Regulation of alpha IIbbeta 3 Function in Human B Lymphocytes*

Weiwei Qi, Elwyn Loh, Gaston Vilaire, and Joel S. BennettDagger

From the Hematology-Oncology Division and the Department of Medicine, the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We studied the function of the platelet integrin alpha IIbbeta 3 using a B lymphocyte model in which alpha IIbbeta 3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alpha IIbbeta 3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alpha IIbbeta 3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galpha i, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alpha IIbbeta 3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alpha IIbbeta 3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galpha i, protein kinase C, and the actin cytoskeleton.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Exposure of a binding site for ligands such as fibrinogen and von Willebrand factor on the platelet integrin alpha IIbbeta 3 is a prerequisite for platelet aggregation (1). The process by which this binding site is exposed has been termed "inside-out" signaling and is initiated when agonist-generated intraplatelet signals induce a conformational change in alpha IIbbeta 3 by interacting with its cytoplasmic tails (2). To study the process of alpha IIbbeta 3 activation in vitro, we have developed a model system in which wild-type and mutant alpha IIbbeta 3 expressed in GM1500 cells, an Epstein-Barr virus-transformed human B lymphocyte line, can be induced to interact with soluble and immobilized fibrinogen by the phorbol ester phorbol 12-myristate 13-acetate (PMA)1 (3, 4). Using this system, we observed that the cytoplasmic tail of alpha IIb is not required for alpha IIbbeta 3 function in lymphocytes, that the conserved GFFKR motif in the alpha IIb tail is required for alpha IIb to interact with beta 3, and that signals interacting with the beta 3 cytoplasmic tail are responsible for the ability of agonists to stimulate alpha IIbbeta 3 function (4).

Phorbol esters such as PMA activate the conventional and novel isoforms of protein kinase C (PKC) (5). While phorbol esters are a potent stimulus for ligand binding to alpha IIbbeta 3 in platelets (6), they bypass the more proximal signaling events that are initiated when agonists bind to their cognate platelet membrane receptors. Most of the known platelet receptors for agonists are seven-transmembrane domain proteins that are coupled to various G proteins (7). Stimulation of these receptors on platelets is known to activate phospholipase Cbeta , generate diacylglycerol and inositol triphosphate, increase cytosolic calcium, and activate several isoforms of PKC (7). We were interested in determining whether stimulation of a G protein-coupled receptor on B lymphocytes would also expose the ligand-binding domain of alpha IIbbeta 3. Honda et al. (8) have reported that stimulation of the human N-formyl peptide chemoattractant receptor (fPR) in murine B lymphocytes induces the alpha 4beta 1-mediated adherence of these cells to VCAM-1. The fPR is a seven-transmembrane domain protein coupled to the G protein Galpha i in leukocytes (9). Accordingly, we coexpressed the human fPR and alpha IIbbeta 3 in GM1500 cells and tested the ability of the transfected cells to interact with immobilized and soluble fibrinogen. We found that like PMA, the chemoattractant peptide formyl Met-Leu-Phe (fMLP), an fPR agonist, increased the avidity of alpha IIbbeta 3 for immobilized fibrinogen and its affinity for soluble fibrinogen. Moreover, using a number of metabolic inhibitors, we outlined an alpha IIbbeta 3 activation pathway involving the G-protein Gi, PKC, and the actin cytoskeleton.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents and Materials-- The anti-FLAG monoclonal antibody (mAb) M1 and the fluorescent Ca2+ indicator fluo-3 AM were obtained from IBI-Kodak and Molecular Probes, respectively. Immulon 2 flat bottom microtiter plates were purchased from Dynatech Laboratories. PMA, fMLP, pertussis toxin (PTX), bovine serum albumin, ionomycin, Arg-Gly-Asp-Ser (RGDS), and cytochalasin D were purchased from Sigma. Human fibrinogen was obtained from Enzyme Research Labs. Bisindolylmaleimide I (BIM I), bisindolylmaleimide V (BIM V), and genistein were purchased from Calbiochem. Recombinant C3 exoenzyme was purchased from Upstate Biotechnology. Lipofectin reagent and Opti-MEM media were obtained from Life Technologies, Inc. G418 was purchased from Mediatech. Piceatannol and hygromycin were obtained from Boehringer Mannheim.

Coexpression of the Human fPR and alpha IIbbeta 3 in Human B Lymphocytes-- Because GM1500 cells express beta 3, introduction of a cDNA for alpha IIb results in the expression of alpha IIbbeta 3 on the cell surface (4). Therefore, to coexpress the fPR and alpha IIbbeta 3 in GM1500 cells, a cDNA for alpha IIb in the plasmid pREP4 containing a gene for resistance to the antibiotic hygromycin (3) and a FLAG octapeptide-tagged cDNA for the human fPR in the plasmid pRc/CMV containing a gene for resistance to the antibiotic neomycin (a gift of Drs. James J. Campbell and Eugene Butcher, Stanford University) (10) were sequentially introduced into 7.5 × 106 cells by electroporation (250 V and 960 microfarads). Stable co-transfectants were selected by growth in RPMI media containing 20% fetal calf serum and both G418 (750 µg/ml) and hygromycin (200 µg/ml). The simultaneous presence of alpha IIbbeta 3 and the fPR on the lymphocyte surface was confirmed by flow cytometry after staining the cells with either the alpha IIbbeta 3-specific mAb A2A9 (11) or the anti-FLAG mAb M1, followed by staining with fluorescein (FITC)-conjugated goat anti-murine IgG. Flow cytometry was performed using a FACScan flow cytometer (Becton-Dickinson) as described previously (12).

To ensure that the expressed fPR was functional, 5 × 107 cells were loaded with 5 µM fluo-3 AM at room temperature for 30 min. The cells were then incubated with either 30 nM ionomycin or 100 nM fMLP for 30 s and Ca2+ flux-induced fluorescence was measured with a FACScan flow cytometer as described previously (8).

Measurement of alpha IIbbeta 3 Function in Human B Lymphocytes-- The ability of alpha IIbbeta 3 expressed by lymphocytes to interact with fibrinogen was tested by measuring agonist-stimulated lymphocyte adherence to immobilized fibrinogen (3) and agonist-stimulated binding of soluble FITC-fibrinogen using flow cytometry as described previously (4).

To measure lymphocyte adherence to fibrinogen, the wells of microtiter plates were coated with 10 µg/ml purified human fibrinogen in 50 mM NaHCO3 buffer, pH 8.0, containing 150 mM NaCl. Unoccupied protein-binding sites on the wells were blocked with 5 mg/ml bovine serum albumin dissolved in the same buffer. 1.5 × 105 B lymphocytes, metabolically labeled overnight with [35S]methionine, were suspended in 100 µl of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl2, 0.1% glucose, and 1% bovine serum albumin, stimulated with either PMA or fMLP, and added to the protein-coated wells. Following an incubation for 30 min at 37 °C without agitation, the plates were vigorously washed four times with the suspension buffer and adherent cells were dissolved using 2% SDS. The SDS solutions were counted for 35S in a liquid scintillation counter.

To measure the binding of soluble FITC-fibrinogen to agonist-stimulated lymphocytes, purified human fibrinogen (13) was labeled with FITC using a CalbiochemTM-FITC Labeling Kit as described by the manufacturer. Fibrinogen labeled with FITC in this manner remained monomeric as assessed by gel-filtration chromatography, supported platelet aggregation as well as unlabeled fibrinogen, and was 95% clottable with thrombin (13). 1.5 × 105 B lymphocytes were then suspended in 100 µl of 10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl, 1 mM CaCl2, and 1% bovine serum albumin (suspension buffer) and incubated with 0.25 µM FITC-fibrinogen in the presence or absence of PMA or fMLP for 30 min at room temperature. The cells were washed once with suspension buffer and resuspended in a fixation solution consisting of 10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl and 0.37% formalin. Following a 10-min incubation on ice, the cells were again washed once with the suspension buffer, and analyzed by flow cytometry as described previously (4).

Effect of Botulinum C3 Exoenzyme on alpha IIbbeta 3 Function in GM1500 Cells-- Recombinant C3 exoenzyme from Clostridium botulinum was introduced in GM1500 cells using Lipofectin Reagent. 1 × 107 GM1500 cells were suspended in 1 ml of Opti-MEM media containing 40 µg of Lipofectin and 12 µg of C3 exoenzyme and incubated for 2 h at 37 °C. The cell suspension was then divided into two aliquots. One aliquot was resuspended in complete media for 1 h and the ability of these cells to adhere to fibrinogen was tested as described above. ADP-ribosylation of the small GTPase RhoA during the incubation was examined using the second aliquot as described previously (14). Briefly, the cells were washed with Opti-MEM, resuspended in 50 µl of 20 mM Tris-HCl buffer, pH 7.5, containing 0.25 M sucrose, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 2 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride, and homogenized by sonication. Following centrifugation of the homogenate at 1000 × g for 5 min, 15 µl of the supernatant were incubated with 50 ng of C3 exoenzyme and 10 µM [32P]NAD+ (Amersham) for 1 h at 30 °C in 100 mM Tris-HCl buffer, pH 8.0, containing 20 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, and 5 mM MgCl2 in a total reaction volume of 100 µl. Sufficient SDS and dithiothreitol were added to make their final concentrations 3% and 200 mM, respectively, and the solution was heated at 100 °C for 3 min. Following 0.1% SDS-10% polyacrylamide gel electrophoresis, ADP-ribosylated RhoA was visualized by autofluorography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Introduction of a Functional fPR into GM1500 Cells-- Previously, we demonstrated that the phorbol ester PMA induces the adherence of B lymphocytes expressing alpha IIbbeta 3 to immobilized fibrinogen (3). To determine whether adherence could also be induced by stimulating a receptor on the lymphocyte surface, we introduced plasmids containing a cDNA for the human fPR tagged at its 5' end with the FLAG epitope and a cDNA for alpha IIb into GM1500 B lymphocytes and selected for cells that stably expressed both proteins using the antibiotics G418 and hygromycin. As shown in Fig. 1A, the simultaneous presence of alpha IIbbeta 3 and the fPR on the lymphocyte surface was confirmed by flow cytometry after staining the cells with the alpha IIbbeta 3-specific mAb A2A9 (11) and the anti-FLAG mAb M1. The cells also stained with a FITC-labeled derivative of fMLP (data not shown).


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Fig. 1.   Co-expression of alpha IIbbeta 3 and the human fPR in GM1500 cells. cDNAs encoding a FLAG epitope-tagged human fPR and alpha IIb were introduced sequentially into GM1500 cells by electroporation. Stable co-transfectants were selected by growth in the presence of both G418 and hygromycin as described under "Experimental Procedures." The presence of the fPR and alpha IIbbeta 3 on the surface of the selected cells was confirmed by flow cytometry after staining the cells with the class-matched control antibody OKT3, the alpha IIbbeta 3-specific mAb A2A9, and the anti-FLAG mAb M1, followed by staining with FITC-conjugated goat anti-murine IgG (A). Function of the expressed fPR was confirmed by loading the cells with 5 µM fluo-3 AM at room temperature for 30 min. The cells were then incubated with either 30 nM ionomycin or 100 nM fMLP for 30 s and Ca2+ flux-induced fluorescence was measured with a flow cytometer (B). The effect of PTX on fMLP-stimulated Ca2+ flux was determined by preincubating the cells with 100 ng/ml PTX for 2 h.

Measurement of Ca2+ Flux in GM1500 Cells Expressing the fPR-- fMLP induces a Ca2+ flux in murine B lymphocytes expressing the human fPR (8). To verify that the fPR we expressed was functional, transfected GM1500 cells were loaded with the fluorescent Ca2+ indicator fluo-3 AM and exposed to either 30 nM ionomycin or 100 nM fMLP. The resulting change in fluo-3 fluorescence was then measured by flow cytometry. As shown in Fig. 1B, ionomycin and fMLP induced comparable changes in fluo-3 fluorescence, confirming that fPR stimulation could induce a flux of Ca2+ in the transfected cells (Fig. 1B). Moreover, the fMLP-stimulated Ca2+ flux was inhibited by preincubating the cells for 2 h with 100 ng/ml PTX, indicating that the fPR in GM1500 cells is coupled to a PTX-sensitive G protein.

Comparison of PMA and fMLP-stimulated alpha IIbbeta 3 Function-- Next, we compared the ability of fMLP and PMA to stimulate the adherence of lymphocytes expressing alpha IIbbeta 3 to immobilized fibrinogen. As shown in Fig. 2A, there was a concentration-dependent increase in lymphocyte adherence following exposure of the cells to fMLP. In multiple experiments, maximal adherence was observed at 300 nM fMLP, but was never more than one-half to two-thirds that induced by 200 ng/ml PMA. Moreover, no additive effect was seen when cells were stimulated simultaneously with fMLP and PMA, suggesting that these agonists were acting through the same signaling pathway.


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Fig. 2.   Comparison of PMA- and fMLP-stimulated lymphocyte adherence to immobilized fibrinogen. A, 1.5 × 105 B lymphocytes, metabolically labeled with [35S]methionine and co-expressing alpha IIbbeta 3 and the fPR, were added to the wells of microtiter plates coated with fibrinogen at 10 µg/ml and incubated for 30 min at 37 °C in the presence or absence of 200 ng/ml PMA, the indicated concentrations of fMLP, or both 200 ng/ml PMA and 1000 nM fMLP together. Lymphocyte adherence to the immobilized fibrinogen was quantitated as described under "Experimental Procedures." B, the effect of soluble fibrinogen on lymphocyte adherence to immobilized fibrinogen stimulated by 300 nM fMLP was measured by performing the assay described in A in the presence of increasing concentrations of soluble fibrinogen. The data in A and B are expressed relative to the adherence of PMA-stimulated cells in the absence of soluble fibrinogen; adherence of the PMA-stimulated cells is designated 1.0. The data are also expressed as the mean and S.E. of quadruplicate determinations.

The adherence of PMA-stimulated lymphocytes to immobilized fibrinogen is inhibited by soluble fibrinogen (4). As shown in Fig. 2B, the adherence of fMLP-stimulated cells also decreased as the concentration of soluble fibrinogen in the suspending buffer increased. Moreover, like cells stimulated by PMA (4), inhibition was maximal at a concentration of soluble fibrinogen of 40-50 µM.

Platelet stimulation induces a change in the affinity of alpha IIbbeta 3 for ligands and enables it to bind soluble fibrinogen (13). Previously, we found that PMA stimulation enabled transfected lymphocytes expressing alpha IIbbeta 3 to bind soluble fibrinogen (4). To determine if fPR stimulation would do likewise, we stimulated lymphocytes coexpressing alpha IIbbeta 3 and the fPR with either 200 ng/ml PMA or 300 nM fMLP and used flow cytometry to compare the binding of FITC-fibrinogen. As we had seen previously (4), the fluorescence histogram of PMA-stimulated lymphocytes was shifted substantially to the right of the histogram of unstimulated cells, indicating that fibrinogen was bound to the stimulated cells (Fig. 3A). The fluorescence histogram of fMLP-stimulated lymphocytes was also shifted to the right, although again fMLP was a less potent agonist than PMA (Fig. 3B). Preincubating both sets of stimulated cells with the mAb A2A9 prevented the shifts in fluorescence, confirming that the FITC-fibrinogen was bound to alpha IIbbeta 3. Moreover, like the binding of fibrinogen to stimulated platelets (13, 15), fibrinogen binding to PMA and fMLP-stimulated lymphocytes reached saturation at a fibrinogen concentration of approx 200 µg/ml and was abolished by either a 15-fold excess of unlabeled fibrinogen or 200 µM RGDS (data not shown).


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Fig. 3.   FITC-fibrinogen binding to PMA- and fMLP-stimulated lymphocytes. Purified human fibrinogen was labeled with fluorescein isothiocyanate as described under "Experimental Procedures." GM1500 cells expressing alpha IIbbeta 3 were suspended in a 10 mM sodium phosphate buffer, pH 7.4, containing 1 mM CaCl2 and incubated with 0.25 µM fluorescein-labeled fibrinogen in the absence or presence of 200 ng/ml PMA (A) or 300 nM fMLP (B) for 30 min at room temperature. The cells were then washed with suspension buffer, fixed in buffer contain 0.37% formalin, and examined by flow cytometry. The specificity of FITC-fibrinogen binding was determined by performing the incubation in the presence of the inhibitory mAb A2A9 at a concentration of 50 µg/ml.

Effect of Signaling Pathway Inhibitors on alpha IIbbeta 3 Function in Lymphocytes-- Because PTX inhibits fMLP-stimulated Ca2+ flux in GM1500 cells expressing the fPR, we asked whether PTX would inhibit fMLP-stimulated alpha IIbbeta 3 function in these cells. As expected, preincubating the cells with PTX had no effect on PMA-stimulated adherence to fibrinogen (Fig. 4). However, PTX reduced fMLP-stimulated adherence to nearly baseline levels. On the other hand, no inhibition was observed when the cells were stimulated with both fMLP and PMA, indicating that PMA was able to bypass the PTX effect. Identical results were seen when FITC-fibrinogen binding, rather than cell adherence, was used as the indicator of alpha IIbbeta 3 function (data not shown).


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Fig. 4.   Inhibition of PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen by PTX. To determine the effect of PTX on alpha IIbbeta 3-mediated lymphocyte adherence to fibrinogen, GM1500 cells coexpressing the fPR and alpha IIbbeta 3 were preincubated with either buffer or 100 ng/ml PTX for 2 h. Lymphocyte adherence to immobilized fibrinogen stimulated by 200 ng/ml PMA, 100 nM fMLP, or both agonists together was measured as described in the legend to Fig. 2 and under "Experimental Procedures." The data are expressed as the mean and S.E. of quadruplicate determinations.

The fMLP- and interleukin 8-stimulated adherence of murine B cells mediated by alpha 4beta 1 is unaffected by inhibiting PKC (16). To determine the effect of PKC inhibitors on fMLP-stimulated alpha IIbbeta 3 function in human B cells, we incubated transfected GM1500 cells overnight with nanomolar concentrations of either the high affinity PKC inhibitor BIM I (17) or the low affinity inhibitor BIM V (18) and measured agonist-stimulated lymphocyte adherence to fibrinogen. As expected, BIM I reduced PMA-stimulated adherence to baseline levels, whereas the same concentrations of BIM V had no effect (Fig. 5). However, to our surprise, BIM I, but not BIM V, also reduced fMLP-stimulated adherence to baseline levels. Identical results were seen when FITC-fibrinogen binding was measured instead of adherence (data not shown). Thus, these experiments suggest that fPR stimulation regulates alpha IIbbeta 3 function in human B cells via a signaling pathway that includes PKC.


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Fig. 5.   Inhibition of PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen by the PKC inhibitors BIM I and BIM V. The effect of BIM I (black-square) and BIM V () on alpha IIbbeta 3-mediated lymphocyte adherence to fibrinogen was determined by incubating GM1500 cells coexpressing the fPR and alpha IIbbeta 3 overnight with the indicated concentrations of the inhibitors. Lymphocyte adherence to immobilized fibrinogen was stimulated by either 200 ng/ml PMA or 100 nM fMLP and measured as described in the legend to Fig. 2 and under "Experimental Procedures." The data are expressed as the mean and S.E. of quadruplicate determinations.

Stimulation of integrin function is associated with the activation of a number of protein tyrosine kinases (19). To determine whether tyrosine phosphorylation regulates alpha IIbbeta 3 function in lymphocytes, we incubated transfected GM1500 cells overnight with micromolar concentrations of the tyrosine kinase inhibitor genistein (20) and measured agonist-stimulated lymphocyte adherence to fibrinogen. As shown in Fig. 6, genistein concentrations as high as 150 µM had no effect on either PMA- or fMLP-stimulated lymphocyte adherence. A small degree of inhibition (29 and 16%, respectively) was observed at a genistein concentration of 300 µM, a concentration at which genistein also affects the activity of serine/threonine kinases (20). Activation of the non-receptor tyrosine kinase Syk is an early event after platelet stimulation by agonists such as collagen and thrombin (21, 22), although in Epstein-Barr virus-transformed lymphocytes, Syk activity is constitutively inhibited by the Epstein-Barr virus-encoded protein LMP2 (23). Nevertheless, to determine whether residual Syk activity in GM1500 cells could regulate alpha IIbbeta 3 function, we incubated transfected cells with the Syk inhibitor piceatannol (24) and measured both PMA and fMLP-stimulated lymphocyte adherence to fibrinogen. As shown in Fig. 6, 30 µg/ml piceatannol, a concentration that completely inhibited collagen-induced platelet aggregation, had no effect on alpha IIbbeta 3 function in cells stimulated by either PMA or fMLP.


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Fig. 6.   Effect of protein tyrosine kinase inhibitors on PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen. GM1500 cells coexpressing the fPR and alpha IIbbeta 3 were incubated overnight with either the protein tyrosine kinase inhibitor genistein at 150 µM or the Syk inhibitor piceatannol at 30 µg/ml. Lymphocyte adherence to immobilized fibrinogen was stimulated by either 200 ng/ml PMA (black-square) or 100 nM fMLP () and measured as described in the legend to Fig. 2 and under "Experimental Procedures." The data are expressed as the mean and S.E. of quadruplicate determinations. square , no agonist.

Role of the Actin Cytoskeleton in Regulating alpha IIbbeta 3 Function in Lymphocytes-- Inhibitors of actin polymerization impair beta 1 and beta 2 integrin function in leukocytes, suggesting that the actin cytoskeleton regulates integrin function in these cells (14, 16, 25). Whether the actin cytoskeleton regulates alpha IIbbeta 3 function in platelets is less certain, although it was recently reported that the actin polymerization inhibitor cytochalasin E inhibits thrombin-stimulated alpha IIbbeta 3 function (26). To determine if the actin cytoskeleton regulates alpha IIbbeta 3 function in GM1500 cells, we measured the effect of cytochalasin D on unstimulated and PMA-stimulated lymphocyte adherence to fibrinogen. We found that increasing concentrations of cytochalasin D inhibited PMA-stimulated adherence, with few adherent cells remaining at a cytochalasin D concentration of 10 µM (Fig. 7). By contrast, submicromolar concentrations unexpectedly, but consistently, increased unstimulated adherence, such that adherence in the presence 0.01 µM cytochalasin D was 2-3-fold greater than in its absence. We also examined the effect of cytochalasin D on FITC-fibrinogen binding to both PMA-stimulated and unstimulated GM1500 cells. As shown in Fig. 8A, 0.1 µM cytochalasin D, but not 0.01 µM cytochalasin D, completely inhibited FITC-fibrinogen binding to PMA-stimulated cells. Conversely, whereas cytochalasin D concentrations of 0.1 µM or greater did not influence the interaction of FITC-fibrinogen with unstimulated lymphocytes, 0.01 µM cytochalasin D consistently induced FITC-fibrinogen binding to these cells, albeit to a limited degree (Fig. 8B).


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Fig. 7.   Effect of cytochalasin D on PMA-stimulated and unstimulated lymphocyte adherence to fibrinogen. The adherence assay described in the legend to Fig. 2 and under "Experimental Procedures" was performed in the presence of the indicated concentration of cytochalasin D using either unstimulated GM1500 cells expressing alpha IIbbeta 3 or the same cells stimulated with 200 ng/ml PMA. The data shown are the mean and S.E. of three separate experiments. PMA-stimulated adherence in the absence of cytochalasin D was designated 100% adherence. , no PMA; black-square, PMA.


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Fig. 8.   Effect of cytochalasin D on FITC-fibrinogen binding to PMA-stimulated and unstimulated lymphocytes. The effect of the indicated concentrations of cytochalasin D (CD) on the binding of FITC-fibrinogen to either PMA-stimulated (A) or unstimulated (B) GM1500 cells expressing alpha IIbbeta 3 was determined using flow cytometry as described in the legend to Fig. 3 and under "Experimental Procedures." The dashed line in B corresponds to the peak fluorescence intensity of unstimulated cells incubated with 10 µM cytochalasin D. The data shown are representative of three separate experiments.

The Rho family of small GTPases regulates a number of cellular functions such as shape, motility, and adhesion by reorganizing the actin cytoskeleton (27). In lymphocytes, inhibiting RhoA with C3 exoenzyme from C. botulinum impairs agonist-stimulated cell adhesion mediated by alpha Lbeta 2 (14) and alpha 4beta 1 (16). C3 exoenzyme has also been reported to inhibit thrombin-induced platelet aggregation (28). To determine whether RhoA plays a role in the regulation of alpha IIbbeta 3 function in GM1500 cells, we introduced C3 exoenzyme into the cells using Lipofectin and measured its effect on PMA and fMLP-stimulated adherence to fibrinogen. To verify that C3 exoenzyme had ADP-ribosylated RhoA in the Lipofectin-treated cells, an aliquot of these cells was homogenized and re-exposed to the enzyme in the presence of [32P]NAD+. As shown in Fig. 9A, there was a substantial reduction in the incorporation of 32P into RhoA from cells that had been treated with Lipofectin in the presence of C3 exoenzyme compared with cells that had been treated with Lipofectin in its absence. This indicates that RhoA in the former cells had been ADP-ribosylated during the first incubation with C3 exoenzyme, rendering it resistant to ADP-ribosylation during the second. Nevertheless, as shown in Fig. 9B, inhibiting RhoA with C3 exoenzyme had essentially no effect on the ability of the cells to adherence to fibrinogen following stimulation with either low or high concentrations of PMA and fMLP.


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Fig. 9.   Effect of C3 exoenzyme on PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen. 12 µg of C3 exoenzyme was introduced into 1 × 107 GM1500 cells coexpressing the fPR and alpha IIbbeta 3 using Lipofectin as described under "Experimental Procedures." A, inhibition of RhoA was determined by incubating homogenates of cells incubated with Lipofectin in the absence (lane 1) or presence of C3 exoenzyme (lane 2) with 50 ng of C3 exoenzyme and 10 µM [32P]NAD+ (Amersham) for 1 h at 30 °C. 32P-Labeled RhoA was then visualized by 0.1% SDS-10% polyacrylamide gel electrophoresis and autofluorography. B, the effect of RhoA inhibition on PMA- and fMLP-stimulated lymphocyte adherence to fibrinogen was measured using the adherence assay described in the legend to Fig. 2 and under "Experimental Procedures." Control cells were incubated with Lipofectin in the absence of C3 exoenzyme and C3 cells were incubated with Lipofectin in the presence of 12 µg of C3 exoenzyme. The data are expressed as the mean and S.E. of quadruplicate determinations.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To delineate signaling pathways that can convert alpha IIbbeta 3 from an inactive to a ligand binding conformation, we have expressed recombinant alpha IIbbeta 3 in human B lymphocytes. Initially, we found that the phorbol ester PMA not only induced the adherence of these cells to fibrinogen, but enabled alpha IIbbeta 3 to bind soluble fibrinogen (4). In the current work, we asked whether an extracellular agonist acting through its cognate membrane receptor could induce alpha IIbbeta 3 function in these cells. Stimulation of G protein-coupled receptors in platelets results in alpha IIbbeta 3 activation (7). Similarly, stimulation of these receptors leads to integrin activation in lymphocytes and other cells of the hematopoietic lineage (9). We found that by coexpressing alpha IIbbeta 3 with the human formyl peptide receptor, a seven-transmembrane domain G protein-coupled receptor (9), we could induce B cell adherence to immobilized fibrinogen and soluble fibrinogen binding to alpha IIbbeta 3 using the chemoattractant peptide fMLP. Moreover, we found that the signaling pathway initiated by fPR stimulation involved PKC and similar to signaling initiated by PMA, resulted in a change in the actin cytoskeleton.

The human fPR is normally expressed in phagocytic cells where it transduces signals by activating the PTX-sensitive G proteins Galpha i2 or Galpha i3 (9). We found that PTX inhibited the fMLP-stimulated interaction of co-transfected GM1500 cells with both immobilized and soluble fibrinogen, implying that Galpha i activation can regulate alpha IIbbeta 3 function in these cells. Whether Galpha i can also regulate alpha IIbbeta 3 function in platelets is uncertain. However, PTX inhibits thrombin-stimulated phosphoinositide hydrolysis in saponin-permeabilized human platelets, suggesting that at least the thrombin receptor in human platelets can couple to Galpha i (29). On the other hand, platelets from Galpha q-deficient mice fail to aggregate in response to thrombin, ADP, collagen, arachidonic acid, and U46619, despite normal levels of Galpha i, indicating that PTX-insensitive G protein Galpha q couples agonist receptors to alpha IIbbeta 3 in murine platelets (30). Nonetheless, it is possible that the difference between human lymphocytes and murine platelets may simply reflect a difference in the types of signaling pathways that are present in human and murine cells, similar to a difference in the types of thrombin receptors expressed by human and murine platelets (31, 32).

The ability of inhibitors of PKC (33) to inhibit platelet aggregation and/or ligand binding to alpha IIbbeta 3 implies that protein phosphorylation by PKC regulates alpha IIbbeta 3 activity. Moreover, the PKC activator PMA is a potent stimulus for ligand binding to alpha IIbbeta 3 on both human platelets (6) and Galpha q-deficient murine platelets (30). The identity of the proteins phosphorylated by PKC in platelets to regulate alpha IIbbeta 3 function is unknown. The beta 3 cytoplasmic tail has been found to contain phosphorylated threonine residues after platelet stimulation by thrombin, PMA, or the thromboxane analogue U46619 (34). However, the fraction of beta 3 containing phosphorylated threonine in both resting and stimulated platelets was low and unlikely to affect the function of more than a few alpha IIbbeta 3 heterodimers. We found that the fMLP-stimulated interaction of alpha IIbbeta 3 with either immobilized or soluble fibrinogen in B cells was prevented by the specific PKC inhibitor BIM I. Thus, activation of PKC, either directly with PMA or via fPR activation of Galpha i, is sufficient to induce alpha IIbbeta 3 function in lymphocytes. On the other hand, Laudanna et al. (16) observed that inhibiting PKC had no effect on fMLP-stimulated alpha 4beta 1 function in murine B cells (16). Again, it is possible that this difference simply reflects differences between human and murine cells. It is also possible that at least two signaling pathways can be initiated by fPR stimulation and that these pathways can differentiate between beta 1 and beta 3 integrins. In support of this possibility, Weber et al. (35) found that signaling pathways arising from the receptors for the chemoattractants RANTES, MCP-3, and C5a in eosinophils can differentially regulate the function of alpha 4beta 1 and alpha Lbeta 2. Whether PKC-independent pathways also exist in B cells that can regulate alpha IIbbeta 3 function remains to be determined.

Phosphorylation of tyrosines 747 and 759 in the beta 3 cytoplasmic tail has also been detected after thrombin-stimulated platelet aggregation (36). However, this phosphorylation was not observed after thrombin stimulation in the absence of aggregation, suggesting that it is a consequence of ligand binding to alpha IIbbeta 3 ("outside-in" signaling), rather than being part of the process of alpha IIbbeta 3 activation. Blystone et al. (37) also detected phosphorylation of beta 3 Tyr-747 after monocytes were exposed to Mn2+ or platelets were treated with either Mn2+ or thrombin. When they expressed alpha vbeta 3 heterologously in K562 cells, they found Tyr-747 phosphorylation necessary, but not sufficient, to support either PMA and thrombin-stimulated cell adhesion, and like Tyr-747 phosphorylation in platelets (36), required ligand binding to alpha vbeta 3. We found that the protein tyrosine kinase inhibitor genistein had no effect on the ability of alpha IIbbeta 3 in B lymphocytes to interact with fibrinogen. Similarly, we found that piceatannol, an inhibitor reportedly specific for the tyrosine kinase Syk found in lymphocytes and platelets (38), had no effect on either PMA- or fMLP-stimulated alpha IIbbeta 3 function in lymphocytes. Thus, our data, combined with the inability to detect significant amounts of phosphorylated serine or threonine on the beta 3 of stimulated platelets (34), suggest that phosphorylation of alpha IIbbeta 3 is not required to regulate its interaction with ligands.

The biochemical events that follow PKC activation in lymphocytes and platelets are uncertain. However, one consequence of PKC-mediated signaling is regulation of membrane-cytoskeletal interactions (39). For example, Kucik and co-workers (40) found that exposing human B lymphocytes to PMA increased the diffusion of alpha Lbeta 2 in the plane of the lymphocyte membrane and augmented lymphocyte adherence to ICAM-1. Similar effects were observed following exposure of the lymphocytes to low concentrations of cytochalasin D. These data suggest that cytoskeletal constraints, released by either PKC activation or cytochalasin D, maintain alpha Lbeta 2 in a low avidity state. Lub et al. (41) extended these observations by showing that maximum alpha Lbeta 2-mediated lymphocyte adherence required both alpha Lbeta 2 clustering and an increase in its affinity for ICAM-1. We found that micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of lymphocytes expressing alpha IIbbeta 3 with either immobilized or soluble fibrinogen. Conversely, we found that nanomolar concentrations of cytochalasin D actually induced fibrinogen binding to alpha IIbbeta 3 on unstimulated cells. Thus, our observations suggest that both PKC and the actin cytoskeleton play a role in regulating both the avidity and affinity of alpha IIbbeta 3 for ligands. How this might occur is uncertain. Platelet stimulation results in a conformational change in alpha IIbbeta 3 that increases its affinity for ligands (42, 43). It is also associated with the disassembly of polymerized actin, followed by actin reassembly and a change in platelet morphology (44). It is conceivable that low concentrations of cytochalasin D can initiate actin disassembly, resulting in increases in both integrin mobility and affinity. Fox et al. (44) found that a variable amount of the alpha IIbbeta 3 in detergent lysates of unstimulated platelets was recovered with fragments of the membrane skeleton and was redistributed to a detergent-insoluble fraction containing a network of cytoplasmic actin filaments after ligand binding. They also observed that high concentrations of cytochalasin E inhibited the binding of the activation-dependent mAb PAC1 to alpha IIbbeta 3 on ADP and thrombin-stimulated platelets (26). Thus, it is possible that the membrane skeleton in platelets, or in our case in lymphocytes, interacts with the cytoplasmic tails of alpha IIbbeta 3 to constrain the integrin in a low affinity configuration. Relief of this constraint by agonists (or cytochalasins) could then be responsible for an augmented interaction of alpha IIbbeta 3 with immobilized fibrinogen and for its ability to bind soluble ligands.

Platelet stimulation is also associated with the formation of clusters of ligand-occupied alpha IIbbeta 3 on the platelet surface (26). However, ligand valency does not appear to be a factor in the ability of alpha IIbbeta 3 to recognize soluble ligands (45). Moreover, alpha IIbbeta 3 is a univalent receptor (11) and electron microscopy of fibrinogen bound to alpha IIbbeta 3 suggests that a fibrinogen molecule can only bind to one alpha IIbbeta 3 heterodimer on the surface of a given platelet (46). Hence, it is unlikely that clustering of alpha IIbbeta 3 alone can account for its ability to bind soluble fibrinogen. Nevertheless, it is possible, and even likely, that agonist-induced clustering of alpha IIbbeta 3 contributes to the augmented adherence of stimulated lymphocytes and platelets to immobilized fibrinogen.

Activity of members of the Ras family of small GTPases has major effects on cytoskeletal organization. For example, microinjection of activated forms of the small GTPases cdc42, Rac, and Rho into Swiss 3T3 fibroblasts results in the formation of filopodia, lamellopodia, and stress fibers, respectively (27). Similar effects have been reported in macrophages (47). Moreover, the small GTPase RhoA appears to play an important role in regulating integrin function in leukocytes, and perhaps in platelets as well. For example, inhibiting RhoA with C3 exoenzyme prevented PMA-stimulated alpha Lbeta 2-mediated homotypic lymphocyte aggregation (14) and the fMLP-stimulated alpha 4beta 1-mediated adherence of murine lymphocytes to VCAM-1 (16). Morii and co-workers (28) have reported that C3 exoenzyme completely inhibits thrombin and PMA-stimulated human platelet aggregation, although they found little correlation between the extent of RhoA inhibition by the C3 exoenzyme and the extent to which platelet function was impaired. On the other hand, in preliminary studies, Leng et al. (48) did not observe the reported effect of C3 exoenzyme on platelet aggregation. We found that C3 exoenzyme had no effect on either PMA and fMLP-stimulated lymphocyte adherence to fibrinogen, despite substantial ADP-ribosylation of RhoA. Thus, RhoA is unlikely to transduce the signals that regulate alpha IIbbeta 3 function, at least in our system. A more likely candidate is Rac1. Microinjection of activated Rac1 into Swiss 3T3 cells induces the submembranous accumulation of actin and the formation of integrin-containing focal complexes at the cell margin (49). Furthermore, activation of Rac1 by the exchange factor Tiam1 in T-lymphoma cells induces the formation of submembranous actin filaments, membrane ruffling, and an invasive phenotype (50). Thus, it is conceivable that a Rac-mediated reorganization of the membrane skeleton in lymphocytes, and by extrapolation in platelets, could be an intermediary step in the regulation of alpha IIbbeta 3 function by agonists.

In summary, we have shown that the ability of integrin alpha IIbbeta 3, expressed in B lymphocytes, to interact with immobilized and soluble fibrinogen can be stimulated by the G protein-coupled formyl peptide receptor. Moreover, our studies begin to define a signaling pathway that includes a PTX-inhibitable G-protein, PKC, and the actin cytoskeleton.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL40387 and HL51258.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.

Dagger To whom correspondence should be addressed: Hematology-Oncology Div., 1005 Stellar-Chance Laboratories, 422 Curie Blvd., Philadelphia, PA 19014. Tel.: 215-662-4028; Fax: 215-662-7617.

1 The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; fPR, formyl peptide receptor; fMLP, formyl Met-Leu-Phe; mAb, monoclonal antibody; PTX, pertussis toxin; RGDS, Arg-Gly-Asp-Ser; BIM I, bisindolylmaleimide I; BIM V, bisindolylmaleimide V; FITC, fluorescein isothiocyanate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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