Two Signaling Mechanisms for Activation of alpha Mbeta 2 Avidity in Polymorphonuclear Neutrophils*

Samuel L. JonesDagger §, Ulla G. Knaus, Gary M. Bokochpar , and Eric J. BrownDagger **

From the Dagger  Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri 63110 and the Departments of  Immunology and par  Cell Biology, Research Institute of Scripps Clinic, La Jolla, California 92037-1092

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin alpha Mbeta 2, assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of alpha Mbeta 2 by immune complexes, but we have found no role for it in alpha Mbeta 2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of alpha Mbeta 2, but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of alpha Mbeta 2. One, induced by Fcgamma R ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of alpha Mbeta 2.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phagocytes are essential cells in host defense of metazoan organisms because they prevent the systemic spread of invading pathogens. Phagocytic cells such as monocytes and polymorphonuclear leukocytes (PMN)1 circulate throughout tissues to be able to initiate a rapid response to injury and infection. At sites of inflammation and infection, these cells perform many functions, including ingestion and killing of invading organisms, generation of inflammatory mediators, and initiation of an immune response. The acquisition of these effector functions required for successful host defense is called phagocyte activation. Adhesion is required to develop the full effector phenotype in phagocytes and, indeed, in other leukocytes as well (reviewed in Refs. 1 and 2). We have used human PMN as a model cell to study how adhesion regulates this phenotypic change and the critical role of leukocyte integrins in this process. PMN express beta 1, beta 2, and beta 3 integrins, but integrins other than the beta 2 family (also known as LeuCAM or CD18 integrins) are present in low number. In particular, the CD18 integrin alpha Mbeta 2 plays a central role in PMN activation at sites of inflammation (3-9). PMN integrins including alpha Mbeta 2 bind poorly to their ligands unless the cells are exposed to inflammatory stimuli such as chemokines, bacterial products, cytokines, complement fragments, or immune complexes (10-15). These stimuli cause an increase in integrin avidity through a process called "inside-out" signaling. The molecular pathways of inside-out signaling are uncertain, but increases in receptor affinity (16-19), receptor clustering (20), cytoskeletal reorganization (19, 21, 22), and association with guanine nucleotide exchange factors (23) may all be involved in the enhancement of beta 2 integrin avidity.

Phosphatidylinositol-3 kinase (PI 3-kinase) has been implicated in the inside-out signaling for integrin activation (24). PI 3-kinase phosphorylates phosphatidylinositols (PI) at the D3 position, producing PI 3-phosphate, PI (3,4)-bisphosphate, and PI (3,4,5)-trisphosphate (PIP3) (25). Five isoforms of mammalian PI 3-kinase have been discovered which appear to be products of distinct genes but have overlapping patterns of expression (26-29). All known isoforms of mammalian PI 3-kinase share sensitivity to the pharmacologic agent wortmannin (26, 30, 31), which inhibits PI 3-kinase activity by binding to the lipid-binding domain of the catalytic subunit (32, 33). Whereas wortmannin specifically inhibits PI 3-kinase activity at concentrations in the low nanomolar range (26, 30, 31), higher concentrations inhibit PI 4-kinase and myosin light chain kinase activity (34, 35). A second agent called LY294002 inhibits PI 3-kinase activity of the p85/p110 isoforms by binding the ATP-binding site of p110, but it has no effect on PI 4-kinase activity at doses up to 100 µM (36). These pharmacologic agents have been extremely useful in delineating cellular activities in which PI 3-kinase has a role, including regulation of adhesion. Wortmannin has been shown to inhibit beta 1 integrin-mediated adhesion to fibronectin of stem cell factor-treated mast cells (37) and CD-2 transfected HL-60 cells (38); thrombin- and Fcgamma RII-induced, beta 3 integrin-mediated aggregation of platelets (39-41); and beta 2 integrin-dependent homotypic adhesion of IL-2-treated lymphocytes (42). Use of PDGF and CD28 receptor mutants that no longer bind PI 3-kinase has provided strong evidence that PI 3-kinase is important for regulating PDGF- and CD28-induced adhesion in mast cells and HL-60 cells, respectively (43, 44). Furthermore, expression of dominant negative mutants of the p85 subunit of PI 3-kinase blocks CD7-induced activation of beta 1 integrins in human T cells (45). Although these data strongly suggest that PI 3-kinase activity is an important early event in inside-out signaling regulating integrin-mediated adhesion, the mechanism by which PI 3-kinase regulates adhesion is not clear nor is the generality of the requirement for PI 3-kinase in integrin activation. PDGF receptors, for example, can activate beta 1 integrins by PI 3-kinase-dependent and -independent mechanisms (44).

Whether PI 3-kinase has a role in alpha Mbeta 2 activation in PMN is not known. Fcgamma R-induced phagocytosis (46), fMLP-induced respiratory burst activity (32, 33, 47), and PDGF-induced chemotaxis (48) in PMN are inhibited by wortmannin. Since each of these events depends on activated integrins, these data suggest the hypothesis that PI 3-kinase is a component of the inside-out signaling pathway regulating integrin activation in PMN. However, PMN contain an intracellular pool of alpha Mbeta 2 which is rapidly expressed at the plasma membrane upon activation (15, 49). While this intracellular pool is not required for PMN binding to endothelia or for aggregation (50-52), it is necessary for optimal adhesion (53). The role of PI 3-kinase in regulating the expression of this intracellular pool at the plasma membrane is unknown.

We tested the importance of PI 3-kinase in regulating integrin activation in PMN in two well characterized experimental systems for activating beta 2 integrin-dependent adhesion. Fcgamma R ligation activates alpha Mbeta 2 through initiation of a tyrosine kinase cascade, whereas fMLP requires a heterotrimeric G-protein to initiate signaling in PMN. Our results suggest that two pathways exist for activating beta 2 integrin-dependent adhesion in PMN. The Fcgamma R-initiated pathway is dependent on PI 3-kinase activity and is inhibited by cytochalasin D, whereas the fMLP-induced increase in alpha Mbeta 2 avidity is independent of PI 3-kinase and unaffected by cytochalasin D. Fcgamma R-mediated enhancement of alpha Mbeta 2 expression is inhibited by wortmannin, but increased expression is not required for adhesion. Importantly, both pathways activate Pak1, a recently described serine/threonine kinase implicated in membrane ruffling and focal adhesion formation (54). Fcgamma R-induced activation of Pak1 is PI 3-kinase-dependent, whereas fMLP-induced activation of Pak1 is independent of PI 3-kinase, potentially placing Pak1 in a common pathway leading to activation of alpha Mbeta 2 avidity. These data demonstrate that there is more than one molecular pathway for inside-out signaling and suggest that the effects of tyrosine kinase cascades, and G protein-dependent signaling on integrin function may be mediated by distinct mechanisms that converge on a common pathway involving Pak1.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
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References

Reagents-- Cytochalasin D, PMA, fMLP, dimethyl sulfoxide, rabbit polyclonal anti-BSA antiserum, C5a, BSA, poly-L-lysine, glutaraldehyde, fluorescein isothiocyanate-conjugated F(ab')2 sheep anti-mouse IgG antibody, MBP, and EGTA were from Sigma. [gamma -32P]ATP was from ICN (Irvine, CA). Phosphate-buffered saline and 10× stock HBSS were from Life Technologies, Inc. Protein A-conjugated Sepharose, Ficoll-Paque, and Dextran T500 were obtained from Amersham Pharmacia Biotech (Uppsala Sweden). IL-8 was purchased from Calbiochem (San Diego, CA), and pertussis toxin was obtained from List Biologicals (Campbell, CA). 1 M stock Hepes and 7.5% sodium bicarbonate were from BioWhittaker (Walkersville, MD). Wortmannin and LY294002 were obtained from LC Laboratories (Woburn, MA). Fetal calf serum (FCS) was from Hyclone (Logan, UT). Calcein and Celltracker Green CMFDA were from Molecular Probes (Eugene OR). Tissue culture plates and 96-well Immulon 2 plates were from Becton-Dickinson (Franklin Lakes, NJ) and Dynatech (Chantilly, VA), respectively. Monoclonal Abs 3G8 (anti-Fcgamma RIII) (55), IV.3 (anti-Fcgamma RII) (56), IB4 (anti-beta 2, CD18) (57), W6/32 (anti-class I HLA) (58), and B6H12 (anti-IAP, CD47) (59) were purified, and F(ab')2 was prepared as described (60). CBRM1/5 (61) concentrated tissue culture supernatant and anti-Pak1 polyclonal antiserum (62) were prepared as described.

Preparation of PMN Suspensions-- Human PMN were isolated from whole blood exactly as described (63) except hypotonic lysis was not performed. PMN were greater than 98% viable as indicated by the exclusion of trypan blue dye. Cells were suspended in HBSS (Hanks' buffered salts solution with 20 mM Hepes and 8.9 mM sodium bicarbonate) with 1.0 mM Mg2+ and 1 mM Ca2+ (HBSS2+) or HBSS with 0.5 mM Mn2+ for adhesion assays and flow cytometry.

Adhesion Assay-- Purified human PMN (1 × 107/ml) were incubated with 2 µg/ml calcein in HBSS for 30 min at RT. The cells were washed once and resuspended in HBSS2+ at 2 × 106/ml. For adhesion experiments in the presence of Mn2+, the cells were washed in HBSS with 2 mM EGTA once, HBSS2+ or HBSS + 0.5 mM Mn2+ once, and resuspended in HBSS2+ or HBSS + 0.5 mM Mn2+. Cells were treated with wortmannin or LY294002 at the indicated concentration or Me2SO as a control for 15 min at 37 °C or with pertussis toxin (2 µg/ml) or control buffer in HBSS + 1% human serum albumin for 2 h at 37 °C. For antibody inhibition experiments, cells were incubated with 10 or 25 µg/ml of the appropriate antibody for 15 min at RT. 1 × 105 cells were added per well to Immulon 2 plates coated with BSA and a 1:25 dilution of rabbit anti-BSA to form IC or 5% FCS as described (64). For PMA or fMLP-stimulated adhesion, PMA (50 µg/ml final), fMLP (100 nM final), or Me2SO control was added to the cells after allowing them to settle onto FCS-coated wells for 6 min at RT. The cells were incubated at 37 °C for the indicated time. The fluorescence (485 nm excitation and 530 nm emission wavelengths) was measured using a fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA) before and after washing twice with 150 µl of phosphate-buffered saline. 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 (data not shown).

Flow Cytometry-- Purified PMN (4 × 106/ml in HBSS2+) were treated with wortmannin (100 nM) or Me2SO for 15 min at 37 °C. For experiments with pertussis toxin, 1 × 107 cells/ml were incubated with pertussis toxin (2 µg/ml) or control buffer for 2 h at 37 °C and then washed. 2 × 106 cells were then treated with 30 µl insoluble IC (IIC) prepared exactly as described (65), fMLP (100 nM), C5a (50 nM), IL-8 (100 nM), or PMA (50 µg/ml) at 37 °C for 10 min, placed on ice, washed once with ice-cold wash buffer (phosphate-buffered saline, 1% FCS, 0.1% sodium azide), and resuspended in 100 µl of wash buffer plus primary antibody (25 µg/ml). Cells were incubated with primary antibody for 40 min on ice and then washed twice. After incubation with fluorescein isothiocyanate-conjugated F(ab')2 sheep anti-mouse IgG secondary antibody at a 1:50 dilution in 200 µl of wash buffer for 20 min on ice, cells were washed twice, and the relative fluorescence of gated PMN was measured using a EPICS XL (Coulter, Miami, FL) flow cytometer. For Mn2+ experiments, cells were treated with wortmannin, washed, resuspended in HBSS2+ or HBSS + 0.5 mM Mn2+, and incubated for 10 min at 37 °C and then placed on ice. Primary antibody was added directly to the cells (25 µg/ml) for 40 min on ice, washed twice, and incubated with secondary antibody as above. All washes were done with HBSS containing appropriate divalent cations.

Pak1 Kinase Assays-- Purified PMN were suspended at 1 × 107 cells/ml in HBSS2+. After pretreatment with wortmannin, LY294002, pertussis toxin, or control buffer as described above, 7.5 × 106 cells were added to 6-well plates coated with IC or FCS as described (66) or stimulated in suspension with fMLP (100 nM). After incubating at 37 °C, the cells were lysed by adding cold 2× lysis buffer (1% Nonidet P-40, 150 mM NaCl, 5 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM NaVO4, 5 mg/ml leupeptin and aprotinin, and 1 mM diisopropyl fluorophosphate, final concentration) for 30 min on ice. Pak1 was immunoprecipitated from the lysates with 5 µl of rabbit anti-Pak1 antiserum and 40 µl of a 1:1 slurry of Protein A-Sepharose for 2 h at 4 °C. The immunoprecipitates were washed four times with lysis buffer and two times with reaction buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl2). Kinase reactions were performed with the Pak1 immunoprecipitates by adding 30 µl of reaction buffer with 2.5 µg of MBP to the beads, incubating for 10 min at RT, followed by 10 µl of reaction buffer containing 100 µM cold ATP and 0.5 µCi of [gamma -32P]ATP (4500 Ci/mmol) for a final ATP concentration of 25 µM. The reactions were incubated for 20 min at 30 °C, after which the reaction was stopped with 50 µl of SDS-polyacrylamide gel electrophoresis sample buffer containing 10% SDS. Phosphorylation of MBP was detected by SDS-polyacrylamide gel electrophoresis, transfer to polyvinylidene difluoride membranes, and autoradiography. For each experiment, Pak1 protein was immunoblotted using anti-Pak1 antiserum (1:1000) primary antibody, horseradish peroxidase-conjugated goat anti-rabbit antiserum (20 µg/ml) secondary antibody, and enhanced chemiluminescence substrate (ECL, Pierce) to ensure that equivalent amounts of kinase protein were added to each in vitro kinase reaction.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PI 3-Kinase Activity Is Required for Fcgamma R-induced Activation of alpha Mbeta 2-dependent Adhesion-- PI 3-kinase activity is required for adhesion of a variety of cell types to fibronectin (37, 38), agonist-induced aggregation and up-regulation of activated alpha IIbbeta 3 expression in platelets (39-41), and PDGF-induced chemotaxis in PMN (48), suggesting that PI 3-kinase activity is important for integrin activation. Fcgamma R-induced phagocytosis in PMN is blocked by wortmannin (46), suggesting that PI 3-kinase activity may be required for Fcgamma R-induced signal transduction and effector functions in PMN. We used two pharmacologic inhibitors of PI 3-kinase, wortmannin and LY294002, to test the hypothesis that PI 3-kinase activity is required for Fcgamma R-induced, beta 2 integrin-dependent adhesion to IC in PMN.

Adhesion of control PMN to IC was maximal by 15 min and sustained for up to 40 min (Ref. 8 and Fig. 1A). PMN pretreated with wortmannin or LY294002 initially adhered to IC-coated surfaces identically to control cells, even at inhibitor concentrations up to 1 and 200 µM, respectively (Fig. 1, A and C, and data not shown). However, after 10 min, adhesion of both wortmannin- and LY294002-treated PMN to IC decreased until there was no specific, IC-dependent adhesion by 40 min (Fig. 1, A and C). Similar kinetics of adhesion were obtained with PMN pretreated with wortmannin for 30 min. Non-adherent, wortmannin-treated PMN excluded trypan blue dye, demonstrating that they were viable. Although the wortmannin and LY294002-treated PMN cells initially (<10 min) spread as well as control cells on IC, after 10 min spreading decreased until by 40 min the cells were completely round (data not shown). In contrast, PMA-induced adhesion to, and spreading on, FCS were not affected at any dose of either PI 3-kinase inhibitor (Fig. 1, B and D). Wortmannin and LY294002 inhibited sustained (>10 min) adhesion to IC in a dose-dependent manner, with IC50 of 5 nM and 8 µM, respectively (Fig. 1, B and D). The IC50 for inhibition of sustained adhesion for each compound is comparable to the IC50 for inhibition of in vitro PI 3-kinase activity in anti-p85 immunoprecipitates from pretreated PMN in our system and others (Ref. 33 and data not shown), and published doses that inhibit fMLP-induced respiratory burst activity, in vitro PI 3-kinase activity, and PIP3 accumulation in PMN (32, 33, 36, 47) and is 6-fold less than that reported to inhibit Fcgamma R-mediated phagocytosis in PMN (46).


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Fig. 1.   Wortmannin and LY294002 inhibit sustained adhesion to IC-coated surfaces in a dose-dependent manner. The kinetics of adhesion (A and C) and the PI 3-kinase inhibitor dose responses (B and D) are presented. PMN (2 × 106 cells/ml) loaded with calcein were treated with control Me2SO or the indicated concentration of wortmannin (wort) (A and B) or LY294002 (C and D) for 10 min at 37 °C and then allowed to adhere to 96-well plates coated with BSA-anti-BSA immune complexes (IC) or FCS for the indicated times (A and C) or 45 min (B and D) at 37 °C. Some PMN were treated with 50 ng/ml PMA at the time of addition to FCS-coated wells. The data are the mean ± S.E. of triplicate wells, reported as attachment index (AI), the percentage of cells that remain adherent after washing as described under "Materials and Methods." The PI 3-kinase inhibitors blocked sustained adhesion to IC-coated surfaces. Data are representative of three separate experiments.

Previous work in our lab had shown that adhesion of PMN to IC-coated surfaces occurs in two phases (8). Initial (<10 min) adhesion is independent of beta 2 integrins as shown by PMN from patients with leukocyte adhesion deficiency or normal PMN treated with anti-beta 2 antibodies. Sustained adhesion (>10 min), however, requires the beta 2 integrin alpha Mbeta 2. The kinetics of adhesion of wortmannin-treated PMN were identical to PMN treated with anti-beta 2 and anti-alpha M F(ab')2 antibody fragments (Fig. 2 and data not shown). This demonstrates that PI 3-kinase inhibitors specifically block the alpha Mbeta 2-dependent phase of PMN adhesion to IC. Other inhibitors of adhesion, including cytochalasin, bromophenacyl bromide, W7, myosin light chain kinase inhibitors ML-9 and KT 5926, or the myosin inhibitor 2,3-butanedione monoxime, inhibited both phases of PMN adhesion (data not shown). Initial adhesion was inhibited partially by anti-Fcgamma RII antibody Fab fragments and completely by a combination of anti-Fcgamma RII and anti-Fcgamma RIII antibodies (data not shown).


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Fig. 2.   Wortmannin specifically blocks the alpha Mbeta 2-dependent phase of PMN adhesion to IC. PMN were incubated with the Me2SO control, wortmannin (100 nM), anti-beta 2 mAb IB4 F(ab')2 (10 µg/ml), or the control anti-integrin-associated protein (CD47) mAb B6H12 F(ab')2 (10 µg/ml) for 10 min at 37 °C. Cell adhesion was measured as in Fig. 1. Anti-beta 2 and wortmannin both inhibit the second phase of PMN adhesion to IC. The data are representative of three separate experiments.


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Fig. 3.   fMLP- and PMA-induced adhesion to FCS is PI 3kinase-independent. A and B, PMN were treated with control Me2SO, wortmannin (wort) (100 nM), anti-beta 2 mAb IB4 F(ab')2 (10 µg/ml), or the control anti-IAP mAb B6H12 F(ab')2 (10 µg/ml) for 10 min at 37 °C prior to measurement of adhesion to FCS-coated wells. Vehicle control (A and B), 100 nM fMLP (A), or 50 ng/ml PMA (B) were added to the appropriate wells to activate alpha Mbeta 2-mediated adhesion. Adhesion was measured as described in Fig. 1 after the indicated times. C, PMN were treated with control buffer, wortmannin (100 nM) or LY294002 (25 µM), or PT(2 µg/ml) prior to measurement of adhesion to IC (30 min) or fMLP- or PMA-induced adhesion to FCS (3 min). Wortmannin and LY294002 significantly inhibited adhesion to IC but did not affect fMLP- or PMA-stimulated adhesion to FCS (<0.05). PT significantly inhibited fMLP- but not PMA- or IC-induced adhesion (p < 0.05). The data are representative of three separate experiments.

fMLP- and PMA-induced Adhesion Is PI 3-Kinase-independent-- PI 3-kinase could be required for integrin-dependent adhesion because it is involved in an Fcgamma R-initiated signaling pathway which results in an alteration in alpha Mbeta 2 avidity or because it is involved in the cytoskeletal rearrangements which are required for increased adhesion. To distinguish these possibilities, we assessed the role of PI 3-kinase in PMA and fMLP-induced PMN adhesion. PMN do not adhere to surfaces coated with FCS in the absence of alpha Mbeta 2 activation but adhere strongly when stimulated with agonists (Fig. 3, A and B). In contrast to its effect on sustained PMN adhesion to IC, wortmannin had no effect on either PMA or fMLP-induced adhesion. fMLP (100 nM)-stimulated adhesion to FCS was maximal by 3 min, decreased by 10 min, but remained above base-line adhesion for at least 30 min (Fig. 3A). Wortmannin treatment had no significant effect on fMLP-induced adhesion at any time point. In confirmation of earlier reports (32, 33, 47), 10 nM wortmannin completely inhibited fMLP-induced respiratory burst activity in PMN (data not shown). Furthermore, wortmannin pretreatment inhibited PI 3-kinase activity in anti-p85 and anti-p110gamma immunoprecipitates with IC50 of 5 and 20 nM, respectively, demonstrating the efficacy of wortmannin treatment (data not shown). Likewise, PMA (50 ng/ml) induced significant adhesion by 3 min which continued to increase until 30 min and was unaffected by wortmannin (Fig. 3B). Identical results were obtained for fMLP- and PMA-induced adhesion to the alpha Mbeta 2 ligand fibrinogen (data not shown). Like wortmannin, LY294002 had no effect on fMLP- or PMA-induced adhesion (data not shown). The PKC inhibitor Gö6976 inhibited fMLP, PMA, and IC-induced adhesion (data not shown).

The fMLP receptor is a seven transmembrane receptor associated with a pertussis toxin (PT)-sensitive G protein, as are many receptors implicated in integrin regulation (9, 11, 67-69). To determine whether the PI 3-kinase-independent pathway for adhesion required the G protein, the effect of PT on fMLP-induced adhesion was assessed. PT pretreatment completely inhibited fMLP-induced adhesion but had no effect on IC- or PMA-induced adhesion (Fig. 3C). To determine whether this difference in sensitivity to PI 3-kinase inhibitors reflected a difference in mechanism or a difference between FCS and IC-coated surfaces, the effect of fMLP and PMA on wortmannin sensitivity of sustained adhesion to an IC-coated surface was assessed (Fig. 4, A and B). Stimulation of wortmannin-treated PMN adherent to IC for 7 min with PMA or fMLP induced sustained adhesion that was equivalent to control PMN at 40 min (Fig. 4, A and B). Furthermore, both fMLP and PMA restored the spread phenotype of wortmannin-treated PMN on IC (data not shown). These data demonstrate that PI 3-kinase activity is not required for adhesion per se and support the hypothesis that PI 3-kinase is an essential component of the signaling cascade initiated by Fcgamma R ligation resulting in alpha Mbeta 2 activation. Whereas PMA directly activates PKC and thus could bypass wortmannin inhibition by this mechanism, fMLP binds to a heterotrimeric G protein-coupled receptor which initiates several signaling cascades of its own. Thus, fMLP-induced adhesion to FCS or IC-coated surfaces is a wortmannin- and LY294002-insensitive pathway for integrin regulation.


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Fig. 4.   fMLP and PMA rescue sustained adhesion or wortmannin-treated PMN to IC. PMN were treated with Me2SO control or wortmannin (wort) (100 nM) (A and B), or anti-beta 2 mAb IB4 F(ab')2 (B), or the control anti-IAP mAb B6H12 F(ab')2 (B). After initial incubation with IC-coated surfaces (A and B), 100 nM fMLP (A), or 50 ng/ml PMA (B) were added and the plates incubated further for 30 min at 37 °C, and adhesion was measured as in Fig. 1. fMLP and PMA overcome wortmannin inhibition of sustained adhesion to IC. The data are representative of three separate experiments. Identical results were obtained when LY294002 (25 µM)-treated PMN were used.

PI 3-Kinase-dependent and -independent Up-regulation of alpha Mbeta 2 Integrin-- When PMN are activated two processes occur that can potentially affect adhesion. Cell surface beta 2 integrin expression increases as integrins stored in intracellular granules are released to the cell membrane by degranulation. Integrins already present on the cell surface also are activated, causing an increase in avidity for ligand which is independent of any increase in receptor expression. To determine whether one or both of these steps was affected by the distinct fMLP and IC signaling pathways and to determine whether one or both was required for sustained adhesion, we examined these processes independently. We first tested whether Fcgamma R-induced up-regulation of beta 2 integrin expression is affected by wortmannin using flow cytometry to detect surface expression of beta 2 on PMN stained with anti-beta 2 integrin antibody F(ab')2.

Stimulation of PMN in suspension with IC, fMLP, or PMA all caused a significant increase in expression of beta 2 (Fig. 5A). Wortmannin (100 nM) inhibited IC-induced beta 2 expression but had no effect on fMLP and PMA-induced beta 2 expression (Fig. 5A). Interestingly, cytochalasin D also specifically inhibited IC- but not fMLP- or PMA-induced up-regulation of beta 2 expression (Fig. 5A). Cytochalasin D also inhibits the increase in [Ca2+]i (where [Ca2+]i means intracellular calcium concentration) induced by IC but not by fMLP (70). Since increased [Ca2+]i is required for enhanced alpha Mbeta 2 expression (71), this may be the mechanism for the inhibitory effect of cytochalasin D. Identical results were obtained for alpha Mbeta 2 expression specifically using the alpha M-specific antibody, OKM1 (data not shown). Thus, sensitivity to cytochalasin D is another distinguishing feature of the signaling pathways activated by IC and fMLP. IC, fMLP, or PMA did not increase expression of HLA (Fig. 5B) or alpha Lbeta 2 integrin (LFA-1, as measured by expression of alpha L, data not shown); in fact PMA caused HLA and alpha L expression to decrease. Although cytochalasin D slightly decreased HLA and alpha L expression in fMLP- and PMA-treated PMN, possibly by enhanced internalization of these receptors, neither wortmannin nor cytochalasin D treatment had any effect on HLA or alpha L expression in IC-stimulated PMN. Thus, Fcgamma R-stimulated expression of alpha Mbeta 2 requires PI 3-kinase and the actin cytoskeleton, whereas fMLP- and PMA-stimulated expression are unaffected by the PI 3-kinase inhibitors which block IC-induced up-regulation of alpha Mbeta 2.


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Fig. 5.   IC-induced up-regulation of beta 2 integrin expression is PI 3-kinase-dependent and requires the actin cytoskeleton, whereas fMLP- and PMA-induced up-regulation do not require PI 3-kinase activity or the actin cytoskeleton. PMN were treated with Me2SO control, wortmannin (100 nM), or cytochalasin D (CytD) (10 µg/ml) for 10 min at 37 °C and then activated in suspension with insoluble IC (IIC), fMLP, or PMA for 10 min at 37 °C. The cells were subsequently stained with anti-beta 2 antibody IB4 F(ab')2 (A) or anti-HLA antibody W6/32 (B) to quantitate receptor expression by flow cytometry. Data are presented as receptor expression normalized to expression on unstimulated PMN. Each point represents the mean ± S.E. relative fluorescence from three separate experiments. Insoluble IC, fMLP, and PMA all significantly (p < 0.05) increased anti-beta 2 staining. Wortmannin and cytochalasin D significantly inhibited IIC-induced anti-beta 2 staining (p < 0.05) but had no effect on fMLP- or PMA-induced anti-beta 2 staining (A). Identical results were obtained when the cells were stained with the anti-alpha M antibody OKM1.

PI 3-Kinase-dependent and -independent Pathways for alpha Mbeta 2 Activation-- We next examined the role of PI 3-kinase in activation of surface-expressed alpha Mbeta 2. We used a monoclonal antibody (CBRM1/5) that specifically recognizes a neoepitope on activated alpha Mbeta 2 to detect activated alpha Mbeta 2 on the surface of stimulated PMN (61). IC, fMLP, and PMA all increased expression of the CBRM1/5 epitope (Fig. 6A and data not shown). However, wortmannin and cytochalasin D inhibited the increased expression of activated alpha Mbeta 2 only on PMN stimulated with IC and not on fMLP- or PMA-stimulated PMN (Fig. 6A and data not shown). Thus, both Fcgamma R-induced up-regulation of alpha Mbeta 2 expression and activation are PI 3-kinase-dependent and also require the actin cytoskeleton. In contrast, fMLP and PMA-induced up-regulation of alpha Mbeta 2 expression and activation are PI 3-kinase-independent and cytochalasin-insensitive.


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Fig. 6.   IC-induced activation of alpha Mbeta 2 is PI 3-kinase-dependent and requires the actin cytoskeleton, whereas G protein-linked receptor-induced activation of alpha Mbeta 2 is PI 3-kinase-independent and cytochalasin-insensitive. A, PMN were treated with Me2SO control, wortmannin (100 nM), cytochalasin D (CytD) (10 µg/ml), or pertussis toxin (PT) (2 µg/ml) and then activated with IIC or fMLP (100 nM) in suspension for 10 min at 37 °C. B, PMN pretreated with control buffer, wortmannin, or PT were stimulated as in A with IIC, fMLP (100 nM), IL-8 (100 nM), or C5a (50 nM). alpha Mbeta 2 activation was assessed by quantitating CBRM1/5 (25 µg/ml) binding with flow cytometry. Data are presented as the fluorescence of the PMN population normalized to the fluorescence of control PMN. Each point represents the mean ± S.E. relative fluorescence from three separate experiments. Insoluble IC, fMLP, IL-8, and C5a significantly (p < 0.05) increased CBRM1/5 binding. Wortmannin significantly inhibited IIC-induced CBRM1/5 binding (p < 0.05) but had no significant effect on fMLP-, C5a-, IL-8-induced CBRM1/5 binding (A and B). Pertussis toxin significantly inhibited fMLP-, C5a-, and IL-8-induced CBRM1/5 binding but had no significant effect on IIC-induced CBRM1/5 binding (A and B). PMA-induced CBRM1/5 binding was unaffected by any inhibitor (data not shown).

To determine whether the PI 3-kinase-independent pathway for integrin regulation required the G protein, the effect of PT on fMLP-induced CBRM1/5 was assessed. PT (2 µg/ml) completely inhibited fMLP-induced CBRM1/5 expression but had no effect on CBRM1/5 expression induced by IIC (Fig. 6A). We next assessed the mechanisms by which ligands for other seven transmembrane receptors regulate integrin avidity. Like fMLP, C5a and IL-8 increased CBRM1/5 binding to PMN. For both C5a and IL-8, CBRM1/5 binding was unaffected by wortmannin but was inhibited by PT (Fig. 6B). Thus, ligation of these different G protein-coupled receptors activated alpha Mbeta 2 in PMN in a wortmannin-insensitive manner.

Activation of alpha Mbeta 2 Avidity Is Necessary and Sufficient for Sustained Adhesion to IC-- Whereas both Fcgamma R-induced increases in beta 2 expression and activation of the alpha Mbeta 2 activation epitope require PI 3-kinase activity, it is well established that receptor activation is required, but increased receptor expression is not necessary for alpha Mbeta 2 adhesion in other systems (50-52, 61). To determine the requirements for sustained adhesion to IC, we tested the effect of CBRM1/5 (61) on adhesion. Like wortmannin and the anti-beta 2 antibody, CBRM1/5 inhibited sustained adhesion to IC but had no effect on the initial beta 2-independent adhesion (Fig. 7) Thus, activated alpha Mbeta 2 is necessary for sustained adhesion.


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Fig. 7.   Activated alpha Mbeta 2 is necessary for sustained adhesion of PMN to IC. PMN were treated with Me2SO control, wortmannin (100 nM), anti-beta 2 mAb IB4 (10 µg/ml), anti-activated alpha Mbeta 2 mAb CBRM1/5 (25 µg/ml), or the control anti-HLA mAb W6/32 (25 µg/ml). Cells were allowed to adhere to IC or FCS-coated surfaces, and adhesion was assessed as in Fig. 1. CBRM1/5 inhibits adhesion to IC with kinetics identical to anti-beta 2 or wortmannin. The data are representative of three separate experiments.

To determine whether increased alpha Mbeta 2 expression is required, PMN were treated with the ion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which prevents increased beta 2 expression by inhibiting degranulation (52). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid had no effect on sustained adhesion to IC (data not shown). Moreover, sustained adhesion to IC was perfectly normal in the absence of extracellular Ca2+, even in cells treated with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (data not shown). Since these cells have very low [Ca2+]i, up-regulation of receptor expression is deficient (71). These data suggested that increased receptor expression was not required for sustained adhesion to IC. To examine this definitively, we used Mn2+ to induce alpha Mbeta 2 activation. Mn2+ activates the high avidity state of many integrins, including alpha Mbeta 2 (72), without inside-out signaling, possibly by altering the conformation of the receptor to increase ligand affinity. Treatment of PMN with Mn2+ increased expression of the CBRM1/5 epitope (Fig. 8A) but had no effect on total beta 2 integrin expression (Fig. 8B). Mn2+-induced expression of the CBRM1/5 epitope was unaffected by wortmannin (Fig. 8A). Mn2+ treatment rescued sustained adhesion of wortmannin-treated PMN to IC (Fig. 8C). The combination of anti-beta 2 and CBRM1/5 antibodies completely inhibited Mn2+-induced adhesion to IC and FCS, indicating that the mechanism of adhesion to these substrates remained beta 2 integrin-dependent. Thus, alpha Mbeta 2 activation is necessary and sufficient for sustained adhesion to IC. These data also show that wortmannin does not inhibit adhesion to IC by affecting beta 2 integrin outside-in signaling.


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Fig. 8.   Mn2+ activation of alpha Mbeta 2 rescues sustained adhesion of wortmannin-treated PMN to IC. A and B, PMN (1 × 106 cells) were pretreated with control Me2SO or wortmannin (100 nM), washed with HBSS containing 2 mM EGTA, and then resuspended in HBSS with Mn2+ (0.5 mM) or Ca2+/Mg2+ (1 mM each). PMN were then incubated with anti-activated alpha Mbeta 2 antibody CBRM1/5 (A) or anti-beta 2 antibody IB4 (B) and analyzed by flow cytometry. Data are presented as the fluorescence of the PMN population normalized to the fluorescence of control PMN. Each point represents the mean ± S.E. relative fluorescence from three separate experiments. Mn2+ significantly increased CBRM1/5 binding (p < 0.05) (A) but had no effect on total beta 2 expression (B). Wortmannin pretreatment did not significantly effect CBRM1/5 or anti-beta 2 binding. Mn2+ had no significant effect on anti-HLA binding (data not shown). C, adhesion to IC after 40 min of PMN pretreated with wortmannin or control was measured in the presence of anti-beta 2 mAb IB4 (10 µg/ml), CBRM1/5 (25 µg/ml), both anti-beta 2 and CBRM1/5 (10 and 25 µg/ml, respectively), or the control anti-HLA W6/32. Adhesion was quantitated as in Fig. 1. These data are representative of three separate experiments.

Pak1 Is Activated by IC and fMLP-- The small GTPases Rac and Cdc42 have been found to be important for regulation of the actin cytoskeleton and formation of integrin complexes in several cell types (73). We investigated the possibility that Rac and/or Cdc42 regulated integrin activation in PMN by examining the activation of the Rac1/Cdc42 effector Pak1 which has been implicated in the regulation of the actin cytoskeleton and formation of focal adhesions (54). Both fMLP and adhesion to IC activated Pak1 in PMN (Fig. 9, A and B). The kinetics of Pak1 activation in response to fMLP and IC was identical to the kinetics of adhesion induced by these stimuli (Fig. 9, C and D). Pak1 activation induced by fMLP or adhesion to IC was unaffected by pretreatment with anti-beta 2 F(ab')2, indicating that Fcgamma R ligation activates Pak1 independently of beta 2 integrins, suggesting that Pak1 activation may be a component of inside-out signaling (Fig. 9E). Importantly, we found that IC-induced Pak1 activation was dependent on PI 3-kinase activity and independent of a PT-sensitive G protein (Fig. 9, A and B). In contrast, fMLP-induced Pak1 activation was inhibited completely by PT but was unaffected by wortmannin (Fig. 9, A and B). Both wortmannin and LY294002 inhibited IC-induced Pak1 activation with IC50 of 5 nM and 5 µM (data not shown), identical to the IC50 for inhibition of sustained adhesion to IC. These data demonstrate that while Fcgamma R and the fMLP receptor induce initially distinct signals, the signaling pathways initiated by these receptors converge on the Rac/Cdc42-activated kinase Pak1.


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Fig. 9.   Pak1 activation induced by adhesion to IC is PI 3-kinase-dependent, whereas fMLP-induced Pak1 activity does not require PI 3-kinase. A, PMN were treated with wortmannin (W) (100 nM) and allowed to adhere to IC or FCS for 10 min or stimulated in suspension with control (C) Me2SO or fMLP (100 nM) for 1 min. The kinase activity of immunoprecipitated Pak1 was assayed using MBP as a substrate (A, top). Western blot analysis of the Pak1 immunoprecipitates with anti-Pak1 antiserum demonstrates that equivalent amounts of protein were used for the kinase reactions (A, bottom). B, PMN were treated with control buffer, wortmannin (100 nM), or PT (2 µg/ml) and allowed to adhere to IC or FCS for 10 min or were stimulated in suspension with fMLP (100 nM) for 1 min. The kinase activity of immunoprecipitated Pak1 was assayed as in A. Phosphorylation of MBP was quantitated by densitometry. Each point represents the mean ± S.E. of three separate kinase reactions. C, PMN were allowed to adhere to IC or FCS, and Pak1 kinase activity after various times was assayed as in A. Phosphorylation of MBP was quantitated by densitometry. D, PMN were treated with 100 nM fMLP or vehicle control in suspension, and Pak1 activity was quantitated after various times as in C. E, after treatment with Me2SO control, wortmannin (Wort) (100 nM), anti-beta 2 mAb IB4 F(ab')2, or the control anti-IAP mAb B6H12 F(ab')2 PMN were allowed to adhere to IC for 10 min, and (E, top) Pak1 kinase activity was assessed as in A. Western blot of Pak1 immunoprecipitates demonstrate that equivalent amounts of Pak1 protein were used for the kinase reactions (E, bottom). These data are representative of three separate experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A hallmark of neutrophil activation is the requirement for cell adhesion to achieve full functional capacity. The beta 2 integrins, particularly alpha Mbeta 2, have a central role in this adhesion-dependent activation, as shown by the multiple, profound functional defects of PMN from patients with leukocyte adhesion deficiency (3-8). Many lines of investigation have demonstrated that unactivated PMN are poorly adherent, and integrin-mediated adhesion is rapidly and reversibly induced by activation stimuli (11, 12, 14). This regulation, which has been called inside-out signaling, is a key event in PMN transendothelial migration, in motility through extracellular matrix, and in induction of effector functions at the site in inflammation. Whereas alpha  and beta  chain domains required for inside-out signaling have been studied in some detail for several integrins (74-77), and integrin clustering and alterations in association with cytoskeleton have been described as consequences of inside-out signaling (19-22), the molecular details of the signal transduction cascade involved in integrin activation remain unclear.

We have identified two inside-out signaling pathways for activation of alpha Mbeta 2 integrin-dependent adhesion, an Fcgamma R-induced, PI 3-kinase-dependent pathway and an fMLP-induced PI 3-kinase-independent pathway. PMA-induced adhesion is also PI 3-kinase-independent, either because PMA-activated PKC is downstream of PI 3-kinase or because it is in the PI 3-kinase-independent pathway. Gö6976, an inhibitor of the classical calcium-dependent PKC, inhibits adhesion to IC as well as fMLP- and PMA-induced adhesion (data not shown), suggesting that PKC is in a common pathway. Although we cannot definitively rule out the possibility that the effect of wortmannin and LY294002 in our experiments is a result of inhibition of other enzymes, the fact that two inhibitors with distinct mechanisms of action cause the same biologic effect argues strongly for specificity. Furthermore, the wortmannin and LY294002 IC50 for inhibition of adhesion and Pak1 activity induced by IC were 5 nM and 5 µM, respectively. These values agree with our own and published values for inhibition of PI 3-kinase activity in PMN and other cells (32, 33, 36, 40, 41, 45, 47, 78-80) and are 30- and 10-fold less than values reported for myosin light chain kinase and PI 4-kinase, respectively (34, 35).

Recently, several groups have suggested an important role for PI 3-kinase in inside-out signaling by lymphocyte receptors including the IL-2 receptor, CD2, CD7, and CD28 (38, 42, 43, 45), by Fcgamma RIIA or thrombin receptors on platelets (39-41), and by stem cell factor receptors on mast cells (37). These studies generally have had as their integrin targets beta 1 and beta 3 integrins, which may differ significantly from leukocyte beta 2 integrins in their mechanisms for regulation (23, 67). The finding that a PI 3-kinase-independent pathway exists for activation of beta 1-dependent adhesion by the PDGF receptor (44) and that PMA and beta 1 integrin-activating antibodies induce adhesion in wortmannin-treated HL60 cells (38) suggest that PI 3-kinase is an element in the signal transduction cascade for beta 1 integrin activation. Our demonstration that beta 2 integrin activation by the physiologic agonist fMLP as well as PMA can occur in PMN in which PI 3-kinase has been inhibited further establishes that PI 3-kinase is a component of inside-out signal transduction and is not required for adhesion itself.

G protein-coupled receptors can activate PI 3-kinase and use it to stimulate PIP3 production, respiratory burst activity, and activation of Raf-1 and ERK1/2 (33, 47, 81). In other cells, trimeric G protein-linked receptors activate the p110gamma isoform of PI 3-kinase by both the alpha  and beta gamma subunits of the heterotrimeric G protein (31). The wortmannin sensitivity of this isoform may be less than the classical p85/p110 heterodimeric kinase, but the IC50 for wortmannin is still less than 50 nM (31). Indeed, in our system, wortmannin pretreatment inhibited PI 3-kinase activity in anti-p110gamma immunoprecipitates with an IC50 of 20 nM (data not shown). Wortmannin has been shown to inhibit completely fMLP-induced PIP3 production in PMN with an IC50 of 5 nM (32, 33). Wortmannin inhibited fMLP-induced respiratory burst activity in our system with an IC50 of 2 nM (data not shown). Thus, activation of PI 3-kinase by fMLP is entirely wortmannin-sensitive in PMN; hence, wortmannin insensitivity demonstrates that PI 3-kinase activation is not required for the activation of alpha Mbeta 2. This second, PI 3-kinase-independent, pathway for activating alpha Mbeta 2 can be initiated by several ligands for seven transmembrane receptors, including fMLP, C5a, and IL-8. This result is consistent with the finding that wortmannin does not inhibit fMLP- or IL-8-induced chemotaxis (48). Interestingly, wortmannin does inhibit PDGF-induced chemotaxis in PMN (48). These data suggest that Fcgamma R and PDGF receptor, which activate tyrosine kinase cascades, utilize PI 3-kinase for integrin activation, whereas G protein-linked receptors do not. Thus, whether PI 3-kinase is involved in integrin activation may depend on whether the initial stimulus initiates a tyrosine kinase cascade. However, PI 3-kinase is not absolutely required for all tyrosine kinase-initiated integrin activation, because PDGF receptor mutants that cannot bind PI 3-kinase but are able to bind phospholipase Cgamma are perfectly capable of activating beta 1 integrins in mast cells (44). At physiologic concentrations of PDGF, PDGF receptor-initiated activation of Erk1 and Erk2 is inhibited by wortmannin, but at higher concentrations of PDGF, activation of Erk1/2 can occur by a PI 3-kinase-independent, phospholipase Cgamma - and PKC-dependent pathway, suggesting that PI 3-kinase activity is critical at low but not high signal strength (82).

Many PMN responses to immune complexes require alpha Mbeta 2, including sustained adhesion (8, 83). This study demonstrates that the recruitment of alpha Mbeta 2 function requires PI 3-kinase activity from Fcgamma R ligation. In our experiments, alpha Mbeta 2 activation was measured by quantitating binding of the mAb CBRM1/5. CBRM1/5 recognizes a neoepitope induced in a subset of alpha Mbeta 2 upon activation by agonists that induce adhesion (61). The characteristics of this subset remain unknown; however, it is clear that this subset of receptors is required for agonist-induced adhesion to alpha Mbeta 2 ligand (61). Although adhesion to IC induced both increased surface expression of the integrin and the conformational change associated with binding of CBRM1/5, increased surface expression was not required for sustained adhesion. In contrast, the conformational change in alpha Mbeta 2 recognized by CBRM1/5 was required for sustained adhesion to IC. This is similar to the conclusions about the role of alpha Mbeta 2 in PMN-endothelial adhesion (52) and in PMN aggregation (50, 51) and makes alpha Mbeta 2 activation similar to activation of alpha Lbeta 2, another beta 2 integrin which exhibits regulated adhesion without changes in surface expression (84). Thus, while PI 3-kinase is involved in activating regulated secretion in PMN which results in increased plasma membrane expression of alpha Mbeta 2, its role in sustaining adhesion to immune complexes requires only induction of integrin activation. Sustained PMN adhesion to IC leads in turn to enhanced generation of LTB4 (8), superoxide (83), and mediators of inflammation.

Our data demonstrate that Fcgamma R and seven transmembrane receptor ligation induce distinct pathways that converge into a common pathway for activation of alpha Mbeta 2 avidity. A potential effector of this common pathway is the Rac/Cdc42-activated kinase Pak1. Fcgamma R-induced Pak1 activation is dependent on PI 3-kinase activity, whereas fMLP activation of Pak1 is independent of PI 3-kinase, consistent with their effects on alpha Mbeta 2 activation. For both IC and fMLP, Pak1 activation is independent of alpha Mbeta 2 ligation as assessed by antibody inhibition, suggesting it is upstream of integrin activation. Cdc42 and Rac regulate focal complex formation and adhesion in fibroblasts and the macrophage cell line Bac1.2F5 (85, 86), consistent with the possibility that they regulate integrin avidity. Since there are several effector pathways initiated by both Rac and Cdc42, it is possible our data reflect a requirement for these small GTPases rather than for Pak1 itself. Our suggestion that Pak1 regulates integrin activation is supported by the finding that expression of an activated form of Pak1 in Swiss 3T3 cells causes large focal adhesions to form and actin accumulation in lamellipodia (54). Interestingly, PDGF and insulin receptor-induced actin cytoskeletal re-organization mediated by Rac are inhibited by wortmannin, while LPA and bombesin responses, which signal via G protein-linked receptors, are not (87), again suggesting that tyrosine-kinase pathways generally activate Rac and Pak1 by a PI 3-kinase-dependent mechanism while the G protein-dependent receptor-initiated pathway does not. It is intriguing as well that cytohesin-1, a cytosolic regulator of beta 2-mediated adhesion which binds to the beta 2 cytoplasmic tail, is a guanine nucleotide exchange factor for Arf-1 (85, 88). We suggest that PI 3-kinase is a necessary effector of tyrosine kinase-mediated but not seven transmembrane receptor-mediated integrin regulation in PMN. These cascades converge at activation of Pak1 through the small GTPases Rac and Cdc42. This model predicts that the rapid, reversible activation of integrin-mediated adhesion that is necessary for chemotaxis and transendothelial migration induced by chemoattractants is controlled by a distinct proximal pathway from that which activates the sustained, integrin-mediated adhesion necessary for PMN effector functions such as Fcgamma R-mediated phagocytosis.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health.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.

§ Supported by a Physician Scientist Award.

** To whom correspondence should be addressed: Division of Infectious Diseases, Washington University School of Medicine, Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2125; Fax: 314-362-9230.

1 The abbreviations used are: PMN, polymorphonuclear neutrophil(s); IC, immune complexes; IIC, insoluble immune complexes; fMLP, formylmethionylleucylphenylalanine; IAP, integrin-associated protein (CD47); HLA, human leukocyte antigen; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; BSA, bovine serum albumin; FCS, fetal calf serum; IL, interleukin; mAb, monoclonal antibody; PT, pertussis toxin; HBSS, Hanks' buffered salts solution; PIP3, PI (3,4,5)-trisphosphate; PDGF, platelet-derived growth factor; RT, room temperature; PKC, protein kinase C.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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