Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

Mimi Takami1,2, Roman Herrera3, and Lilli Petruzzelli1,4

1 Department of Internal Medicine, Divisions of 4 Hematology/Oncology and 2 Gastroenterology, University of Michigan Medical Center and Department of Veterans Affairs Medical Center, Ann Arbor 48109; and 3 Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Activated neutrophils display an array of physiological responses, including initiation of the oxidative burst, phagocytosis, and cell migration, that are associated with cellular adhesion. Under conditions that lead to cellular adhesion, we observed rapid tyrosine phosphorylation of an intracellular protein with an approximate relative molecular mass of 92 kDa (p92). Phosphorylation of p92 was inducible when Mac-1 was activated by phorbol 12-myristate 13-acetate, the beta 2-specific activating antibody CBR LFA-1/2, or interleukin-8 (77 amino acids). In addition, tyrosine phosphorylation of p92 was dependent on engagement of Mac-1 with ligand. Several observations suggest that this event may be an important step in the signaling pathway initiated by Mac-1 binding. p92 phosphorylation was specifically blocked with antibodies to CD11b, the alpha -subunit of Mac-1, and was rapidly reversible on disengagement of the integrin ligand interaction. Integrin-stimulated phosphorylation of p92 created binding sites that were recognized in vitro by the SH2 domains of c-CrkII and Src. Our observations suggest that neutrophil adhesion mediated through the binding of the beta 2-integrin Mac-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interactions via SH2 domains, a key process shared with growth factor signaling pathways.

integrins; adhesion; CD11b; CD18


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF CIRCULATING NEUTROPHILS by a variety of agonists leads to protease release, reduction of molecular oxygen to super oxide anion, initiation of the respiratory burst, and reorganization of the actin-based cytoskeleton (61, 68, 76). Activated neutrophils aggregate, undergo chemotaxis, and phagocytize particles (60, 72, 76). In each of these physiological responses, cell adhesion has proven to be a critical event (44, 56, 57, 67, 71). Full activation of neutrophils is dependent on the expression, activation, and binding of adhesion receptors of the beta 2-integrin family, specifically leukocyte function-associated antigen (LFA)-1 and Mac-1, and requires that the cells make contact with a surface coated with extracellular matrix proteins, plasma proteins, serum proteins, or other cells (56). Intercellular adhesion molecule (ICAM)-1 serves as a common ligand for both LFA-1 and Mac-1. In addition, Mac-1 recognizes fibrinogen, factor Xa, and iC3b as well as unidentified ligands on the endothelial cell surface (1, 19, 42, 83).

Adhesion through the beta 2 family of integrins is a complex process in which there is enhanced avidity of the beta 2-integrins for their ligands (18, 35, 40, 74, 75). Integrin binding to ligand is stimulated through an inside-out signaling mechanism triggered by the engagement of well-characterized receptors such as those for the chemokines interleukin (IL)-8, C5a, and N-formylmethionylleucyl phenylalanine (FMLP) and for cytokine receptors such as tumor necrosis factor (TNF) (18, 25, 35, 75). Enhanced adhesion through LFA-1 and Mac-1 also can be achieved by direct activation of protein kinase C (PKC) with phorbol esters (18, 25, 35, 75). Engagement of these inflammatory receptors initiates a signal transduction cascade that results in inside-out activation of the integrin and enhanced adhesion of the cell. This increase in avidity is independent of a change in the level of cell surface expression of the beta 2-integrin (69, 80).

The signaling pathways activated in response to inflammatory signals or phorbol 12-myristate 13-acetate (PMA) treatment have yet to be elucidated. Direct phosphorylation of the integrin subunits has been demonstrated (11, 12, 28). CD11b is constitutively phosphorylated, whereas phosphorylation of the beta -subunit (CD18) has been demonstrated in response to PMA. In addition, phosphoamino acid analysis of CD11/18 in PMA- or FMLP-treated monocytes has revealed predominantly phosphoserine residues and only a small portion of phosphothreonine and phosphotyrosine residues (12, 77). The role of phosphorylation of the integrin Mac-1 in regulating adhesion has not been established (11, 32).

Subsequent to integrin binding to ligand, it has become evident that a signal transduction cascade is initiated that may affect changes in cytoskeletal structure and gene expression required for the physiological response of the cell (14). Protein phosphorylation appears to be one of the earliest signal transduction events detected in response to integrin binding (14). Early studies that focused on beta 1- and beta 3-integrins demonstrated that a signaling cascade is initiated by integrin activation and binding to ligand and is at least in part mediated by the activation of protein tyrosine kinases (10, 13, 33, 45, 47, 81). beta 2-Integrin-dependent adhesion results in the modulation of protein tyrosine phosphorylation of cellular proteins. In granulocytes, beta 2-integrin-dependent adhesion is associated with tyrosine phosphorylation of the Src family tyrosine kinase Fgr, paxillin, and a set of uncharacterized proteins of p70, p115, and p140 (8, 22, 23, 30). Phosphorylation may depend on the alpha -subunit, as suggested by the observation that phosphorylation of paxillin in TNF-treated neutrophils is dependent on Mac-1 but not LFA-1 binding (26). Ligation of beta 2-integrins on polymorphonuclear neutrophils (PMN) also induces an increase in the intracellular calcium concentration (31, 36, 58) and the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], possibly by tyrosine phosphorylation of phospholipase C (PLC)-gamma and D-myo-inositol-trisphosphate 3-kinase, respectively (31, 48). Antibody cross-linking of beta 2-integrins on adherent human neutrophils has been shown to trigger activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav (84). In the promegakaryocytic cell line MO7e, ligation of beta 2-integrins leads to cytoskeleton-dependent tyrosine phosphorylation of Cbl (51), and lymphocyte adhesion through LFA-1 results in phosphorylation of p130cas and its association with CrkII (64). These findings suggest that intracellular signaling in response to beta 2-integrin activation involves the stimulation of protein tyrosine kinases and leads to the regulation of protein-protein interaction via SH2 domains, a key process shared with growth factor signaling pathways (62).

The complex role of outside-in signals initiated by engagement of the beta 2-integrin in the physiological response of the neutrophil remains to be elucidated. It has become evident that signaling cascades involving tyrosine phosphorylation of intracellular proteins are important for mediating the physiological responses of neutrophils to activating agents (4, 65, 82). Neutrophils contain distinct pools of tyrosine kinase activity (5, 7, 41), and studies have indicated that discrete sets of proteins undergo tyrosine phosphorylation in response to granulocyte-macrophage colony-stimulating factor (37, 38, 46), IL-3 (38, 46), FMLP (6, 7, 24), leukotriene B4 (6), calcium ionophore (6), PMA (6), and TNF (22, 23, 67). In addition, tyrosine kinase inhibitors have been shown to inhibit superoxide production and chemotaxis in neutrophils, but they do not affect degranulation or actin polymerization (7, 24, 55). Involvement of these intracellular signals in adhesion is supported by the observation that inhibitors of tyrosine kinases and of D-myo-inositol-trisphophate 3-kinase block the beta 2-integrin-dependent spreading of PMN (22).

In the present study, we have explored the signal transduction cascade initiated by adhesion through the beta 2-integrin family members in human neutrophils. We have found that activation of the beta 2-integrin Mac-1, but not LFA-1, leads to tyrosine phosphorylation of a novel protein, p92. Tyrosine phosphorylation of p92 is dependent on integrin-ligand binding and cellular adhesion. In addition, the phosphorylation event is rapidly reversible, blocked by protein tyrosine kinase inhibitors, and dependent on an intact actin cytoskeleton.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Antibodies and materials. The beta 2-specific stimulatory antibody CBR LFA-1/2 and the anti-CD18 antibody CBR LFA-1/7 are mouse anti-human monoclonal antibodies (MAbs) that were isolated and purified as described previously (63). The anti-phosphotyrosine antibody 4G10 was purchased from Upstate Biotechnologies (Lake Placid, NY). Mouse MAbs directed against p130cas, focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase (PI 3-kinase), Crk, and cortactin were purchased from Transduction Laboratories (Lexington, KY). The Syk(sc-573), Vav(sc-132), and ZAP-70(sc-574) rabbit polyclonal antibodies, as well as protein A/G-agarose, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The inhibitory anti-CD11b (Mac-1 alpha -subunit) A44 MAb was the generous gift of Dr. Robert Todd III. Leupeptin, aprotinin, staurosporine, Triton X-100, and PMA were obtained from Calbiochem (La Jolla, CA). Cytochalasin B, benzamidine, FMLP, and fibrinogen were purchased from Sigma (St. Louis, MO). Genistein was purchased from GIBCO-BRL (Grand Island, NY).

Purification of neutrophils. Human neutrophils were isolated from whole blood after dextran sedimentation, Ficoll gradient centrifugation, and hypotonic lysis of red blood cells, as previously described (20). Neutrophils (1 × 107 cells/ml) were resuspended in Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES and 2 mM MgCl2, as described previously (17).

Cell binding to immobilized ligand. Fibrinogen (1 ml; 0.5 mg/ml) or BSA (1%) in phosphate-buffered saline (PBS) was adsorbed to 35-mm polystyrene petri dishes (Falcon 1008; Becton Dickinson, Lincoln Park, NJ) for 1.5 h at room temperature. Unbound protein was aspirated, and the plate was rinsed with PBS containing 1% Tween 20. After 2 min, the plates were washed six times with PBS. Neutrophils (1 ml; 1 × 107 cells/ml) were layered onto each plate, and cells were stimulated by the addition of PMA (50 ng/ml), CBR LFA-1/2 (10 µg/ml), IL-8 (77 amino acids, 200 ng/ml; Peprotec, Princeton, NJ), or MnCl2 (2 mM) at 37°C for 2-60 min, as indicated in legends for Figs. 1-11. After stimulation, adherence was confirmed visually by light microscopy. Unbound cells were aspirated and collected by centrifugation for 2 min in a microfuge at 2,000 rpm. Bound cells were removed by incubation with lysis buffer (50 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 3 mM sodium vanadate, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM benzamidine, 1 µg/ml leupeptin, 0.5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and lysates were added to the corresponding unbound cells. After 10 min on ice, samples were centrifuged at 13,000 rpm in an Eppendorf microfuge at 4°C for 15 min, and the supernatant was collected. Adhesion was blocked by preincubation with A44 ascites (1:100). Additional studies included incubation with cytochalasin B (2 µM) for 10 min, staurosporine (5 µg/ml) for 30 min, or genistein (50 µg/ml) for 30 min at 37°C. For experiments in which diisopropylfluorophosphate (DFP) was used, cells were preincubated for 10 min with 1 mM DFP and simulated in the presence of DFP; alternatively, 1 mM DFP was added to lysis buffer only.


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Fig. 1.   Activation of neutrophil adhesion to fibrinogen. A: human neutrophils were labeled with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) for 30 min, and adhesion assays were performed as indicated in MATERIALS AND METHODS. Polymorphonuclear neutrophils (PMN) were incubated in the absence of stimuli (Neg) or with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), CBR LFA-1/2 (AB; 15 µg/ml), or human interleukin-8 (IL-8; 77 amino acids, 200 ng/ml) for 30 min at 37°C. B: in blocking studies, cells were preincubated with control monoclonal antibodies (MAb) TS1/22 (10 µg/ml) or A44 (1:100 ascites) for 30 min at room temperature. Stimulation was subsequently performed at 37°C. Binding was performed in triplicate, and data are reported as means ± SE.



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Fig. 2.   Tyrosine phosphorylation in adherent neutrophils. A: PMN were incubated in the absence of stimuli (-) or with PMA, CBR LFA-1/2 (15 µg/ml), or human IL-8 (77 amino acids, 200 ng/ml) for 30 min at 37°C. Lysates were prepared as described and then immunoprecipitated with anti-phosphotyrosine antibody (4G10), and the immunoblot was probed with 4G10. Molecular mass markers are indicated at left. P, PMA; A, antibody CBR LFA-1/2; I, IL-8. B: cell lysates were prepared from unstimulated neutrophils or from those stimulated with PMA, CBR LFA-1/2, or IL-8 at 37°C. In addition, either neutrophils were preincubated with 1 mM diisopropylfluorophosphate (DFP) and then stimulated or stimulated cells were lysed in buffer containing 1 mM DFP. External controls were prepared before the experiments from lysates of unstimulated (-) PMN or from PMN stimulated with PMA. Lysates were subjected to immunoprecipitation and immunoblot with 4G10 as described. Ext Con, external control.



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Fig. 3.   Time course of activation and adhesion of neutrophils. A: time course of p92 phosphorylation. PMN were stimulated with PMA (P), activating antibody (A), or IL-8 (I) for 2, 10, 30, or 60 min as indicated. Lysates were prepared and analyzed as described in Fig. 2. B: time course of neutrophil adhesion. BCECF-labeled human PMN were incubated in the absence of stimulus (Neg) or with PMA, activating antibody CBR LFA-1/2 (AB), or IL-8 for 10, 30, or 60 min. Bound cells were quantified as described in MATERIALS AND METHODS. Binding was performed in triplicate, and data are reported as means ± SE.



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Fig. 4.   p92 does not comigrate with known signaling molecules. Lysates were prepared from untreated PMN (-) or from those stimulated with PMA and then immunoprecipitated with specific antibodies to proteins indicated for each lane (A and B). Immunoprecipitated proteins were subjected to SDS-PAGE (7.5%) and transferred to nitrocellulose. The immunoblot was probed with 4G10. Controls shown in the first and second lane of each gel demonstrate p92 immunoprecipitated with anti-phosphotyrosine (Ptyr) antibody. The presence of Syk (C), Vav (D), and paxillin (Pax) (E) in the lysates derived from cells treated with PMA (P), CBR LFA-1/2 (A), and IL-8 (I) is demonstrated. PI3K, phosphatidylinositol 3-kinase; IP, immunoprecipitate; WB, Western blot.



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Fig. 5.   p92 does not comigrate with CD18. Cell lysates were prepared from neutrophils stimulated with either PMA (P) or CBR LFA-1/2 (A). Lysates were precipitated with 4G10 (anti-Ptyr) or CBR LFA-1/7 [M; monoclonal antibody (MAb) to CD18], and immunoblots were probed with the indicated MAb. Purified LFA-1 was included (CD18). The samples and gels were run in the absence of reducing agents.



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Fig. 6.   Mac-1 specificity of p92 phosphorylation. A: PMN were incubated in the absence of stimuli (lane 1) with PMA (P; 50 ng/ml; lane 2) or CBR LFA-1/2 (A; 15 µg/ml; lane 3) for 30 min at 37°C. Cells depicted in lanes 4 and were preincubated with the anti-Mac-1 MAb (A44) at 1:100 of ascites before stimulation with either PMA or CBR LFA-1/2. Lysates were prepared as described and, after immunoprecipitation with 4G10, they were subjected to SDS-PAGE (7.5%) and transferred to nitrocellulose. The immunoblot was probed with 4G10. B: inhibition of LFA-1-dependent adhesion. Experiment was performed as in A except that TS1/22 (10 µg/ml) was used as a blocking antibody in lanes 4 and 5.



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Fig. 7.   Ligand-specificity of IL-8-stimulated integrin-dependent phosphorylation in neutrophils. A: neutrophils were labeled with BCECF as described and incubated in the absence of stimuli (Con) or with IL-8. Cells were incubated on polystyrene plates coated with purified human fibrinogen (Fib), heat-inactivated BSA, or 1% Tween 20 for 30 min at 37°C. Unbound cells were removed by aspiration, and fluorescently labeled bound cells were quantitated as described in MATERIALS AND METHODS. B: PMN, stimulated with PMA (P), the activating MAb CBR LFA-1/2 (A), or IL-8 (I), were incubated on polystyrene plates coated with fibrinogen, heat-inactivated BSA, or 1% Tween 20, as indicated. Cell lysates were prepared as described and immunoprecipitated with anti-phosphotyrosine antibody, 4G10. Immunoprecipitates were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose, and filters were immunoblotted with 4G10 as described. C: PMN, unstimulated (-) or stimulated with PMA (P), activating antibody CBR LFA-1/2 (A), or 2 mM MnCl2 (Mn), were incubated on polystyrene plates coated with fibrinogen or fibronectin, as indicated. Lysates were prepared, and protein tyrosine phosphorylation was analyzed as described in B.



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Fig. 8.   Reversibility of p92 tyrosine phosphorylation. Cells were incubated in the absence of stimuli (lane 1) or in the presence of PMA (50 ng/ml; lanes 2-4), or the activating antibody CBR LFA-1/2 (lanes 5-7). Samples presented in lanes 3 and and lanes 6 and were incubated in the presence of EDTA (10 mM). Samples presented in lanes 4 and 7 were subsequently washed 3 times in Hanks' balanced salt solution (HBSS) containing 2 mM CaC12 and 2 mM MgCl2 to remove EDTA and replete divalent cations, and cells were restimulated with PMA or activating antibody as indicated. Lysates were prepared, and protein tyrosine phosphorylation was analyzed as in Fig. 7. Wash restim, washed and restimulated.



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Fig. 9.   Effect of cytochalasin B on the tyrosine phosphorylation of p92. Cells were incubated in the absence of stimuli (-) and in the presence of PMA (P) or activating antibody CBR LFA-1/2 (A). For studies depicted in lanes 4 and 5, cells were preincubated in 2 µM cytochalasin B (Cyto) and stimulated in the presence of this inhibitor for 30 min at 37°C. Cell lysates were prepared as described and immunoprecipitated with anti-phosphotyrosine antibody, 4G10. Immunoprecipitates were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose, and filters were immunoblotted with 4G10 as described.



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Fig. 10.   Effect of kinase inhibitors on neutrophil adhesion and tyrosine phosphorylation. A: effect of inhibitors on adhesion of neutrophils to fibrinogen. Cells were labeled with BCECF as described, incubated with the indicated inhibitor, and stimulated with PMA, CBR LFA-1/2 (AB), or IL-8 (IL8). Control experiments were performed in the presence of the carrier DMSO. For inhibitor studies, cells were preincubated in inhibitor for 30 min and subsequently stimulated in the presence of inhibitor for 30 min at 37°C. Unbound cells were removed by washing, and bound cells were quantitated as described. Staur, staurosporine (5 µg/ml); Gen, genistein (50 µg/ml). B: effect of inhibition of protein kinase activity on phosphorylation of p92. Cells were incubated in the absence of stimuli (-) or in the presence of PMA (P), CBR LFA-1/2 (A), or IL-8 (I). Inhibitors are indicated for each lane: Stau, staurosporine (5 µg/ml); Gen, genistein (50 µg/ml); and BIM-1, bisindolylmaleimide-I (5 µg/ml). Cells were preincubated with inhibitors for 30 min at room temperature and then stimulated as indicated in the presence of inhibitors for 30 min at 37°C. Control samples were incubated in the presence of DMSO, the solvent. Cell lysates were prepared as described and immunoprecipitated with anti-phosphotyrosine antibody, 4G10. Immunoprecipitates were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose, and filters were immunoblotted with 4G10 as described.



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Fig. 11.   Association of phosphorylated p92 with Src-SH2 and Crk-SH2 in vitro. A: cells were incubated in the absence of stimuli (-) or in the presence of PMA (P) or CBR LFA-1/2 (A). The sample represented in lane 6 was coincubated with the Mac-1 blocking antibody A44. Cell lysates were prepared as described, and samples depicted in lanes 1-3 were immunoprecipitated with the antibody 4G10. Samples represented in lanes 4-6 were precipitated with glutathione-S-transferase (GST) Src-SH2 fusion protein. Precipitates were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose, and filters were immunoblotted with 4G10 as described. B: cells were incubated in the absence (-) of stimuli or with PMA (P), CBR LFA-1/2 (A), or IL-8 (I). Cells were then treated with either HBSS alone or HBSS containing EDTA. Lysates were precipitated with GST-Crk-SH2 fusion protein ascribed to glutathione-Sepharose beads (PPT), subjected to 7.5% SDS-PAGE, and transferred to nitrocellulose, and filters were immunoblotted with 4G10 as described above.

For binding to fibronectin, 1 × 107 PMN were layered onto 12-well fibronectin-coated plates (Collaborative Research). Cells were stimulated with 2 mM MnCl2 for 30 min at 37°C and processed as described above.

Immunoprecipitation. Solubilized cell extracts were incubated with antibody, as indicated in legends for Figs. 1-11, in the presence of 30 µl of protein A/G-agarose for 1.5 h at 4°C. The immunoprecipitates were washed twice with lysis buffer and boiled for 5 min in 20 µl of 2× SDS sample buffer (50 mM Tris, pH 6.8, 20% glycerol, 2% SDS, and 5% beta -mercaptoethanol). After SDS-PAGE, the proteins were transferred to nitrocellulose, and the filter was analyzed by immunoblotting with anti-phosphotyrosine antibody (4G10; 1:1,000 dilution). Bound antibody was detected with horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence (ECL) according to the manufacturer's directions (Amersham, Arlington Heights, IL).

Binding to glutathione-S-transferase-SH2 domains. The glutathione-S-transferase (GST) fusion protein containing the SH2 domain of CrkII and Src was the generous gift of Drs. Birge and Hanafusa (9). The GST fusion protein containing the SH2 domain of Abl was obtained from Dr. Alan Saltiel. Production and purification of GST-SH2 fusion proteins were performed as described previously (49). Binding was carried out by incubating the GST fusion proteins bound to glutathione-Sepharose (Pharmacia Biotech, Uppsala, Sweden) with the cell extracts prepared as described in Cell binding to immobilized ligand (40 µl of 1:1 slurry/ml of lysate). After 1.5 h at 4°C, the beads were washed with lysis buffer, and the bound proteins were analyzed by SDS-PAGE, followed by transfer to nitrocellulose, immunoblotting, and detection by ECL (Amersham), as described in Immunoprecipitation.

Neutrophil adhesion. Purified human fibrinogen (0.5 mg/ml in PBS) was adsorbed to polystyrene 96-well plates (Linbro/Titertek; ICN, Aurora, OH) for 1.5 h, as described in Cell binding to immobilized ligand. Human neutrophils (2 × 106/ml) were labeled by preincubation with 1.7 ug/ml of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min. PMN were washed in HBSS and preincubated in the presence of the Mac-1 blocking antibody A44 (1:100 ascites), LFA-1 blocking antibody TS1/22 (10 µg/ml), staurosporine (5 µg/ml), genistein (50 µg/ml), or cytochalasin B (2 µM), as indicated in legends for Figs. 1-11. Neutrophils were added to wells containing PMA (50 ng/ml), CBR LFA-1/2 (15 µg/ml), human IL-8 (77 amino acids, 200 ng/ml), or FMLP (1 µM) with or without inhibitor and centrifuged at 200 g for 1 min. The total fluorescent content of the cells in each well was assessed in a Fluorescent Concentration Analyzer (Cytofluor; PerSeptive Biosystems), and plates were incubated as indicated in legends for Figs. 1-11. Unbound cells were removed by the addition of HBSS and aspiration with a 20-gauge needle four times at 90° intervals around the well at room temperature. Bound cells were quantitated in the Fluorescent Concentration Analyzer, and data are expressed as percentages of bound-to-total input cells per well. Each binding condition was assessed in triplicate.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Activation of Mac-1 on human neutrophils induces cellular adhesion to immobilized ligand. Adhesion of neutrophils is a complex event that can be achieved by stimulation of a number of cell surface receptors, involves multiple integrin family members, and may involve more than one ligand. Our studies are aimed at elucidating the signaling events activated upon integrin-dependent adhesion of neutrophils. We have focused on three stimulatory mechanisms: activation of the neutrophil through the chemokine IL-8, which has been shown to be critical in transendothelial migration; through PMA-induced activation of PKC; and through direct stimulation of integrin binding by the beta 2-integrin-activating antibody CBR LFA-1/2. Human neutrophils isolated from whole blood were stimulated to bind to purified human fibrinogen, a ligand specific for the predominant beta 2-integrin receptor on neutrophils, Mac-1. Figure 1 demonstrates that PMA, the beta 2-activating antibody CBR LFA-1/2, and IL-8 stimulate neutrophil adhesion more than 10-fold above that of control conditions (Fig. 1A). Adhesion was blocked by the Mac-1-specific blocking antibody A44, but not by TS1/22, an LFA-1 blocking MAb (Fig. 1B). These results demonstrate that activation of Mac-1 on human neutrophils through inside-out signals initiated by IL-8, activation of PKC by PMA, or directly with beta 2-activating antibody leads to integrin-mediated cellular adhesion that is Mac-1 dependent.

Activation of beta 2-integrin on human neutrophils induces tyrosine phosphorylation of p92. Signaling events triggered by activation and binding of Mac-1 during adhesion in human PMN were examined by analyzing protein tyrosine phosphorylation in response to IL-8, PMA, and CBR LFA-1/2. Cell adhesion to fibrinogen, under each condition of stimulation, was associated with enhanced tyrosine phosphorylation of a predominant protein migrating at a relative molecular weight of 92,000 daltons (Mr approx  92 kDa; p92) (Fig. 2A). We more closely examined whether p92 represented a proteolytic product by incubating cells with DFP or adding DFP to the cell lysate as an alternative method to inhibit proteolysis. Preincubation of the cells with DFP results in the accumulation of some higher molecular weight phosphorylated species and an increase in phosphorylation of p92 in the absence of added stimuli. Inclusion of DFP in the lysis buffer alone results in a similar pattern of tyrosine phosphorylation to that observed under our standard lysis conditions (Fig. 2B).

Phosphorylation of p92 was detected at near-maximal levels as early as 2 min after stimulation with PMA. In contrast, antibody-stimulated p92 phosphorylation, detectable at 2 min, was significantly enhanced by 30 min of stimulation (Fig. 3A). IL-8-stimulated p92 phosphorylation was minimally detectable at 2 min, enhanced at 10 min, and diminishing by 30 min of stimulation. Adhesion was observed by 10 min of stimulation (Fig. 3B). At the later time points, in response to IL-8 stimulation, adhesion was preserved, but phosphorylation of p92 was no longer observed. This may reflect the modulation of different signaling pathways during cellular adhesion.

In an attempt to identify p92, we screened for several proteins known to play a role in tyrosine kinase intracellular signaling cascades. Stimulated and unstimulated cell extracts were immunoprecipitated with antibodies to cortactin (p85), p130cas (p130), Vav (p95), PI 3-kinase (p85), Syk (p72), Zap-70 (p70), paxillin, (p69), and Stat (p91/p84). None of these proteins exhibited differential phosphorylation after beta 2-integrin binding, and none comigrated with the p92 band (Fig. 4, A and B). In addition, no protein species of 92 kDa was detected in the absence of cell lysate (data not shown).

Syk (Fig. 4C), Vav (Fig. 4D), and paxillin (Fig. 4E) were present in the lysates isolated from neutrophils, as has been demonstrated previously (23, 84). Variable recovery of Syk may reflect differences in solubility upon activation of the cells. We also controlled for the possibility that p92 is a phosphorylated form of the integrin itself by immunoprecipitating with CBR LFA-1/7, which recognizes the beta -subunit. The complexes were then separated with SDS-PAGE and analyzed by immunoblot with anti-phosphotyrosine MAb and CBR LFA-1/2. p92 did not comigrate with proteins in the CBR LFA-1/7 immunoprecipitate or with the CD18 band from purified LFA-1 (Fig. 5).

Engagement of Mac-1 is required for phosphorylation of p92. Because human neutrophils express both Mac-1 and LFA-1 on their cell surface (74), we examined whether the phosphorylation of p92 was specific for binding through Mac-1, LFA-1, or both. We studied the effect of the blocking MAbs A44 (16) and TS1/22, which recognize specifically the alpha m (Mac-1) or the alpha L (LFA-1) subunits, respectively. Neutrophils were preincubated with either of these MAbs and then treated with PMA, CBR LFA-1/2, or IL-8. The MAb to Mac-1 inhibited cellular adhesion when examined in the adhesion assays (Fig. 1B). Corresponding with inhibition of adhesion, treatment with A44 resulted in reduced tyrosine phosphorylation of p92 (Fig. 6A). In contrast, the MAb to LFA-1 inhibited neither cellular adhesion nor phosphorylation of p92 (Figs. 1B and 6B).

IL-8 stimulates Mac-1-mediated neutrophil adhesion and tyrosine phosphorylation of p92 in a ligand-specific manner. Neutrophils bind to the endothelial layers in an integrin-dependent manner in response to secretion of the chemokine IL-8 (34). In our experiments, we utilized IL-8 as a physiological stimulator of neutrophil adhesion. To further characterize the specificity of the IL-8 response, we compared neutrophil adhesion to fibrinogen (a Mac-1-specific ligand), BSA, and Tween 20. In response to treatment with IL-8, neutrophils adhered to fibrinogen but not to BSA- or Tween 20-coated wells (Fig. 7A). In addition, p92 phosphorylation in response to IL-8 was observed only in those neutrophils that were adherent to fibrinogen (Fig. 7B), whereas it is observed in response to antibody or PMA on all three surfaces. Similarly, neutrophils treated with Mn2+ to stimulate beta 1-integrin adhesion did not exhibit enhanced tyrosine phosphorylation of p92 when tested on plates coated with fibronectin, a beta 1-specific ligand (Fig. 7C). This suggests that p92 phosphorylation is dependent on beta 2-mediated adhesive interactions.

Phosphorylation of p92 is reversible upon cell deadhesion. Divalent cations play a critical role in adhesion of beta 2-integrins to ligand, and in the absence of the divalent cation Mg2+, regulated adhesion through beta 2-integrin family members does not occur (2, 21, 52, 53). When cells were stimulated to adhere with PMA or activating antibody, phosphorylation of p92 was observed. These cells were then treated with EDTA to chelate Mg2+ and disrupt adhesion (as observed by light microscopy). Under these conditions, tyrosine phosphorylation of p92 was markedly diminished (Fig. 8). EDTA-treated cells were then washed in HBSS containing Mg2+ and Ca2+ and restimulated to adhere with PMA or activating antibody; coinciding with adhesion was the reappearance of tyrosine phosphorylated p92 (Fig. 8).

Inhibition of tyrosine phosphorylation of p92 by kinase inhibitors and cytochalasin B. Neutrophils undergo dramatic cell shape changes and cytoskeletal reorganization in response to activating agents (61, 73). Disruption of the cytoskeleton by cytochalasin B has been shown to inhibit both spreading and the respiratory burst in TNF-stimulated PMN (43). To address whether an intact cytoskeleton is important for tyrosine phosphorylation of p92, we studied its phosphorylation pattern in cells treated with cytochalasin B in the presence of the activating antibody CBR LFA-1/2 or in response to PMA. Cytochalasin B treatment of PMN prevented both antibody- and PMA-mediated adhesion and tyrosine phosphorylation of p92 compared with the control experiment with DMSO alone (Fig. 9). In addition, we examined cellular adhesion and tyrosine phosphorylation of p92 in stimulated neutrophils incubated in the presence of the protein tyrosine kinase inhibitor genistein and the PKC inhibitor staurosporine. Under these conditions, we observed inhibition of PMA-, antibody-, and IL-8-stimulated cellular adhesion (Fig. 10A). Tyrosine phosphorylation of p92 in response to PMA or antibody stimulation was diminished in the presence of genistein, staurosporine, or the more specific PKC inhibitor bisindolylmaleimide I (BIM) (Fig. 10B). In response to IL-8, BIM does not appear to affect phosphorylation of p92 (Fig. 10B). These data indicate that to initiate an outside-in signaling cascade and ultimately mediate cellular adhesion, Mac-1 requires an intact cytoskeleton and a complex signaling pathway possibly involving a balance of protein kinase and phosphatase activity.

Tyrosine phosphorylated p92 associates with Src-SH2 and CrkII-SH2 domains in vitro. The initiation of an outside-in signaling cascade through beta 1-integrins on platelets and fibroblasts and through beta 2-integrins on lymphocytes established a relationship among Src family tyrosine kinases, integrin activation, and the cytoskeleton (14). To address whether p92 forms a part of a signaling complex, we examined whether tyrosine phosphorylation p92 contains a recognition motif that forms a protein complex through an interaction with SH2 domains of known Src family members. We utilized a series of SH2 domain GST fusion proteins to probe extracts of neutrophils treated with either PMA or the beta 2-activating antibody CBR LFA-1/2. We found that when cells were stimulated with either PMA or MAb, phosphorylated p92 associated with Src-SH2 (Fig. 11A). In addition, tyrosine phosphorylated p92 specifically associated with the CrkII-SH2 domain (Fig. 11B) but not the Crk II-SH3 domain (data not shown). The interaction of p92 with the Crk-SH2 domain was lost when cellular adhesion was disrupted by treatment with EDTA (Fig. 11B). Similarly, the association with Src-SH2 was disrupted when cells were treated with the Mac-1 blocking MAb A44 (Fig. 11A). The Abl-SH2 and Grb-SH2 GST constructs did not precipitate tyrosine phosphorylated p92, thus controlling for nonspecific interactions with the GST-construct (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The outside-in signaling pathways generated by the beta 2-integrins in neutrophils upon interaction with their ligands are in the process of being defined and are the focus of the present study. Leukocyte integrins have been reported to signal an increase in intracellular calcium, inositol phospholipid hydrolysis (31, 36, 58, 67, 79), and tyrosine phosphorylation of a number of intracellular proteins, including Fgr (8), paxillin (23), PLC-gamma (31, 39), p130cas (64), and a set of as yet uncharacterized proteins: p70, p115, and p140 (22).

Here, we report the tyrosine phosphorylation of a protein of Mr approx  92 kDa (p92) in human neutrophils in response to stimulation of both the inside-out signaling pathways with IL-8 or phorbol esters and the direct activation of Mac-1, but not LFA-1, by an MAb directed against the beta -subunit (Fig. 2A). One approach that we have taken is to initially concentrate the tyrosine phosphorylated proteins in cell lysates by anti-phosphotyrosine immunoprecipitation in an attempt to enhance sensitivity of detection. By using this method we enhance our ability to examine a protein that is a minor species when total cell lysates are utilized.

This protein is likely not a proteolytic product of a larger molecular weight species (Fig. 2B). Although preincubation of PMN with DFP resulted in the accumulation of higher molecular weight phosphoproteins as well as an increase in the background phosphorylation, we were still able to detect enhanced phosphorylation of p92 in response to stimulation. Inclusion of DFP in the lysis buffer alone yielded a pattern of tyrosine phosphorylation similar to that observed under our standard lysis conditions (Fig. 2). Preincubation of neutrophils with DFP has been shown to interrupt various physiological responses of the cell, including elastase activity in azuorophil granules (66), plasminogen-independent fibrinolytic activity (29), and extracellular cleavage of protein S (59). In addition, treatment of neutrophils with DFP may interfere with key signals induced by calcium ionophore and responsible for activation of NADPH-oxidase (50). Therefore, the diminished level of tyrosine phosphorylation observed in response to PMA, but not MAb, in our DFP preincubation experiments may represent an effect of DFP on other signal transduction pathways involved in PMA-stimulated adhesion in neutrophils and raises the possibility that key proteolytic steps are required in the activation pathway. The preservation of p92 under our standard lysis conditions and in the presence of DFP, as an additional serine protease inhibitor, suggests that p92 is likely not a cleavage product of a higher molecular weight phosphoprotein in the lysate.

p92 appears to be a phosphoprotein distinct from CD18 that has previously been shown to be phosphorylated in response to stimulation of cells with PMA. Phosphorylation of CD18 occurs primarily on serine and threonine residues and minimally on tyrosine residues (11). The predominant phosphorylation site on the CD18 polypeptide in response to phorbol ester stimulation is Ser-756; however, mutation of this site with concomitant loss of phosphorylation of CD18 does not alter the ability of the integrin to become activated by phorbol ester (32). Phosphorylation of threonine residues is rapid and transient and is detectable only after inhibition of phosphatases with okadaic acid (78). The relationship between phosphorylation of CD18 on threonine residues and adhesive capability of the integrin has not yet been established (78). In our experiments, p92 is phosphorylated on tyrosine residues and does not comigrate with purified CD18 on SDS-PAGE (Fig. 5). In addition, this phosphorylation event appears to be closely associated with and dependent on cellular adhesion (Fig. 8).

Tyrosine phosphorylation of p92 is rapid in onset, occurring within 2 min of stimulation with either PMA or activating antibody (Fig. 3A). The tyrosine phosphorylation of p92 is specifically dependent on Mac-1 activation, binding, and subsequent cellular adhesion, while it is independent of interactions involving LFA-1 or beta 1-integrin, known to be on the surface of the neutrophil (Figs. 1B, 6A, 6B, and 7C). Mac-1 has been established as the adhesion receptor that plays a critical role in neutrophil adhesion and activation (40). MAbs directed against Mac-1 inhibit phorbol ester-induced neutrophil aggregation, chemotaxis, and adherence to protein-coated glass and plastic (3, 16, 54) and, as we show in the present study, also inhibit the tyrosine phosphorylation of p92 (Fig. 6).

The leukocyte integrins appear to be capable of rapid activation and deactivation (18). This ability is likely essential for leukocyte migration through endothelium, during which activated leukocytes adhere to endothelial cells and subsequently deadhere and transmigrate. To place our observations in this physiological context, we studied intracellular signaling events in response to rapid adhesion and deadhesion of neutrophils by disrupting adhesion through chelation of divalent cations, in particular Mg2+, required for ligand binding (2, 21, 52, 53). We analyzed tyrosine phosphorylation in adherent cells, and when cation-dependent adhesion was disrupted, we observed a concomitant loss of tyrosine phosphorylation of p92 (Fig. 8). In parallel, we noted a loss in association of p92 with the Crk-SH2 domain (Fig. 11). When the same cells were then washed free of EDTA and the divalent cations replaced, we observed a return of the adhesive properties of the cell, along with a reappearance of the tyrosine phosphorylated form of p92. Rapid phosphorylation and dephosphorylation of p92 suggests that this process is tightly coupled to integrin dependent adhesion and that there may be a protein tyrosine phosphatase closely integrated with this system.

Tyrosine phosphorylation of p92 is inhibited by cytochalasin B, indicating that cytoskeletal rearrangement is important to this event (Fig. 9). This is consistent with the finding, by Fuortes et al. (22) that protein tyrosine-phosphorylation resulting from treatment of adherent neutrophils with TNF is reversible by cytochalasin. It has been suggested that the tyrosine phosphoproteins may be protected from dephosphorylation when associated with the cytoskeleton (22, 27). In contrast, we have previously observed that the tyrosine phosphorylation of p130cas is not blocked by cytochalasin. Phosphorylation of p130cas was observed in a lymphocyte cell line undergoing homotypic aggregation through LFA-1-ICAM-1 interaction, and this type of association may be less dependent on cytoskeletal rearrangement than the Mac-1-mediated cellular adhesion and spreading observed in neutrophils. Protein tyrosine phosphorylation mediated through beta 1-integrins, which tend to participate in focal adhesions at the cell surface, also has been shown to be inhibited with cytochalasin (27, 47, 81).

Mac-1-mediated neutrophil adhesion in response to PMA, IL-8, or activating MAb is inhibited by the tyrosine kinase inhibitor genistein and the PKC inhibitor staurosporine (Fig. 10A). The effect of inhibition of these pathways varies depending on the stimulus examined. PKC inhibitors markedly reduce the level of phosphorylation in response to MAb and PMA. In contrast, there appears to be less of an effect of inhibition of this pathway on IL-8-stimulated phosphorylation of p92 (Fig. 10A). Genistein treatment results in a moderate decrease in phosphorylation of p92. Inhibition by these agents was observed whether cells were stimulated with PMA or by an activating antibody that bypasses inside-out signaling to activate the integrin (63). Inhibition by genistein further supports the role of initiation of a tyrosine kinase cascade upon adhesion of Mac-1 with its ligand(s). The effects of staurosporine raise at least two possible explanations: 1) that preservation of PKC activity is required to establish integrin-dependent tyrosine phosphorylation of p92, or 2) that staurosporine inhibits a kinase critical to either the activation of integrin binding or outside-in integrin signals. Nonetheless, PMA or MAb stimulation of Mac-1-mediated cellular adhesion, as well as tyrosine phosphorylation of p92, is dependent on a kinase cascade and, ultimately, is due to activation of a tyrosine kinase. The IL-8-stimulated p92 phosphorylation event is rapid in onset and transient (Fig. 3A), and it may involve a complex signaling cascade that includes the dynamic interactions between phosphatases and kinases.

We have utilized IL-8 as a physiological stimulator of neutrophil adhesion and activation. IL-8 has been shown to be an important chemokine that plays a critical role in neutrophil transmigration across both endothelial and epithelial barriers (15, 34, 70). IL-8 activates the beta 2-integrin through inside-out signals, and, as shown in Fig. 2A, IL-8 stimulation of neutrophils results in tyrosine phosphorylation p92 upon engagement of the integrin with ligand. Thus the tyrosine phosphorylation of p92 seems to be an outside-in signal common to the three mechanisms of integrin activation utilized in this study: stimulation by PMA, external activation of the integrin with the beta 2-activating antibody, and integrin activation through the chemokine receptor stimulated by IL-8. Whether or not the activation of PKC is an important element of the inside-out signals linking IL-8 stimulation to integrin activation in this system remains to be investigated. The IL-8 response, in terms of both neutrophil adhesion and tyrosine phosphorylation of p92, appears to be more ligand specific than the PMA- or CBR/LFA-1/2-stimulated responses, as shown in Fig. 7. Thus this physiological stimulus may render a more subtle and easily perturbed response than that elicited by pharmacological agents.

The results presented here indicate that the tyrosine phosphorylation of p92 may be an integral component of the outside-in signal transduction cascade initiated by activation and binding of Mac-1 on human neutrophils. We have demonstrated two in vitro associations with SH2 domains present in proteins known to play a role in tyrosine phosphorylation and receptor signaling. We have shown these associations to be influenced by integrin-ligand binding and subsequent cellular adhesion; however, it remains to be determined whether these associations are truly part of the Mac-1 signaling cascade. p92 does not appear to cross-react with antibodies to several known signaling molecules of similar molecular weight (Fig. 4) and, thus, may be a novel intracellular protein. Future studies need to focus on identification of this protein.


    ACKNOWLEDGEMENTS

We are grateful to Lisa Cummins for preparation of this manuscript.


    FOOTNOTES

L. Petruzzelli is supported by Department of Veterans Affairs Grant 12A78, National Institute of Allergy and Infectious Diseases Grant AI-01376-01, and the American Society of Hematology Scholar Award. M. Takami is supported by a fellowship from the Crohn's and Colitis Foundation and an award from the Gastrointestinal Peptide Research Center of the University of Michigan.

Address for reprint requests and other correspondence: L. Petruzzelli, Dept. of Internal Medicine, Division of Hematology/Oncology, Univ. of Michigan Medical Center, MSRB III, Rm. 5301B, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 (E-mail: lpetruzz{at}umich.edu).

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.

Received 7 May 1999; accepted in final form 18 November 2000.


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DISCUSSION
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Am J Physiol Cell Physiol 280(5):C1045-C1056