Activation of SHIP by NADPH Oxidase-stimulated Lyn Leads to Enhanced Apoptosis in Neutrophils
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m110005200
ISSN1083-351X
AutoresShyra J. Gardai, Ben B. Whitlock, Cheryl D. Helgason, Dan Ambruso, Valerie A. Fadok, Donna L. Bratton, Peter M. Henson,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoNeutrophils undergo rapid spontaneous apoptosis. Multiple antiapoptotic stimuli can inhibit this process via activation of the Akt pathway. However, despite no such effect singly, combined anti- and proapoptotic stimuli inhibit Akt activity, leaving the cells susceptible to accelerated apoptosis. The blockade of Akt activation depended on reduced phosphoinositide 3,4,5-trisphosphate levels but not decreased phosphatidylinositol 3-kinase activity, thus implicating the involvement of an inositol phosphatase. Evidence for SHIP involvement was provided by SHIP localization to membrane receptors and subsequent activation along with the observed inability of SHIP −/− neutrophils to exhibit enhanced apoptosis with the stimulus combination. Activation of SHIP was found to depend on Lyn activation, and this, in turn, required NADPH oxidase. Neutrophils from chronic granulomatous disease patients and Lyn −/− mice no longer responded to the combined stimuli. Thus, we propose a role for oxidants and Lyn in SHIP regulation and suggest a novel mechanism for regulating neutrophil apoptosis. Neutrophils undergo rapid spontaneous apoptosis. Multiple antiapoptotic stimuli can inhibit this process via activation of the Akt pathway. However, despite no such effect singly, combined anti- and proapoptotic stimuli inhibit Akt activity, leaving the cells susceptible to accelerated apoptosis. The blockade of Akt activation depended on reduced phosphoinositide 3,4,5-trisphosphate levels but not decreased phosphatidylinositol 3-kinase activity, thus implicating the involvement of an inositol phosphatase. Evidence for SHIP involvement was provided by SHIP localization to membrane receptors and subsequent activation along with the observed inability of SHIP −/− neutrophils to exhibit enhanced apoptosis with the stimulus combination. Activation of SHIP was found to depend on Lyn activation, and this, in turn, required NADPH oxidase. Neutrophils from chronic granulomatous disease patients and Lyn −/− mice no longer responded to the combined stimuli. Thus, we propose a role for oxidants and Lyn in SHIP regulation and suggest a novel mechanism for regulating neutrophil apoptosis. Once they have been released from the bone marrow, neutrophils have a remarkably short life span of only a few hours, with 80% undergoing apoptosis within 16–20 h of in vitro culture (1Savill J.S. Wylie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1351) Google Scholar). This rapid induction of apoptosis is thought to be the result of a preprogrammed, or spontaneous, apoptotic sequence that, in the absence of additional signals, leads to surface changes on the cell and clearance from the circulation (2Haslett C. Clin. Sci. (Lond.). 1992; 83: 639-648Crossref PubMed Scopus (317) Google Scholar, 3Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. 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Protective stimuli include integrin ligation, a wide variety of chemokines and other chemoattractants, GM-CSF, 1The abbreviations used are:GM-CSFgranulocyte-macrophage colony-stimulating factorPI 3-kinasephosphatidylinositol 3-kinaseTNFtumor necrosis factorDHRdihydrorhodamine-123LPSlipopolysaccharideDPIdiphenyleneiodonium chloridePtdInsphosphatidylinositolPIP3phosphatidylinositol 3,4,5-trisphosphatePIP2phosphatidylinositol 4,5-bisphosphateCGDchronic granulomatous disease and even bacterial lipopolysaccharide (9Coxon A. Rieu P. Barkalow F.J. Askari S. Sharpe A.H. von Adrian U.H. Arnaout M.A. Mayadas T.N. Immunity. 1996; 5: 653-666Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 10Sweeney J.F. Nguyen P.K. Omann G. Hinshaw D.B. J. Surg. Res. 1999; 81: 108-112Abstract Full Text PDF PubMed Scopus (17) Google Scholar). In contrast, proapoptotic stimuli commonly encountered within an inflammatory lesion would include TNFα, Fas ligand, and a variety of oxidants (11Takeda Y. 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This clearance, in turn, results in the production of anti-inflammatory molecules and the subsequent resolution of the inflammatory reaction (3Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2563) Google Scholar). granulocyte-macrophage colony-stimulating factor phosphatidylinositol 3-kinase tumor necrosis factor dihydrorhodamine-123 lipopolysaccharide diphenyleneiodonium chloride phosphatidylinositol phosphatidylinositol 3,4,5-trisphosphate phosphatidylinositol 4,5-bisphosphate chronic granulomatous disease Apoptotic processes in neutrophils can be regulated to extend the cells' life span by three major pathways including PI 3-kinases and Akt, mitogen-activated protein kinases of the extracellular signal-regulated kinase subgroup (15Tudan C. Jackson J.K. Blanis L. Pelech S.L. Burt H.M. J. Immunol. 2000; 165: 5798-5806Crossref PubMed Scopus (32) Google Scholar), and activation of NF-κB (16Ward C. 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Henson P.M. J. Cell Biol. 2000; 151: 1305-1320Crossref PubMed Scopus (112) Google Scholar). The serine/threonine kinase Akt has been shown to inhibit both spontaneous and stress-induced apoptosis and appears to be responsible for regulating growth factor-mediated cell survival (20Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). Akt is activated by a variety of growth factors via PI 3-kinase-generated phosphatidylinositols (20Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). PI 3-kinase phosphorylates phosphoinositides (PI) on the 3′-position of the inositol ring to generate PtdIns 3-monophosphate, PtdIns 3,4-bisphosphate, and PtdIns 3,4,5-trisphosphate (21Kappeller R. Cantley L.C. Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar). Generation of PtdIns 3,4,5-trisphosphate allows for recruitment of Akt to the plasma membrane via its PH domain (22Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (795) Google Scholar). Once at the plasma membrane, PDK1 and an as yet unknown serine/threonine kinase phosphorylate Akt, thereby inducing its activation (22Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (795) Google Scholar). Active Akt is thought to inhibit apoptosis in a variety of ways both upstream and downstream of mitochondrial perturbation. It can inhibit caspase 9 activity, phosphorylate proapoptotic Bcl-2 family members such as Bad, or regulate activation of transcription factors such as CREB, NF-κB, and members of the Forkhead family (20Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar, 22Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (795) Google Scholar, 23Du K. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). This makes Akt a prime candidate for regulation in the circumstance of paradoxical anti- and proapoptotic signal combinations. Prevention of Akt activation in this circumstance could occur by the inhibition of PI 3-kinase or the activation of a phosphatidylinositol phosphatase, each leading to reduced Akt activation and translocation. SHIP (SH2-containing inositol 5-phosphatase) is a 145-kDa protein highly expressed in hematopoetic cells (24Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). It specifically hydrolyzes the 5′-phosphate from PtdIns 1,3,4,5-tetraphosphate and PtdIns 3,4,5-trisphosphate, the predominant product of PI 3-kinase (25Krystal G. Damen J.E. Helgason C.D. Huber M. Hughes M.R. Kalesnikoff J. Lam V. Rosten P. Ware M.D. Yew S. Humphries R.K. Int. J. Biochem. Cell Biol. 1999; 31: 1007-1010Crossref PubMed Scopus (39) Google Scholar). SHIP overexpression has been shown to inhibit Akt activity, whereas SHIP null cells exhibit sustained Akt activity (26Liu Q. Sasaki T. Kozieradzki I. Wakeham A. Itie A. Dumont D.J. Penninger J.M. Genes Dev. 1999; 13: 786-791Crossref PubMed Scopus (288) Google Scholar). SHIP has also been shown to down-regulate prosurvival and proliferative signals in vivo as reviewed in Ref. 27Liu L. Damen J.E. Ware M. Hughes M. Krystal G. Leukemia. 1997; 11: 181-184Crossref PubMed Scopus (44) Google Scholar. Stimulation of B cells by the antigen receptor activates PI 3-kinase and its downstream target Akt and results in proliferation (28Otero D.C. Omori S.A. Rickert R.C. J. Biol. Chem. 2001; 276: 1474-1478Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In contrast, co-clustering with the B-cell IgG receptor, FcγRII, results in decreased activation of Akt and inhibition of BCR-induced proliferation (29Carver D.J. Aman M.J. Ravichandran K.S. Blood. 2000; 96: 1449-1456Crossref PubMed Google Scholar, 30Chacko G.W. Tridandapani S. Damen J.E. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar). The down-regulation of Akt is due to SHIP recruitment from the cytosol to the membrane ITIM domain of FcγRII upon concomitant BCR and FcγRII ligation (31Tridandapani S. Kelley T. Pradhan M. Cooney D. Justement L.B. Coggeshall K.M. Mol. Cell. Biol. 1997; 17: 4305-4311Crossref PubMed Google Scholar). The mechanism by which SHIP is regulated remains controversial, although it is known that the molecule must be recruited from the cytoplasm to the plasma membrane for activation (32Phee H. Jacob A. Coggeshall K.M. J. Biol. Chem. 2000; 275: 19090-19097Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The hypothesis to be examined herein is whether the combination of antiapoptotic and stress stimuli activates SHIP and subsequently blocks Akt-derived antiapoptotic pathways in neutrophils, resulting in enhanced apoptosis. Our data support the notion that both antiapoptotic and stress signals can each activate PI 3-kinase and Akt on their own but that the combination of these stimuli leads to the complete abrogation of Akt activity via a decrease in PIP3 levels independent of alterations in PI 3-kinase activity. The blockade of Akt appears to be due to NADPH oxidase-derived oxidants and their ability to activate Lyn, which can aid in the recruitment of SHIP to the plasma membrane. Once at the plasma membrane, SHIP appears to associate with the β2-integrin as well as other antiapoptotic signaling receptors, and once there it can modulate PIP3 levels and subsequently apoptosis. The following antibodies were obtained from commercial sources and were confirmed to bind to human PMN by flow cytometry. Anti-HLA-ABC W6/32 was obtained from DAKO (Carpinteria, CA). Anti-αM VIM12 was obtained from Caltag (Burlingame, CA). Anti-Akt, anti-SHIP, and anti-Lyn antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Akt and anti-phosphotyrosine (4G10) monoclonal antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Unless specified in the figure legends, mAbs were used at a final concentration of 1 μg/ml. Human and murine TNFα were obtained from R & D Systems (Minneapolis, MN) and used at a final concentration of 1000 units/ml. Human anti-Fas IgM clone CH11 was obtained from Upstate Biotechnology and used at a concentration of 1 μg/ml to induce apoptosis. Murine anti-Fas clone Jo2 was obtained from Pharmingen (San Diego, CA), was used at 1 μg/ml, and was cross-linked with an anti-Armenian hamster secondary antibody obtained from Jackson Immunochemicals (Bar Harbor, ME) at a 1:1 ratio. The PI 3-kinase inhibitor (LY294002; 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) was obtained from Calbiochem and used at 100 nm. The NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) and catalase were obtained from Sigma and used at 5 μg/ml or 1000 units/ml, respectively. The Src family kinase inhibitor PPI was obtained from Alexis (San Diego, CA) and used at 10 μg/ml. Phosphatidylinositol and phosphatidylserine were obtained from Avanti Polar Lipids (Alabaster, AL). [3H]Inositol 1,3,4,5-tetrakisphosphate was obtained from American Radiolabeled Chemicals (St. Louis, MO). PtdIns 4,5-bisphosphate and PtdIns 4-monophosphate were obtained from Sigma. Human neutrophils were purified from whole blood as described previously (33Haslett C. Guthrie L.A. Kopaniak M.M. Johnston R.B., Jr. Henson P.M. Am. J. Pathol. 1985; 119: 101-110PubMed Google Scholar). Neutrophil purity was >95% with typically ≤2% eosinophils. Following isolation, neutrophils were suspended at 2 × 106cells/ml in RPMI supplemented with 1% LPS-free bovine serum albumin (Sigma; A-1933) and incubated in conical polypropylene tubes (Sarstedt, as control; Sparks, NV). Neutrophils, with or without β2-integrin reagent VIM12 (1 μg/ml) or anti-HLA (1 μg/ml), were allowed to apoptose for 4 h in the presence or absence of anti-Fas IgM (1 μg/ml) or TNFα (1000 units/ml). Apoptosis was assessed as by Whitlock et al. (4Whitlock B.B. Gardai S. Fadok V. Bratton D. Henson P.M. J. Cell Biol. 2000; 151: 1305-1320Crossref PubMed Scopus (112) Google Scholar). Akt activity was measured using an enzyme activity kit (Upstate Biotechnology) with some modification of the provided protocol. Cells were resuspended at 7.5 × 106/ml cells and stimulated with either 10 ng of GM-CSF (R & D Systems) or 100 ng/ml LPS (List Biological) for 5 min or 1 μg/ml VIM12 or anti-HLA for 15 min in the presence or absence of 1 μg/ml anti-Fas IgM or 1000 units/ml TNFα. Cells were lysed in ice-cold radioimmune precipitation buffer (supplemented with 15 μg/ml leupeptin and aprotinin, 1 mmphenylmethylsulfonyl fluoride, and 0.2 mm sodium orthovanadate), and Akt activity was measured as by Whitlock et al. (4Whitlock B.B. Gardai S. Fadok V. Bratton D. Henson P.M. J. Cell Biol. 2000; 151: 1305-1320Crossref PubMed Scopus (112) Google Scholar). Western blots were carried out the same way, except immunoprecipitates were boiled in Lammeli buffer, run on a 10% SDS-PAGE gel, and probed with a 1:1000 sheep anti-phospho-Akt antibody (Upstate Biotechnology). Blots were stripped as by Frasch et al. (8Frasch S.C. Nick J.A. Fadok V.A. Bratton D.L. Worthen G.S. Henson P.M. J. Biol. Chem. 1998; 273: 8389-8397Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and reprobed with an anti-Akt antibody for equal loading. Lyn samples were stimulated for 10 min with the indicated stimuli and then lysed in 500 μl of lysis buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 30 mm sodium pyrophosphate, 50 mm NaF, 1 mm Na3VO4, 1% Nonidet P-40 with 1 mm phenylmethylsulfonyl fluoride and 15 μg/ml leupeptin and aprotinin) for 10 min on ice. Lysates were then cleared by centrifugation, and supernatants were transferred to 25 μg of protein A-Sepharose (Zymed Laboratories Inc.) beads containing 1 μg of rabbit anti-Lyn antibody (Santa Cruz Biotechnology) for 4 h with rotation. Immunoprecipitates were then boiled, run on a 10% gel, transferred, and then probed with 1:1000 anti-phosphotyrosine 4G10 (Upstate Biotechnology) overnight. SHIP samples were lysed according to Liu et al. (27Liu L. Damen J.E. Ware M. Hughes M. Krystal G. Leukemia. 1997; 11: 181-184Crossref PubMed Scopus (44) Google Scholar). SHIP was imunoprecipitated, run on a 7% gel, and probed with anti-CD11b clone 44 (Sigma) or anti-phosphotyrosine 4G10 overnight. Immunoblots were visualized with ECL detection reagents (Amersham Biosciences). All immunoblots were stripped as by Frasch et al. (8Frasch S.C. Nick J.A. Fadok V.A. Bratton D.L. Worthen G.S. Henson P.M. J. Biol. Chem. 1998; 273: 8389-8397Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and then reprobed with either anti-Lyn or anti-SHIP to confirm equal protein levels. Neutrophils were [32P]orthophosphate-labeled as described previously (34Vlahos C.J. Matter W.F. FEBS. 1992; 309: 242-248Crossref PubMed Scopus (53) Google Scholar). Briefly, neutrophils were suspended at 1 × 108/ml in buffer A (30 mm Hepes, pH 7.2, 110 mm NaCl, 10 mm KCl, 1 mm MgCl2, 10 mm glucose) and 1 mCi/ml [32P]orthophosphate (PerkinElmer Life Sciences). Cells were incubated at 37 °C for 90 min, at which point they were washed three times in buffer A. Labeled cells were then treated as in the apoptosis studies for 10 min. Reactions were stopped with the addition of 3 ml of chloroform/methanol (1:2, v/v) followed by 4 ml of chloroform, 2.4 m HCl (1:1, v/v). The resultant organic phase was removed, and the aqueous phase was washed four times. The combined organic phases were evaporated to dryness under N2. Dried lipids were resuspended in 20 μl of chloroform/methanol (2:1, v/v) and run on 20 × 20-cm silica gel 60 plates (Whatman) impregnated with 1% potassium oxalate. Plates were developed in chloroform/acetone/methanol/acetic acid/water (80:30:26:24:14, v/v/v/v/v). Radiolabeled phospholipids were detected by autoradiography. Standard PtdIns/PtdIns-4-P/PtdIns-3,4-P2 were co-chromatographed and visualized by I2 vapor. Neutrophils were resuspended at 5 × 106/ml and treated as above for 10 min, at which time they were lysed in 500 μl of lysis buffer containing 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 10% glycerol, 1 mm MgCl2, 1 mmCaCl2, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 15 μg/ml leupeptin, and aprotinin at 4 °C for 20 min. Lysates were transferred to protein A-Sepharose (Zymed Laboratories Inc., San Francisco, CA) beads containing 1 μg of anti-PI 3-kinase antibody (Upstate Biotechnology clone UB93–3) for 2 h, at which point they were spun down and washed three times with kinase buffer (50 mmHEPES (pH 7.25), 0.5 mm EDTA, and 5 mmMgCl2). Reaction were then performed as described previously (24Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Briefly, immunoprecipitates were resuspended in 40 μl of the wash buffer containing 25 μm ATP and 20 μCi of [γ-32P]ATP. Reactions were initiated with 10 μl of PtdIns sonicated in 20 mm HEPES buffer (pH 7.4). Reactions were allowed to go for 10 min at 37 °C and then stopped with 20 μl of 6 n HCl and 160 μl of chloroform/methanol (1:1, v/v). Samples were allowed to sit for 10 min and then spun for 10 min to separate the organic and aqueous phase. The lower organic phase was applied to silica 60 TLC plate and developed in CHCl3/MeOH/H2O/NH4OH (60:47:11.3:2). Plates were dried, and the kinase reaction was visualized by autoradiography. Preparation of [32P]PtdIns 3,4,5-trisphosphate and SHIP assays were carried out as described before (24Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar, 32Phee H. Jacob A. Coggeshall K.M. J. Biol. Chem. 2000; 275: 19090-19097Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, samples were stimulated as in the Akt activity assay for 10 min, at which time samples were lysed and immunoprecipitated with anti-SHIP for 2 h at 4 °C. Samples were then incubated with 25 μl of [32P]PIP3 or 50 μm3H-labeled PtdIns 1,3,4,5-P4 in reaction buffer for 20 min. Reactions were stopped with 100 μl of chloroform/methanol (1:1) and 100 μl of 2 m KCl. Dried phospholipids were dissolved in 30 μl of chloroform/methanol (1:1) and separated on TLC as in the metabolic labeling. 32P samples were visualized by autoradiograph, with the position of [32P]PtdIns 3,4,5-trisphosphate and [32P]PtdIns 3,4-bisphosphate determined by the mobility of 32P in vitro labeled phosphatidylinositol and PIP2 standards.3H samples were washed, dried, and resuspended and ran as above; positions were confirmed with I2 visualization of commercial standards as well as the position of [32P]PtdIns 3,4,5-trisphosphate and [32P]PtdIns 3,4-bisphosphate prepared standards. Inositol 1,3,4,5-tetraphosphate was excised from TLC plates solublized in HCl/methanol (1:1) for 1 h and then counted in a scintillation counter as described by Siddiqi et al. (35Siddiqi A.R. Srajer G.E. Leslie C.C. Biochim. Biophys. Acta. 2000; 1497: 103-114Crossref PubMed Scopus (57) Google Scholar). Mouse peritoneal exudate neutrophils were isolated following the methods of Savige et al. (36Savige J.A. Saverymuttu S.H. Pinching A.J. Clin. Exp. Immunol. 1984; 58: 737-744PubMed Google Scholar). Cytospun samples revealed a highly enriched neutrophil population (>90%) with occasional macrophages. Typical yields were ∼20 × 106 peritoneal neutrophils/mouse for SHIP and Lyn +/+ and 35 × 106 peritoneal neutrophils/mouse for SHIP and Lyn −/− mice. Neutrophils were resuspended at 2 × 106 cells/ml and incubated with 1 μg/ml of the murine activating anti-CD18 antibody, clone M18/2 (37Driessens M.H. van Hulten P. Zuurbier A., La Riviere G. Roos E. J. Leukocyte Biol. 1996; 60: 758-765Crossref PubMed Scopus (29) Google Scholar), from Pharmingen (San Diego, CA), in the presence or absence of murine TNFα, anti-Fas (Jo2), which was cross-linked with secondary antibody. Cells were incubated for 4 h before apoptosis was assessed with the human neutrophils. Dihydrorhodamine-123 (DHR; Molecular Probes, Inc., Eugene, OR) was performed as described by Vowells and colleagues (38Vowells S.J. Sekhsaria S. Malech H.L. Shalit M. Fleisher T.A. J. Immunol. Methods. 1995; 178: 89-97Crossref PubMed Scopus (394) Google Scholar). Briefly, neutrophils were preloaded with 1.0 × 10−5 nm dihydrorhodamine-123 for 5 min in a 37 °C shaking water bath prior to the addition of stimuli. Following a 2-h incubation at 37 °C, cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry (FL-2, FACScalibur; Becton Dickinson). CGD blood was obtained with the help of Dr. Dan Ambruso. Blood was drawn from three individuals, each displaying different mutations. One patient displayed a gp15 X-linked form, and the other two had an autorecessive mutation in either p67 or p22 of NADPH oxidase. All patients were deficient in NADPH oxidase-derived oxidants as tested by superoxide generation by Dr. Ambruso. Neutrophils were isolated as described above. Averages and SD values were calculated from at least three experiments. Statistical analysis was carried out using the JMP statistical programs (SAS Institute, Cary, NC). Details concerning the statistical tests used for each experiment can be found in the figure legends. Several cytokines and membrane receptor signals, such as β2-integrin activation, GM-CSF, LPS, and even low doses of TNFα (1 μg/ml) can activate Akt and thus inhibit neutrophil apoptosis (6Lee A. Whyte M.K. Haslett C. J. Leukocyte Biol. 1993; 54: 283-288Crossref PubMed Scopus (673) Google Scholar). However, previous observations have shown that neutrophils incubated simultaneously with the antiapoptotic β2-integrin-activating antibody VIM12 along with the stress stimuli TNFα, anti-Fas IgM, or UV irradiation no longer exhibited inhibition of apoptosis but rather an enhancement of this process (Fig. 1 A and Ref. 4Whitlock B.B. Gardai S. Fadok V. Bratton D. Henson P.M. J. Cell Biol. 2000; 151: 1305-1320Crossref PubMed Scopus (112) Google Scholar). Each stress stimulus alone resulted in a marked increase in neutrophil apoptosis compared with control, although we observed that lower concentrations of TNFα had an antiapoptotic effect. On the other hand, combining the opposing stimuli resulted in an increase in apoptosis above each stress stimulus alone. The Akt/protein kinase B pathway is the common antiapoptotic signaling pathway stimulated by β2-integrin activation, LPS, GM-CSF, and TNFα. Blockade of this pathway upon the combination of antiapoptotic and stress stimuli could result in the enhanced apoptosis seen in our system. Incubation of neutrophils with either 100 nm GM-CSF or 100 ng LPS for 5 min or with 1 μg/ml of the β2-integrin activation antibody VIM12 for 15 min increased Akt activity above base line (Fig. 1 B). Stress stimuli alone, either TNFα or anti-Fas IgM, also increased Akt activity. However, the combination of anti-apoptotic and stress stimuli resulted in the complete prevention of Akt activation. This effect on Akt was not seen when cells were treated with a control antibody against HLA, which neither stimulated Akt activity on its own nor attenuated anti-Fas IgM- or TNFα-generated Akt activity. Akt phosphorylation (Fig. 1 B, inset) correlated with its activity in that Akt phosphorylation reverted to control levels upon combining VIM12 with anti-Fas IgM or TNFα. Since Akt activation requires recruitment of Akt to PIP3 lipids in the plasma membrane via its PH domain (20Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar), the effect of pro- and antiapoptotic stimulus combinations on PIP3 levels was assessed in [32P]orthophosphate-labeled neutrophils. Neutrophils were labeled and then incubated with the antiapoptotic β2-integrin activation stimulus in the presence or absence of the stress stimuli. All of the individual stimuli alone enhanced PIP3 production above control; however, upon the combination of antiapoptotic and stress stimuli, the levels of PIP3 significantly decreased back to control levels (Fig.2 A). The combination of anti-HLA and TNFα or anti-Fas IgM showed no reduction in PIP3 levels (data not shown). The reduction in PIP3 suggested either that the combined stimuli inhibited PI 3-kinase activity or that a phosphatidylinositol phosphatase was activated. To examine whether the reduction in PIP3 levels was due to down-regulated PI 3-kinase, PI 3-kinase activity was measured directly after stimulation with VIM12 in the presence or absence of TNFα or anti-Fas IgM. Control (unstimulated) cells showed moderate PI 3-kinase activity in accordance with reported observations (34Vlahos C.J. Matter W.F. FEBS. 1992; 309: 242-248Crossref PubMed Scopus (53) Google Scholar). This activity was markedly enhanced in the cells incubated with the antiapoptotic or the stress stimuli alone. Importantly, the stimulation of PI 3-kinase was not inhibited or decreased upon the combination of antiapoptotic β2-integrin activation and stress stimuli (Fig. 2 B). However, cells incubated with the PI 3-kinase inhibitor LY98062 (100 nm) showed almost complete inhibition of activity (data not shown). Since PI 3-kinase activity was not altered but PIP3 levels were decreased when the two stimulus types were combined, activation of a phosphatidylinositol phosphatase was examined. Candidates included SHIP and PTEN. SHIP recruitment to the plasma membrane was examined d
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