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Originally published online as doi:10.1189/jlb.0405192 on January 24, 2008

Published online before print January 24, 2008
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(Journal of Leukocyte Biology. 2008;83:964-971.)
© 2008 by Society for Leukocyte Biology

Tonic protein kinase A activity maintains inactive β2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase

Clayton D. Chilcoat*, Yousuf Sharief* and Samuel L. Jones*,{dagger},1

* Department of Clinical Sciences and
{dagger} Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA

1 Correspondence: Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA. E-mail: sam_jones{at}ncsu.edu


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ABSTRACT
 
Activation of β2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase β2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of β2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on β2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated β2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of β2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of β2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.

Key Words: PMN • PKA • ROCK • avidity • adhesion • cytoskeleton


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INTRODUCTION
 
Neutrophils are rapidly recruited into infected tissues, where they are activated to combat pathogens. Adhesion is an important regulator of the transition to an activated phenotype [1 , 2 ]. This adhesion is mediated by cell surface receptors, called integrins, which bind ligands on other cells and in the extracellular matrix at sites of inflammation [1 , 2 ]. The β2 integrins {alpha}Lβ2, {alpha}Xβ2, and {alpha}Mβ2 are the most abundant integrins expressed on the neutrophil cell surface [1 ]. In quiescent neutrophils, β2 integrins are in a low-affinity state and will bind ligand-poorly [1 , 3 ]. When the neutrophil receives an extracellular proinflammatory signal, the β2 integrins become activated to a high-affinity state and subsequently, bind strongly to their specific ligands [3 ]. In addition, when neutrophils are stimulated, β2 integrins are released from cytoskeletal constraints to allow the receptors to form clusters at sites of adhesion [4 5 6 ]. This up-regulation of integrin-ligand affinity and receptor clustering increases overall cellular strength of binding and is termed "avidity activation" [3 , 7 ]. Neutrophil stimulation with proinflammatory mediators also mobilizes preformed β2 integrins (particularly {alpha}Mβ2) from secretory vesicles to the cell surface to increase overall cell surface expression [8 9 10 ]. This increase in overall integrin cell surface expression also contributes to the avidity of cellular adhesion. The biochemical pathways initiated by proinflammatory mediators that induce avidity activation are often referred to as "inside-out signals."

Protein kinase A (PKA; cAMP-dependent protein kinase) is a key regulator of neutrophil integrin activation and adhesion. We and others [12 13 14 15 16 17 18 19 ] demonstrated that treatment of neutrophils with a cell-permeant analog of cAMP or substances that increase intracellular cAMP concentrations (and therefore, PKA activity [11 ]) inhibit neutrophil adhesion and adhesion-dependent functions. We demonstrated that inhibition of PKA activity rescues neutrophils from the inhibitory effect of cAMP [12 , 13 ]. Moreover, we showed that inhibition of PKA in otherwise unstimulated neutrophils was sufficient to induce activated β2 integrins and adhesion [12 ]. Our data suggest that a constitutive or "tonic" PKA activity in quiescent neutrophils plays a role in maintaining neutrophil integrins in the inactivated state, thus preventing inappropriate integrin and subsequent functional activation. This tonic PKA activity is modulated by cAMP levels and must be overcome for integrin activation to occur. However, the effectors of PKA in this mechanism remain undefined.

The acto-myosin cytoskeleton plays a critical role in avidity activation of integrins and adhesion [4 5 6 , 20 21 22 23 24 ]. Moreover, PKA regulates many actin cytoskeletal functions, positioning that actin cytoskeleton as a possible effector of PKA in the mechanism-regulating integrin function and adhesion in neutrophils. We have recently shown that inhibition of PKA prevents F-actin reorganization and subsequent neutrophil polarization in response to a chemoattractant, and exposure of otherwise unstimulated neutrophils to a point source of the PKA inhibitor KT5720 polarizes the neutrophil cytoskeleton and is sufficient to activate migration toward the source of the PKA inhibitor [25 ]. These findings demonstrate that PKA has a fundamental role in regulating the actin cytoskeleton and thus, may be a target for PKA in the mechanism maintaining inactive β2 integrins in unstimulated neutrophils.

Myosin light chain (MLC) is a subunit of the myosin complex that regulates the contractile activity in nonmuscle cells. Phosphorylation of MLC serine 19 (Ser 19) is required for formation of the actin-myosin complex and myosin contractility and results in stabilization and contraction of the actin cytoskeleton [26 ]. MLC can be phosphorylated on Ser 19 by MLC kinase (MLCK) or Rho kinase (ROCK) [27 , 28 ]. These kinases have been implicated in adhesion regulation in leukocytes—MLCK activity important for attachment at the leading-edge pseudopod and ROCK activity required for the detachment of the trailing-edge uropod [29 , 30 ]. The calmodulin (CaM)-binding site of MLCK includes a PKA phosphorylation site, and its phosphorylation inhibits MLCK activity, resulting in decreased MLC phosphorylation [31 ]. PKA further influences MLC phosphorylation by decreasing the activity of the Rho/ROCK pathway. ROCK increases MLC phosphorylation by directly phosphorylating MLC Ser 19 [28 ] and by inhibiting by MLC phosphatase activity [32 ]. These data suggest that tonic PKA activity in neutrophils may act by decreasing MLCK or ROCK activity, either of which, when liberated from the inhibition, could contribute to MLC phosphorylation.

Based on the known role for the acto-myosin cytoskeleton in β2-integrin-mediated adhesion and the role for PKA in regulating MLC phosphorylation, we hypothesized that in unstimulated neutrophils, a tonic level of PKA inhibits β2-integrin activation via inhibition of MLC phosphorylation. We tested our hypothesis by first determining whether inhibition of tonic PKA activity was sufficient to induce MLC phosphorylation in unstimulated neutrophils. We then determined whether MLC phosphorylation, adhesion, {alpha}Mβ2-integrin activation, and increased β2-integrin cell surface expression associated with loss of tonic PKA activity required MLCK or ROCK activity.


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MATERIALS AND METHODS
 
Reagents
Powdered PBS and HBSS were purchased from Gibco-BRL (Grand Island, NY, USA). Ficoll-Paque Plus, dextran, and ECL detection reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Calcein was purchased from Molecular Probes (Eugene, OR, USA). KT5720, ML-7, and the MLCK inhibitory peptide MLC (11–19) amide [33 ] were purchased from Alexis Corp. (San Diego, CA, USA). A scrambled control version of the MLC (11–19) amide inhibitory peptide was synthesized by SynPep Corp. (Dublin, CA, USA). The MLCK inhibitor peptide 18 [34 ] was purchased from Calbiochem (La Jolla, CA, USA). Bovine FCS was obtained from HyClone (Logan UT, USA). Diisopropylfluorophosphate (DFP) and the MLCK inhibitory peptide were purchased from Calbiochem. The bicinchinonic acid (BCA) assay kit was purchased from Pierce (Rockford, IL, USA). Heparin was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ, USA). Polyacrylamide was purchased from National Diagnostics (Atlanta, GA, USA). The SDS, 2-ME, Triton-X-100, and sodium azide were purchased from Fisher Biotech (Fair Lawn, NJ, USA). The polyvinylidene fluoride (PVDF) membrane was purchased from Millipore (Billerica, MA, USA). Aprotinin, leupeptin, PMSF, pepstatin, iodoacetamide, BSA, monoclonal mouse anti-MLC IgM, HRP-conjugated anti-mouse IgM, and FITC-conjugated F(ab')2 sheep anti-mouse IgG were purchased from Sigma Chemical Corp. (St. Louis, MO, USA). Mouse antiphospho-MLC IgG and HRP-conjugated anti-mouse IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-β2-integrin mAb IB4 [35 ] was purified, and F(ab')2 was prepared as described [36 ]. The mAb CBRM1/5 binds a neoepitope on affinity-activated {alpha}Mβ2 integrins [37 ] and was obtained from eBioscience (San Diego, CA, USA).

Neutrophil isolation
Heparinized whole blood was collected from healthy adult donors via cephalic venipuncture. The blood was mixed with dextran (final concentration, 1.4% dextran) and then allowed to settle for 60 min, after which time, the leukocyte-rich plasma was collected and layered onto 5 mL Ficoll-Paque Plus in 15 mL conical tubes. The plasma was centrifuged at 1800 RPM for 20 min at room temperature. The residual erythrocytes were lysed using a hypotonic saline lysis solution, and the remaining neutrophils were resuspended in HBSS containing 20 mM HEPES and 8.9 mM sodium bicarbonate without calcium or magnesium (HBSS), yielding neutrophils that were greater than 98% pure and greater than 99% viable (data not shown).

Adhesion assay
Ninety-six-well microtiter plates (Immulon 2HB, Dynex Technologies, Chantilly, VA, USA) were coated with 50 µL 5% FCS for 2 h. Neutrophil adhesion was quantified as described previously [38 ]. Neutrophils were suspended in HBSS at a concentration of 1 x 107 cells/mL. Neutrophils were then incubated with 1 µg/mL calcein at room temperature for 30 min. Following incubation with calcein, the cells were washed once with HBSS. The neutrophils were then resuspended in HBSS containing 1 mM Ca2+ and 1 mM Mg2+ (HBSS++) at a concentration of 2 x 106 cells/mL and treated with vehicle control or various inhibitors at the indicated concentration for 30 min at 37°C. The neutrophil suspension (50 µL) was then added to the appropriate wells of a substrate-coated microtiter plate and incubated at room temperature for 10 min to allow the neutrophils to settle to the bottom of the wells. KT5720 or vehicle control was then added, and the plates incubated for 30 min at 37°C to allow adhesion to occur. Total intracellular calcein fluorescence of each well ({lambda}=485 nm excitation; {lambda}=530 nm emission) was measured using an fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) before and after washing with 150 µL PBS. Percent adhesion was calculated by dividing the fluorescence after washing by the fluorescence before washing. In preliminary experiments, fluorescence was linearly related to cell number (data not shown).

Flow cytometry
Neutrophils were suspended in HBSS++ at a concentration of 4 x 106/mL and pretreated with the indicated inhibitor for 30 min, followed by treatment with vehicle control or KT5720 at the indicated concentrations for 30 min at 37°C. The neutrophils were then washed and resuspended with chilled PBS containing 1% FCS and 0.1% sodium azide (wash buffer). The neutrophils were then incubated with the indicated primary antibody for 40 min on ice, washed twice, and resuspended with chilled wash buffer. The neutrophils were subsequently incubated with FITC-conjugated anti-mouse F(ab')2 for 20 min on ice. The neutrophils were washed twice with chilled wash buffer to remove any unbound antibodies, and the neutrophils were then suspended in PBS. The relative fluorescence of 10,000-gated neutrophils was then measured using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA, USA).

MLC phosphorylation analysis
Neutrophils were suspended in HBSS++ at a concentration of 4 x 106 cells/mL and treated with vehicle control, KT5720, and MLCK inhibitors at the indicated concentrations for 30 min at 37°C. The neutrophils were centrifuged at 6000 RPM for 10 min and resuspended in chilled lysis buffer containing 0.5% Triton-X-100, 10 ug/mL aprotinin/leupeptin, 1 mM PMSF, 0.1 mg/mL pepstatin, 1 mM iodoacetamide, and 5 mM DFP. The neutrophils were agitated on ice for 30 min and then centrifuged at 14,000 RPM and 4°C for 10 min. The supernatant was collected, an aliquot of each sample was saved for total protein quantification via BCA assay, and the remainder of each sample was diluted with an equal volume of Laemmeli buffer with 5% 2-ME and boiled for 5 min. The equal total protein content of each lysate was loaded on 12% SDS-polyacrylamide gels and subsequently resolved via SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with BSA and then probed with mouse antiphospho-MLC IgG or mouse anti-MLC IgM. Unbound antibody was removed via washing of the membrane, and the membrane was then probed with an appropriate HRP-conjugated secondary antibody, which was detected via chemiluminescence using the ECL detection kit and exposure of Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY, USA). The films were digitized using a ScanJet 5100C (Hewlett-Packard, Palo Alto, CA, USA), and the OD of each band was determined using ONE-Dscan 2.05 for Windows (Scanalytics, Inc., Fairfax, VA, USA).

Statistical analysis
Significance of difference between data points was determined using Student’s t-test. Findings were considered significantly different with P < 0.05.


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RESULTS
 
PKA negatively regulates MLC phosphorylation via a MLCK-dependent pathway
Previous studies have determined that PKA can phosphorylate and thereby inactivate MLCK [31 ]. Our hypothesis predicts that inhibition of PKA activity in unstimulated neutrophils will increase MLCK activity, resulting in increased MLC phosphorylation. To determine if MLC phosphorylation is regulated by PKA in neutrophils, neutrophils were isolated from peripheral blood and treated with the PKA inhibitor KT5720 at varying concentrations for 30 min at 37°C, and MLC phosphorylation was determined using Western blot analysis. Treatment of neutrophils with KT5720 at concentrations greater than 5 µM significantly increased MLC phosphorylation in a dose-dependent manner (Fig. 1A and 1B ). The ability of KT5720 to increase MLC phosphorylation in neutrophils was abolished by pretreatment with the specific MLCK inhibitor ML-7 or a cell-permeant MLCK inhibitory peptide (KKRAARATS amide) derived from amino acids 11–19 of MLC (Fig. 1C and 1D) . Treatment with a control, scrambled version of the MLCK inhibitory peptide (AKAKRRSAT amide) had no effect on KT5720-induced MLC phosphorylation. An alternative mechanism by which decreasing PKA activity may increase MLC phosphorylation is via activation of ROCK, which inhibits MLC phosphatase activity. The ability of KT5720 to increase MLC phosphorylation in neutrophils was not affected by pretreatment with the specific ROCK inhibitor Y-27632 (Fig. 1E) at a concentration that inhibits ROCK-dependent functions in neutrophils (ref. [30 ] and our data not shown), demonstrating that ROCK activity is not essential for MLC phosphorylation induced by inactivation of PKA.


Figure 1
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  		Figure 1. Treatment of neutrophils with the PKA inhibitor KT5720 results in MLCK-dependent phosphorylation of MLC. (A) Neutrophils were treated with the indicated concentration of KT5720 or vehicle alone for 30 min at 37°C. Separate aliquots of lysates prepared from each sample containing equal amounts of protein were resolved via SDS-PAGE and blotted with antiphospho-MLC to detect Ser 19 phosphorylation (Phospho) or anti-MLC to detect total (Whole) MLC. VC, Vehicle control. (B) The relative densities (normalized to the unstimulated vehicle control group) of each phospho-MLC band were determined and reported as fold induction of MLC phosphorylation. Each point represents the mean ± SE of three experiments. KT5720 significantly increased MLC phosphorylation at all concentrations tested (*, P<0.05). (C) Neutrophils were pretreated with vehicle control, ML-7 (10 µM), MLC (10–19) peptide (10 µM), or scrambled control peptide (10 µM) for 30 min. Cells were then treated with vehicle or KT5720 (10 µM) for 30 min. Separate aliquots of lysates prepared from each sample containing equal amounts of protein were resolved via SDS-PAGE and blotted with antiphospho-MLC to detect Ser 19 phosphorylation or anti-MLC to detect total MLC. (D) The relative densities (normalized to the unstimulated vehicle control group) of each phospho-MLC band were determined and reported as fold induction of MLC phosphorylation. Each point represents the mean ± SE of three separate experiments. ML-7 and the MLC (10–19) inhibitory peptide significantly (*, P<0.05) decreased KT5720-induced MLC phosphorylation. (E) Neutrophils were pretreated with vehicle control or Y-27632 (10 µM) for 30 min followed by vehicle or KT5720 (10 µM) for 30 min at 37°C. Separate aliquots of lysate prepared from each sample containing equal amounts of protein were resolved via SDS-PAGE and blotted with antiphospho-MLC to detect Ser 19 phosphorylation or anti-MLC to detect total MLC. Pretreatment with Y-27632 did not affect KT5720-induced MLC phosphorylation.

PKA regulates β2-integrin-mediated neutrophil adhesion via a MLCK-dependent pathway
Our data demonstrate that MLCK activity is essential for the increase in MLC phosphorylation associated with inhibition of PKA. We next determined whether the ability of PKA inhibitors to induce β2-integrin-dependent adhesion was dependent on MLCK activity. We investigated this using an assay for β2-integrin-dependent adhesion used previously in our laboratory [20 , 39 , 40 ]. As we demonstrated previously [12 ], the PKA inhibitor KT5720 significantly induced adhesion to the β2-integrin substrate FCS in a dose-dependent manner (Fig. 2A and 2B ). To determine whether MLCK activity was necessary for KT5720-induced adhesion, neutrophils were treated MLCK inhibitors prior to KT5720 treatment. Pretreatment of the neutrophils with ML-7 significantly inhibited the ability of a range of concentrations of KT5720 to stimulate adhesion (Fig. 2A) . The ability of ML-7 to inhibit KT5720-induced adhesion was dose-dependent (Fig. 2B) , with an IC50 of 2–3 µM. ML-7 also inhibits PKC, albeit at a 100-fold higher concentration than required to inhibit MLCK activity. The PKC inhibitor staurosporine (100 nM, 1 µM, or 10 µM) did not affect KT5720-induced adhesion in our assay (data not shown). As a control for the specificity of ML-7, we examined the effect of the MLCK inhibitory peptide on KT5720-induced neutrophil adhesion. Pretreatment with the MLCK inhibitory peptide MLC (11–19) amide significantly inhibited β2-integrin-mediated adhesion in neutrophils treated with KT5720 (25 µM; Fig. 2C ) with an IC50 of ~0.1 µM (data not shown). A control, scrambled version of the MLC (11–19) amide peptide had no effect on KT5720-induced neutrophil adhesion at any tested concentration. Treatment with the cell-permeant MLCK inhibitory peptide Pep18 [34 ] also significantly inhibited KT5720-induced adhesion. These data demonstrate that MLCK activity is necessary for the ability of the PKA inhibitor KT5720 to induce adhesion in neutrophils.


Figure 2
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  		Figure 2. KT5720-induced neutrophil adhesion is inhibited by dependency on MLCK activity. (A) Calcein-loaded neutrophils were pretreated with the MLCK inhibitor ML-7 (10 µM) or vehicle control, added to FCS-coated wells, and then treated with vehicle or the indicated concentration of KT5720 and allowed to adhere for 30 min. Pretreatment with ML-7 significantly inhibited KT5720-induced adhesion at all concentrations (*, P<0.05). (B) Neutrophils were pretreated with the indicated concentration of ML-7 or vehicle control. Cells were then treated with vehicle or KT5720 (25 µM) and allowed to adhere to FCS-coated plastic for 30 min. Pretreatment with 5 and 10 µM ML-7 significantly inhibited KT5720-induced adhesion (*, P<0.05). (C) Neutrophils were treated with the MLCK inhibitory peptides Pep18 or MLC (11–91) amide or a control-scrambled version (Scr pep) of the MLC (11–19) peptide. Cells were then treated with vehicle or KT5720 (KT; 10 µM) and allowed to adhere to FCS-coated plastic for 30 min. Pep18 and MLC-11–19) amide treatment significantly inhibited KT5720-induced adhesion (*, P<0.05). The scrambled control peptide did not significantly affect adhesion. (D) Neutrophils were pretreated with vehicle control, ML-7 (10 µM), or the ROCK inhibitor Y-27632 (10 µM). Cells were then treated with vehicle or KT5720 (10 µM) and allowed to adhere to FCS-coated plastic for 30 min. ML-7, but not Y-27632, significantly inhibited KT5720-induced adhesion (*, P<0.05). The data are reported as percent adhesion, the percentage of cells that remained after washing, as described in Materials and Methods. Each point represents the mean ± SE of triplicate wells. Data presented are the best representations of at least three separate experiments performed on different donors. The variability in overall adhesion observed in this figure is attributed to donor differences.

We examined the role for ROCK in the mechanism of KT5720-induced adhesion by pretreating the neutrophils with Y-27632 prior to treatment with KT5720. Unlike MLCK inhibitor pretreatment, Y-27632 did not significantly affect KT5720-induced adhesion (Fig. 2D) . These data demonstrate that ROCK activity is not necessary for adhesion induced by inhibition of PKA.

PKA regulates β2-integrin avidity activation via a MLCK-dependent pathway
β2 integrins must be activated for neutrophil adhesion to occur. Adhesion induced by PKA inhibitors is associated with an increase in the number of β2-integrin molecules on the cell surface, resulting from mobilization of preformed integrins from intracellular stores, as well as an increase in the number of high-affinity {alpha}Mβ2 integrins expressed on the cell surface [12 ]. We determined whether the increase in total and high-affinity β2 integrins induced by inhibition of PKA required MLCK activity. We used flow cytometry to assess the total expression of β2 integrins on the cell surface, as measured by binding of the anti-β2-integrin antibody IB4, and expression of high-affinity {alpha}Mβ2, as measured by binding of the mAb CBRM1/5, which recognizes a neoepitope formed when {alpha}Mβ2 shifts to the high-affinity conformation [37 ].

As we have shown previously, treatment of neutrophils with KT5720 (25 µM) resulted in a significant increase in the number of β2 integrins and high-affinity {alpha}Mβ2 integrins on the cell surface (Fig. 3A and 3B ). Pretreatment with ML-7 (10 µM) by itself had no effect on β2-integrin cell surface expression. However, pretreatment with ML-7 significantly inhibited the ability of KT5720 to increase β2-integrin cell surface expression (Fig. 3A) . Pretreatment with ML-7 also significantly inhibited the ability of KT5720 to increase the cell surface expression of high-affinity {alpha}Mβ2 (Fig. 3B) .


Figure 3
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  		Figure 3. KT5720-induced up-regulation of total β2-integrin expression and high-affinity {alpha}Mβ2-integrin expression on the cell surface are MLCK-dependent. Neutrophils were treated with vehicle control or 10 µM ML-7 for 30 min at 37°C. The neutrophils were then treated with vehicle or the indicated concentration of KT5720 for 30 min. Cells were stained as described in Materials and Methods with (A) the anti-β2-integrin antibody IB4 or (B) the antibody CBRM1/5, which specifically binds to an {alpha}Mβ2-affinity activation-associated neoepitope. The neutrophils were washed, and the fluorescence of 10,000-gated neutrophils was then measured. Cell surface expression of β2 integrins and activated {alpha}Mβ2 are presented as the relative fluorescence intensity of each treatment group normalized to the mean fluorescence intensity (MFI) of the unstimulated vehicle control group. Each point represents the mean ± SE of six separate experiments. ML-7 treatment significantly inhibited KT5720-induced cell surface β2-integrin expression and activated {alpha}Mβ2 (*, P<0.05).

KT5720 treatment may increase expression of high-affinity {alpha}Mβ2 indirectly by increasing the overall number of {alpha}Mβ2 integrins on the surface available for activation or directly by inducing the conformational change associated with transition to the high-affinity state. Inhibition of degranulation with the ion channel blocker 4,49-diisothiocyanostilbene-2,29-disulfonic acid (DIDS) abolishes the increase in total β2-integrin expression induced by a number of integrin activators in neutrophils without affecting β2-integrin-dependent adhesion [39 , 41 ]. We used DIDS to determine whether the increase in high-affinity {alpha}Mβ2 induced by KT5720 is a result of an overall increase in cell surface expression of the integrins or activation of integrins already at the cell surface. Pretreatment with DIDS significantly inhibited the increase in total cell surface β2-integrin expression associated with treatment with KT5720 and PMA (Fig. 4A ). DIDS pretreatment also significantly inhibited the number of high-affinity {alpha}Mβ2 integrins on the cell surface in KT5720-treated neutrophils (Fig. 4B) . In contrast, high-affinity {alpha}Mβ2-integrin expression on the cell surface in PMA-stimulated cells was not significantly affected by DIDS pretreatment (Fig. 4B) .


Figure 4
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  		Figure 4. KT5720-induced activation of {alpha}Mβ2 requires mobilization of β2 integrins from intracellular stores. Neutrophils were pretreated with DIDS to inhibit degranulation for 30 min and then treated with vehicle, KT5720 (25 µM), or PMA for 30 min. Cells were stained as described in Materials and Methods with (A) the anti-β2-integrin antibody IB4 or (B) the antibody CBRM1/5, which specifically binds to an {alpha}Mβ2 activation-associated neoepitope. The neutrophils were washed, and the fluorescence of 10,000-gated neutrophils was then measured. Cell surface expression of β2 integrins and activated {alpha}Mβ2 are presented as the relative fluorescence intensity of each treatment group normalized to the MFI of the untreated vehicle control group. Each point represents the mean ± SE of three separate experiments. DIDS treatment significantly inhibited KT5720- and PMA-induced cell surface β2-integrin expression and KT5720-induced cell surface expression of high-affinity {alpha}Mβ2 (*, P<0.05).


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DISCUSSION
 
Little is known about how integrins are maintained in the inactive state in circulating quiescent neutrophils to prevent inappropriate adhesion and cellular activation. We demonstrated previously that a tonic level of PKA activity is necessary to maintain inactive β2 integrins in neutrophils [12 ]. Indeed, inhibition of PKA activity is sufficient to increase the number of high-affinity {alpha}Mβ2 integrins on the cell surface, increase cell surface β2-integrin expression, and induce adhesion and spreading. However, the downstream targets of PKA in the mechanism maintaining inactive β2 integrins and preventing adhesion are not known.

MLCK is a PKA substrate that is involved in regulating leukocyte integrin activation, adhesion, and migration [29 ]. MLCK activity is inhibited by PKA phosphorylation, which blocks CaM binding to the enzyme, a step necessary for activation of MLCK [31 ]. In neutrophils, MLCK appears to be involved in regulating adhesion at the trailing-edge uropod during migration [42 ]. In lymphocytes, MLCK localizes to the leading edge of polarized, migrating cells and is required for β2-integrin-mediated attachment to substrate as well as migration [29 ]. The cytoskeleton has a role in regulating adhesion, not only by governing shape change but also, by affecting the stability of integrin-mediated adhesions and integrin signaling [21 , 22 , 43 44 45 ]. Thus, there is evidence implicating MLCK and the acto-myosin cytoskeleton in the mechanism regulating the state of leukocyte integrin activation and activation-associated integrin functions.

We hypothesized that MLCK is an effector of PKA in the mechanism maintaining inactive β2 integrins in quiescent neutrophils. The data presented here support this hypothesis. Inhibition of PKA activity with KT570 is sufficient to induce phosphorylation of MLC at the MLCK phosphorylation site Ser 19. Although the principal function of MLCK is to phosphorylate MLC, other kinases may phosphorylate this site as well [31 , 46 ]. It is notable that the ability of KT5720 to induce MLC phosphorylation was abolished by two specific inhibitors of MLCK: ML-7 and a cell-permeant MLCK inhibitory peptide. The cell-permeant MLCK inhibitory peptide used for our experiments was derived from amino acids 11–19 of MLC, which contains the phosphorylation site of MLCK, and acts as a competitive inhibitor of MLCK [33 ]. ML-7 and the MLCK inhibitory peptide inhibit MLCK at the active site [33 , 47 ], demonstrating that it is indeed the MLCK kinase activity that is required for KT5720-induced MLC phosphorylation. It is notable that the ability of ML-7 to inhibit KT5720-induced adhesion was maximal at 10 µM, less than the concentration required to completely inhibit T cell attachment to a β2-integrin substrate.

As was demonstrated for KT5720-induced MLC phosphorylation, adhesion, increased β2-integrin surface expression, and increased cell surface expression of high-affinity {alpha}Mβ2 induced by the PKA inhibitor KT5720 were dependent on MLCK activity. A role for MLCK in adhesion and up-regulation of β2-integrin cell surface expression can easily be envisioned. The acto-myosin cytoskeleton is involved with the shape changes associated with cellular adhesion, integrin adhesion receptor clustering, and degranulation, leading to mobilization of preformed β2 integrins to the cell surface. It is not certain, however, how MLCK or the acto-myosin cytoskeleton is involved with KT5720-induced, β2-integrin-affinity activation observed here.

β2-integrin-affinity activation involves a conformational change that creates a high-affinity binding site for the ligand [3 ]. One possible role for MLCK in controlling the number of high-affinity {alpha}Mβ2 integrins on the cell surface is to directly regulate the conformational change associated with the shift to a high-affinity integrin. However, we demonstrate that the increase in high-affinity {alpha}Mβ2 associated with KT5720 treatment requires mobilization of β2 integrins from intracellular stores, suggesting that the primary mechanism by which inhibition of PKA activity increases cell surface expression of high-affinity {alpha}Mβ2 is by increasing the overall expression of the integrin on the surface available for activation. The fact that the proportional increase in total β2-integrin expression and high-affinity {alpha}Mβ2 in KT5720-treated cells is roughly similar supports this model. Thus, it is more likely that the role of MLCK and MLC phosphorylation in controlling the number of high-affinity {alpha}Mβ2 integrins is to regulate the myosin motor that enables degranulation. Inhibition of PKA activates MLCK, resulting in MLC phosphorylation and activation of the myosin motor, driving degranulation and mobilization of β2 integrins to the cell surface so that more {alpha}Mβ2 integrins are available to be activated to the high-affinity conformation. This would be consistent with the known role for MLCK in degranulation in other cells [48 49 50 ]. However, our data do not rule out a dual role for MLCK in regulating total β2-integrin expression and the affinity of cell surface {alpha}Mβ2.

In addition to regulating cell surface expression of β2 integrins, MLCK may have a role mobilizing integrins on the cell surface from cytoskeletal constraints and/or clustering integrins at sites of adhesion where integrin activation occurs. Although initial clustering of β2 integrins requires release of the receptors from cytoskeletal linkages, once the receptors have clustered, the cytoskeleton reorganizes to assemble structures at the site of adhesion called podosomes, where signaling molecules are concentrated [21 , 22 , 43 44 45 ]. It may be at this point that MLCK is required to maintain integrin clustering and in the high-affinity conformation during adhesion. This would be consistent with previous studies in which the actin cytoskeleton was found to have a role in regulating integrin mobility and adhesion stability [4 , 21 , 23 , 51 ].

PKA increases MLC phosphatase activity directly or by reducing the inhibitory effect of the Rho/ROCK pathway [31 ]. Thus, it is possible that PKA inhibitor activation of integrins and adhesion in neutrophils operates, not by activating MLCK but by inactivating MLC phosphatase, resulting in a net increase in MLC phosphorylation. Our data argue strongly against an indirect effect of PKA on MLC phosphatase via ROCK. Inhibition of ROCK activity did not affect KT5720-induced MLC phosphorylation or adhesion. However, we cannot rule out a direct effect of inhibition of PKA on MLC phosphatase activity. Inhibition of MLC phosphatase by treating neutrophils with calyculin-A alone resulted in MLC phosphorylation (data not shown). However, calyculin-A treatment inhibited KT5720-inducted adhesion as well as adhesion induced by PMA and immobilized immune complexes (data not shown). The significance of this finding is not clear and may be a result of a lack of specificity of calyculin-A for MLC phosphatase. Unfortunately, we do not have reagents specific enough for MLC phosphatase to further explore this possibility in primary neutrophils.


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ACKNOWLEDGEMENTS
 
This work was supported by the American Heart Association grant #0365446U.

Received April 13, 2005; revised December 12, 2007; accepted December 21, 2007.


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