* Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, and Departments of Medecine and
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
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Key Words: tyrosine phosphorylation • phagocytosis • gout • CD32
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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 
(TNF-) [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
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 FcRIII-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.
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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.
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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.
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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 FcRII-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).
 View larger version (59K): [in a new window] |
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.
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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.
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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.
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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).
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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.
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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).
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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.
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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 FcRIIIb, 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)- 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.
Received December 22, 2000; revised May 14, 2001; accepted May 16, 2001.
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