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(Journal of Leukocyte Biology. 2001;70:659-668.)
© 2001 by Society for Leukocyte Biology

Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals

Philippe Desaulniers*, Maria Fernandes*, Caroline Gilbert*, Sylvain G. Bourgoin*,{dagger} and Paul H. Naccache*

* Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, and Departments of Medecine and
{dagger} Physiology, Faculty of Medecine, Université Laval, Québec, Canada

Correspondence: Dr. Paul H. Naccache, CHUL, Room T1-49, 2705, Boulevard Laurier Ste-Foy, Québec, G1V 4G2, Canada. E-mail: paul.naccache{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine-phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross-linking CD32 or by the addition of fMet-Leu-Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.

Key Words: tyrosine phosphorylation • phagocytosis • gout • CD32


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The polymorphonuclear neutrophilic leukocyte (neutrophil) is one of the major actors in the innate response to infection. To perform the functions of phagocytosis and bacterial lysis, the neutrophil is equipped with a wide array of granule-bound proteases and cytolytic enzymes. This renders the neutrophil very efficient in host defense but also potentially dangerous if not properly regulated. In inflammatory diseases such as rheumatoid arthritis and gout, the neutrophil is thought to be largely responsible for tissue damage caused by the excessive release of its cytolytic enzymes into the synovial fluid as well as the aberrant production and secretion of pro-inflammatory chemokines and cytokines [1 ].

Acute gouty arthritis is characterized by the deposition of monosodium urate microcrystals (MSU) in the synovial fluid and is associated with intense pain and swelling of the joint [2 , 3 ]. Studies have shown that the initial interaction between synovial cells and MSU crystals is crucial to the development of acute gouty attacks. The synovial response leads to the accumulation of immune cells, which release a variety of pro-inflammatory signals such as interleukin (IL)-1ß [4 ], IL-6 [5 ], IL-8 [6 ], tumor necrosis factor {alpha} (TNF-{alpha}) [7 ], prostaglandin E2 [8 , 9 ], leukotriene B4 [10 ], and crystal chemotactic factor (CCF) [11 ]. These signals are likely to be responsible for the recruitment of several immune cells and neutrophils in particular. The interaction of neutrophils with MSU crystals results in the release of lysosomal enzymes [12 ], oxygen-derived free radicals [13 14 15 16 17 18 19 ], eicosanoids, IL-1 [20 21 22 ], and IL-8 [20 , 23 ]. These mediators amplify and perpetuate the inflammatory reaction, which can lead, if unchecked, to tissue damage (as reviewed in ref. [1 ]).

The transduction signals underlying the interactions between MSU crystals and neutrophils are not fully understood. There is evidence that MSU crystals interact with the Fc receptor for IgG (Fc{gamma}R)IIIb (CD16) in association with the CD11b/CD18 integrin complex [24 ]. A significant increase in tyrosine phosphorylation of many proteins [25 , 26 ], including Syk [24 ] and Cbl [27 ], has been described in response to MSU crystals. Several other responses, including increases in the cytoplasmic concentration of free calcium, activation of phosphatidylinositol-3-kinase [19 ], and phospholipases D (PLD) [28 , 29 ] and A2 [30 ], are also induced by MSU crystals. The majority of these responses are regulated by tyrosine kinases, as evidenced by the inhibitory effects of tyrosine kinase inhibitors [19 , 29 , 31 , 32 ]. The ability of these compounds to limit neutrophil responses to MSU crystals indicates that tyrosine-phosphorylation pathways play a central role in orchestrating them.

The cytoplasmic tyrosine kinase Syk is known to be implicated in the stimulation of cells by Fc receptors in macrophages [33 , 34 ], mastocytes, and neutrophils [34 ]. Syk contains two tandem SH2 domains that associate during activation with tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of immune receptors such as those present on some Fc receptors (but not CD16) in neutrophils, on the associated chains of the T-cell receptor (TCR) and B-cell receptor (BCR) [34 ], and on a catalytic domain that enables Syk to phosphorylate itself and other proteins. The tyrosine kinase Syk has also been shown to associate with the cytoplasmic portion of CD18 [35 ]. The crucial role of Syk in phagocytosis has been established using syk-/- macrophages and piceatannol-treated neutrophils that lose the capacity to ingest antibody-covered erythrocytes [33 , 36 , 37 ]. The transfection of COS cells with a chimeric Fc{gamma}RIII-Syk protein is enough to confer a phagocytic capacity upon these cells [38 ]. The mutation or deletion of the Syk kinase domain greatly diminishes the phagocytic capacity of the transfected COS cells [38 ]. Other studies have implicated Syk in integrin signaling and spreading [39 40 41 ]. Conversely, CD11b/CD18 stimulation did not elicit Syk tyrosine phosphorylation [42 ]. Finally, Syk is linked to the activation and phosphorylation of a wide variety of proteins, including PI3K [37 , 43 ], Vav [44 ], Cbl [45 ], paxillin [46 ], tubulin [47 ], and Syk itself [48 ].

The present study aimed to clarify the role of Syk in the activation of neutrophils by MSU crystals. We first characterized the effects of the microcrystals on the tyrosine-phosphorylation status and enzymatic activity of Syk. We also used piceatannol, which was described as a Syk inhibitor [37 ], to determine the implication of Syk in various responses of human peripheral blood neutrophils to MSU crystals, namely tyrosine phosphorylation, activation of PLD, production of superoxide anions, and calcium mobilization. The results obtained support the hypothesis that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The enhanced chemiluminescence (Renaissance) reagents used for immunoblotting were purchased from DuPont Pharmaceuticals (Mississauga, Ontario, Canada). Piceatannol (El-227) was purchased from Biomol (Plymouth Meeting, PA). Diisopropylfluorophosphate (DFP), cytochrome C, and Nonidet P-40 (NP-40) were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). SAM68-GST (sc-4249) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Dextran, Sephadex G-10, and glutathione protein A-Sepharose were purchased from Pharmacia (Baie d’Urfé, Québec, Canada). Ficoll-Paque and the Mg2+-free Hank’s balanced salt solution (HBSS) were obtained from Wisent Canadian Laboratories (St-Bruno, Québec, Canada).

Antibodies
F(ab')2 fragments of Ab IV.3 (hybridoma obtained from American Type Culture Collection, Manassas, VA) were prepared essentially as described in the Pierce catalog (Rockford, IL). Briefly, the antibodies (Abs) were digested with pepsin (as pepsin beads), and intact Abs were eliminated by adding protein A and protein G beads. The integrity of the F(ab')2 fragments was verified by their ability to label intact, human neutrophils as determined by flow cytometry. Affinity-purified F(ab')2 goat anti-mouse immunoglobulin G (IgG) F(ab')2 (115-006-072) and peroxidase-labeled anti-mouse (115-095-072) or anti-rabbit IgG (711-035-152) Abs were obtained from Jackson ImmunoResearch (West Grove, PA). The monoclonal anti-Syk (MAB88906), used for immunoprecipitation and immunoblotting, was purchased from Chemicon International Inc. (Tenecula, CA). The anti-phosphotyrosine Ab (UBI-05-321, clone 4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). The F(ab')2 anti CD-16 (3G8) (028-2) was purchased from Medarex Research Reagents (Annandale, NJ). The polyclonal anti-Cbl (sc-170) and anti-SAM68 (sc-333) were obtained from Santa Cruz Biotechnology.

Neutrophil purification
Blood was obtained from the peripheral vein of healthy adults. The neutrophils were obtained by means of 2% Dextran sedimentation followed by standard techniques of Ficoll gradients and hypotonic lysis of erythrocytes. The cells were resuspended in HBSS containing 1.6 mM calcium and no magnesium (pH 7.4).

Tyrosine phosphorylation
Neutrophil suspensions (4x107 cells/ml) were incubated at 37°C with MSU crystals (3 mg/ml) for 10 min and F(ab')2 IV.3 anti-CD32 fragments (2.5 µg/ml) for 1 min at 37°C, followed by ligation with F(ab')2 anti-F(ab')2 (25 µg/ml) for 1 min at 37°C and F(ab')2 3G8 anti-CD16 (25 µg/ml) for 15 min on ice, followed by an incubation with F(ab')2 anti-F(ab')2 (150 µg/ml) for 30 s or with fMet-Leu-Phe (fMLP; 10-7 M) for 30 s. The concentrations of the primary and the cross-linking antibodies were determined empirically from concentration-response curves using flow cytometry and amplitude of the tyrosine-phosphorylation response, respectively. The reactions were stopped by the addition of cell aliquots to an equal volume (100 µL) of boiling 2x Laemmli sample buffer [1x is 62.5 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol blue] and were boiled for 7 min. Samples were then subjected to 7.5–20% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA). Immunoblotting was performed using the 4G10 antiphosphotyrosine antibody at a final dilution of 1/4000 and revealed by the Renaissance Plus detection system as previously described [49 ]. Piceatannol, when added, was incubated with cells at 107 cells/ml before being resuspended in the same incubation medium (because there are indications of reversible activity of piceatannol) at 4 x 107 cells/ml and then stimulated with the appropriate stimuli.

Immunoprecipitation
Neutrophils were stimulated as described above but lysed in a denaturing lysis buffer (1x is 62.5 mM Tris-HCl, pH 6.8, 3% SDS, 1.5% ß-mercaptoethanol, 8.5% glycerol, 2.5 mM orthovonadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.025% bromophenol blue) as previously described [50 ]. These denatured lysates were filtered through Sephadex G-10 columns to remove the denaturing and the reducing agents; then NP-40, orthovonadate, aprotinin, and leupeptin (final concentrations, 1%, 2mM, 10 µg/ml, and 10 µg/ml, respectively) were added. Lysates were incubated for 90 min with anti-Syk Abs previously bound to protein A-Sepharose (1 µg anti-Syk Abs for 50 µl of a 30% slurry of protein A-Sepharose beads). The cells were centrifuged at 13,000 g for 5 min, and Sepharose-A beads coupled to anti-Syk were added to the supernatants and incubated at 4°C for 2 h. The beads were washed three times with 1% NP-40 buffer [137 mM NaCl, 1 mM ethylenediaminetetraacetate (EDTA), 2 mM DFP, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM orthovanadate, 50 µg/ml soybean trypsic inhibitor, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] before being submitted to immunoblot analysis with an anti-phosphotyrosine antibody. Cells were also lysed under native conditions in a hypotonic buffer (10 mM NaCl, 50 mM Tris, pH 8.0, 3 mM DFP, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM orthovanadate, 50 µg/ml soybean trypsic inhibitor, and 0.5 mM PMSF) [46 ], sonicated for 5 s at constant minimum intensity in a Branson Sonifier 450, and then centrifugated at 13,000 g for 5 min at 4°C. The supernatants were incubated with anti-Syk Abs coupled to protein A-Sepharose beads as described above, and the buffer is rendered isotonic by the addition of NaCl (137 mM) and is also more stringent with the addition of 1% NP-40. The beads were then washed three times in the isotonic 1% NP-40 buffer before being analyzed by immunoblot with an anti-phosphotyrosine antibody.

Autophosphorylation assay
Neutrophils were lysed and immunoprecipitated under native conditions as described above. The immunoprecipitates were washed three times in the 1% NP-40 isotonic buffer and then washed three times in kinase buffer (50 mM HEPES, pH 7.6, 10 mM MnCl2, 2 mM MgCl2, 1 mM pNPP, and 10 µM orthovanadate) as described by Fernandez and Suchard [46 ]. The beads were resuspended in kinase buffer with 100 µM adenosine 5'-triphosphate (ATP) and incubated at 37°C for 10 min, which corresponds to the maximum of tyrosine phosphorylation, before addition of an equal volume of boiling 2x sample buffer. The samples were then analyzed by immunoblot using an anti-phosphotyrosine antibody.

Kinase assay
This was done exactly like the autophosphorylation assay described above except that 1 µg SAM68-GST was added to the Syk immunoprecipitates just before the addition of 50 µM ATP. After 10 min of incubation, the beads were pelleted by centrifugation precipitate, and the supernatant was harvested and added to a small volume of 1% NP-40 isotonic buffer containing 30 µl gluthatione-Sepharose 4B beads. The supernatant was incubated for 1 h and then washed three times in 1% NP-40 isotonic buffer before being analyzed by Western blot with an anti-phosphotyrosine antibody or an anti-SAM68 antibody.

Calcium mobilization
The cells (107 cells/ml) were incubated for 30 min at 37°C with 1 µM fura-2/AM. The neutrophils were washed once in HBSS to remove the extracellular probe, resuspended at 5 x 106 cells/ml, and transferred to the thermostatted (37°C) cuvette compartment of a spectrofluorimeter (SLM 8000, Aminco, Urbana, IL). The fluorescence of the cells was monitored at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The internal calcium concentrations were calculated as described by Grynkiewicz et al. [51 ].

PLD measurements
Neutrophils were pre-labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 cells) for 90 min as described previously [29 ]. The cells were then washed and resuspended at 8 x 106 cells/ml. Samples of the cell suspensions (0.5 ml) were pre-incubated at 37°C for 5 min and pretreated with piceatannol (40 µM) before stimulation with MSU crystals (3 mg/ml) for 15 min. The incubations were stopped by adding 1.8 ml cold chloroform/methanol/HCl (50:100:1, vol/vol/vol) and unlabeled phosphatidylethanol (PEt) as a standard. The lipids were extracted, dried under nitrogen, and spotted on pre-washed silica gel 60 thin-layer chromatographic (TLC) plates. PEt was separated from the other lipids with the solvent mixture chloroform/methanol/acetic acid (65:15:2, vol/vol/vol). Lipids were visualized by Coomassie brilliant blue-staining, and the different lipid classes were scraped off the plates. Radioactivity in PEt was monitored by liquid scintillation counting, and the results were corrected for background radioactivity and quenching.

Superoxide production measurement
Neutrophils (106 cells/ml) were incubated for 5 min at 37°C with cytochrome C (final concentration 62.5 µM). After appropriate stimulation with the MSU crystals at 1.5 mg/ml for 5 min at 37°C, the reactions were stopped on ice after a brisk agitation. The cells were then centrifugated at 600 g at 4°C for 10 min. The optical density of the supernatants was read at 540 and 550 nm in a Perkin-Elmer spectrophotometer, and the amount of superoxide produced was calculated from the difference between these two readings from those of untreated cells using an extinction coefficient of 21.1. The data shown are the increase of superoxide production compared with untreated cells.

Microscopy
The neutrophils (2x107 cells/ml) were incubated with MSU (1 mg/ml) for 30 min in 6-well plates at 37°C before being observed with a polarized light microscope. Wherever indicated, the cells were pre-incubated with piceatannol (40 µM) for 10 min at 37°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of piceatannol on the tyrosine-phosphorylation response induced by MSU crystals
Neutrophils (4x107 cells/ml), pre-incubated or not with piceatannol (4–100 µM, 10 min at 107 cells/ml), were stimulated with MSU crystals (3 mg/ml) for 10 min. The reactions were stopped by direct transfer of cell-suspension aliquots to an equal volume of boiling 2x sample buffer and were analyzed by immunoblot using an anti-phosphotyrosine antibody. The results of a representative experiment are illustrated in Figure 1 . The addition of MSU crystals, as previously described [25 , 26 ], increased the tyrosine phosphorylation of proteins with apparent molecular masses of 130, 118, 80, 70, and 60 kDa with major phosphorylated substrates at 118 and 70 kDa. The tyrosine kinase Syk has been identified as one of the tyrosine-phosphorylated substrates that migrate at 70 kDa [24 ]. Pre-incubation of neutrophils with increasing concentrations of piceatannol inhibited the pattern of tyrosine phosphorylation in a concentration-dependent manner, from a mild inhibition at 4–40 µM to a practically complete inhibition at 100 µM. All the major bands of tyrosine phosphorylation, including those at 70 and 118 kDa, were inhibited by piceatannol. All further experiments were done with 40 µM piceatannol, because we observed some inhibition at that concentration, and there was indication of nonspecific impact on the Src family kinase Lyn at 100 µM [36 ].



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  		Figure 1. Effect of piceatannol on the tyrosine phosphorylation induced by MSU crystals. The cells (107 cells/ml) were incubated with 1 mM DFP and the indicated concentrations of piceatannol for 10 min at 37°C and then stimulated at 4 x 107 cells/ml with MSU crystals (3 mg/ml) for 10 min. The reactions were stopped by transfer of cell aliquots to an equal volume of 2x sample buffer and boiled for 7 min. The samples were analyzed by SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody. The results shown are from a single experiment representative of three independent experiments.

 
Effect of piceatannol on the tyrosine-phosphorylation pattern induced by ligation of CD32 and CD16 and the addition of fMLP
As a result of the evidence indicating that MSU crystals interact with at least a subset of Fc{gamma}Rs in human neutrophils [24 ], the effects of piceatannol on the tyrosine-phosphorylation patterns induced by ligation of CD32 and CD16 were investigated next. Previous studies in monocytes [52 , 53 ] and Fc{gamma}RII-transfected cells [54 ] have implicated the tyrosine kinase Syk in the responses to cross-linking of CD32. Neutrophils (107 cells/ml) were pre-incubated with piceatannol (40 µM) for 10 min at 37°C and then resuspended at 4 x 107 cells/ml. CD32 and CD16 were then cross-linked as described in Materials and Methods for 1 min and 30 s, respectively. Immunoblot analysis with an anti-phosphotyrosine antibody showed that the cross-linking of CD32 induced robust increases in the levels of tyrosine phosphorylation in stimulated cells (Fig. 2A ). The addition of piceatannol did not diminish the pattern of tyrosine phosphorylation induced by ligation of CD32, although slight delays (30 s) in the initial kinetics of tyrosine phosphorylation were noted in some cell populations (unpublished results). The ligation of CD16 also resulted in a strong increase in tyrosine phosphorylation (Fig. 2A) . The addition of piceatannol largely inhibited the pattern of tyrosine phosphorylation induced by ligation of CD16. We also observed that fMLP-induced tyrosine phosphorylation is not inhibited by piceatannol (Fig. 2B) , although some minor kinetic delays were also noted (unpublished results).



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  		Figure 2. Effect of piceatannol on the tyrosine phosphorylation induced by ligation of CD32 or CD16 and fMLP. The cells were incubated at 107 cells/ml with 1 mM DFP and piceatannol (40 µM) for 10 min at 37°C and then stimulated at 4 x 107 cells/ml by cross-linking CD32 and CD16 (A) or by addition of fMLP (B) as described in Materials and Methods. The control cells (Ctrl) were incubated only in the presence of HBSS. The reactions were stopped by transfer of cell aliquots to an equal volume of 2x sample buffer and boiled for 7 min. The samples were analyzed by SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody. The results shown are from a single experiment representative of three separate experiments.

 
Effect of piceatannol on the tyrosine phosphorylation of Syk induced by ligation of CD32 and MSU crystals
Neutrophils (107 cells/ml) were pre-incubated with piceatannol (40 µM) for 10 min at 37°C and then resuspended to 4 x 107 cells/ml before being stimulated by ligation of CD32 (1 min) or the addition of MSU crystals (10 min). The reactions were stopped in the denaturing lysis buffer in the case of the CD32 cross-linked samples or by sonication in the hypotonic buffer described in Materials and Methods for the MSU-stimulated samples. Similar results were obtained by immunoprecipitation with sonication in the hypotonic buffer, although the denaturing lysis buffer better preserved the tyrosine phosphorylation of Syk in response to the ligation of CD32 (unpublished results). Following immunoprecipitation with an anti-Syk antibody, as described in Materials and Methods, the immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody (Fig. 3 ). Nonstimulated cells (ctrl) showed little, if any, basal tyrosine phosphorylation of Syk. In contrast, ligation of CD32 caused an increase in the tyrosine phosphorylation of Syk that was not affected by piceatannol (Fig. 3A) . MSU crystals also increased the level of tyrosine phosphorylation of Syk (Fig. 3B) . This response to MSU crystals, in contrast to that of CD32 cross-linking, was markedly inhibited by piceatannol. Equal loading of Syk was verified by reprobing an identically loaded membrane with an anti-Syk antibody. These data are derived from separate experiments. Although the relative intensity of tyrosine phosphorylation of Syk resulting from the ligation of CD32 or from the addition of MSU crystals varied to some extent between donors, MSU crystal-elicited responses were inhibited consistently by piceatannol, and those to CD32 cross-linking were not.



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  		Figure 3. Effect of piceatannol on the tyrosine phosphorylation of Syk induced by ligation of CD32 or addition of MSU crystals. The cells (107 cells/ml) were incubated with or without piceatannol (40 µM) for 10 min at 37°C. The cells were resuspended at 4 x 107 cells/ml, incubated with 1 mM DFP for 10 min at 37°C, and then stimulated by cross-linking CD32 (A) or upon the addition of MSU crystals (3 mg/ml; B). An equivalent volume of HBSS was added to the unstimulated cells (ctrl). The reactions were stopped in the denaturing lysis buffer in the case of the CD32 cross-linked samples or sonicated in the hypotonic buffer for the MSU crystal-stimulated samples. Immunoprecipitation with anti-Syk antibodies (1 µg) was carried out as described in Materials and Methods. Immunoprecipitates were subjected to electrophoresis on two identical gels. After transfer onto PVDF membranes, one membrane was probed with an anti-phosphotyrosine antibody (pY), and the second identical membrane was probed with an anti-Syk antibody (Syk). The data shown are representative of three independent experiments.

 
Effect of piceatannol on the in vitro autophosphorylation of Syk
The ability of piceatannol to inhibit the in vitro autophosphorylation activity of Syk was tested directly. Unstimulated neutrophils were sonicated in the hypotonic lysis buffer, and Syk was immunoprecipitated as described in Materials and Methods. The beads were then washed three times in lysis buffer and three additional times in kinase buffer before resuspension in kinase buffer containing 50 µM ATP and varying quantities of piceatannol and were incubated for 10 min at 37°C. The level of tyrosine phosphorylation of Syk was then monitored by immunoblotting with an anti-phosphotyrosine antibody. As can be seen in Figure 4 , Syk derived from unstimulated cells autophosphorylated itself on tyrosine residues in the presence of ATP (Fig. 4 , lanes 1 and 2). The addition of piceatannol decreased the kinase activity of Syk in a concentration-dependent manner (Fig. 4 , lanes 3–7). A Syk reblot demonstrated that equal amounts of Syk were loaded in each lane.



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  		Figure 4. Inhibition of the autophosphorylation of Syk by piceatannol. Cells (4x107 cells/ml) were sonicated in the hypotonic buffer. The lysates were processed for immunoprecipitation with an anti-Syk antibody (1 µg) as described in Materials and Methods. The immunoprecipitates were washed three times in the 1% NP-40 isotonic buffer and then three times in the kinase buffer and incubated with the indicated concentrations of piceatannol before the addition of ATP (100 µM), except for the control (Ctrl) sample, which did not contain ATP. Immunoprecipitates were subjected to electrophoresis on two identical gels. After transfer onto PVDF membranes, one membrane was probed with an anti-phosphotyrosine antibody (pY) and the second identical membrane was probed with an anti-Syk antibody (Syk). The data shown are representative of three independent experiments.

 
Effect of piceatannol on the kinase activity of Syk
We used a SAM68-GST construct to monitor the effects of piceatannol on the kinase activity of Syk toward an exogenous substrate. SAM68-GST was added, with or without piceatannol (40 µM), to Syk immunoprecipitates from unstimulated cells in the kinase buffer before the addition of ATP. SAM68-GST was harvested and purified as described in Materials and Methods. The level of tyrosine phosphorylation of SAM68-GST was monitored by immunoblotting with an anti-phosphotyrosine antibody. As shown in Figure 5A , SAM68 was tyrosine-phosphorylated by Syk, and this phosphorylation was inhibited by the addition of piceatannol. We next immunoprecipitated Syk from unstimulated (Ctrl) or MSU crystal-stimulated neutrophils (3 mg/ml, 10 min) and monitored the ability of immunoprecipitated Syk to phosphorylate SAM68-GST. The results of these experiments are illustrated in Figure 5B . These data show that the kinase activity of Syk toward SAM68-GST derived from MSU crystals was significantly higher than that of Syk immunoprecipitated from unstimulated cells.



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  		Figure 5. Tyrosine phosphorylation of SAM68-GST by Syk. (A) Unstimulated cells (4x107 cells/ml) were sonicated in the hypotonic buffer as described in Materials and Methods. The lysates were processed for immunoprecipitation with an anti-Syk antibody (1 µg). The immunoprecipitates were washed three times in the 1% NP-40 isotonic buffer and then three times in the kinase buffer and incubated with 40 µM piceatannol and 1 µg SAM68-GST before the addition of ATP (50 µM). (B) Cells (4x107 cells/ml) were unstimulated (Ctrl) or stimulated with MSU crystals (3 mg/ml) for 10 min before being sonicated in the hypotonic lysis buffer. The lysates were processed for immunoprecipitation with an anti-Syk antibody (1 µg) in the 1% NP-40 isotonic buffer as described above. After the kinase assay, SAM68-GST precipitates were subjected to electrophoresis. The membranes were probed with an anti-phosphotyrosine antibody (pY) and then reprobed with an anti-SAM68 antibody (SAM).

 
Functional significance of the activation of Syk in MSU crystal-stimulated neutrophils
Having established that Syk was involved in the response of human neutrophils to the addition of MSU crystals, the potential downstream effector systems of this tyrosine kinase were then studied. We first investigated the effect of piceatannol on the mobilization of intracellular calcium. As shown in Figure 6 , and as previously demonstrated [31 ], significant increases in the concentration of cytoplasmic-free calcium are evident in response to the addition of MSU crystals. Preincubation of neutrophils with piceatannol (40 µM) inhibited the mobilization of intracellular calcium induced by MSU crystals by as much as 70%.



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  		Figure 6. Effect of piceatannol on the mobilization of calcium induced by MSU crystals in human neutrophils. The cells (107 cells/ml) were loaded with 1 µM Fura-2/AM as described in Materials and Methods. Piceatannol (40 µM) was added for the last 10 min of the incubation with Fura-2/AM. The arrow represents the time of addition of the MSU crystals (0.3 mg/ml). The data shown are representative of three independent experiments.

 
MSU crystals have also been shown to stimulate the activity of PLD in human neutrophils [29 ]. As shown in Figure 7 , piceatannol markedly reduced the stimulation of PLD activity induced in human neutrophils by MSU crystals. The effects of piceatannol on the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by MSU crystals were then examined. As summarized in Figure 8 , piceatannol decreased, in a concentration-dependent manner, the oxidative response to MSU crystals.



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  		Figure 7. Effect of piceatannol on MSU crystal-induced PLD activity, which was monitored as described in Materials and Methods. The cells were pre-incubated with piceatannol (Pic; 40 µM, 10 min) before being stimulated by the addition of 3 mg/ml MSU crystals. Mean ± SE of three independent experiments.

 


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  		Figure 8. Effect of piceatannol on the production of superoxide anions induced by MSU crystals. The cells (107 cells/ml) were pre-incubated with varying concentrations of piceatannol for 10 min. The cells (106 cells/ml) were then incubated at 37°C for 5 min with cytochrome C (62.5 µM) before stimulation with MSU crystals (1.5 mg/ml). The stimulations were stopped after 5 min by transfer to 4°C. Levels of O2- were measured as described in Materials and Methods. The data are the means ± SE from four independent experiments. * = P < 0.05 (Student’s t-test).

 
The effects of piceatannol on the interaction of MSU crystals with human neutrophils were next directly visualized by polarized light microscopy. MSU crystals (1 mg/ml) were added to neutrophil suspensions for 30 min at 37° before being observed under polarized light (Fig. 9 ). The untreated cell samples show that a small percentage of the cells were in direct contact with the crystals. Several cells (indicated by arrows) appear to have internalized MSU crystals as evidenced by their adoption of oval-like shapes approximating the contours of the apposed crystals. The morphological changes seen in control cells were largely absent in piceatannol-treated cells, which retained the circular, symmetric morphology of unstimulated cells. No evidence of internalization of MSU crystals was observed in piceatannol-treated cells.



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  		Figure 9. Microscopic observations of neutrophil-MSU crystal interactions. Cells (1.5x107 cells/ml) were incubated with piceatannol (40 µM) for 10 min before the addition of MSU crystals (1 mg/ml). After 30 min, the cells were then transferred to a 6-well plaque and visualized under a polarized light microscope. (A) Untreated cells with MSU crystals; (B) piceatannol-treated cells with MSU crystals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The involvement of the tyrosine kinase Syk in the mediation of the responses of human neutrophils to MSU crystals, the etiological agent of gout, was investigated. The results of these studies indicate that the interaction of MSU crystals with neutrophils leads to the stimulation of Syk by mechanisms that differ pharmacologically from those called upon subsequent to the ligation of CD32. Moreover, evidence was also obtained indicating that the activation of Syk lies up-stream of the initiation of the mobilization of calcium and of the stimulation of PLD and the NADPH oxidase activities. Finally, morphological data were also obtained, which support the hypothesis that Syk is involved in the internalization of crystals by human neutrophils.

The interaction of MSU crystals with human neutrophils has been shown to be mediated by CD16 and CD11b/CD18 [24 ] and to be associated with a pattern of tyrosine phosphorylation, which is qualitatively and pharmacologically distinct from that induced by soluble neutrophil agonists [25 , 26 ]. Although relatively few MSU crystal-responsive, tyrosine-phosphorylated substrates have been identified to date, one of them is the tyrosine kinase Syk [24 ]. Syk has been closely linked to the mediation of the activity of several Fc receptors [33 , 34 , 55 ], in particular to that of CD32 [49 , 53 , 56 ], and to the control of the phagocytic process [33 , 34 , 37 , 38 ].

Several lines of evidence were obtained indicating that Syk is centrally involved in the responses of human neutrophils to MSU crystals. These include the following observations: Tyrosine-phosphorylated Syk was recovered from neutrophil lysates derived from MSU crystal-stimulated cells; the activity of Syk derived from MSU crystal-stimulated cells toward an exogenous substrate (SAM68-GST) was higher than that of Syk isolated from control cells; piceatannol decreased the level of tyrosine phosphorylation induced by MSU crystals (including the stimulation of the tyrosine phosphorylation of Syk itself); piceatannol inhibited the mobilization of calcium and the activation of PLD and NADPH oxidase stimulated by MSU crystals; and piceatannol inhibited the morphological evidence of internalization of MSU crystals by neutrophils.

The specifics of the interactions of MSU crystals with human neutrophils are still poorly understood. However, the use of blocking antibodies has recently provided support for the hypothesis that the microcrystals interacted, probably fortuitously, with Fc{gamma}RIIIb, the glycosylphosphatidylinositol-linked CD16 isoform constitutively expressed on the surface of human neutrophils [57 ]. The ability of piceatannol to inhibit the tyrosine-phosphorylation response induced in human neutrophils subsequent to the cross-linking of CD16 is consistent with this hypothesis. Previously, it had been postulated that MSU crystals interacted specifically with CD16 and not with CD32. The present observation that piceatannol affects the tyrosine-phosphorylation responses to CD16 (but not CD32), much as it does that to MSU crystals, further supports this conclusion. However, the apparent lack of effect on the CD32 responses is, at first sight, somewhat unexpected in view of the link previously established between Syk and phagocytosis. However, it should be pointed out that the indices monitored in the present study, namely the whole-cell tyrosine-phosphorylation patterns and the tyrosine-phosphorylation status of Syk, only provide a partial and perhaps incomplete picture of the signaling events. The relative lack of effect of piceatannol on the whole-cell phosphorylation pattern induced by CD32 indicates that this response relies on multiple kinases, with Syk contributing only a quantitatively minor proportion to the overall response and with Src family kinases very likely to be critically involved. These data do not rule out a role for Syk in the functional responses elicited by cross-linking CD32. Indeed, piceatannol concentration dependently inhibited the mobilization of calcium induced by ligation of CD32 (unpublished results).

Piceatannol differentially affected the in situ tyrosine phosphorylation of Syk, strongly inhibiting that induced by MSU crystals and not affecting that observed in response to cross-linking CD32. Syk is known to be tyrosine-phosphorylated through two alternative, possibly sequential, pathways: through the action of an up-stream src-family kinase and autophosphorylation. In the case of MSU crystals, Syk phosphorylation may be a result of, in large part, autophosphorylation events. The lack of effect of piceatannol on the in situ tyrosine phosphorylation of Syk observed subsequently to the ligation of CD32 indicated that this response, contrary to that of MSU crystals, depends to a major extent on kinases other than Syk itself, most likely Src family members.

We used an exogenous substrate, SAM68-GST, to study the activity of Syk. SAM68 was chosen as a substrate because this protein is tyrosine-phosphorylated in response to the addition of MSU crystals and to cross-linking CD32 [58 ]. SAM68 is a 68-kDa protein of the STAR family known to be a substrate for the Src kinase family [59 ], and a sequence analysis points to a strong affinity for RNA [60 ]. It is also established that SAM68 plays a role in the tyrosine kinase pathways by serving as a potential substrate for numerous kinases (Src, Fyn, Lck, Btk, Tec, ZAP70, Jak3) [59 60 61 62 63 64 65 66 67 68 69 ]. We observed a marked tyrosine phosphorylation of SAM68-GST by Syk in the in vitro kinase assay, which was markedly inhibited by the addition of piceatannol. Together with the above-described data, these results show that piceatannol inhibits the autophosphorylation and kinase activity of Syk. We also observed an increase in the ability of Syk isolated from crystal-stimulated cells to tyrosine-phosphorylate SAM68-GST. This indicates that the kinase activity of Syk can be regulated depending on the state of activation of the cells. The implications of Syk-SAM68 interactions on neutrophil activation are, as of yet, unknown. However, these results raise the possibility that Syk, by way of SAM68, may participate in the regulation of RNA translation.

The results obtained support the idea that Syk lies up-stream of the sequence of events leading to multiple signaling responses in human neutrophils stimulated by MSU crystals. Piceatannol nearly abrogated the mobilization of calcium and the activation of the activity of PLD and the NADPH oxidase induced by the microcrystals. The calcium and superoxide responses to calcium pyrophosphate crystals have previously been shown to be inhibited by another tyrosine kinase of undefined kinase specificity, namely methyl 2,5-dihydroxycinnamate [32 ]. The present results indicate that Syk may be the proximal tyrosine kinase involved in these responses. They also suggest that Syk may be involved in the stimulation of a phospholipase C (PLC)-{gamma} upon the addition of MSU crystals, a point that warrants direct examination. The magnitude of the inhibitory effects of piceatannol on these three responses indicates that Syk is a major contributor to the initiation of these pathways.

A series of microscopic observations permitted us to observe that the engulfment of MSU crystals by neutrophils was inhibited by piceatannol. No internalization or even detectable surface-binding of MSU crystals was apparent in piceatannol-treated cells. The absence of binding of MSU crystals to piceatannol-treated cell ligation points to some sort of inside-out signaling, probably dependent on an association of Syk with CD11b/CD18, thereby indicating that Syk may be involved in the earliest stages of the phagocytic process.

Nonetheless, it is important to take note of the potential, nonspecific effects of piceatannol or indeed of any inhibitor. The lack of inhibition of the stimulation of the levels of tyrosine phosphorylation induced by CD32 ligation and the addition of fMLP provide evidence of a certain degree of specificity of piceatannol under the experimental conditions (inhibitor and cell concentrations; time of incubation) used in the present study. However, recent studies have shown that piceatannol might reduce the activation of p38 mitogen-activated protein kinase (MAPK) in response to fMLP, an agonist which does not stimulate the tyrosine phosphorylation of Syk [70 ], and it may also inhibit the kinase activity of Src and FAK [71 ] and the tyrosine phosphorylation of STAT3 and STAT5 [72 ]. Although Syk is not tyrosine-phosphorylated in response to fMLP [70 ], its implication in the responses to this chemotactic factor cannot be ruled out categorically. An association with other kinases or the formation of various active complexes, e.g., with Vav, tubulin, paxillin, Cbl, PI3K, or its serine/threonine phosphorylation, could conceivably impact on the activity of Syk without altering its level of tyrosine phosphorylation. It is perhaps relevant in this respect to point out that the kinase activity of Syk in fMLP-stimulated cells has not been directly monitored as of yet.

The results gathered in this study as well as in others have implicated CD16 in the response to MSU crystals [24 ]. Evidence has pointed to an interaction of CD16 with CD11b/CD18 in MSU recognition [24 ]. Existing models also propose an interaction between CD16 and CD11b/CD18 [73 , 74 ], and the results of previously published experiments show that CD11b/CD18 is essential for CD16-mediated phagocytosis [75 ]. Further studies will be necessary to determine whether CD11b directly interacts with MSU crystals or is simply needed for adequate signaling by CD16. The details of the transmission of the activation signal from CD16 to Syk also remain to be elucidated. However, CD18 has been shown to associate with Syk [35 ] and could, therefore, serve as an intermediary in neutrophil activation mediated by MSU crystals. Although the present data indicate that Syk may play an important role in MSU crystal-induced, neutrophil stimulation, the elucidation of the specifics of the activation of Syk requires further investigation.

In summary, the results of the dual approach used in this study, with the direct observation of Syk activity and with piceatannol, indicate that the tyrosine kinase Syk plays a central role in the mediation of the responses of human neutrophils to MSU microcrystals. Direct immunobiochemical evidence for an activation of Syk was provided. The inhibitory effects of piceatannol on the functional responses to MSU crystals further support this conclusion.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants and fellowships from the Canadian Institutes of Health Research and from the Arthritis Society of Canada. C. G. is supported by a fellowship from the K.M. Hunter Charitable Foundation and the Canadian Institutes of Health Research.

Received December 22, 2000; revised May 14, 2001; accepted May 16, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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