©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Signaling Functions of L-selectin
ENHANCEMENT OF TYROSINE PHOSPHORYLATION AND ACTIVATION OF MAP KINASE (*)

Thomas K. Waddell (1)(§), Lea Fialkow (1)(¶), Chi Kin Chan (1), Takashi Kei Kishimoto (2), Gregory P. Downey (1)(**)

From the (1)Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada and the (2)Department of Immunology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band co-migrated with mitogen- activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab`) fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion.


INTRODUCTION

Neutrophils constitute the first line of defense against invading micro-organisms. Paradoxically their uncontrolled activation also contributes to a variety of diseases, such as ischemia-reperfusion injury, shock, and the adult respiratory distress syndrome(1) , through the unregulated release of large amounts of reactive oxygen intermediates, proteolytic enzymes, and inflammatory cytokines. An early step in their recruitment into sites of inflammation entails the interaction of L-selectin on the neutrophil and P-selectin on the endothelial cell with their respective carbohydrate ligands. This results in rolling of the neutrophil along the endothelial surface under conditions of flow(2) . The next phase is firm adhesion, which requires stimulation of the leukocyte, activation of its integrins, and their association with endothelial cell adhesion molecules including ICAM-1(3) . Transmigration follows, involving additional adhesion molecules, such as platelet-endothelium cell adhesion molecule(4) . Although the function of L-selectin in ligand binding and rolling has been extensively studied, its potential role in neutrophil activation events has only recently been described(5, 6, 7) .

L-selectin is the smallest member of the selectin family of carbohydrate-specific adhesion molecules(8) . Its extracellular region consists of a lectin domain, an epidermal growth factor-like domain, and two repeats of a complement regulatory protein domain. Its intracellular domain is small (17 amino acids), and the predicted primary structure reveals no known catalytic region or binding site consensus sequences. The natural ligands for neutrophil L-selectin have not yet been determined, although the lymphocyte-specific ligands, GlyCAM-1 and CD34, have recently been identified(9, 10) . The ability to bind L-selectin depends both on the protein backbone as well as on post-translational modifications of these ligands(11) . On both lymphocytes and neutrophils, the surface expression of L-selectin is down-regulated after activation by proteolytic cleavage(12) .

Recent studies by ourselves and others have revealed a signaling role for neutrophil L-selectin in the generation of cytosolic calcium transients, potentiation or activation of the oxidative burst(5, 7) , and enhanced tumor necrosis factor and interleukin-8 gene expression (6). The signaling pathways utilized by L-selectin have not yet been identified. Phosphorylation of proteins on tyrosine residues is an extremely important regulatory pathway in a wide variety of cell types (13). Tyrosine phosphorylation in neutrophils occurs rapidly following stimulation with a variety of agonists(14) , and tyrosine kinase inhibitors can block many neutrophil functional responses such as chemotaxis and the oxidative burst(15) . Because of the purported importance of these pathways and evidence that other adhesion molecules, such as integrins, transmit intracellular signals by pathways involving tyrosine phosphorylation(16) , we examined whether L-selectin might transmit intracellular signals by pathways involving tyrosine phosphorylation in neutrophils.


EXPERIMENTAL PROCEDURES

Reagents

Percoll and dextran T500 were obtained from Pharmacia Biotech Inc. (Baie D'Urfe, Quebec, Canada). KRPD()buffer was prepared using reagents from Mallinckrodt (Paris, KY). Gelatin was from Bio-Rad (Hercules, CA). Nonidet P-40 and phenylmethylsulfonyl fluoride were obtained from Boehringer Mannheim (Laval, Quebec, Canada). Leupeptin, pepstatin A, and aprotinin were obtained from ICN Biochemicals (Mississauga, Ontario, Canada). Ethanolamine, 2-mercaptoethanol, Trizma (Tris base), HEPES-buffered RPMI 1640, cytochalasin B and D, fMLP, herbimycin A, bovine serum albumin, superoxide dismutase, cytochrome c, PMA, sulfatides, and Type I and Type II cerebrosides were purchased from Sigma. EDTA was obtained from BDH (Toronto, Ontario, Canada). DFP was from Calbiochem (La Jolla, CA). Genistein was from Upstate Biotechnology, Inc. (Lake Placid, NY). Paraformaldehyde was from J. B. E. M. Services (Pointe Claire-Dorval, Quebec, Canada).

Antibodies

The murine anti-human L-selectin antibodies, DREG 200, DREG 56 (whole molecule and F(ab`) fragment), and DREG 55 (all IgG) were prepared as described previously(17) . DREG 200 was proteolytically cleaved using immobilized papain (Calbiochem) to generate monovalent Fab fragments, which were purified by protein A affinity chromatography (Bio-Rad Affi-Gel MAPS II System). Control mAb included: IB4, an IgG mouse anti-human CD18 provided by Dr. D. Chambers (San Diego Regional Cancer Center); 4B2, an IgG anti-CD45 provided by R. Sutherland (The Toronto Hospital); B-E3, an IgG anti-CD10 (Serotec Canada, Toronto, Ontario, Canada); and an IgG anti-human PKC (Upstate Biotechnology, Inc.). Secondary, cross-linking goat anti-mouse IgG antibodies were affinity-purified cross-adsorbed F(ab`) fragments from Cappel Research Products (Organon Teknika, Durham, NC). Anti-phosphotyrosine antibodies were from two sources: (i) affinity-purified polyclonal anti-phosphotyrosine from Transduction Laboratories (Lexington, KY) and (ii) affinity-purified monoclonal anti-phosphotyrosine (4G10 hybridoma) from Upstate Biotechnology, Inc. A monoclonal anti-MAP kinase antibody (raised against a peptide corresponding to amino acids 325-345 of p44) from Zymed (San Francisco, CA) was used for immunoblotting. Polyclonal anti-MAP kinase antibody, anti-ERK-2 (SC-154), obtained from Santa Cruz Biotechnology (Santa Cruz, CA) was used for immunoprecipitation. Blocking antibodies against the neutrophil Fc receptor were obtained from Medarex (Annandale, NJ). Fab fragments of the monoclonal IV.3, a mouse IgG2b against human FcRII, and F(ab`) fragments of 3G8, a mouse IgG1 against FcRIII, were used. ST-AR 9, which is a F(ab`) fragment of rabbit anti-mouse labeled with FITC, was used to stain unconjugated primary antibodies (Serotec Canada).

Cell Isolation

Human neutrophils (>98% pure) were isolated from citrated whole blood obtained by venipuncture, using dextran sedimentation and discontinuous plasma-Percoll gradient techniques as described previously(18) . The separation procedure required 2 h, and the cells were used immediately after isolation at a cell density of 8 10/ml. The functional integrity and nonactivated state of neutrophils isolated in this manner have been extensively validated in previous publications(18) .

Flow Cytometry

Surface expression of L-selectin, CD18, FcRII, and HLA class I was measured using indirect immunofluorescence and flow cytometry. Unless otherwise specified, cells were labeled with primary mAb for 30 min on ice, washed, and resuspended in PBS containing 0.5% bovine serum albumin and 20 mM glucose with FITC-labeled secondary antibody (1:500). After a 30-min incubation on ice, cells were washed and resuspended in PBS with 1.5% paraformaldehyde. Stained cells were analyzed on a FACScan (Becton-Dickinson, Palo Alto, CA) using FL1 detector (488 nm excitation and 530 nm emission wavelengths). Cells were gated on the forward and right angle light scatter to exclude debris and cell clumps. Typically, 10,000 cells were analyzed per condition.

SDS-PAGE and Immunoblotting

Aliquots of cells were either stimulated with sulfatides or control lipids in KRPD at 37 °C or resuspended in HEPES-buffered RPMI 1640 and incubated with anti-L-selectin or control mAb for 20 min at 37 °C. The latter cells were washed and resuspended in KRPD (which contained, in mM, NaCl 122, NaHPO 3.1, NaHPO 12.5, KCl 4.8, MgSO 1.2, CaCl 1, glucose 11). After stimulation, the cells were rapidly sedimented, resuspended in boiling 2% SDS sample buffer, and boiled for 15 min. Thirty µl of cell lysate (the equivalent of 8 10 cells) of each sample and molecular weight standards were then subjected to SDS-polyacrylamide gel electrophoresis through a 4-20% gradient polyacrylamide gel (Novex Experimental Technology, San Diego, CA). Following electrophoresis, the proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH) and blocked overnight at 4 °C in blocking buffer (0.25% gelatin, 10% ethanolamine, in 0.1 M Tris, pH 9.0). The membrane was then incubated in antibody buffer (0.25% gelatin and 0.05% Nonidet P-40 in 0.15 M NaCl, 5 mM EDTA, and 0.05 M Tris, pH 7.5) containing 0.2 µg/ml polyclonal anti-phosphotyrosine Ab (Transduction Laboratories) for 2 h at room temperature. Next, the membrane was washed three times in antibody buffer, incubated with a 1:5000 dilution of peroxidase-conjugated polyclonal anti-rabbit secondary Ab (Amersham, Oakville, Ontario, Canada) for 1 h at room temperature, and washed three additional times with antibody buffer alone. The membrane was then visualized using ECL (Amersham Corp.) according to the manufacturer's instructions. Similar results were obtained using the 4G10 monoclonal anti-phosphotyrosine Ab (Upstate Biotechnology, Inc.) and peroxidase-conjugated polyclonal anti-mouse Ab (Amersham) (data not shown). To strip the membrane for reprobing with anti-MAP kinase, the membrane was washed in water and then incubated in stripping buffer (62.5 mM Tris, pH 6.8, 2 mM EDTA, and 100 mM 2-mercaptoethanol) for 30 min at 50 °C. The membrane was again blocked overnight and blotted as described previously, except that the primary antibody used was a 1:5000 dilution of anti-MAP kinase (Zymed, San Francisco, CA) which is reactive against both the p42 and p44 isoforms of MAP kinase and the secondary Ab was peroxidase-conjugated anti-mouse (Amersham).

MAP Kinase Immunoprecipitation and Gel Renaturation Kinase Assay

Cells were pretreated with 2.5 mM DFP for 30 min at room temperature, washed, and resuspended at a concentration of 2 10 cells/ml in HEPES-buffered RPMI. After stimulation, the cells were sedimented and resuspended in boiling lysis buffer (0.15 M NaCl, 5 mM EGTA, 10 mM sodium pyrophosphate, 10 mM NaF, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µM pepstatin A, 10% glycerol, 25 mM Tris, pH 7.5) containing 1% SDS and boiled for 15 min. The lysate was diluted with 9 parts lysis buffer without SDS but containing 1% Triton X-100, 0.5% Nonidet P-40, and 1% sodium deoxycholate. The lysate was sonicated for 15 min and then centrifuged in a microcentrifuge for 15 min at 4 °C. The lysate was precleared by incubation with 50 µl prewashed protein A/G-agarose beads (Santa Cruz catalog number SC-2003) for 1 h at 4 °C. The supernatant was transferred to a fresh tube, and 50 µl of polyclonal anti-MAP kinase Ab (Santa Cruz) was added for 2 h at 4 °C. This mixture was then added to 50 µl of prewashed protein A/G beads and incubated overnight at 4 °C. The immune complexes were washed twice with washing buffer (25 mM Tris, pH 7.4, 2 mM EDTA, 150 mM NaCl) and then boiled for 5 min in Laemmli sample buffer. The beads were sedimented and the supernatants analyzed by SDS-PAGE as described previously except that the equivalent of 2 10 cells were loaded per lane. For immunoblotting, the monoclonal anti-phosphotyrosine (4G10) from Upstate Biotechnology, Inc. was used because of diminished nonspecific binding compared with the polyclonal Ab.

MAP kinase activity was determined using a gel renaturation assay, using myelin basic protein as the substrate(19) . Polyacrylamide (10%) gels were cast, incorporating 0.54 mg/ml myelin basic protein within the gel. After electrophoresis of the immunoprecipitate (the equivalent of 2 10 cells/lane) or whole cell lysate (the equivalent of 5 10 cells/lane), the SDS was removed by washing the gel twice in 50 mM Tris, pH 8.0, 20% isopropyl alcohol for 30 min each time. This was followed by a 1-h incubation in 50 mM Tris, pH 8.0, 5 mM 2-mercaptoethanol and then two 30-min incubations in 6 M guanidine HCl, 50 mM Tris, pH 8.0, 5 mM 2-mercaptoethanol. The gel was renatured by incubation in five changes of buffer (50 mM Tris, pH 8.0, 5 mM 2-mercaptoethanol, and 0.04% Tween 40) over 12-18 h at 4 °C. The kinase reaction was carried out by incubation of the gel in kinase buffer (25 mM HEPES, pH 8.0, 2 mM dithiothreitol, 0.1 mM EGTA, 5 mM MgCl, 25 µM ATP, and 25 µCi [-P] ATP) for 1 h at 21 °C. The gel was then washed repeatedly with 5% trichloroacetic acid containing 1% sodium pyrophosphate until the washes contained minimal radioactivity. The gel was then dried and exposed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) followed by conventional autoradiography using KODAK X-Omat film.

Measurement of Cytosolic Calcium Concentration

Neutrophils were loaded by incubation for 20 min at 37 °C in HEPES-buffered RPMI containing 2 µM Fura-2 AM. Cells were washed and resuspended in Ca measuring buffer (in mM, NaCl 140, KCl 5, HEPES 10, glucose 10, MgCl 1, pH 7.4, and CaCl 1). Fura-2 fluorescence was measured in a Hitachi F2000 fluorometer using an excitation wavelength of 335 nm and an emission wavelength of 495 nm. The fluorescence was calibrated using the ionomycin/Mn technique(20) .

Measurement of Superoxide Production

Superoxide production by neutrophils was determined as described(5) . Neutrophils were treated with the indicated stimuli at 37 °C in KRPD containing cytochrome c (75 µM) for 10 min. Reference tubes were treated identically except superoxide dismutase (30 units/ml) was added before stimulation. The absorbance at 550 nm was used to calculate the superoxide dismutase-inhibitable reduction of cytochrome c.


RESULTS

To determine whether tyrosine phosphorylation in human neutrophils could be modulated by L-selectin, cells were incubated with primary anti-L-selectin (DREG 55, 56, or 200) or control mAb. This treatment resulted in an increase in tyrosine phosphorylation of several cellular proteins, including prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa ( Fig. 1and Fig. 3A, lane 1). This response was not seen when cells were treated with a variety of mAb which bind to the neutrophil surface in amounts approximately equal to the anti-L-selectin mAbs, as confirmed by flow cytometry (data not shown). To ensure that the enhanced tyrosine phosphorylation observed after ligation of L-selectin by Ab was not an artifact of the antibodies used, we studied the effects of sulfatides, sulfated glycolipids which are potential ligands of L-selectin. Sulfatides clearly can function as a ligand for the L-selectin molecule in vitro(21) and have been shown to induce Ca transients and increased tumor necrosis factor and interleukin-8 mRNA in intact human neutrophils(6) . Fig. 1B illustrates that exposure of neutrophils to sulfatides resulted in a dose-dependent increase in total cellular tyrosine phosphorylation. This effect was not seen after treatment with Type I or Type II cerebrosides which have the same glycolipid structure but lack the sulfate group and therefore do not bind L-selectin(21) . Tyrosine phosphorylation was markedly diminished (Fig. 2A) if neutrophils were pretreated with chymotrypsin, which cleaves L-selectin (6, 22) but not other neutrophil surface antigens from the cell surface (Fig. 2B), prior to sulfatides treatment. Although the magnitude of the response to sulfatides was greater than that due to mAb-mediated L-selectin ligation, the phosphorylation patterns and the kinetics of the response after mAb-mediated ligation of L-selectin or stimulation with sulfatides were indistinguishable. In both cases the response was rapid, occurring within 30 s, and persisted for at least 20 min (data not shown).


Figure 1: Ligation of L-selectin increases tyrosine phosphorylation of several cellular proteins. A, human neutrophils (8 10/ml) were incubated with or without mAb at 30 µg/ml for 20 min at 37 °C, washed, and resuspended. After 2 min the cells were sedimented and resuspended in boiling sample buffer and analyzed by SDS-PAGE and anti-phosphotyrosine immunoblotting as described under ``Experimental Procedures.'' Increased tyrosine phosphorylation is seen after treatment with the anti-L-selectin mAbs, DREG 56 and DREG 200, but not after treatment with IB4 (anti-CD18), 4B2 (anti-CD45), or B-E3 (anti-CD10). B, sulfatides increase tyrosine phosphorylation in human neutrophils. Neutrophils were incubated alone or with 400 µg/ml of either Type I or Type II cerebrosides (nonsulfated glycolipids) or with increasing amounts of sulfatides (sulfated glycolipids). Sulfatides caused a dose-dependent increase in tyrosine phosphorylation, not seen even after treatment with 400 µg/ml of the control lipids.




Figure 3: The increased response after L-selectin ligation is specific for phosphotyrosine and is inhibited by the tyrosine kinase inhibitor, genistein. A, neutrophils were incubated with either the anti-L-selectin Ab, DREG 56, or the L-selectin ligand, sulfatides, before analysis by SDS-PAGE and phosphotyrosine immunoblotting as described under ``Experimental Procedures.'' After immunoblotting of parallel samples, the nitrocellulose membrane was divided and incubated in the primary anti-phosphotyrosine Ab in the presence or absence of 1 mMO-phosphotyrosine. While some nonspecific bands are visible at 15, 20, 29, and 50 kDa in all lanes, the majority of the higher molecular mass bands observed above 32.5 kDa were specific and were not seen in the presence of excess phosphotyrosine. B, neutrophils were incubated with genistein at the indicated dose for 30 min at 37 °C, washed, and treated with 400 µg/ml sulfatides or cytochalasin B and fMLP for 2 min. Cells were sedimented, boiled in sample buffer, and analyzed by SDS-PAGE and immunoblotting. The response to sulfatides is inhibited in a dose-dependent fashion by genistein.




Figure 2: Chymotrypsin pretreatment inhibits the response to sulfatides and selectively cleaves L-selectin. A, neutrophils were incubated with 10 units/ml chymotrypsin at 37 °C for 10 min. After washing, the cells were untreated or stimulated with either sulfatides or fMLP. Pretreatment with chymotrypsin by itself has no effect on tyrosine phosphorylation (lanes 1 and 2) but does significantly decrease the response to sulfatides (lanes 3 and 4). The response to fMLP is unaffected (lanes 5 and 6). B, neutrophils were incubated with chymotrypsin as in A and then incubated with saturating amounts of the indicated primary antibody for 30 min on ice. Next the cells were washed, stained with FITC-labeled secondary antibody, and fixed before analysis on a FACScan. Decreased binding of anti-L-selectin antibody is seen after chymotrypsin treatment, whereas the binding of antibodies against CD18, FcRII, and HLA class I is unaffected. Without chymotrypsin treatment is shown as lightly stippled, whereas chymotrypsin-pretreated cells are shown with the dark line.



To confirm that the detection system was specific for tyrosine phosphorylation, the nitrocellulose membrane was incubated with primary Ab in the presence of 1 mMO-phosphotyrosine. As shown in Fig. 3A, excess phosphotyrosine significantly reduced the intensity of the majority of higher molecular weight bands, demonstrating that the response to both anti-L-selectin antibodies and the L-selectin ligand, sulfatides, indeed represents phosphorylation of tyrosine residues. The nonspecific reaction with lower M proteins was due to nonspecificity of the primary Ab, since no bands were observed when the blot was developed in the absence of primary Ab (data not shown). Similar increases in tyrosine phosphorylation, especially at 40-42 kDa, were also observed when an alternate anti-phosphotyrosine antibody, 4G10, was used (data not shown). The response to sulfatides was inhibited, in a dose-dependent manner, by the tyrosine kinase inhibitor, genistein, suggesting that the tyrosine phosphorylation response was due, at least in part, to activation of protein tyrosine kinases (Fig. 3B).

The increase in tyrosine phosphorylation seen after incubation of the cells with whole anti-L-selectin mAb might have been due to binding of the Fc portion of the mAb to Fc receptors on the neutrophil(23) . This is not likely since isotype-matched control mAb, which also bound to the neutrophil as confirmed by flow cytometry (data not shown), did not result in enhanced tyrosine phosphorylation (Fig. 1A). However, to exclude this possibility, cells were treated with F(ab`) fragment of DREG 56. Fig. 4A demonstrates that binding of F(ab`) fragments resulted in a clear increase in tyrosine phosphorylation with a pattern similar to that induced by whole Ab, although the magnitude of this response was smaller. Both whole immunoglobulin and F(ab`) fragments are bivalent molecules and are theoretically capable of cross-linking two adjacent L-selectin molecules which might lead to intracellular signals. Alternatively, univalent ligation of L-selectin might be sufficient. To distinguish between these possibilities, DREG 200 was subjected to proteolytic cleavage into monovalent Fab fragments and then purified to remove contaminating Fc fragments and whole IgG. Fig. 4B illustrates that binding of these Fab fragments was also able to induce an increase in tyrosine phosphorylation, albeit of lesser magnitude than after L-selectin ligation by whole mAb. In addition, pretreatment of neutrophils with saturating amounts of blocking antibodies to both neutrophil Fc receptors, FcRII and FcRIIIB, did not inhibit tyrosine phosphorylation induced by anti-L-selectin mAbs (Fig. 4C). Taken together, these data suggest that univalent ligation of L-selectin is sufficient to induce tyrosine phosphorylation and that neither cross-linking nor Fc-mediated events are required.


Figure 4: Increased tyrosine phosphorylation is not due to FcR binding. A, neutrophils were incubated without antibody or with either whole IgG or the F(ab`) fragment of DREG 56 (anti-L-selectin). Increased tyrosine phosphorylation was still observed after treatment with the F(ab`) fragment. B, Fab fragments also increase tyrosine phosphorylation. The anti-L-selectin mAb, DREG 200, was proteolytically cleaved by papain to yield Fab fragments. Neutrophils were incubated without mAb or with either whole DREG 200 or the Fab fragment before analysis by SDS-PAGE and immunoblotting. C, blockade of Fc receptors does not prevent anti-L-selectin-induced tyrosine phosphorylation. Neutrophils were preincubated with saturating amounts of antibodies against FcRII (IV.3 Fab) and/or FcRIII (3G8 F(ab`)) for 30 min on ice before stimulation with the anti-L-selectin mAb DREG 200. Treatment with the anti-FcR antibodies alone does not increase tyrosine phosphorylation, and the response to anti-L-selectin mAb is unchanged.



In previous studies(5) , we observed that binding of primary antibody alone was insufficient to increase intracellular calcium or enhance the oxidative burst: cross-linking with secondary Ab was required. Accordingly, the effect of cross-linking L-selectin on tyrosine phosphorylation was examined. Cells were treated with primary anti-L-selectin mAb, washed, and resuspended at 37 °C before addition of goat anti-mouse Ig (F(ab`) fragment. After addition of the cross-linking antibody, total tyrosine phosphorylation was never increased and a decrease in phosphorylation of the band in the 40-kDa region was reproducibly observed (Fig. 5).


Figure 5: Cross-linking of L-selectin using secondary Ab does not increase tyrosine phosphorylation. Neutrophils were incubated with or without the anti-L-selectin mAb, DREG 56, for 20 min at 37 °C. After washing the cells were resuspended and treated either with or without secondary, cross-linking goat anti-mouse (GAM) Ab. After 2 min the cells were sedimented, resuspended in boiling sample buffer, and analyzed as before. Increased tyrosine phosphorylation is seen after L-selectin ligation (lane 3) which is not increased after cross-linking (lane 4). One particular band at approximately 40 kDa was consistently dephosphorylated after cross-linking.



Antibody-mediated cross-linking of platelet integrins leads to rapid increases in tyrosine phosphorylation of several cellular proteins (24). This increase is inhibitable by pretreatment with cytochalasin, suggesting that integrins transmit their signals via the actin cytoskeleton. However, as illustrated in Fig. 6, cytochalasin D, at doses up to 5 µM, did not inhibit tyrosine phosphorylation induced by binding of anti-L-selectin antibodies. These data suggest that, unlike induction of tyrosine phosphorylation in platelets by integrin cross-linking(25) , enhancement of tyrosine phosphorylation by L-selectin does not require cytoskeletal reorganization in neutrophils.


Figure 6: Tyrosine phosphorylation is not inhibited by cytochalasin D. Neutrophils were incubated in the indicated concentration of cytochalasin D (CYTO D) for 10 min prior to addition of the anti-L-selectin mAb DREG 56. After incubation with or without mAb, the cells were sedimented and resuspended in boiling sample buffer. SDS-PAGE and immunoblotting was performed as above. Increased tyrosine phosphorylation is observed after L-selectin ligation (lane 2), which is not inhibited by either 2 or 5 µM cytochalasin D (lanes 3 and 4, respectively).



As noted previously, prominent tyrosine phosphorylated bands were seen in the 40-42 kDa range after treatment with either anti-L-selectin antibodies or sulfatides. The MAP kinase pathway has recently been shown to be activated by chemoattractants in human neutrophils(26, 27) and MAP kinase itself is one of the most prominent phosphorylated bands in chemoattractant-activated neutrophils(28) . Because MAP kinase consists of 42- and 44-kDa isoforms, consistent with the bands observed here, we examined whether the MAP kinase was phosphorylated after ligation of L-selectin. After treatment with anti-L-selectin antibodies (Fig. 7A) or sulfatides (Fig. 7B), increased tyrosine phosphorylation of 40-42 kDa protein species were observed. The nitrocellulose membrane was stripped and reprobed with anti-MAP kinase mAb. Fig. 7illustrates that MAP kinase co-migrated with the tyrosine-phosphorylated band at an estimated molecular mass of 42 kDa.()


Figure 7: The prominent 42-kDa band co-migrates with MAP kinase. Neutrophils were treated with either DREG antibodies (A) or sulfatides or cytochalasin B and fMLP (B) as described previously. After SDS-PAGE and immunoblotting, the nitrocellulose membranes were probed with anti-phosphotyrosine (lanes 1-4 in A, lanes 1-3 in B). The same membrane was then stripped and re-probed with anti-MAP kinase (lanes 5-8 in A, lanes 4-6 in B). Both DREG 200 and sulfatides increase tyrosine phosphorylation of several cellular proteins, including a 42-kDa band which appears to co-migrate with MAP kinase.



To prove more definitively that MAP kinase itself was tyrosine-phosphorylated, MAP kinase was immunoprecipitated, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane which was probed with monoclonal anti-phosphotyrosine antibody. Fig. 8illustrates that after treatment of cells with sulfatides, MAP kinase was phosphorylated on tyrosine. This phosphorylation was not seen if cells were incubated with chymotrypsin, which cleaves L-selectin but not other surface antigens such as FcRII, HLA class I, or CD18 (Fig. 2B) from the cell surface(6, 22) , prior to treatment with sulfatides (lane 3). Similar negative results were also obtained using Type I and Type II galactocerebrosides as negative controls (data not shown). To determine if MAP kinase was activated following L-selectin ligation, gel renaturation kinase assays were performed using both whole cell lysate and MAP kinase immunoprecipitates. Cells were treated with control cerebrosides, sulfatides, or cytochalasin B and fMLP before immunoprecipitation of MAP kinase from the cell lysate. Samples of whole cell lysate or immunoprecipitate were resolved on SDS-PAGE gels which incorporated the MAP kinase substrate, myelin basic protein. After renaturation of the protein within the gel, kinase activity was detected by incubating the gel with [P]ATP. As Fig. 9A demonstrates, sulfatides, but not control cerebrosides, increased the MAP kinase activity in both whole cell lysates and MAP kinase immunoprecipitates. Using densitometric analysis, the kinase activity induced by sulfatides was 20% of the response induced by cytochalasin B and fMLP, consistent with the lower intensity of MAP kinase phosphorylation seen in Fig. 8. Similarly, treatment of cells with anti-L-selectin mAbs but not control mAbs resulted in activation of MAP Kinase activity (Fig. 9B).


Figure 8: Sulfatides increase tyrosine phosphorylation of MAP kinase. Neutrophils were pretreated with DFP for 30 min, washed, and resuspended at 2 10 cells/ml in HEPES-buffered RPMI. Cells were then treated with chymotrypsin 10 units/ml in PBS (lane 3) or PBS alone (lane 1, 2, and 4). After 10 min cells were treated with PBS alone, 400 µg/ml sulfatides, or cytochalasin B followed by fMLP. After 2 min the cells were sedimented, lysed, and boiled and MAP kinase was immunoprecipitated. The immunoprecipitate was resolved on SDS-PAGE and immunoblotted with monoclonal anti-phosphotyrosine. Sulfatides caused an increase in tyrosine phosphorylation of a 42-kDa band in the MAP kinase immunoprecipitate (lane 2), which was not seen if L-selectin was enzymatically cleaved by chymotrypsin pretreatment (lane 3). The response to cytochalasin B and fMLP is shown as a positive control.




Figure 9: Sulfatides or anti-L-selectin mAb cause activation of MAP kinase activity. A, neutrophils were treated with sulfatides or cytochalasin B and fMLP as described in the legend to Fig. 8. After immunoprecipitation of MAP kinase, the immunoprecipitate was resolved on 10% polyacrylamide gels containing the MAP kinase substrate, myelin basic protein. The proteins within the gel were then renatured and incubated with [-P]ATP, washed, and exposed to autoradiographic film. Sulfatides caused an increase in the kinase activity of the MAP kinase immunoprecipitate (lane 4), whereas treatment with the control cerebrosides (lane 2 and 3) did not. The response of cells treated with cytochalasin B and fMLP is shown as a positive control. B, neutrophils were treated with mAb as in Fig. 1 or sulfatides (lane 6) as above. Anti-L-selectin antibodies (lanes 2 and 3), but not mAb against CD18 (IB4) or CD45 (4B2) stimulate MAP kinase activity.



To determine if enhanced tyrosine phosphorylation was necessary for any downstream response to L-selectin ligation, cellular tyrosine kinases were inhibited with the chemically unrelated tyrosine kinase inhibitors, genistein, and herbimycin A. Both compounds inhibited the cytosolic calcium transient in response to sulfatides (Fig. 10A and data not shown). Such transient increases in cytosolic calcium have previously been shown to be required for potentiation of the oxidative burst(5) . In addition, tyrosine kinase inhibition with genistein decreased the amount of superoxide radical released from neutrophils stimulated with sulfatides (Fig. 10B). Genistein did not nonspecifically inhibit the NADPH oxidase, since the oxidative burst induced by PMA was unaltered in its presence.


Figure 10: Tyrosine phosphorylation is important and the tyrosine kinase inhibitor, genistein, inhibits downstream events induced by sulfatides. A, cells were incubated in 150 µM genistein or equivalent levels of the vehicle, DMSO, simultaneously with Fura-2 AM for 30 min at 37 °C. After washing, the cells were resuspended in Ca measuring buffer and stimulated with 100 µg/ml sulfatides and subsequently fMLP (10M). Genistein selectively inhibits the response to sulfatides but does not impair the calcium transient induced by fMLP. B, neutrophils were incubated in genistein as in A and resuspended in KRPD with cytochrome c. Production of O was measured as described under ``Experimental Procedures.'' Sulfatides induce a measurable oxidative burst which return to control levels in cells pretreated with genistein. Genistein does not significantly affect the response to PMA. Note broken y axis to accommodate the much larger response to PMA. , MeSO; , genistein.




DISCUSSION

The recruitment of neutrophils to inflammatory sites is important physiologically in host defense and pathophysiologically in disease states involving inflammatory injury. Recruitment proceeds via several distinct steps, which involve separate adhesion receptor-ligand interactions between the neutrophil and the endothelial cell. In certain circumstances, engagement of L-selectin may be the first signal encountered by the migrating neutrophil. In this study, additional evidence for such a signaling role for L-selectin is provided. This may be in addition to the signaling or co-signaling role for tethered platelet activating factor which, when present, may lead to priming or activation of the neutrophil(29) .

Using both monoclonal anti-L-selectin antibodies and a possible ligand for L-selectin (sulfatides), we have shown that ligation of L-selectin on the surface of the neutrophil results in tyrosine phosphorylation of a variety of cellular proteins. Prominent tyrosine-phosphorylated bands were found at molecular masses 40-42, 55-60, 70-72, and 105-120 kDa. The binding of antibodies against other surface antigens did not enhance tyrosine phosphorylation, indicating the specificity of this response. The magnitude of enhanced tyrosine phosphorylation after antibody-mediated ligation of L-selectin was less than that observed in cells treated with the chemoattractant, fMLP. One possible explanation for this observation is that fMLP may be a more potent stimulus than L-selectin ligation. Alternatively, ligation of L-selectin by mAb may only partially mimic the interaction of L-selectin with its natural ligand. Sulfatides induced larger increases in tyrosine phosphorylation and MAP kinase activity than did ligation of L-selectin by mAb. For P-selectin the binding sites for natural ligand and sulfatides are overlapping(30) . This mimicry of the natural ligand by sulfatides might explain the larger response to sulfatides.

In the current study, we have carefully tried to exclude the potential confounding effects of Fc receptor activation, since tyrosine phosphorylation is a known consequence of Fc receptor engagement in neutrophils(31) . Indeed, preliminary experiments using whole IgG rabbit anti-mouse secondary Ab to cross-link anti-L-selectin resulted in large increases in tyrosine phosphorylation (data not shown). However, this response was not specific for L-selectin as it was also seen after cross-linking a variety of other cell surface antigens and was dependent upon the presence of the Fc portion of the secondary antibody. Therefore, to rule out Fc-mediated signaling, F(ab`) fragments of DREG 56 were tested for their ability to induce tyrosine phosphorylation. While the response was of lower magnitude, these fragments were capable of inducing increases in tyrosine phosphorylation. Similar supporting evidence was also obtained using Fab fragments of DREG 200. In addition, blocking neutrophil Fc receptors with combined treatment with IV.3 and 3G8, mAbs against FcRII and FcRIII, respectively, did not inhibit the response to anti-L-selectin mAbs. Furthermore, chymotrypsin did not remove FcRII from the cell surface but did inhibit the response to sulfatides, supporting our conclusion that L-selectin, and not FcRII, is the actual signal-transducing molecule.

In previous studies, binding of anti-L-selectin antibody alone was insufficient to induce Ca transients or potentiate the oxidative burst(5, 6) , whereas in the present study, tyrosine phosphorylation occurred after binding of whole anti-L-selectin mAb alone. One possibility was that bivalent antibodies were cross-linking two adjacent L-selectin molecules which might have been sufficient to transduce intracellular signals. However, this did not appear to be required, since Fab fragments of anti-L-selectin antibodies induced similar responses, implying that univalent ligation of L-selectin was capable of induction of tyrosine phosphorylation. We speculate that L-selectin-mediated signaling may be a two-step process. Certain intracellular signals are transmitted by univalent ligation, including but perhaps not limited to tyrosine phosphorylation, whereas other signals, including Ca transients, require subsequent cross-linking of L-selectin. Surprisingly, deliberate cross-linking with secondary anti-mouse antibodies did not cause an increase in phosphorylation. In fact, such treatment appeared to cause dephosphorylation, at least of the 40-kDa band. This may have been due to proteolytic cleavage of L-selectin, resulting in termination of the signal, as antibody-mediated cross-linking has been reported to cause shedding of L-selectin from the surface of the neutrophil(32) . More specifically, protein tyrosine kinases regulated by L-selectin might be deactivated by cleavage and shedding of L-selectin with termination of the presumed ``on'' signal. However, it was not possible to test this hypothesis, since the cleavage of L-selectin cannot be inhibited by any known protease inhibitors(12) . Alternatively, cross-linking may induce activation of a protein tyrosine phosphatase. It is not currently known if this dephosphorylation has any physiological consequences.

In the current study, treatment of cells with cytochalasin did not prevent L-selectin-induced tyrosine phosphorylation. These results, on first consideration, appear to be contrary to other reports in the literature. However, using the hypothesis advanced by Fuortes et al.(33) , it may be possible to reconcile our data with reports that integrin cross-linking in platelets results in cytochalasin-sensitive tyrosine phosphorylation(24, 34) . In the study by Fuortes et al.(33) , neutrophils adherent to physiologic substrates and stimulated with tumor necrosis factor underwent rapid and prolonged increases in tyrosine phosphorylation. This effect required integrin-dependent adherence to physiologic substrates. Most of the tyrosine-phosphorylated proteins were concentrated at points of contact with the substratum as determined by immunofluorescence microscopy. In their study, tyrosine phosphorylation was not inhibited by cytochalasin but rather tyrosine dephosphorylation was enhanced. They speculated that this was because an intact cytoskeleton served to sequester phosphoproteins and protect them from phosphatases. Returning to the studies with platelets(24, 34) , ligation of integrins may have activated tyrosine kinases and subsequent aggregation and cytoskeletal reorganization may have protected tyrosine-phosphorylated proteins from phosphatases. Treatment with cytochalasin, by preventing cytoskeletal reorganization and protection from phosphatases, would be predicted to result in diminished tyrosine phosphorylation. In our studies with suspended neutrophils, any effect of cytochalasin on dephosphorylation was not apparent (perhaps because the cells were in suspension at low density which did not allow aggregation or adherence to a substrate). This might explain our findings that tyrosine phosphorylation was not affected by cytochalasin. Alternatively, the signaling pathways after L-selectin ligation and integrin cross-linking may be distinct and differ in their sensitivity to cytochalasins.

An intact cytoskeleton may be required for some L-selectin functions, such as those occurring later or distally in the signaling pathways but may not required for others, such as very early or proximal events. In previous studies, cytochalasin did not abrogate the potentiation of the oxidative burst that occurred after L-selectin cross-linking(5) . However, cytochalasin was able to inhibit leukocyte rolling and binding to HEV cells(25) . In the latter study, mutated L-selectin with a deletion of the cytoplasmic tail was similarly unable to mediate rolling or cell adhesion to HEV endothelium. At least two possible explanations for these data exist: that there is an interaction of components of the cytoskeleton with the cytoplasmic tail of L-selectin (or associated molecule) or that the cytoplasmic domain of L-selectin has a role in signaling and there is a later requirement for an intact cytoskeleton during rolling and adhesion. In this regard, there is data to support association between selectins and signaling molecules: the CD3 chain co-immunoprecipitates with L-selectin in lymphocytes (35) and P-selectin co-immunoprecipitates with pp60 in platelets(36) .

Ligation of L-selectin by monoclonal antibodies or by sulfatides resulted in similar patterns of enhanced tyrosine phosphorylation. Sulfatides were used in these experiments largely to show that the observed effects were not an artifact of the mAb used. However, the potential significance of sulfatide-induced responses warrants further consideration. Sulfatides are a heterogeneous group of sulfated galactosyl-ceramides, with fatty acyl groups of varying lengths. They are found in a variety of tissues, including brain, tumors, and on leukocytes themselves. They are related to CD57, which is found on endothelial cells. Although both CD57 and sulfatides are ligands for L-selectin in vitro(21, 37) , their function in vivo is not yet known. They are almost certainly not the inducible ligand on non-lymphoid endothelium. There is another incompletely characterized ligand for L-selectin in the central nervous system which has been localized to myelin(38) . It is noteworthy that the central nervous system ligand is chemically quite distinct from the endothelial ligand. The L-selectin ligand in the central nervous system is resistant to sialidase, but sensitive to organic solvents, suggesting that it has a lipid rather than a protein backbone(38) . Sulfatide or a closely related sulfated glycolipid might fulfil these criteria. Sulfatides on or released by the neutrophil are also ligands for P-selectin. Sulfatides will inhibit rolling of neutrophils on purified P-selectin, suggesting that sulfatides bind to P-selectin on or close to the functional site(39) . Indeed, the sulfatide binding site on P-selectin has recently been mapped to a separate but overlapping site from the carbohydrate binding site(30) . It has even been suggested that binding of sulfatides may cause a conformational change in P-selectin leading to ligand release. Thus, the physiological role for sulfatides may be to regulate selectin disengagement that would allow cellular transmigration. Whether sulfatides are indeed important ligands for L-selectin in vivo and whether they function in an adhesion promoting or inhibiting fashion is yet to be determined. It is plausible that changes in tyrosine phosphorylation lead to decreased rather than increased selectin-mediated adhesion.

As seen in Fig. 3B, the responses to fMLP and ligation of L-selectin differ. Following fMLP treatment there is more prominent phosphorylation of protein substrates at approximately 30, 49, and 55 kDa, when compared with L-selectin ligation. The identity of the specific protein substrates, other than MAP kinase, and the significance of these different patterns of phosphorylation remains unknown. The magnitude of increased tyrosine phosphorylation was generally smaller following L-selectin ligation when compared with chemoattractant stimulation. However, it is possible that the amount of tyrosine phosphorylation induced by interaction of L-selectin with its natural ligand under physiologic conditions is larger. A more complete understanding of this will depend upon the isolation and characterization of the inducible endothelial ligand for neutrophil L-selectin.

The significance of the activation of MAP kinase by L-selectin ligation is as yet unknown. MAP kinase has been directly linked to long term responses, such as proliferation(40) , but its role in acute events in neutrophils is unclear. Recent evidence suggests that MAP kinase activation does not occur early enough to account for rapid responses, such as triggering the oxidative burst (41). Activation of MAP kinase leads to phosphorylation, activation, and translocation of cPLA(42, 43) , which has been suggested to play an important role in neutrophil priming and other responses. The pathways upstream of MAP kinase have recently been characterized and can be activated by receptor tyrosine kinases (44) or through G-protein coupled receptors(27) . Activation of MAP kinase by integrins has been recently reported (45) but activation by any selectin appears to be a novel finding.

It is not possible to demonstrate the physiologic relevance of MAP kinase activation in neutrophils, which would require specific inhibition of the enzyme such as by overexpression of dominant negative MAP kinase mutants. However, it does appear that tyrosine phosphorylation is required for downstream signaling events leading to functional neutrophil responses. The tyrosine kinase inhibitors, genistein and herbimycin, significantly inhibited the cytosolic calcium transient induced by sulfatides. We speculate that L-selectin is linked to tyrosine kinases which activate phospholipase C which is found in neutrophils (46) while Ca mobilization after fMLP stimulation is mediated by phospholipase C and is therefore unaffected by tyrosine kinase inhibition. The level of intracellular calcium in neutrophils is an important regulator of a variety of neutrophil processes, such as motility and phagocytosis, exocytosis, priming, and the respiratory burst. We have previously shown that prevention of such calcium transients prevents the potentiation of the oxidative burst seen after L-selectin cross-linking(5) . Treatment with sulfatides but not antibody-mediated L-selectin ligation is sufficient to activate the respiratory burst. Crockett-Torabi et al. (7) have recently reported that cross-linking L-selectin using antibodies adherent to a planar surface does, in fact, activate NADPH oxidase. Sulfatides are poorly soluble in aqueous buffer and form micelles. The detectable oxidative burst after sulfatides might be due to large micelles acting as a planar surfaces or ligand mimcry as discussed earlier. These minor discrepancies notwithstanding, the important observation is that the oxidative burst can be inhibited by genistein. Thus, for L-selectin signaling it appears that tyrosine phosphorylation is necessary for Ca signaling, and both are required for the downstream events, in this case, the oxidative burst.

In summary, ligation of L-selectin by either monoclonal antibodies or the natural ligand sulfatides led to increased tyrosine phosphorylation of several cellular proteins, including MAP kinase. These data confirm the important role of L-selectin as a signaling molecule. Signaling pathways, initiated by the engagement of L-selectin, may lead to enhancement of a variety of neutrophil functions, such as integrin-mediated adhesion, cytokine production, and the respiratory burst.


FOOTNOTES

*
This work was supported by operating grants (to G. P. D.) from the Medical Research Council of Canada, the Ontario Thoracic Society, the Robert O. Lawson Fund of the Toronto Hospital, and the National Sanatorium Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Medical Research Council of Canada Fellowship.

Recipient of a Zeneca Pharma/University of Toronto/Medical Research Council of Canada Fellowship.

**
Recipient of a Career Scientist Award from the Ontario Ministry of Health. To whom correspondence should be addressed: Clinical Sciences Division, Rm. 6264 Medical Sciences Bldg., 1 King's College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8. Tel.: 416-978-8923; Fax: 416-978-8765.

The abbreviations used are: KRPD, Krebs-Ringer phosphate dextrose; fMLP, formylmethionylleucylphenylalanine; DFP, diisopropyl fluorophosphate; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; MAP kinase, mitogen-activated protein kinase; mAb, monoclonal antibody; Ab, antibody; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis.

A lower band of unknown identity (approximate molecular mass 40 kDa) was tyrosine-phosphorylated after L-selectin ligation, but in this experiment, not after treatment with cytochalasin B and fMLP. However, this finding was not reproducible, and in other experiments cytochalasin B and fMLP induced the tyrosine phosphorylation of proteins in this molecular mass range (see Fig. 3B).


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