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• W1970590098 abstract "In human neutrophils, β2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the β2 integrin-induced down-regulation of Rac activities. In contrast, β2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the β2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The β2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by β2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a β2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane. In human neutrophils, β2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the β2 integrin-induced down-regulation of Rac activities. In contrast, β2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the β2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The β2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by β2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a β2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane. In response to inflammatory signals such as tumor necrosis factor-α (TNF), 1The abbreviations used are: TNF, tumor necrosis factor-α; GAP, GTPase-activating protein; GDI, GDP dissociation inhibitor; PI 3-kinase, phosphoinositide 3-kinase; PMN, polymorphonuclear neutrophil; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; mAb, monoclonal antibody; PBS, phosphate-buffered saline; DTT, dithiothreitol; PVDF, polyvinylidene difluoride; NBT, nitro blue tetrazolium; GST, glutathione S-transferase; fMLP, formyl-methionyl-leucylphenylalanine. 1The abbreviations used are: TNF, tumor necrosis factor-α; GAP, GTPase-activating protein; GDI, GDP dissociation inhibitor; PI 3-kinase, phosphoinositide 3-kinase; PMN, polymorphonuclear neutrophil; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; mAb, monoclonal antibody; PBS, phosphate-buffered saline; DTT, dithiothreitol; PVDF, polyvinylidene difluoride; NBT, nitro blue tetrazolium; GST, glutathione S-transferase; fMLP, formyl-methionyl-leucylphenylalanine. polymorphonuclear neutrophils (PMNs) adhere to the surface of endothelium and then crawl forward (diapedesis) and pass between neighboring endothelial cells (transmigration) to reach infected tissues. This is followed by ingestion of the invading microbes, resulting in their dissolution, largely through the release of granule contents into the phagolysosome and generation of oxygen radicals by the membrane-bound NADPH oxidase (1von Andrian U.H. Chambers J.D. McEvoy L.M. Bargatze R.F. Arfors K.E. Butcher E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7538-7542Google Scholar). Several of the above functional responses are mediated by integrin-mediated cell adhesion (2Nathan C.F. J. Clin. Invest. 1987; 80: 1550-1560Google Scholar), including a dynamic rearrangement of the actin cytoskeleton (3Löfgren R. Ng-Sikorski J. Sjölander A. Andersson T. J. Cell Biol. 1993; 123: 1597-1605Google Scholar). Members of the β2 integrin family are the dominating integrins expressed on PMNs. β2 integrins are noncovalently associated heterodimers composed of a common β-chain, CD18, and one of four unique α-chains (CD11a, CD11b, CD11c, or CD11d), with CD11b/CD18 being the most prominent on PMNs (4Sanchez-Madrid F. Nagy J.A. Robbins E. Simon P. Springer T.A. J. Exp. Med. 1983; 158: 1785-1803Google Scholar, 5Gahmberg C.G. Tolvanen M. Kotovuori P. Eur. J. Biochem. 1997; 245: 215-232Google Scholar, 6Arnaout M.A. Blood. 1990; 75: 1037-1050Google Scholar). Engagement of β2 integrins on PMNs triggers activation of various nonreceptor tyrosine kinases, such as Src family members (p58c-fgr (Fgr), p59/61hck (Hck), and p53/56lyn (Lyn)), the non-Src tyrosine kinases p72syk (Syk) and Pyk2 (7Berton G. Yan S.R. Fumagalli L. Lowell C.A. Int. J. Clin. Lab. Res. 1996; 26: 160-177Google Scholar, 8Dib K. Andersson T. Front. Biosci. 1996; 5: d438-d451Google Scholar), and the lipid kinase PI 3-kinase (3Löfgren R. Ng-Sikorski J. Sjölander A. Andersson T. J. Cell Biol. 1993; 123: 1597-1605Google Scholar), and it generates a phospholipase C-γ2-dependent calcium signal (9Hellberg C. Molony L. Zheng L. Andersson T. Biochem. J. 2000; 317: 403-409Google Scholar). GTP-binding proteins of the Rho subfamily, which belongs to the Ras superfamily of small GTPases, are necessary for regulation of PMN functions. For example, the motile potential of PMNs is largely due to the formation of membrane protrusions, a process that requires relocalization and activation of Rac and Cdc42 (as well as other signaling molecules such as PI 3-kinase) at the leading edge of these motile cells (10Worthylake R.A. Burridge K. Curr. Opin. Cell Biol. 2001; 13: 569-577Google Scholar). The requirement for Rho GTPases in these functional events can readily be ascribed to their dynamic regulation of the actin-based cytoskeleton, similar to that described in many other cell models (11Hall A. Science. 1998; 279: 509-514Google Scholar, 12Aspenström P. Curr. Opin. Cell Biol. 1999; 11: 95-102Google Scholar). In PMNs, Rac and Cdc42 are involved in actin nucleation (13Arcaro A. J. Biol. Chem. 1998; 273: 805-813Google Scholar, 14Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Google Scholar); Rac promotes dissociation of gelsolin from actin filaments (13Arcaro A. J. Biol. Chem. 1998; 273: 805-813Google Scholar), and Cdc42 activates WASP proteins and the ARP2/3 complex (15Glogauer M. Hartwig J. Stossel T. J. Cell Biol. 2000; 150: 785-796Google Scholar). In addition to participating in regulation of cytoskeletal dynamics, Rac1 and Rac2 are part of the multicomponent, plasma membrane-bound enzyme NADPH oxidase; however, the exact role that Rac GTPases play in the regulation of the respiratory burst in phagocytic cells has not yet been completely elucidated (16Bokoch G.M. Curr. Biol. 1994; 6: 212-218Google Scholar, 17Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar). These small GTP-binding proteins cycle between a GDP-bound inactive form to a GTP-bound active form (18Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Google Scholar). In their inactive form, Rho GTPases are bound to proteins called GDIs (RhoGDI and LyGDI), which compete more efficiently in vivo for GDP-bound than for GTP-bound Rho GTPases (19Abo A. Webb M.R. Grogan A. Segal A.W. Biochem. J. 1994; 298: 585-591Google Scholar). GDI-bound Rho GTPases are found in the cytosol, because their C-terminal, lipid-modified end is inserted into a hydrophobic pocket of the immunoglobulin-like domain of the GDI molecule, which prevents the Rho GTPases from interacting with the membrane (20Hoffman G.R. Nassar N. Cerione R. Cell. 2000; 100: 345-356Google Scholar). Guanine nucleotide exchange factors, activated by extracellular stimuli, are responsible for the GDP-GTP switch. In their GTP-bound state, these proteins interact with specific effectors to initiate downstream signals and functions. The subsequent hydrolysis of bound GTP to GDP is catalyzed by the family of GTPase-activating proteins (GAPs). We have recently shown that ligation of the β2 integrins on PMNs resulted in activation of Ras (21Zheng L. Sjölander A. Eckerdal J. Andersson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8431-8436Google Scholar) and RhoA (22Dib K. Melander F. Andersson T. J. Immunol. 2001; 166: 6311-6322Google Scholar), but it is not yet known whether other Rho GTPases are also regulated in the course of this event. In the present study, we examined β2 integrin-dependent regulation of Rac and Cdc42 GTPases in adherent PMNs. Antibodies—The antibodies and their sources were as follows. mAb IB4 (mouse anti-human CD18, IgG2a isotype) was originated by Dr. S. Wright (Rockefeller University, New York, NY) (23Wright S.D. Rao P.E. van Voorhiss W.C. Craigmyle L.S. Iida K. Talle M.A. Westberg E.F. Goldstein G. Silverstein S.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 18: 5699-5703Google Scholar); rabbit anti-Rac2 and goat anti-LyGDI antisera were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mAbs anti-Cdc42, anti-Rac1, anti-Rac, anti-p47phox, and anti-p67phox were purchased from Transduction Laboratories (Lexington, KY); peroxidase-conjugated IgGs and IgG2a control antibody were obtained from Dakopatts (Glostrup, Denmark). Chemicals—Protein G-Sepharose was from Oncogene™ (Germany); protein A-Sepharose, Dextran, and Ficoll-Hypaque were purchased from Amersham Biosciences; and the protease inhibitors pefabloc, pepstatin, leupeptin, aprotinin, and antipain were from Roche Applied Science. Benzamidine, LY294002, and wortmanin were obtained from Sigma, the tyrosine kinase inhibitor PP1 was from Alexis Biochemicals, and all electrophoresis reagents were obtained from Bio-Rad. All other chemicals were of analytical grade and were purchased from Sigma. Isolation of Human PMNs—Blood was collected from healthy donors, and PMNs were isolated under endotoxin-free conditions as previously described (24Böyum A. Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 31-50Google Scholar). In short, the blood was subjected to dextran sedimentation followed by a brief hypotonic lysis of erythrocytes. The lysis was stopped by adding 3 ml of buffer A (565 mm NaCl, 2.7 mm KCl, 6.7 mm Na2HPO4 2H2O, 1.5 mm KH2PO4, pH 7.3) and 3 ml of Ringer's modified phosphate buffer, KRG (120 mm NaCl, 4.9 mm KCl, 1.7 mm KH2PO4, 1.2 mm MgSO47H2O, 8.3 mm Na2HPO42H2O, 10 mm glucose, pH 7.3). The cell suspension was then centrifuged on Ficoll-Hypaque (15 ml) and washed twice with KRG buffer. Finally, the pelleted cells were resuspended in a calcium-containing medium (136 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgSO4, 5.0 mm NaHCO3, 1.1 mm CaCl2, 0.1 mm EGTA, 5.5 mm glucose, 20 mm Hepes, pH 7.4). The cell suspension consisted of ∼97% PMNs. Ligation of β2Integrins—For adhesion of PMNs, Petri dishes (Easy Grip™) containing 20 μg/ml fibrinogen in phosphate-buffered saline (PBS) were incubated either overnight at 4 °C or for 2 h at room temperature and then washed twice with PBS and once with calcium-containing medium. PMNs (20 × 106) were subsequently incubated in the fibrinogen-coated dishes at 37 °C in the presence of TNF (20 ng/ml) for different periods of time. For treatment of suspended PMNs, polypropylene tubes (15 ml) were blocked for 2 h with 10% fetal calf serum and then rinsed extensively with PBS and once with calcium-containing medium, after which the cells (106/ml) were incubated in the tubes under gentle rotation at 37 °C in the absence (control cells) or presence of TNF (20 ng/ml) for the indicated periods of time. The reactions were terminated by putting the Petri dishes or tubes on ice. GST Pull-down Assays and Western Blotting—The cDNA of the Rac and Cdc42 binding domain from PAK1B (PAKcrib; amino acids 56–267) was cloned into the bacterial expression vector pGEX-2T and was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (25Edlund S. Landström M. Heldin C.H. Aspenström P. Mol. Biol. Cell. 2002; 13: 902-914Google Scholar). PMNs were lysed in a buffer composed of 50 mm Tris-HCl, pH 7.5, 1% Triton X-100, 100 mm NaCl, 10 mm MgCl2, 5% glycerol, 1 mm Na3VO4, and protease inhibitors (20 μg/ml aprotinin; pepstatin, leupeptin, and antipain (1 μg/ml each); 2.5 mm benzamidine; 2 mm pefabloc). Lysates were centrifuged at 15,000 × g for 10 min, and the Triton X-100-soluble fraction was recovered. A bacterial lysate containing the GST-PAKcrib fusion protein was added to PMNs lysates together with glutathione-Sepharose beads. After 1 h, the beads were collected by centrifugation and washed three times in 25 mm Tris-HCl, pH 7.5, 1% Triton X-100, 1 mm dithiothreitol (DTT), 100 mm NaCl, and 30 mm MgCl2. The beads were then resuspended in Laemmli sample buffer and boiled under reducing conditions for 5 min. The precipitated proteins were subjected to 12% SDS-PAGE and transferred to polyscreen PVDF membranes. The membranes were blocked in PBS supplemented with 0.2% Tween 20 and 3% milk and then incubated for 1 h with a primary antibody (1 μg/ml dilution of anti-Rac2, anti-Cdc42, or anti-Rac Abs or 0.15 μg/ml anti-Rac1 mAb) and thereafter washed three times for 5 min in PBS supplemented with 0.2% Tween 20. The membranes were subsequently incubated for 1 h with peroxidase-conjugated anti-mouse IgGs (1:10,000) in PBS supplemented with 0.2% Tween 20 and 3% milk. The blots were extensively washed, and antibody binding was visualized by enhanced chemiluminescence (ECL). Measurement of NADPH Oxidase Activity—Nitro blue tetrazolium (NBT) is an electron acceptor used to indirectly detect the production of superoxide by PMNs (26Baehner R.L. Nathan D.G. N. Engl. J. Med. 1968; 278: 971-976Google Scholar). Upon electron acceptance, soluble and yellow NBT is converted to blue-black formazan that can be quantitated spectrophotometrically after extraction from cells with N,N-dimethylformamide. Briefly, PMNs (2.5 × 106/ml) in calcium-containing medium were preincubated with 0.2% NBT for 10 min, after which PMNs were plated on fibrinogen-coated plates in the presence of TNF for the indicated time periods. Suspended PMNs were taken as control cells (zero time point). Thereafter, adherent cells (5 × 106) were scraped off, and the total cell suspension was transferred to Eppendorf tubes. The tubes were spun for 3 min, and the resulting pellets were dissolved in 1 ml of N,N-dimethylformamide and left at 56 °C for 1 h. The tubes were then again spun for 3 min, after which the optical densities (515 nm) of cell extracts were determined in a spectrophotometer. The OD values obtained from resting cells ranged between 0.07 and 0.1. Measurement of Rac-GAP Activity in PMN Lysates—PMNs (30 × 106) were lysed in 100 μl of ice-cold lysis buffer (PBS containing 1% Triton X-100, 1 mm EGTA, 5% glycerol, 10 mm benzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm pefabloc, 1 μg/ml pepstatin and antipain), and the lysates were clarified by centrifugation (15,000 × g, 10 min). The Rac-GAP assay was performed essentially as described elsewhere (27Molnár G. Dagher. M.C. Geiszt M. Settleman J. Ligeti E. Biochemistry. 2001; 40: 10542-10549Google Scholar), with minor modifications. Briefly, 10 μl of precleared lysates (containing about 50 μg of proteins) were incubated in 30 μl of Rac-GAP buffer (16 mm Tris-HCl, pH 7.5, 0.1 mm DTT, 1 mg/ml bovine serum albumin, 1 mm GTP) for 5 min at room temperature. Thereafter, the reaction was initiated by adding 4 μl of [γ-32P]GTP-loaded Rac1, and the sample was incubated for 5 min at room temperature under shaking. The reaction was stopped by adding 0.2 ml of ice-cold Rac-GAP buffer and placing the samples on ice for 2 min. Aliquots (50 μl) were filtered through nitrocellulose filters (0.45-mm pore size) under vacuum, and the filters were washed three times with 1 ml of wash buffer (50 mm Tris-HCl, pH 7.7, 5 mm MgCl2). The filters were then air-dried and placed in plastic vials. 5 ml of scintillation mixture (Ready Gel; Beckman) was added, and radioactivity bound to the filters was measured using a scintillation counter. To achieve GTP loading of Rac1, 1–3 μg of affinity-purified histidine-tagged Rac1 (expressed in Sf9 cells) was incubated at room temperature for 5 min in loading buffer (16 mm Tris-HCl, pH 7.5, 20 mm NaCl, 0.1 mm DTT, 5 mm EDTA, 100 nm GTP, and 5 μCi of [γ-32P]GTP with a specific activity of 5000 Ci/mmol). Thereafter, MgCl2 (20 mm) was added to block further nucleotide exchange activity, and the tubes were placed on ice. An aliquot was filtered through a nitrocellulose filter, and the filter was washed three times with wash buffer (see above). The radioactivity remaining on the filters was counted and considered as total bound Rac1 (100%). Pretreatment of PMNs with Anti-β2Integrin Antibodies—PMNs (10 × 106/ml) were resuspended in calcium-containing medium in polypropylene tubes and then incubated for 30 min at 37 °C with 15 μg/ml IB4 antibody or isotype-matched IgG2a monoclonal antibody. Thereafter, the cells were placed on dishes coated with fibrinogen and stimulated with TNF (20 ng/ml). The reactions were terminated by placing the dishes on ice, after which GST-PAKcrib pull-down assays were performed (see above). Immunoprecipitation—PMNs were lysed by adding the following lysis buffer: 100 mm Tris-HCl, pH 7.5, 1% Triton X-100, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 5 mm NaF, 1 mm Na3VO4, and protease inhibitors (20 μg/ml aprotinin; 1 μg/ml each pepstatin, leupeptin, and antipain; 2.5 mm benzamidine; 2 mm pefabloc). Cell lysates were clarified by centrifugation (10 min at 15,000 × g), and LyGDI in the supernatants was immunoprecipitated by exposure to the anti-LyGDI antiserum (3 μg/ml) for 2 h and then to 40 μl of a 50% slurry of protein G-Sepharose for 45 min. The beads were subsequently collected by centrifugation and washed three times in a wash buffer (50 mm Hepes, pH 7.4, 1% Triton X-100, 0.1%, SDS, 150 mm NaCl, 1 mm Na3VO4). The beads were then resuspended in Laemmli sample buffer and boiled under reducing conditions for 5 min. The immunoprecipitated proteins were subjected to electrophoresis on 12% SDS-PAGE and transferred to polyscreen PVDF transfer membranes. To detect Rac in the anti-LyGDI immunoprecipitates, the membranes were incubated with either anti-Rac1 mAb or anti-Rac2 antiserum and subsequently with peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1:10,000), as described under “GST Pull-down Assays and Western Blotting.” Determination of the Translocation of Cytosolic Components of the NADPH Oxidase—PMNs were scraped off from the Petri dishes, suspended in disruption buffer (100 mm Tris-HCl, pH 7.5, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 5 mm NaF, 1 mm Na3VO4, and protease inhibitors (20 μg/ml aprotinin; 1 μg/ml each pepstatin, leupeptin, and antipain; 2.5 mm benzamidine; 2 mm pefabloc)), and then placed in a cell disruption bomb at 4 °C (28Klempner M.S. Mikkelsen R.B. Corfman D.H. André-Schwartz J. J. Cell Biol. 1980; 86: 21-28Google Scholar). The bomb was equilibrated at 1000 p.s.i. for 10 min, after which the pressure was quickly released. The cell suspension was subsequently centrifuged at 10,000 × g for 10 min at 4 °C to pellet nuclei, heavy membrane fractions, and undisrupted cells. The supernatant was further centrifuged at 100,000 × g for 1 h, and the resulting pellet was resuspended in disruption buffer. The protein content was determined (29Schaffner W. Weissmann C. Anal. Biochem. 1973; 56: 502-514Google Scholar), and aliquots were mixed with 2× Laemmli buffer supplemented with 50 mm DTT and boiled. The proteins (5–10 μg) were separated on 12% SDS-PAGE, and immunoblot analysis was performed as described above, using anti-Rac1 mAb, anti-Rac2 antiserum, anti-p47phox, or anti-p67phox mAbs. To measure translocation of Rac proteins to the detergent-insoluble fraction, the Triton X-100-insoluble fraction was resuspended in Laemmli buffer containing 50 mm DTT, and the samples were subjected to sonication. Aliquots were taken to estimate the protein content of each sample. Aliquots of proteins (20–40 μg) were separated by 12% SDS-PAGE and transferred to polyscreen PVDF transfer membranes. The membranes were incubated with either anti-Rac1 mAb or anti-Rac2 antiserum and then with peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1:10,000), as described above. β2Integrin-mediated Regulation of Rac and Cdc42 GTPases in PMNs—To engage β2 integrins on PMNs, the cells were incubated on a surface coated with fibrinogen (a ligand for β2 integrins) (30Yakubenko V.P. Solovjov D.A. Zhang L. Yee V.C. Plow E.F. Ugarova T.P. J. Biol. Chem. 2001; 276: 13995-14003Google Scholar) and exposed to TNF. Stimulation of PMNs with TNF or other cytokines such as leukotriene B4 increases the content of the CD11b/CD18 β2 integrin on the surface of PMNs (31Sengelov H. Kjeldsen L. Diamond M.S. Springer T.A. Borregaard N. J. Clin. Invest. 1993; 92: 1467-1476Google Scholar), and it is presumed that these β2 integrins exhibit augmented avidity for their ligands (32Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Google Scholar). In the present experiments, we measured the relative activities of Rac and Cdc42 in PMNs upon engagement of β2 integrins by using the GST-PAKcrib binding assay, which is based on the knowledge that the GST-PAKcrib fusion protein binds the GTP-bound forms of Cdc42 and Rac but does not bind GTP-bound RhoA (33Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Google Scholar). We found that the amounts of Rac-GTP decreased in a time-dependent manner in PMNs adhering to immobilized fibrinogen in the presence of TNF, and we observed the lowest levels (2.5-fold decrease over controls) after incubation for 20–40 min on a fibrinogen-coated surface (Fig. 1A). PMNs express both Rac1 and Rac2, and the latter represents 95% of total Rac (34Heyworth P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Google Scholar); thus, the results presented in Fig. 1A definitely reflect decreased levels of Rac2-GTP. To corroborate this finding, and to ascertain whether regulation of Rac1 occurs in adherent PMNs, we conducted GST-PAKcrib pull-down assays followed by Western blot analysis using specific anti-Rac1 or anti-Rac2 antibodies. As expected, ligation of the β2 integrins on PMNs induced by exposure to immobilized fibrinogen caused a time-dependent decrease in the level of Rac2-GTP (Fig. 1C) and, with a similar time course, also lowered the amount of Rac1-GTP (Fig. 1B). In parallel experiments, we confirmed that engagement of β2 integrins on PMNs by plating them on fibrinogen in the presence of TNF led to a time-dependent activation of the respiratory burst as assessed by measuring the production of superoxide-induced formazan (Fig. 1D). In contrast to Rac, ligation of the β2 integrins led to a time-dependent activation of Cdc42, and we detected the maximum levels of Cdc42-GTP (2.5-fold increase over controls) in PMNs incubated on fibrinogen for 20–40 min (Fig. 2). However, stimulating suspended PMNs with TNF did not modify the basal activities of Cdc42, Rac1, or Rac2 (Fig. 3A), which confirms that TNF regulates only the activities of Cdc42 and Rac in adherent PMNs. This was further illustrated in our next set of experiments, in which PMNs were preincubated for 30 min at 37 °C with anti-CD18 mAb (IB4) or an isotype-matched control antibody and then placed on a surface coated with fibrinogen and exposed to TNF. The relative activities of Cdc42 and Rac GTPases were subsequently measured using the GST-PAKcrib binding assay (see above). We found that preincubation with anti-CD18 antibody almost totally abolished adhesion-dependent regulation of Cdc42 and Rac activities (Fig. 3B), whereas pretreatment with an isotype-matched control antibody had no significant effect. These results agree well with a study by Nathan (2Nathan C.F. J. Clin. Invest. 1987; 80: 1550-1560Google Scholar) showing that TNF-mediated activation of the NADPH oxidase depended on adhesion to a surface and activation of the β2 integrins (2Nathan C.F. J. Clin. Invest. 1987; 80: 1550-1560Google Scholar).Fig. 3TNF-induced regulation of Cdc42 and Rac GTPases in PMNs requires adhesion and activation of the β2 integrins.A, PMNs in suspension (106/ml) were stimulated with TNF (20 ng/ml) for the indicated times, after which the cells (20 × 106) were lysed. The blots show the amounts of active Cdc42, Rac1, and Rac2 determined by the GST-PAKcrib binding assay, as described in the legend to Fig. 1. The results of the GST pull-down assays are illustrated to the right. B, PMNs in suspension (10 × 106/ml) were preincubated with either an isotype-matched control antibody IgG2a (20 μg/ml) or the anti-β2 integrin antibody (anti-CD18, IB4; 20 μg/ml) for 30 min at 37 °C. Thereafter, the cells were incubated on a fibrinogen-coated surface and stimulated with TNF (20 ng/ml) for 30 min. The PMNs (20 × 106) were lysed, and the amounts of active GTP-bound Cdc42, Rac1, and Rac2 were determined using the GST pull-down assay as described in the legend to Fig. 1. WB, Western blot.View Large Image Figure ViewerDownload (PPT) To further assess the validity of our GST-PAKcrib pull-down assay, we treated PMNs with fMLP (10–7m, 1 min) and found that this substance increased the activity of Cdc42 (196 ± 22% over controls; n = 4, p < 0.01), Rac1 (160 ± 21% over controls; n = 4, p < 0.05) and Rac2 (400 ± 106% over controls; n = 4, p < 0.05), which agrees with the results reported by other investigators (33Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Google Scholar, 35Akasaki T. Hirofumi K. Sumimoto H. J. Biol. Chem. 1999; 274: 18055-18059Google Scholar, 36Geijsen N. van Delft S. Jan A.M Raaijmakers Lammers J-W.J. Collard J.G. Koenderman L. Coffer P.J. Blood. 1999; 94: 1121-1130Google Scholar). Thus, these findings show that the fMLP receptor and the β2 integrins collaborate in regulation of Cdc42 but oppose each other in the regulation of Rac GTPases. In control experiments, we observed that GST-PAKcrib, but not GST, could pull down Cdc42 and Rac GTPases (Fig. 3A), which rules out any nonspecific precipitation of the GTPases by the GST part of the fusion protein. Identification of the Signaling Pathways Involved in β2Integrin-induced Regulation of Rac and Cdc42 GTPases—To explore the difference in adhesion-dependent regulation of Rac and Cdc42, we pretreated PMNs with a variety of pharmacological agents and then incubated them on plates coated with fibrinogen in the presence of TNF. Thereafter, we used the GST-PAKcrib binding assay (described above) to measure the relative amounts of the active forms of Rac1, Rac2, and Cdc42. We pretreated the PMNs with the following agents: wortmanin or LY294002, both of which inhibit PI 3-kinase by distinct mechanisms and are known to block fMLP-induced generation of phosphatidylinositol 3,4,5-trisphosphate and activation of the respiratory burst in PMNs (37Ding J. Vlahos C.J. Liu R. Brown R.F. Bradwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Google Scholar), or PP1, which is a potent and selective inhibitor of Src family tyrosine kinases (38Hanke J.H. Garden J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Google Scholar). We have previously shown that PP1 (3 μm) blocks basal and β2 integrin-induced overall tyrosine phosphorylation of proteins in PMNs (22Dib K. Melander F. Andersson T. J. Immunol. 2001; 166: 6311-6322Google Scholar). In our present study, pretreatment of PMNs with wortmanin or LY294002 significantly reduced, but did not totally block, adhesion-induced up-regulation of Cdc42 activity, and it partly reversed adhesion-elicited down-regulation of the activities of Rac1 and Rac2 (Fig. 4). Interestingly, pre-exposure to PP1 had no impact on activation of Cdc42 caused by ligation of the β2 integrins (Fig. 4, left panel), whereas it partly reverted adhesion-induced down-regulation of Rac1 (Fig. 4, middle panel) and Rac2 activities (Fig. 4, right panel). Ligation of β2Integrins Enhances the Activity of Rac-GAP—To further elucidate the mechanism by which ligation of PMNs β2 integrins leads to down-regulation of Rac activities, we performed experiments to determine whether increased Rac-GAP activity could induce this phenomenon in adherent PMNs. We incubated PMNs lysates for 5 min in the presence of semipurified Rac1 loaded with [γ-32P]GTP; in this assay, a decrease in γ-32P-labeled Rac1 indicates an increas" @default.
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• W1970590098 date "2003-06-01" @default.
• W1970590098 modified "2023-10-15" @default.
• W1970590098 title "Down-regulation of Rac Activity during β2 Integrin-mediated Adhesion of Human Neutrophils" @default.
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• W1970590098 doi "https://doi.org/10.1074/jbc.m302300200" @default.
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