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Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh
Correspondence: Samuel L. Jones, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. E-mail: sam_jones{at}ncsu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mß2 integrin activation. Elevation of cAMP inhibited Mß2 integrin-dependent adhesion of PMN to immune complexes (IC), but not PMA-induced adhesion. The PKA inhibitor KT5720 reversed the ability of cAMP to suppress adhesion to IC. Moreover, inhibition of PKA activity was sufficient to activate Mß2 integrin-dependent adhesion and increase ß2 integrin expression and binding of the monoclonal antibody CBRM1/5, which recognizes activated Mß2 specifically. However, PKA activity was necessary for sustained adhesion. Disruption of A kinase-anchoring, protein-PKA binding with a cell-permeant peptide derived from the AKAP Ht31 also activated adhesion. Unlike pharmacologic inhibition of PKA, AKAP peptide-induced adhesion was PKC dependent and did not affect ß2 integrin expression or CBRM1/5 binding. These data demonstrate that PKA appears to have a dual role in the mechanism regulating Mß2 integrin avidity and adhesion.
Key Words: immune complexes • PMN • Mß2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chains coupled with a ß2 chain. They have as ligands counter receptors expressed on endothelial and epithelial cells, extracellular matrix proteins, products of the coagulation and complement cascades, and other proteins that are present in inflamed tissues.
ß2 integrin ligand binding, and thus adhesion, require a transition from a low avidity to a high avidity state in response to signals generated by immune complexes (IC), bacterial products, chemokines, cytokines, or other inflammatory mediators [1
]. Activation of the high avidity state is a complex mechanism that involves changes in ß2 integrin conformation, which increase affinity for ligand, as well as changes in integrin mobility in the cell membrane and association with cytoskeletal elements. The signaling mechanisms that regulate ß2 integrin avidity are not completely understood. We previously identified two mechanisms of activating ß2 integrin avidity in PMN. The first, induced by ligation of Fc receptors for immunoglobulin G (IgG; Fc
Rs) by IC, is dependent on phosphatidylinositol 3-kinase (PI3K), whereas the second, activated by G-protein-linked serpentine receptor ligation, is PI3K independent [3
]. However, very little is known about how ß2 integrins are maintained in the inactive state in quiescent PMN or are inactivated once the high avidity state is achieved.
In general, cyclic adenosine monophosphate (cAMP) activation of protein kinase A (PKA) inhibits many PMN functions including adhesion and migration [4
5
6
7
]. cAMP-elevating agents inhibit spreading and actin assembly in tumor necrosis factor -stimulated PMN adherent to a ß2 integrin substrate [8
]. Moreover, pharmacological cAMP treatment has been demonstrated to hasten lymphocyte deadhesion from cells expressing the Lß2 integrin ligand intercellular adhesion molecule-1 [9
]. These data suggest that PKA activity inhibits integrin function. In contrast, recent work has identified a necessary role for PKA activity in prolonged adhesion of PMN to immune complexes [10
]. Our hypothesis is that PKA-dependent signaling is an important negative regulator of ß2 integrin avidity activation, which is distinct from its role in sustained adhesion.
We demonstrate in this work that PKA activity is required for cAMP-mediated inhibition of ß2 integrin-dependent adhesion of PMN to IC. Importantly, inhibition of PKA activity is sufficient to activate ß2 integrin-dependent adhesion, total ß2 integrin expression, and binding of a monoclonal antibody (mAb; CBRM1/5), which specifically recognizes a neoepitope induced by activation of Mß2 [11
]. PKA inhibitor-induced activation of adhesion is independent of PI3K and protein kinase C (PKC). However, PKA activity is necessary for sustained adhesion to IC. Treatment of PMN with a cell-permeant peptide derived from the PKA binding site of human thyroid A kinase anchoring protein (AKAP), which inhibits PKA/AKAP binding, also activates ß2 integrin-dependent adhesion. In contrast to adhesion induced by global inhibition of PKA activity, AKAP peptide-induced adhesion is PKC dependent and is not associated with an increase in ß2 expression or CBRM1/5 binding. Moreover, AKAP peptides do not affect sustained adhesion to immune complexes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of PMN suspensions
Human PMN were isolated from whole blood using a dextran sedimentation/Ficoll gradient centrifugation protocol as described [3
]. PMN were >98% viable as indicated by the exclusion of trypan blue dye. Cells were suspended in HBSS with 20 mM HEPES, 8.9 mM sodium bicarbonate, 1.0 mM Mg2+, and 1 mM Ca2+ (HBSS++) for adhesion assays and flow cytometry.
Adhesion assay
Purified human PMN (1x107/ml) were incubated with 2 µg/ml calcein in HBSS for 30 min at room temperature (RT). The cells were washed once and resuspended in HBSS++ at 2 x 106/ml. Cells were treated with inhibitors at the indicated concentration or Me2SO as a control for 20 min at 37°C. For antibody inhibition experiments, cells were incubated with 25 µg/ml of the appropriate antibody F(ab')2 for 15 min at RT. Cells (1x105) were added per well to Immulon 2 plates coated with BSA and a 1:50 dilution of rabbit anti-BSA to form IC or 5% FCS as described [15
]. For PMA or AKAP St-Ht31 peptide adhesion, PMA, St-Ht31-inhibitory peptide, or St Ht31P control peptides were added at the indicated final concentrations to the cells after allowing them to settle onto FCS-coated wells for 6 min at RT. The plates were then incubated at 37°C for the indicated time. The fluorescence (485 nm excitation; 530 nm emission wavelengths) was measured using an fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA) before and after washing twice with 150 µl PBS. Percent adhesion was calculated by dividing the fluorescence after washing by the fluorescence before washing. In preliminary experiments, fluorescence was shown to be linearly related to cell number (unpublished results).
Flow cytometry
Purified PMN (4x106/ml in HBSS++) were treated with inhibitors, peptides, or PMA at the indicated concentrations for 20 min at 37°C. The cells were then placed on ice, washed once with ice-cold wash buffer (PBS, 1% FCS, 0.1% sodium azide), and resuspended in 100 µl wash buffer plus primary antibody (25 µg/ml). Cells were incubated with primary antibody F(ab')2 for 40 min on ice and then washed twice. After incubation with FITC-conjugated sheep anti-mouse IgG secondary antibody F(ab')2 at a 1:50 dilution in 200 µl wash buffer for 20 min on ice, cells were washed twice, and the relative fluorescence of 10,000 gated PMN was measured using a FacsCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mß2 integrin-dependent adhesion in PMNR-induced adhesion to surfaces coated with immobilized IC, which is dependent on Mß2 activation [3
, 16
17
18
]. Treatment with the cell permeant pharmacologic cAMP analog 8-Br-cAMP inhibited adhesion to immobilized IC (Fig. 1
). The nonspecific phosphodiesterase inhibitor pentoxifylline, the phosphodiesterase IV inhibitor rolipram, and the ß2 adrenergic agonists clenbuterol and isoproterenol inhibited adhesion to IC similarly (unpublished results). 8-Br-cAMP increased adhesion to FCS slightly, but this effect was not consistent. In contrast to adhesion to IC, none of these agents had a significant effect on adhesion induced by the phorbol ester PMA (Fig. 1A)
, demonstrating that cAMP elevation does not inhibit PMN adhesion globally and is not toxic to the cells. The effect of cAMP on adhesion to IC was reversed with the specific PKA inhibitor KT5720 (Fig. 1A)
, demonstrating that PKA activity is necessary for cAMP to inhibit ß2 integrin-dependent adhesion to IC.
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Mß2 integrin activationR-induced activation of Mß2 integrin dependent adhesion is consistent with the ability of cAMP signaling to inhibit agonist receptor signal transduction. However, it is also possible that cAMP activation of PKA regulates ß2 integrin function directly in the absence of integrin activator-induced signals. To examine the latter possibility, we determined whether inhibition of PKA activity was sufficient to activate adhesion in unstimulated PMN. KT5720 treatment activated adhesion of PMN to the Mß2 integrin substrate FCS [3
] in the absence of any other stimulus (Figs. 1A
and 2A
). KT5720 stimulated adhesion in a dose-dependent manner with maximal adhesion observed at a concentration of 25 µM (Fig. 2 A ). The PKA inhibitor H89 similarly activated adhesion of PMN to FCS in a dose-dependent manner with maximal adhesion observed at a concentration of 50 µM (Fig. 2B)
. Neither KT5720 nor H89 activated respiratory burst activity in PMN in suspension or adherent to FCS substrates at doses that induced adhesion (data not shown), demonstrating that inhibition of PKA activity does not activate PMN globally. At concentrations that activated adhesion to FCS, KT5720 (25 and 50 µM) inhibited adhesion to IC at 30 min slightly but significantly (P<0.05), but did not affect PMA-induced adhesion at any concentration (Fig. 2A) . H89 inhibited adhesion to IC and PMA-induced adhesion at 30 min modestly but significantly (P<0.05; Fig. 2B
). Like adhesion to FCS induced by other integrin activators, KT5720-stimulated adhesion was inhibited by anti-ß2 integrin mAb IB4 but was unaffected by the control mAb anti-HLA W6/32 (Fig. 3
) or anti-Lß2 TS1/22 (unpublished results).
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Mß2 integrin substrate FCS (Fig. 1B) . Maximal activation of adhesion was observed at St-Ht31 peptide concentrations of 50 µM (Fig. 2C)
. St-Ht31-induced adhesion was inhibited by anti-ß2 mAb (Fig. 3)
. In contrast, the control peptide had no significant effect on adhesion. Treatment with 8-Br-cAMP did not significantly inhibit St-Ht31-induced adhesion of PMN to FCS (Fig. 1B)
. Moreover, St-Ht31 rescued adhesion of 8-Br-cAMP-treated PMN to IC (Fig. 1B)
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PKA has a distinct role in regulating adhesion activation and sustained adhesion
To investigate more carefully the dynamics of adhesion in PMN treated with PKA inhibitors, we determined the kinetics of adhesion in H89-treated PMN during adhesion to IC and FCS. H89 (50 µM) inhibited adhesion to IC modestly, up to 30 min (Fig. 4
). As has been shown previously [10
], H89 inhibited sustained adhesion to IC progressively, compared with controls (Fig. 4)
. H89 inhibition of adhesion to IC was almost complete by 60 min. Adhesion to FCS induced by H89 to FCS was maximal by 30 min and then decreased dramatically thereafter, demonstrating that although inhibition of PKA activity is sufficient to activate ß2 integrin-dependent adhesion, PKA activity is necessary to sustain adhesion beyond 30 min. Unlike pharmacologic PKA inhibitor-induced adhesion, St-Ht31 peptide-induced adhesion did not decrease for up to 60 min, and St-Ht31 treatment had no effect on PMN adhesion to IC at any time point (data not shown).
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Mß2 integrin-dependent adhesion in the absence of any other stimulus, suggesting that inhibition of PKA induces signals that activate ß2 integrins. PI3K and PKC are important signaling molecules involved in signaling pathways that activate ß2 integrin-dependent adhesion in PMN. We therefore investigated whether adhesion induced by inhibition of PKA was dependent on PI3K or PKC activity. Consistent with our previous work [3
], wortmannin, a specific inhibitor of PI3K, and staurosporine, a specific inhibitor of PKC, both significantly inhibited adhesion of PMN to immobilized IC (Fig. 5
). Staurosporine, but not wortmannin, inhibited PMA-induced adhesion. Neither wortmannin nor staurosporine affected KT5720-induced adhesion (Fig. 5)
. Interestingly, cotreatment of PMN with KT5720 and staurosporine or wortmannin inhibited adhesion to IC and (in the case of staurosporine cotreatment) PMA-induced adhesion, to the level of KT5720-induced adhesion to FCS (data not shown). Like KT5720-induced adhesion, St-Ht31-stimulated adhesion of PMN to FCS was insensitive to wortmannin (Fig. 5)
. However, St-Ht31-induced adhesion was inhibited by staurosporine significantly (Fig. 5)
, suggesting that the mechanism of St-Ht31 activation of PMN adhesion was distinct from pharmacological PKA inhibitor-induced adhesion.
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Mß2 that is induced upon activation [11
]. To investigate the mechanism by which PKA inhibitors activate ß2 integrin-dependent adhesion, we determined the effect of KT5720 and St-Ht31 on CBRM1/5 binding. Treatment of PMN with KT5720 at a concentration that activates adhesion (25 µM) induced a 3.5-fold increase in CBRM1/5 binding, equivalent to the potent Mß2 activator PMA (Fig. 6
). KT5720 treatment increased total ß2 integrin expression to a level greater than PMA. In contrast to KT5720 treatment, treatment with the St-Ht31 peptide at concentrations that activate adhesion (50 µM) did not increase ß2 integrin expression or CBRM1/5 binding (Fig. 6)
, further distinguishing the two mechanisms of activating adhesion. Concentrations of St-Ht31 peptide up to 100 µM did not affect CBRM1/5 binding nor did treatment for up to 45 min (data not shown). KT5720, St-Ht-31, or PMA did not increase expression of HLA (Fig. 6)
or of Lß2 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mß2 integrin avidity in stimulated and unstimulated PMN. In general, stimulation of PKA with cAMP inhibits cellular activation by inhibiting agonist receptor signaling [4
, 22
23
24
25
26
]. Our data are consistent with a model in which cAMP stimulation of PKA inhibits FcR signaling during activation of Mß2 avidity. We also demonstrate that inhibition of PKA activity is sufficient to activate Mß2 integrin-dependent adhesion and Mß2 avidity in unstimulated PMN. Thus, prevention of Mß2 integrin activation by PKA is critical for the maintenance of the nonadherent state of quiescent PMN. Our conclusion from this data is that PKA must have an active and direct role in regulating Mß2 avidity even in the absence of activating signals induced by inflammatory stimuli. Like activation of PMN adhesion, the nonadherent state of PMN, such as is necessary in circulating blood, is a regulated process. Activating and inhibitory signals influence the ultimate avidity state of ß2 integrins in PMN even in unstimulated PMN. PKA is a critical element of this inhibitory signal.
As demonstrated here and elsewhere [4
], pharmacological elevation of intracellular cAMP inhibits inflammatory mediator-induced stimulation of ß2 integrin activation, thus preventing the initiation of adhesion. However, cAMP-dependent signals also are capable of promoting de-adhesion [9
], a process that is likely critical at the trailing edge during leukocyte migration. These related but distinct effects of cAMP on the PMN-adhesive phenotype are consistent with the ability of cAMP to inhibit agonist receptor signals that activate ß2 integrin-dependent adhesion and to terminate activating signals, both via PKA-dependent phosphorylation of RhoA [4
, 27
]. We now show that a basal level of PKA activity tips the balance of signals that regulate Mß2 avidity in favor of inhibitory signals in quiescent PMN. It is tempting to speculate that PKA phosphorylation of RhoA is also responsible for inhibiting Mß2 integrin-avidity activation in unstimulated PMN. In contrast to PKA, PKC appears to be an important activator of RhoA, placing RhoA distal to converging, but antagonistic, pathways that regulate leukocyte adhesion. This suggests that PKA and PKC are regulatory elements of inhibiting and activating pathways that converge on RhoA. In our model, RhoA acts as a switch mechanism that ultimately determines ß2 integrin avidity in stimulated and resting PMN (Fig. 7A
). However, PKC activity is not required for adhesion induced by PKA inhibitors. Thus, PKC activation may not be required for RhoA activation and may instead be a mechanism to override PKA-induced inactivation of RhoA. This is consistent with our finding that PMA-induced adhesion is relatively insensitive to cAMP.
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Mß2 avidity and adhesion [28
]. Activation of Mß2 avidity by the L-plastin aminoterminus is dependent on the phosphorylation site Ser5 [28
]. In addition, the kinetics of L-plastin phosphorylation is consistent with a role in sustained adhesion [29
]. These features suggest a model in which PKA-dependent phosphorylation of L-plastin regulates sustained adhesion, perhaps by effects on the structural aspects of the actin cytoskeleton (podosomes) at adhesion sites (Fig. 7B)
. We have found that PKA regulates adhesion activation by at least two mechanisms. Pharmacologic inhibition of PKA activity and St-Ht31 peptide-induced disruption of PKA-AKAP binding activate adhesion, but have distinct dependence on PKC and distinct effects on CBRM1/5 binding and total ß2 integrin expression. Moreover, unlike pharmacological inhibition of PKA, AKAP peptides do not inhibit sustained adhesion. Thus, it appears that PKA has a complex role in regulating adhesion that is determined by kinetic and perhaps spatial factors. It is significant to note that like the St-Ht31-AKAP peptide, low concentrations of cytochalasin D activate PMN ß2 integrin-dependent adhesion but do not affect total ß2 integrin expression or CBRM1/5 binding (data not shown). This adhesion is also PKC-dependent, suggesting that St-Ht31-AKAP peptides activate adhesion by disrupting the actin cytoskeleton.
PI3K is an important component of the FcR-initiated mechanism of ß2 integrin activation and sustained adhesion to IC [3
]. Moreover, IC-induced L-plastin phosphorylation and sustained adhesion are dependent on PKA and PI3K [10
, 29
], suggesting a link between PI3K and PKA in the mechanism of adhesion. However, pharmacologic inhibition of PKA or disruption of AKAP binding with St-Ht31 activates ß2 integrins independently of PI3K. This suggests that the PKA-dependent inhibitory pathway regulating ß2 integrin activation does not involve PI3K downstream of PKA. It is possible, at least for FcR-initiated activation of ß2 integrins, that PI3K has role in regulating PKA function. Our data demonstrating that AKAP peptides do not affect sustained adhesion to IC (unlike pharmacological inhibition of PKA) raise the possibility that PI3K regulates PKA-AKAP binding. PI3K-dependent relocalization of PKA may explain the requirement for PI3K for sustained adhesion to IC and PKA-mediated L-plastin phosphorylation and in part explain the contrasting roles of PKA in avidity activation and maintenance of adhesion.
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ACKNOWLEDGEMENTS |
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Received July 13, 2001; revised January 29, 2002; accepted January 31, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mß2 avidity in polymorphonuclear neutrophils J. Biol. Chem. 273,10556-10566RIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRRII and tyrosine phosphorylation J. Cell Biol. 125,1407-1416 receptor stimulation J. Leukoc. Biol. 55,735-742[Abstract]
RII-mediated adhesion and phagocytosis induce L-plastin phosphorylation in human neutrophils J. Biol. Chem. 271,14623-14630This article has been cited by other articles:
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  C. D. Chilcoat, Y. Sharief, and S. L. Jones Tonic protein kinase A activity maintains inactive {beta}2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase J. Leukoc. Biol., April 1, 2008; 83(4): 964 - 971. [Abstract] [Full Text] [PDF]
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 G. Carmona, E. Chavakis, U. Koehl, A. M. Zeiher, and S. Dimmeler Activation of Epac stimulates integrin-dependent homing of progenitor cells Blood, March 1, 2008; 111(5): 2640 - 2646. [Abstract] [Full Text] [PDF]
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R. E. Eckert and S. L. Jones Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant J. Leukoc. Biol., November 1, 2007; 82(5): 1311 - 1321. [Abstract] [Full Text] [PDF]
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S. L. Jones and Y. Sharief Asymmetrical protein kinase A activity establishes neutrophil cytoskeletal polarity and enables chemotaxis J. Leukoc. Biol., July 1, 2005; 78(1): 248 - 258. [Abstract] [Full Text] [PDF]
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A. Ortiz-Stern and C. Rosales Fc{gamma}RIIIB stimulation promotes {beta}1 integrin activation in human neutrophils J. Leukoc. Biol., May 1, 2005; 77(5): 787 - 799. [Abstract] [Full Text] [PDF]
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 K. Sendide, N. E. Reiner, J. S. I. Lee, S. Bourgoin, A. Talal, and Z. Hmama Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1 J. Immunol., April 1, 2005; 174(7): 4210 - 4219. [Abstract] [Full Text] [PDF]
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M. Ariga, B. Neitzert, S. Nakae, G. Mottin, C. Bertrand, M. P. Pruniaux, S.-L. C. Jin, and M. Conti Nonredundant Function of Phosphodiesterases 4D and 4B in Neutrophil Recruitment to the Site of Inflammation J. Immunol., December 15, 2004; 173(12): 7531 - 7538. [Abstract] [Full Text] [PDF]
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