stimulated with 20 nM ionomycin for p38 MAP kinase activation or 2 µM ionomycin for p42/44 activation over a time course of 0-10 min and immunoblotted. Twenty nanomolar ionomycin stimulation resulted in detectable p38 MAP kinase dual phosphorylation at 30 s, maximal phosphorylation at 1 min, and diminished phosphorylation at 5 and 10 min (Fig. 4A). The p42/44 MAP kinase dual phosphorylation, induced by 2 µM ionomycin stimulation, was absent at 30 s, detectable at 60 s, maximal at 2 min, was sustained for 5 min, and became diminished at 10 min (Fig. 4B). The p38 MAP kinase dual phosphorylation in ionomycin-stimulated PMNs occurs concurrently with the cytosolic Ca2+ flux, while the p42/44 MAP kinase dual phosphorylation appears to be delayed compared with the cytosolic Ca2+ flux.


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Fig. 4.   Time course of ionomycin-induced dual phosphorylation of p38 and p42/44 MAP kinases. Isolated PMNs were treated with 2 µM ionomycin or DMSO control for 0-10 min. The proteins from the whole cell lysates were separated by 10% polyacrylamide electrophoresis and immunoblotted with antibodies to dual phosphorylated p38 (A) and dual phosphorylated p42/44 (B) MAP kinases. Representative of 3 identical experiments.

Attenuation of the ionomycin-induced p42/44 MAP kinase dual phosphorylation by genistein and MEK1 inhibition. Because ionomycin stimulation resulted in dual phosphorylation, tyrosine and threonine, of the p38 and p42/44 MAP kinases, we examined the ability to inhibit this dual phosphorylation by employing specific inhibitors: PD-98059, an inhibitor of MEK1, the kinase that phosphorylates p42/44 MAP kinase; genistein, a tyrosine kinase inhibitor; and SB-203580, a p38 MAP kinase inhibitor (9, 13, 52). PMNs were pretreated with DMSO control, 1 - 10 µM PD-98059, 1 - 10 µM SB-203580, or 100 µM genistein for 30 min and subsequently activated with 2 µM ionomycin. Genistein pretreatment of PMNs inhibited ionomycin-induced dual phosphorylation of p38 MAP kinase (Fig. 5A), whereas the inactive analog daidzein had no affect (data not shown). Further control data indicated that genistein (100 µM) inhibited the FMLP-activated respiratory burst by 62 + 8%, abrogated all of the changes in FMLP-mediated tyrosine protein phosphorylation, and did not decrease PMN viability (trypan blue negativity: 99 + 2% with DMSO, 98 + 3% with 100 µM genistein), similar to other reports (19, 35). Lower genistein doses displayed decreased amounts of inhibition. Further control experiments also showed that the inactive genistein analog daidzein (100 µM) had no affect on the FMLP-activated respiratory burst and did not inhibit FMLP-mediated changes in tyrosine protein phosphorylation. As expected, SB-203580 did not cause inhibition of dual phosphorylation of p38 MAP kinase because it is an inhibitor of this MAP kinase, and, in these experiments, SB-203580 served as a negative control. However, both PD-98059 and genistein pretreatment inhibited the dual phosphorylation of p42/44 MAP kinase phosphorylation (Fig. 5B). In addition, PMNs pretreated with 10 µM PD-98059 did not affect the ionomycin-mediated dual phosphorylation of p38 MAP kinase, nor did preincubation of PMNs with SB-203580 affect dual phosphorylation of p42/44 MAP kinases in response to ionomycin (results not shown). Thus ionophore-stimulated PMNs require genistein-sensitive tyrosine kinases for activation of both p38 and p42/44 MAP kinases as well as MEK1 for activation of p42/44 MAP kinase.


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Fig. 5.   Inhibition of dual phosphorylation of MAP kinases with genistein (G), PD-98058 (PD), and SB-203580. PMNs were pretreated with DMSO, 100 µM genistein, 1 - 10 µM SB-203580 (a selective inhibitor of p38 MAP kinase, A), or 1 - 10 µM PD-98059 (an inhibitor of MAP/extracellular signal-related kinase 1, B) at 37°C for 30 min in the dark. PMNs were subsequently stimulated with 2 µM ionomycin for 1 min, and the resulting lysates were separated by 10% SDS-PAGE and immunoblotted with an antibody to the dual-phosphorylated p38 MAP kinase or the dual-phosphorylated p42/44 MAP kinase. Representative of 3 identical experiments.

Inhibition of ionomycin-induced oxidase activation with MAP kinase inhibitors. Because p38 and p42/44 MAP kinases were phosphorylated in PMNs stimulated with ionomycin, inhibition of these MAP kinases was investigated to see if activation of these enzymes was required for superoxide generation. PMNs were incubated with MAP kinase inhibitors SB-203580 (p38 MAP kinase inhibitor) and PD-98059 (MEK1 inhibitor) or DMSO (control), as described, over a range of concentrations of 0.1-10 µM. The PMNs were then activated with 2 µM ionomycin, and superoxide generation was measured (Table 2). SB-203580 pretreatment inhibited the ionomycin activation of the oxidase over a range of concentrations from 1 to 10 µM by 38 ± 9 to 45 ± 13% (P < 0.05, n = 10). The observed inhibition was similar to SB-203580 inhibition of the FMLP-activated oxidase, 40 + 5% to 57 + 14%, employing the identical PMNs. Conversely, PD-98059 did not inhibit the ionomycin-induced activation of the oxidase at concentrations of 0.1 - 10 µM. Superoxide generation was significantly diminished by p38 MAP kinase inhibition but not by p42/44 MAP kinase inhibition, implicating a role of p38 MAP kinase in ionomycin-induced superoxide generation.

                              
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Table 2.   Inhibition of ionomycin activation of PMN oxidase by p38 MAP kinase and MEK1 antagonists

The effects of Ca2+ chelation on ionomycin-induced dual phosphorylation of p38 and p42/44 MAP kinases. To investigate the role of cytosolic Ca2+ chelation and ionomycin-induced MAP kinase phosphorylation, PMNs were treated with 50 µM BAPTA, a chelator of intracellular Ca2+, or 5 mM EGTA, a chelator of extracellular Ca2+. The dose of BAPTA was chosen at 50 µM because previous work demonstrated that lower concentrations of BAPTA (5-25 µM) were unable to chelate the cytosolic Ca2+ flux in indo 1-loaded PMNs. Moreover, BAPTA (25 µM) pretreatment inhibited the respiratory burst of PAF-primed PMNs stimulated with FMLP by 58 + 9%, while identical PMNs pretreated with 50 µM BAPTA demonstrated inhibition of 88 + 9% (P < 0.05, n = 9 for each group). After preincubation with BAPTA or EGTA, PMNs were stimulated with DMSO, 20 nM ionomycin, 2 µM PAF, or 1 µM FMLP for 60 s. Pretreatment of PMNs with BAPTA totally inhibited the dual phosphorylation of p38 MAP kinase to ionomycin, PAF, and FMLP, whereas EGTA had little effect (Fig. 6A). In contrast, similar experiments done with PMNs stimulated with 2 µM ionomycin for 1 min showed inhibition of p42/44 MAP kinase dual phosphorylation with both BAPTA and EGTA pretreatment, compared with DMSO control (Fig. 6B). Moreover, dual phosphorylation of p42/44 MAP kinase in response to both PAF and FMLP was unaffected by either cytosolic or extracellular Ca2+ chelation. These results suggest that p38 MAP kinase dual phosphorylation requires Ca2+ from intracellular stores, but not extracellular Ca2+ influx, for all agonists employed, including ionomycin and both receptor-linked chemoattractants PAF and FMLP. However, although in ionomycin-treated PMNs the dual phosphorylation of the p42/44 MAP kinase required extracellular Ca2+ influx alone, chemoattractant-stimulated PMNs did not appear to require Ca2+ for dual phosphorylation. Because both FMLP and PAF caused dual phosphorylation of p42/44 MAP kinases despite chelation of cytosolic Ca2+, we postulated that activation of other signaling pathways might result in erk1/erk2 activation. Both FMLP and PAF have been reported to activate protein kinase C (PKC) (44, 48); moreover, activators of PKC do not cause increases in cytosolic Ca2+ concentration (41). To determine whether PKC activation, without altering cytosolic Ca2+ concentration, causes rapid activation of p42/44 MAP kinases, PMNs (1 × 106/well) were treated with 200 ng/ml of PMA over a time course of 30-300 s and lysed. The proteins were separated and probed with an antibody to activated p42/44 MAP kinase. As expected, PMA caused rapid activation (30 s) of erk1/erk2 (Fig. 7). In analogous experiments, PMA was unable to cause p38 MAP kinase activation at these early time points; however, dual phosphorylation of p38 MAP kinase was initiated after 2 min of PMA treatment (results not shown).


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Fig. 6.   Inhibition of p38 and p42/44 MAP kinase dual phosphorylation by Ca2+ chelation. PMNs were pretreated with 50 µM BAPTA (B; 30 min at 37°C in the dark), 5 mM EGTA (E; 2 min), or DMSO vehicle control and subsequently stimulated with 20 nM (A) or 2 µM (B) ionomycin, 2 µM PAF, or 1 µM FMLP for 1 min. The cellular lysates were separated by 10% SDS-PAGE and immunoblotted with an antibody to the dual-phosphorylated p38 or p42/44 MAP kinase antibodies. In addition, densitometry was performed on all 3 blots by employing Scion Image 4.0.2 software from the National Institutes of Health website. In these graphs, densitometry in arbitrary units is graphed as a function of treatment group. Representative of 3 identical experiments.



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Fig. 7.   Phorbol ester-induced dual phosphorylation of p42/44 MAP kinases. PMNs were incubated with DMSO control or 200 ng/ml of phorbol 12-myristate 13-acetate (PMA) for 30 - 600 s. The proteins from the cellular lysates were separated by 10% SDS-PAGE and immunoblotted with an antibody against the dual-phosphorylated form of p42/44 MAP kinase. Representative of 2 experiments.

Inhibition of ionomycin-induced tyrosine phosphorylation by p38 MAP kinase antagonism. Ionomycin is known to cause tyrosine phosphorylation of a number of proteins in PMNs (4). To investigate whether these tyrosine-phosphorylated proteins may be downstream of p38 MAP kinase activation, PMNs were pretreated with DMSO, 1 µM SB-203580 to inhibit p38 MAP kinase, or 10 µM PD-98590 to inhibit p42/44 MAP kinases for 30 min and then stimulated with 2 µM ionomycin for 60 s. The proteins from these whole cell lysates were separated by 5-15% SDS gradient polyacrylamide electrophoresis, transferred to nitrocellulose, and immunoblotted with an anti-phosphotyrosine antibody. Ionomycin caused tyrosine phosphorylation of a number of proteins, but especially those at molecular weights of 72 and 105 kDa, respectively, compared with DMSO-pretreated, buffer-stimulated controls (Fig. 8). Preincubation with SB-203580 inhibited tyrosine phosphorylation of both the 72- and 105-kDa proteins, while pretreatment with PD-98058 had no effect (Fig. 8). Thus ionomycin-induced tyrosine protein phosphorylation in human PMNs demonstrated at least two protein substrates that are dependent on activation of the p38 MAP kinase cascade for phosphorylation of tyrosine residues.


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Fig. 8.   Ionomycin-induced tyrosine protein phosphorylation in PMNs. PMNs were preincubated with 10 µM SB-203580, 10 µM PD-98059, or DMSO for 30 min at 37°C and stimulated with 2 µM ionomycin for 1 min. Proteins from these whole cell lysates were separated by PAGE and immunoblotted with a monoclonal antibody to phosphotyrosine. Sample size of 4 is shown.

Manipulation of cytosolic Ca2+ with thapsigargin. To rule out the possibility that ionophores cause intrinsic activation of non-Ca2+-linked signaling pathways, the cellular effects of thapsigargin were investigated. Thapsigargin is an inhibitor of the endoplasmic reticulum ATPase that causes a release of cytoplasmic Ca2+ (10). Thapsigargin has been reported to rapidly activate p42/44 MAP kinases; thus, for the purpose of these studies, we examined the changes in cytosolic Ca2+, priming and activation of the PMN oxidase, and dual phosphorylation of p38 MAP kinase (58). Thapsigargin (1 µM) elicited a rapid rise in cytosolic Ca2+, followed by a sustained increase in cytosolic Ca2+ (Fig. 9A). The initial rise in cytosolic Ca2+ could not be totally inhibited by pretreatment with EGTA, although it only represented 60-100 nM of a 2,900-nM rise in cytosolic Ca2+ (Fig. 9B). However, the sustained rise in Ca2+, which is a reflection of recruitment of Ca2+ from the extracellular stores, was severely attenuated by preincubation with 5 mM EGTA. Therefore, thapsigargin causes a small but reproducible release of Ca2+ from the calciosomes followed by prolonged Ca2+ influx through extracellular channels.


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Fig. 9.   Thapsigargin-induced changes in cytosolic Ca2+ concentration in indo 1-loaded PMNs. PMNs were loaded with 5 µM indo 1 for 10 min at 37°C. PMNs were pretreated with 5 mM EGTA or buffer control and then stimulated with 1 µM thapsigargin or DMSO control. The changes in cytosolic Ca2+ were measured over real time in a dual-wavelength spectrofluorimeter. A: compares the differences in the changes in cytosolic Ca2+ in PMNs with or without EGTA stimulated with thapsigargin. B: demonstrates the changes in cytosolic Ca2+ in PMNs pretreated with 5 mM EGTA and stimulated with thapsigargin or DMSO control. Representative of 4 identical experiments.

The effects on the PMN oxidase were then investigated, and thapsigargin alone was not sufficient to activate the respiratory burst of quiescent PMNs (thapsigargin: 0.1 + 0.1 vs. DMSO control: 0.1 +0.1 nmol O<UP><SUB>2</SUB><SUP>−</SUP></UP>/min, n = 5). However, thapsigargin pretreatment (5 min) primed the FMLP-activated respiratory burst by 6.2 ± 2.3-fold compared with DMSO-treated control PMNs (n = 5; P < 0.05). Additionally, thapsigargin caused rapid (60 s) dual phosphorylation of p38 MAP kinase (Fig. 10). Thus manipulation of cytosolic Ca2+ with thapsigargin, which primes the PMNs and is not structurally related to ionomycin, resulted in activation of p38 MAP kinase.


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Fig. 10.   Thapsigargin-induced dual phosphorylation of p38 MAP kinase. PMNs were stimulated with 1 µM thapsigargin, 1 µM FMLP (positive control), or DMSO over a time course of 0 - 10 min. Proteins were separated from whole cell lysates by 10% PAGE and immunoblotted with an antibody to the dual-phosphorylated p38 MAP kinase. Representative of 3 identical experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many studies have documented the requirements of Ca2+ in signal transduction and the normal physiological response of PMNs, including oxidase activation, chemotaxis, the increased surface expression of cell adhesion molecules, and bactericidal activity (21, 42). The observed increases in cytosolic Ca2+ concentration resulting from PMN agonist stimulation may arise from extracellular Ca2+ transported into the PMN by a receptor-mediated process or the release of intracellular Ca2+ stores (15, 30, 38). In addition to the role of Ca2+ in PMN physiology, other investigators have demonstrated the relative importance of the p38 and p42/44 MAP kinase pathways in regulating neutrophil function (32, 39, 40). Inhibition of the p38 MAP kinase attenuates antimicrobial PMN functions such as superoxide generation, increased surface expression of cell adhesion molecules, and chemotaxis (39, 46). Conversely, divergent results have been published on the role of the p42/44 MAP kinase pathway in regulating PMN function, especially with regard to superoxide generation, chemotaxis, and degranulation (8, 12, 33). While the importance of intracellular Ca2+ flux has been documented in tyrosine phosphorylation of PMN whole cell extracts (20, 45), it has been suggested that intracellular Ca2+ is not required or necessary for p38 MAP kinase phosphorylation or activation (32). Moreover, to date, studies of the Ca2+ dependence of the p42/44 MAP kinases have not been definitively performed.

Our results suggest a possible role for cytosolic Ca2+ in both p38 and p42/44 MAP kinase activation and divergent requirements of p42/44 and p38 MAP kinase in regulating ionomycin-modulated PMN functions. Ionomycin caused a rapid rise in cytosolic Ca2+ at the priming dose of 20 nM, which was abrogated by BAPTA pretreatment but only partially inhibited by EGTA. This suggests that ionomycin not only transports extracellular Ca2+ into the PMN but also causes release of intracellular Ca2+ stores to activate PMN signal transduction pathways. It was surprising that a nonreceptor-linked PMN agonist may cause Ca2+ release from intracellular stores. Indeed, the mechanism of this ionomycin-induced Ca2+ release from intracellular stores needs to be elucidated, although it may involve an induced permeability of the calciosome membrane by ionophores leading to an increase in cytosolic Ca2+ from intracellular stores.

Ca2+ ionophore stimulation of PMNs resulted in dual phosphorylation of p38 and p42/44 MAP kinases in a differential manner. Ionomycin caused dual phosphorylation of p38 MAP kinase at 20 nM - 2 µM, while dual phosphorylation of p42/44 MAP kinase occurred at 200 nM-2 µM. Identical concentrations of ionomycin that caused dual phosphorylation also caused activation of both p38 and p42/44 MAP kinases, employing immunoprecipitation of the active enzyme followed by in vitro phosphorylation of a known substrate. Thus we have correlated dual phosphorylation of the p38 and p42/44 MAP kinases with enzymatic activity. Moreover, SB-203580 inhibition of p38 MAP kinase attenuated the ionomycin activation of the PMN oxidase and caused dephosphorylation of two different tyrosine-phosphorylated proteins of 72 and 105 kDa. p38 MAP kinase-mediated tyrosine phosphorylation of "downstream" substrates is not surprising, because p38 MAP kinase is at least partially responsible for activation of phospholipase C-gamma by tyrosine phosphorylation in PMNs and is directly involved in the tyrosine phosphorylation of the signal transducer and activator of transcription-1 (STAT1) and the tyrosine phosphatase SHP-2 in COS-7 cells exposed to hyperosmolarity (5, 25). In contrast, inhibition of p42/44 MAP kinase activation did not affect the PMN oxidase or the tyrosine phosphorylation of PMN proteins. Furthermore, there did not appear to be any "cross talk" between the p38 and p42/44 MAP kinase activation pathways, because the MEK1 inhibitor PD-98059 had no effect on dual phosphorylation of p38 MAP kinase nor the downstream tyrosine-phosphorylated substrates. In analogous studies, SB-203580 had no effect on dual phosphorylation of the p42/44 MAP kinase. These results show not only a dependence of p38 MAP kinase activity for oxidase activation and protein tyrosine phosphorylation in PMNs but also provide additional support for other studies that have concluded that p42/44 MAP kinase phosphorylation is not required for activation of the respiratory burst (33, 46). The function of p42/44 MAP kinase activation, which may occur simultaneously to p38 MAP kinase activation, in response to ionomycin, remains unclear.

Cytosolic Ca2+ appears to be required for activation of p38 MAP kinase in response to not only ionomycin but also to both FMLP and PAF. Ionomycin, FMLP, and PAF all demonstrated that increases in cytosolic Ca2+ from calciosomes were prerequisite for p38 MAP kinase activation through BAPTA chelation studies. Furthermore, Ca2+ from the extracellular milieu does not affect p38 MAP kinase activity, as demonstrated by studies with EGTA. In addition, the experiments with thapsigargin provide supportive evidence that changes in cytosolic Ca2+ cause activation of the p38 MAP kinase. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, directly increased cytosolic Ca2+ from both intracellular and extracellular stores and caused priming of the FMLP-activated respiratory burst and dual phosphorylation of p38 MAP kinase similar to ionomycin. In contrast, maximal activation of the p42/44 MAP kinases required changes in cytosolic Ca2+, but it is likely that the source of the Ca2+ was from extracellular "stores", because both EGTA and BAPTA had similar inhibitory effects on p42/44 MAP kinase activation. It is important to note that the dose of BAPTA employed has the capacity to rapidly chelate virtually all of the Ca2+ that accumulates in the cytosol irrespective of its source, either calciosomes or extracellular stores. Conversely, FMLP and PAF activation of p42/44 MAP kinases did not appear to require Ca2+. As demonstrated, PMA rapidly activated erk1/erk2 in PMNs, presumably due to direct stimulation of PKC. Moreover, PMA and other direct PKC activators do not cause cytosolic Ca2+ flux in PMNs (29, 43). Because both FMLP and PAF have been shown to rapidly translocate and activate PKC and because direct PKC stimulation in PMNs results in rapid activation of p42/44 kinase activation, one may infer that chemoattractants activate erk1/erk2 through PKC.

We conclude that ionomycin affects PMN function through signal transduction pathways involving changes in cytosolic Ca2+ from both extracellular and intracellular stores. Ionomycin-induced changes in cytosolic Ca2+ concentration mediate differential phosphorylation and activation of the p38 and p42/44 MAP kinase cascades, whereby p38 MAP kinase requires intracellular Ca2+ for activation, but p42/44 MAP kinase may require extracellular Ca2+ for maximal dual phosphorylation. Signal transduction induced by a Ca2+ ionophore in PMNs demonstrates the importance of cytosolic Ca2+ for p38 MAP kinase activation. Data have also been provided that directly demonstrate the requirements of cytosolic Ca2+ for p38 MAP kinase activation in response to different stimuli, including PAF and FMLP.


    ACKNOWLEDGEMENTS

This work was supported by the Bonfils Blood Center; Clinical Associate Physician Award M01-RR00069 from the General Clinical Research Centers Program, National Centers for Research Resources; National Heart, Lung, and Blood Institute Grant HL-59355, and a National Blood Foundation grant.


    FOOTNOTES

Address for reprint requests and other correspondence: C. C. Silliman, Bonfils Blood Center, 717 Yosemite Circle, Denver, CO 80230 (E-mail: christopher.silliman{at}uchsc.edu).

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

Received 14 August 2000; accepted in final form 27 February 2001.


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