Soluble E-selectin Induces Monocyte Chemotaxis through Src Family Tyrosine Kinases*

Cellular adhesion molecules such as E-selectin function to recruit leukocytes into the inflammatory lesions of diseases such as rheumatoid arthritis (RA) and atherosclerosis. Monocytes are the key components of the cellular infiltrates present in these disorders. We hy-pothesized that soluble E-selectin (sE-selectin) might mediate the chemotaxis of monocytes. In this report, we show that sE-selectin induced normal human peripheral blood monocyte migration in the nanomolar range in a concentration-dependent manner. Neutralization studies using RA human joint synovial fluids and anti-E-selectin antibody showed a mean 31% reduction in RA synovial fluid-mediated monocyte chemotaxis ( p < 0.05), indicating that sE-selectin is a major monocyte recruiter in RA. Next, we investigated the role of tyrosine phosphorylation pathways in sE-selectin-induced monocyte chemotaxis. Human peripheral blood monocytes stimulated with sE-selectin showed a time-dependent increase in the tyrosine phosphorylation of a broad range of cellular proteins, predominantly in the molecular size range of Src family kinases (50–60 kDa) and mitogen-activated protein kinases (MAPKs). Western blot analysis of Src family kinases showed a time-de-pendent increase in Src, Hck, and Lyn phosphorylation. The pretreatment of monocytes with the Src inhibitor Lyn the blots m M Tris and m M 2-mercaptoethanol and reprobed with rabbit polyclonal anti-Lyn or polyclonal anti-Hck antibody, Activation Assay— Ras activation studied using a Ras activation kit stimulated each time point monocytes each The activation In the step, precipitated m each monocyte immobilized run on activated Ras samples was then a specific mouse monoclonal anti-Ras antibody different bands with antibody to detect activated Hck or Lyn kinase. Alternatively, the were probed for total kinase. sE-selectin induced the tyrosine of A and in which was markedly inhibited by with These are representative of three independent experiments.

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory conditions such as rheumatoid arthritis (RA) 1 or atherosclerosis (1,2). E-selectin is a single-chain 115-kDa glycoprotein with a lectin-like N-terminal domain, an epidermal growth factor (EGF)-like motif, and a variable number of repeat units homologous to the consensus repeats of complement binding proteins (3). The lectin domain of E-selectin plays a major role in ligand recognition and binds to sialyl Le X on leukocytes as well as on endothelial cells (4 -6). Soluble isoforms of these adhesion molecules are rapidly shed from the cellular surfaces on cellular activation (1). We have found elevated levels of soluble Eselectin (sE-selectin) in patients with RA (7). In contrast to the earlier hypothesis that soluble adhesion molecules serve an anti-inflammatory role, our laboratory has shown a unique role for sE-selectin as a proinflammatory, angiogenic mediator (8).
The infiltration of monocytes into the synovial tissue is a key factor in amplifying and perpetuating RA. The functions of elevated levels of sE-selectin and the mechanism of action of sE-selectin are still poorly understood. One of the primary mechanisms by which leukocytes are activated might be through the activation of protein tyrosine kinases at the cell surface (9,10). The Src family of tyrosine kinases is activated during a number of biological functions including monocyte interaction with endothelial cells (11), endothelial cell differentiation (12), and cardiac motility (13). A number of pathways lead to the activation of mitogen-activated protein kinases (MAPKs). MAPKs are a family of 38 -45 kDa proteins that exist in a dephosphorylated form in quiescent cells and can be activated in response to various growth factors and chemoattractants (14,15). MAPKs are activated by MAPK/ERK kinase (MEK), which in turn is activated by MEK kinase (MEKK). One of the extensively studied MEKKs is Raf kinase (16). Raf kinase is in turn activated by binding to Ras-GTP (17), and Src is an important upstream kinase linked to Ras-Raf activation (12,18).
In this study, we examined the role and mechanism of action of sE-selectin in mediating monocyte recruitment. We report that sE-selectin is a potent chemotactic factor for monocytes, and its neutralization in RA synovial fluids, led to a significant decrease in RA synovial fluid-mediated monocyte migration. Furthermore, our results suggest that sE-selectin mediates signaling in monocytes through a Src-Ras-MAPK pathway. The inhibition of Src kinase by the Src-specific inhibitor, PP2, abolished sE-selectin-mediated monocyte chemotaxis. These results suggest that sE-selectin mediates chemotaxis through the Src pathway, and this could thus be a potential target for modulating monocyte recruitment-driven diseases.
Patient Sample Preparation-Synovial fluids were obtained from patients with RA who met the American College of Rheumatology criteria for RA (19). All specimens were obtained with Institutional Review Board approval.
Isolation of Human Mononuclear Cells (MN) and Monocytes-MN were isolated from the peripheral blood (PB) of normal healthy volunteers using Accu-Prep. PB was collected from normal healthy adult donors in heparinized tubes. After collecting a buffy coat, MN were purified under sterile conditions by centrifugation on Accu-Prep at 400 ϫ g for 30 min at room temperature. MN were collected at the interface, washed twice with phosphate-buffered saline, and resuspended in HBSS with calcium and magnesium at 2.5 ϫ 10 6 cells/ml. Cell viability was determined by trypan blue exclusion. The viability of MN used was Ͼ98%, and the purity was Ͼ99%. The method of monocyte separation was described previously (20). Briefly, 4 ml of MN (5 ϫ 10 7 cells) was gently layered over 8 ml of isolation buffer (1.65 ml of 10ϫ HBSS in 10 ml of Percoll, pH 7.0). After centrifugation at 400 ϫ g for 25 min at room temperature, monocytes at the interface were collected. The viability of monocytes determined by trypan blue exclusion was found to be Ͼ98%, and the purity was Ͼ90%.
Cell Lysis and Immunoblotting-Monocytes (1 ϫ 10 7 cells/ml) were incubated in 24-well plates for 3 h in HBSS (with calcium and magnesium) prior to stimulation with sE-selectin for various time points. At the end of each time period, supernatants were gently aspirated, and cells were lysed in extraction buffer containing 100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM NaP 2 O 4 , 2 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors (protease inhibitor mixture tablets, 1 tablet/10 ml) (Roche Molecular Biochemicals). For experiments with signaling inhibitors, monocytes were preincubated with the respective inhibitor before activation with sE-selectin. The protein content of different samples was quantitated using a bicinchoninic acid (BCA) protein assay kit. Cell lysates were mixed at 1:1 with Laemmli's sample buffer and boiled for 5 min. 15 g of each sample was subjected to 10% SDS-polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred from the gel onto nitrocellulose membranes using a Tris-glycine buffer. To block nonspecific binding, membranes were incubated with 3% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The blots were incubated in respective primary antibody in TBST ϩ 3% bovine serum albumin at 4°C overnight. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000) or with goat anti-rabbit IgG (1:10,000) for 45 min at room temperature. An enhanced chemiluminescence detection (ECL) system (Amersham Pharmacia Biotech) was used to detect specific protein bands. The different bands were then scanned and quantitated using an imaging densitometer (Bio-Rad).
Immunoprecipitation-Cell lysates were precleared with rabbit nonimmune serum prebound to sheep anti-rabbit IgG-agarose (Sigma). Lyn and Hck kinases were immunoprecipitated from 100 g of each precleared sample by incubation at 4°C for 16 h with 2 g of polyclonal rabbit anti-Lyn (Upstate Biotechnology) or 3 g of polyclonal rabbit anti-Hck antibody (Santa Cruz Biotechnology), respectively. The immune complexes were further incubated for 2 h at 4°C after the addition of sheep anti-rabbit IgG-agarose conjugate. Immunopellets were washed three times with lysis buffer, reconstituted in 40 l of 2ϫ Laemmli sample buffer, and boiled for 5 min. These samples were subsequently run on 10% SDS-PAGE as described above. The presence of activated Lyn and Hck kinases was detected by probing the blots with mouse monoclonal anti-phosphotyrosine antibody (4G10 clone) (Upstate Biotechnology). The immunoblots were stripped for 30 min at 55°C in 67 mM Tris at pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol and reprobed with rabbit polyclonal anti-Lyn or rabbit polyclonal anti-Hck antibody, respectively.
Ras Activation Assay-Ras activation was studied using a Ras activation kit (Upstate Biotechnology). Monocytes were stimulated with sE-selectin (50 nM) for different time periods. At each time point monocytes were extracted with cell lysis buffer, and the protein content in each sample was quantitated. The Ras activation assay involved two steps. In the second step, the presence of activated Ras was immunoprecipitated from 100 g of each monocyte lysate sample with an immobilized Raf-1-Ras binding domain and subsequently run on 10% SDS-PAGE as described above. The presence of activated Ras in samples was then detected by probing with a specific mouse monoclonal anti-Ras antibody (1 g/ml). The different bands were then scanned and quantitated using an imaging densitometer.
Monocyte Chemotaxis-Monocyte chemotaxis was performed using 48-well chemotaxis chambers (Neuroprobe, Cabin John, MD) with a polyvinylpyrolidone-free polycarbonate filter as described previously (21). In brief, 25 l of stimulant or buffer was added to the bottom wells of the chambers. A 5-m membrane was placed in the assembly, and 40 l of monocytes at 2.5 ϫ 10 6 cells/ml was placed in the top of wells. The chemotaxis chambers were incubated for 2 h at 37°C with 5% CO 2 . The filters were removed, and the membranes were fixed in methanol and stained with Diff-Quik (VWR Scientific Products, Chicago, IL). Each test group was assayed in quadruplicate. Three high power (ϫ400) microscope fields were counted in each replicate well, and the results were expressed as cells per high power field. In synovial fluid neutralization studies, 1:2 diluted synovial fluid was preincubated with anti-sE-selectin antibody (5 g/ml) (Biogen) or corresponding control antibody (mouse IgG 1 ) for 1 h at 37°C. In the signaling inhibitor studies, monocytes (2.5 ϫ 10 6 cells/ml) were preincubated with their respective inhibitors for 1 h at 37°C. After washing twice with HBSS containing calcium and magnesium, the cells were adjusted to 2.5 ϫ 10 6 cells/ml and then assayed in response to different concentrations of sE-selectin. The results are expressed as the mean and S.E. of three high power fields (ϫ400) per replicate well, and each test group was assayed in quadruplicate. The incubation of monocytes with the Src inhibitor PP2 at 1 M did not alter cell viability. For all assays, controls included HBSS (negative control), fMLP (positive control), and Me 2 SO (a vehicle for the Src inhibitor).
Statistical Analysis-Data were analyzed using Student's t test, and p values less than 0.05 were considered significant.

sE-selectin Is Chemotactic for Monocytes in
Vitro-Our laboratory has shown that sE-selectin levels are up-regulated in synovial fluids from patients with RA (7). To examine the hypothesis that sE-selectin might be chemotactic for monocytes, we studied monocyte chemotaxis in response to sE-selectin in a modified Boyden chamber. A representative assay from five experiments is shown in Fig. 1. sE-selectin-induced chemotaxis in a concentration-dependent manner and was significantly higher at 10, 50, and 100 nM sE-selectin concentrations as compared with the negative control (p Ͻ 0.05).
Contribution of sE-selectin to RA Synovial Fluid Chemotactic Activity for Monocytes-We have shown earlier that RA syno-sE-selectin-mediated Monocyte Chemotaxis vial fluid is potently chemotactic for monocytes (22), and we have also demonstrated that high levels of sE-selectin are present in the RA synovial fluid (7). To examine the contribution of sE-selectin present in the RA synovial fluid to the chemotaxis of normal PB monocytes, a 1:2 dilution of RA synovial fluid was preincubated with isotype-matched control antibody or anti-E-selectin antibody for 1 h at 37°C. The incubation of RA synovial fluid with anti-E-selectin antibody significantly decreased monocyte chemotaxis (129 Ϯ 3.2, 193 Ϯ 4.4, and 90 Ϯ 7.5) as compared with the isotype control antibody in the respective samples (225 Ϯ 5.9, 270 Ϯ 13.7, and 134 Ϯ 4.1) (Table I). These data indicate that sE-selectin contributes significantly to the RA synovial fluid chemotactic activity for monocytes.
Signaling Mechanism Involved in sE-selectin-mediated Chemotaxis-We next investigated the signaling pathways involved in the sE-selectin-mediated monocyte chemotaxis. sEselectin (50 nM) induced protein tyrosine phosphorylation in monocytes in a time-dependent manner showing two peaks. Western blot analysis showed a first peak at 30 s and a second peak at 30 min. Proteins phosphorylated at tyrosine residues corresponding to molecular sizes of 130 -140, 70, 60 -65, 50 -55, 42-44, 35-40, and 25 kDa were detected (Fig. 2) by using a mouse monoclonal antibody (4G10), which specifically reacts with tyrosine phosphorylated proteins.
As the predominant bands (50 -65 kDa) observed with the 4G10 antibody corresponded to the molecular sizes of the Src family kinases, we next studied the role of Src family kinases Src, Hck, and Lyn in sE-selectin-mediated signaling in monocytes. We first performed a time course study of Src kinase activation followed by a Src inhibition study with the Src inhibitor PP2. Western blot analysis of activated Src with a phosphorylation state-specific antibody (Tyr(P) 418 ) showed a biphasic activation of Src with the first peak at 30 s and a second peak at 30 min (Fig. 3). The pretreatment of monocytes with the Src inhibitor PP2 (1 M) for 30 min prior to stimulation with sE-selectin partially inhibited Src phosphorylation (Fig. 4). We next studied the role of two other Src family kinases (Hck and Lyn) in sE-selectin-mediated signaling in monocytes. Hck and Lyn kinases were immunoprecipitated from sE-selectin-stimulated monocytes by polyclonal rabbit anti-Hck or polyclonal rabbit anti-Lyn antibodies, respectively.
The activation of Hck and Lyn kinases was determined by subjecting these immunoprecipitates to SDS-PAGE and Western blotting with anti-phosphotyrosine antibody (4G10). Hck kinase showed a time-dependent increase in tyrosine phosphorylation (Fig. 5A), whereas Lyn kinase showed a minor peak at 30 s and a major peak at 30 min (Fig. 5B). The pretreatment of monocytes with PP2 markedly inhibited the phosphorylation of both Hck and Lyn kinases (Fig. 5). The total protein phosphotyrosine profile analysis in monocytes pretreated with PP2 prior to sE-selectin stimulation showed inhibition of the phosphorylation of proteins in the MAPK region (35-45 kDa) in addition to the inhibition of Src family kinase region (60 -70 kDa) (Fig. 2B). . Results are expressed as the mean and S.E. of three high power fields (ϫ400) per replicate well. Each test group was assayed in quadruplicate. This is a representative assay from five independent experiments. HBSS was used as a negative control, and fMLP (100 nM) was used as a positive control. sE-selectin showed a dose-dependent increase (p Ͻ 0.05) in monocyte chemotaxis as compared with the negative control HBSS. * represents a significant difference (p Ͻ 0.05) between the sE-selectin group and the negative control HBSS. FIG. 2. Protein tyrosine phosphorylation pattern in monocytes stimulated with sE-selectin (A) or monocytes pretreated with the Src inhibitor PP2 prior to sE-selectin stimulation (B). Monocytes were stimulated with sE-selectin (50 nM) for various amounts of time as indicated (NS, nonstimulated; m, minutes) or first pretreated with PP2 (1 g) for 30 min and then stimulated with sE-selectin. Cells were lysed with lysis buffer, and the protein content of each sample was quantitated. 15 g of each sample was resolved by 10% SDS-PAGE and probed with mouse monoclonal antibody 4G10 (750 ng/ml) to detect the phosphorylated proteins. Experiments shown in A and B were repeated five and three times, respectively, with essentially identical results. sE-selectin induced the tyrosine phosphorylation of a number of proteins. An increase in tyrosine phosphorylation was observed as early as 30 s, peaked at 30 min, and then tapered off by 1 h. The pretreatment of monocytes with PP2 prior to sE-selectin stimulation showed the inhibition of the tyrosine phosphorylation of proteins in the Src region (50 -60 kDa) and MAPK region (35-45 kDa).

sE-selectin-mediated Monocyte Chemotaxis
To test the hypothesis that the sE-selectin signal from Src family kinases might be acting through MAP kinases, we studied the time course of extracellular signal-related kinase (ERK1/2) and p38 MAPK activation in monocytes. ERK1/2 showed a biphasic activation profile as observed with Src kinase with the first peak occurring at 1 min and the second peak at 30 min (Fig. 6). However, p38 MAPK did not show phosphorylation until 5 min after stimulation and showed a single peak at 30 min (Fig. 7). Next, we studied the role of the Src inhibitor PP2 on ERK1/2 and p38 MAPK activation. The pretreatment of monocytes with the Src inhibitor PP2 prior to stimulation with sE-selectin revealed 89 and 83% inhibition of ERK1/2 and p38 MAPK, respectively, as compared with sE-selectin-stimulated monocytes (Figs. 8 and 9) at 30 min. Similar levels of the inhibition of ERK1/2 and p38 MAPK were observed at other time points.
sE-selectin is a glycoprotein and contains an EGF-like motif (3), so we tested whether sE-selectin mediates its action through the EGF receptor. Our results show that sE-selectin does not induce the phosphorylation of the EGF receptor (data not shown). We also studied the phosphorylation profile of focal adhesion kinase, a substrate for the Src family kinases. However, we failed to detect the phosphorylation of focal adhesion kinase in sE-selectin-stimulated monocytes (data not shown). These data indicate that in contrast to Src family kinases (Src, Hck, and Lyn) and MAPKs (ERK1/2 and p38), the EGF receptor and focal adhesion kinase pathways are not involved in sE-selectin-mediated monocyte signaling.
A number of studies have shown that Raf kinase is a predominant MEK kinase (16, 23) that in turn is activated by binding to Ras-GTP (17). Src family kinases have also been shown to be linked to MAPK through Ras/Raf kinases (12, 18).  figure (m, minutes). Cell extracts were prepared with cell lysis buffer, and the protein content of each sample was quantitated. 5 g of each sample was subjected to 10% SDS-PAGE and probed with rabbit polyclonal anti-pSrc antibody (0.5 g/ml). sE-selectin induced the tyrosine phosphorylation of Src in a time-dependent manner showing a biphasic activation pattern. The first activation peak occurred at 30 s, and the second peak occurred at 30 min. This blot is representative of three independent experiments.

FIG. 4. Inhibition of sE-selectin-mediated Src activation by the Src-specific inhibitor PP2.
Monocytes were pretreated with the Srcspecific inhibitor PP2 (1 M) for 30 min at 37°C before being stimulated with sE-selectin for different time points. Cell extracts were prepared with lysis buffer at each time point, and the protein concentration in each sample was quantitated. 15 g of each sample was subjected to 10% SDS-PAGE and probed with rabbit polyclonal anti-pSrc antibody (0.5 g/ml). The pretreatment of monocytes with the Src-specific inhibitor PP2 markedly decreased Src phosphorylation by sE-selectin. This blot is representative of three independent experiments.

sE-selectin-mediated Monocyte Chemotaxis
To study the activation of Ras/Raf kinases, we first immunoprecipitated activated Ras with a Raf-1-Ras binding domain conjugated to agarose beads and subsequently probed with mouse monoclonal anti-Ras antibody. sE-selectin stimulation of monocytes showed a time-dependent activation of the Ras/ Raf pathway, which peaked at 30 min (Fig. 10).
Next, to examine the functional role of Src kinase in sEselectin-induced monocyte chemotaxis, we performed monocyte chemotaxis in the presence and absence of the Src inhibitor PP2 (1 M). Monocytes were pretreated with PP2 for 30 min at 37°C prior to performing the chemotaxis assay. fMLP served as the positive control, and HBSS served as the negative control. Fig. 11 shows a representative assay from three independent experiments. The pretreatment of monocytes with the Src inhibitor PP2 significantly inhibited (p Ͻ 0.05) monocyte chemotaxis as compared with sE-selectin alone, thereby suggesting that sE-selectin could be mediating monocyte chemotaxis through the Src pathway. DISCUSSION Monocyte recruitment into the synovial tissue is a key step in RA pathogenesis because activated monocytes and/or macrophages can function as antigen-presenting cells, and these cells also have the ability to secrete a variety of inflammatory mediators including prostaglandins (24), nitric oxide (25), interleukin-1, tumor necrosis factor-␣ (26), interleukin-8 (27), monocyte chemotactic protein-1 (28), and macrophage inflammatory protein-1␣ (29). We and others have reported a number of putative monocyte/macrophage chemoattractants in the RA synovial fluid, such as monocyte chemotactic protein-1 and macrophage inflammatory protein-1␣ (29,30), which may be involved in monocyte/macrophage recruitment into the RA synovial tissue.
In this report, we show that sE-selectin is a potent chemotactic agent for monocytes in the nanomolar range. We have FIG. 7. p38 MAPK activation in monocytes stimulated with sE-selectin. Monocytes were stimulated with sE-selectin (50 nM) at various time points as indicated in Fig. 6 (m, minutes). Cells were extracted with cell lysis buffer, and the protein content of each sample was quantitated. 15 g of each sample was subjected to 10% SDS-PAGE and probed with rabbit polyclonal anti-p38 antibody (1:1000 dilution). The sE-selectin stimulation of monocytes induced the enhanced tyrosine phosphorylation of p38 MAPK in a time-dependent fashion as compared with the control nonstimulated monocytes. This blot is representative of three independent experiments. previously shown elevated levels of sE-selectin (in the nanomolar range) in the RA synovial fluid (7). Thus, it is likely that at the concentrations found in the RA joints, sE-selectin might be acting in large part to recruit and activate monocytes. To address the contribution provided by the sE-selectin to RA synovial fluid chemotactic activity for monocytes, we immunodepleted RA synovial fluid sE-selectin by using neutralizing mouse monoclonal anti-E-selectin antibodies. The neutralization of RA synovial fluid sE-selectin significantly decreased monocyte chemotaxis (mean of 31%, p Ͻ 0.05) as compared with the matched isotype control antibody. Thus, these results suggest a novel function for sE-selectin as a chemotactic factor for monocytes in the RA synovial fluid.
Recent studies have shown that intact, transmembrane Eselectin can transduce signals in endothelial cells during inflammatory leukocyte recruitment (31). Hu et al. (23) showed that the cross-linking of endothelial cell surface E-selectin with antibodies leads to the up-regulation of mRNA for c-fos, an early response gene, through the Ras-Raf-MAPK pathway. However, we are not aware of any studies regarding the signaling pathway used by sE-selectin to mediate its biological function. In this regard, we investigated sE-selectin-mediated signaling in normal PB monocytes. sE-selectin induced the tyrosine phosphorylation of a number of monocyte proteins corresponding to molecular sizes of 130 -140, 70 -74, 60 -65, 50 -55, 42-44, 35-40, and 20 -25 kDa in a time-dependent manner. Marked increases in tyrosine phosphorylation were detected as early as 30 s after stimulation and peaked at 30 min, which is consistent with a typical time course of protein tyrosine phosphorylation (13,32). We next investigated the role that Src family kinases play in sE-selectin-mediated signaling. The Src family of tyrosine kinases is reported to be activated during a number of biological phenomena including monocyte adherence to endothelial cells (11), integrin-mediated signaling in monocytes and/or macrophages (33), endothelial cell differentiation (12), and cardiac contractility (13). In our studies, sE-selectin up-regulated the phosphorylation of Src family kinases (Src, Hck, and Lyn) in a time-dependent manner. The pretreatment of monocytes with PP2 for 30 min prior to sEselectin stimulation partially inhibited Src phosphorylation, whereas Hck and Lyn phosphorylation was markedly inhibited. These results indicate that the Src family kinases may be an important mediator in sE-selectin-mediated signaling in monocytes.
We next studied the role of PP2 on the total cellular phosphotyrosine pattern. The pretreatment of monocytes with PP2 prior to stimulation with sE-selectin showed the partial inhibition of a 60-kDa band. However, other Src family kinases (50 -60 kDa) were substantially inhibited, thus corroborating our other findings that PP2 has a partial effect on sE-selectinmediated Src phosphorylation, whereas it markedly inhibited Hck and Lyn kinases. In addition to the Src family kinases, PP2 also showed substantial inhibition of the tyrosine phosphorylation of proteins in the MAPK region (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). MAPK is a key signaling central point at which a number of pathways converge (34). MAPK is involved in a number of basic physiological phenomena, including cell migration, cell cycle, and apoptosis, in addition to playing an important role in the signaling of a number of important genes such as chemokines (35) and adhesion molecules (36,37). We next determined whether sE-selectin-mediated signaling involves the activation of MAPK. sE-selectin-stimulated monocytes showed the timedependent phosphorylation of ERK1/2 and p38 MAPK. ERK1/2 activation was observed as early as 30 s and persisted until 30 min. However, p38 activation was detectable from 5 min onwards. To test the hypothesis that the signal from Src might be going through MAPK, we pretreated monocytes with the Src inhibitor PP2 and then studied the phosphorylation of ERK1/2 and p38. Pretreatment with the Src inhibitor (PP2) showed 89 and 83% inhibition of ERK1/2 and p38, respectively, at 30 min after sE-selectin stimulation. These results suggest that MAPKs (ERK1/2 and p38) are activated in monocytes by sEselectin through the Src kinase pathway. Schmid-Alliana et al. (38) have demonstrated that microtubule depolymerization by colchicine in human monocytes induces the selective production of interleukin-1 through the Src, Ras, Raf-1, and MAPK pathway. A similar signaling cascade involving Src, Ras, and MAPK was reported by Jalali et al. (35) in vascular endothelial cells stimulated with shear stress.
Raf kinase is one of the best characterized MEKKs (16). Raf kinase is activated by binding to Ras-GTP, which then phosphorylates MEK. Ras kinase has been shown to be activated by Src kinase (12,35). To test whether Ras and Raf kinases are also activated during the sE-selectin activation of monocytes, we immunoprecipitated Ras with the Raf-1-Ras binding domain linked to agarose beads and then probed with mouse monoclonal anti-Ras antibody. sE-selectin stimulation showed a time-dependent activation of Ras, thereby suggesting a possible link between Src and MAPK in sE-selectin signaling through Ras-Raf kinase.
We next determined whether this Src-MAPK signaling cascade has functional relevance and whether it is essential for sE-selectin-mediated monocyte chemotaxis. PB monocytes were pretreated with the Src-specific inhibitor PP2 at a concentration of 1 M for 30 min at 37°C before performing chemotaxis in response to sE-selectin. PP2 completely blocked sE-selectin-mediated chemotaxis. These data suggest that sEselectin uses the Src pathway to mediate monocyte chemotaxis.
In summary, we have demonstrated a novel function for sE-selectin as a monocyte chemotactic agent. sE-selectin accounted for a significant proportion of the monocyte chemotactic activity in the RA synovial fluid. We have further shown that sE-selectin uses the Src-Ras-MAPK signaling pathway to mediate its biological function. Thus, sE-selectin may be a potent recruiter of monocytes in the RA joint and may contrib- FIG. 11. Src inhibitor PP2 inhibits monocyte chemotaxis mediated by sE-selectin. Monocytes were preincubated with the Src inhibitor PP2 (1 M) for 30 min at 37°C and then assayed in a 48-well chemotaxis chamber in response to various concentrations of sE-selectin. Results are expressed as the mean and S.E. of three high power fields (ϫ400) per replicate well. Each test group was assayed in quadruplicate. This is a representative assay of three independent experiments. HBSS-and fMLP-induced chemotaxis were tested as negative and positive controls, respectively. Error bars represent S.E., and * represents a significant difference (p Ͻ 0.05) between the sE-selectin group and negative control. # represents a significant inhibition (p Ͻ 0.05) of chemotaxis in the sE-selectin ϩ PP2 (Src inhibitor) group as compared with sE-selectin alone.