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Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-07-2346.
PHAGOCYTES
From the Department of Laboratory Medicine, University
of California, San Francisco, CA; the Department of Physiology,
Semmelweis University, Budapest, Hungary; and the Division of Allergy,
La Jolla Institute for Allergy and Immunology, San Diego, CA.
The Syk tyrosine kinase is essential for immunoreceptor and
multiple integrin functions as well as being implicated in signaling from G-protein-coupled receptors (GPCR) in cell lines, transfection systems, and pharmacologic studies. In contrast, using Syk-deficient primary cells, we show here that Syk does not play a major functional role in chemoattractant/chemokine signaling in neutrophils and mast
cells. syk Syk is a nonreceptor tyrosine kinase most abundant
in cells of the hematopoietic lineages. Its 2 tandem SH2 domains are
involved in the high-affinity binding to the dually phosphorylated
tyrosine residues of the immunoreceptor tyrosine-based activation
motifs (ITAMs) in multiple surface receptor complexes, thus recruiting and activating Syk. Concomitant phosphorylation of intracellular substrates of Syk will initiate downstream signaling events and cellular responses.1
Together with the related kinase ZAP-70, Syk long has been known to be
essential for immunoreceptor (B-cell receptor,2,3 T-cell
receptor,2,4-7 and FC
receptor8-10) function and for signaling from the
FC-receptor homolog GpVI,11 which serves as
the collagen receptor of platelets. More recently, we and others have
shown that Syk also is required for integrin signaling in neutrophils,12 platelets,13 and monocytic
cells.14 These observations, together with the recent
reports showing that Syk is expressed in multiple nonhematopoietic cell
types,15-17 support the emerging concept that Syk has a
broad function in multiple biologic processes beyond
immunoreceptor signaling and the mechanisms of adaptive immunity. The
function of Syk in integrin signaling also may be responsible for the
perinatal lethality of syk In contrast to the broad consensus about the role of Syk in
immunoreceptor and integrin function, its possible involvement in
G-protein-coupled receptor (GPCR) signaling is unclear and controversial. In correlative studies, several groups have shown activation of Syk in response to GPCR activation,18-24 and
Syk also appeared to be physically associated with the
Gs More importantly, several groups attempted to test the functional role
of Syk in GPCR signaling. In the first such report,26 overexpression of the dual SH2 domains of Syk in a rat basophilic cell
line failed to have any dominant-negative effect on the release of
arachidonic acid (AA) initiated through activation of a transfected muscarinic acetylcholine receptor (mAChR). In contrast, Wan et al18,19 showed impaired activation of mitogen-activated
protein (MAP) kinases through transfected mAChR in a
syk-deficient DT-40 chicken B-cell line. More recently, the
tyrosine kinase inhibitor piceatannol has been used to study the role
of Syk in multiple processes, including GPCR signaling. Piceatannol
inhibited the activation of the MAP kinase cascade by angiotensin II in
a JTC-27 rat liver-derived cell line.21 In our own
experiments with human neutrophils,27 piceatannol
inhibited the degranulation and the activation of the p38 MAP kinase
pathway in response to the bacterial chemoattractant formyl-Met-Leu-Phe
(fMLP), though the lack of concomitant activation of Syk made this
observation difficult to interpret. In studies on chemokine signaling
in the MonoMac6 human monocytic cell line, piceatannol inhibited the
activation of JNK1 by monocyte chemoattractant protein-1
(MCP-1)22 and the activation of extracellular
signal-related kinase (ERK) by soluble fraktalkine.23 In
rat peritoneal mast cells, piceatannol inhibited the release of AA in
response to the synthetic basic secretagog analog
c48/80.24
The overall impression from the above results would be that Syk is
widely required for multiple GPCR-mediated signaling processes and may
serve a central role in coupling these receptors to the different MAP
kinase pathways. Even the first functional report26 may be
consistent with this, since the dominant-negative truncation mutant
used in that study only interferes with the immunoreceptor tyrosine-based activation motif (ITAM)-mediated activation of Syk but not with a potential ITAM-independent mechanism that could be
involved in GPCR signaling. Such a mechanism has been suggested for the
integrin-mediated activation of Syk.28
There are, however, several issues that may raise doubts about the
conclusions of the above functional studies. The few genetic studies
were performed using transfection of mAChR into lymphocytic or mast
cell lines. The potentially very high expression level and the
nonendogenous nature of the receptors in the host cells, as well as the
long-term in vitro propagation of the cell lines used, temper the
physiologic relevance of those findings. The results with piceatannol
are even more problematic, since the specificity of this compound
toward Syk is highly questionable.27,29,30 In fact,
besides Syk, piceatannol also inhibits Src-family kinases as well as
the focal adhesion kinase (FAK) family,29 both of which seem to be involved in mediating GPCR signaling
events.31-33 Taken together, these results
indicate that despite the large number of reports dealing with the role
of Syk in GPCR signaling, this issue is still unresolved.
In this work we tested the effects of ligation of endogenous GPCRs in
syk Animals
Preparation of bone marrow neutrophils and neutrophil assay
conditions
To determine the amount of Syk- and Src-family kinases in wild-type and
syk All neutrophil assays were performed at 37°C in Hanks balanced salt
solution (HBSS) supplemented with 20 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4). To avoid activation of integrin-mediated
pathways, most experiments (except for adherent superoxide release and
migration assays) were performed in the absence of Mg2+
salts. Cells were kept at room temperature in Ca2+-free and
Mg2+-free medium until use and were preincubated for 10 minutes at 37°C in the assay medium prior to activation. Where
indicated, 10 µM cytochalasin B (CB; Sigma) was included in
this step. Cells were stimulated by the bacterial chemoattractant fMLP
(Sigma, St Louis, MO), the inflammatory mediators leukotriene
B4 (LTB4) and recombinant human C5a (both from
Sigma), or the murine chemokines MIP-1 Effector functions of neutrophils Superoxide release was measured by a cytochrome c reduction test as described.12 In case of experiments in suspension, plates were precoated with 10% FCS to further minimize the integrin-mediated adhesion of the cells.Release of the primary granule marker elastase was measured by a real-time fluorimetric assay37 using the fluorigenic elastase substrate peptide N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (Sigma). In this assay, the elastase activity in the extracellular space at any time point is proportional to the instantaneous slope of the increase in the fluorescence of the reaction product 7-amino-4-methylcoumarin. Suspensions containing 106/mL cells were preincubated and then supplemented with 20 µM elastase substrate peptide. The fluorescence of the samples was then followed at 380 nm excitation and 460 nm emission with constant stirring, using a Hitachi F-4500 spectrofluorimeter (Tokyo, Japan). Percent release of elastase was calculated by subtracting the elastase activity present prior to stimulation, and expression of the such obtained value in percent of the total cellular elastase activity, which was determined by treating the cells with 0.02% cetyltrimethyl-ammonium-bromide detergent (CTAB; Sigma) at the end of each run. Release of the secondary granule marker lactoferrin was measured by enzyme-linked immunosorbent assay (ELISA), as described previously,34 from supernatants of cells stimulated in 96-well polypropylene plates (Greiner, from Applied Scientific, South San Francisco, CA) for 10 minutes. Neutrophil migration Neutrophil migration was determined using a NeuroProbe 96-well migration chamber (NeuroProbe, Gaithersburg, MD) with uncoated polycarbonate membrane of 8 µm pore size, in the presence of 0.1% BSA. Neutrophils were prelabeled with 5 µM CellTracker Green (Molecular Probes, Eugene, OR). The bottom wells were filled with the indicated concentrations of the chemoattractants, and 200 µL of 5 × 106/mL prelabeled neutrophils were added to the top wells. After 45 minutes, the medium in the top wells was aspirated, and any remaining cells were wiped off the top of the filter. The bottom plate (with the filter still on top) was then centrifuged for 5 minutes at 400 × g. The filter was then separated, rinsed in PBS, and dried. The bottom wells were incubated for 20 minutes with 100 nM phorbol myristate acetate (PMA) (Sigma) to facilitate permanent adhesion of transmigrated neutrophils, washed, and the cells lysed with 0.1% Triton X-100 in PBS. The fluorescence of the filter (representing cells that transmigrated but remained adherent to the bottom of the membrane) and that of the wells of the bottom plate (representing cells that transmigrated and then fell from the filter) was determined by a PerSeptive Biosystems Cytofluor II fluorescence plate reader (Foster City, CA) at 485 nm excitation and 530 nm emission wavelengths and added together to obtain total transmigration. The values then were expressed in percent of the fluorescence of cells originally loaded to each well.In preliminary experiments, CD18 Intracellular signaling in neutrophils The intracellular Ca2+ concentration was determined by a ratiometric fluorescence assay.38 Cells were preincubated with 5 µM Fura-2-acetoxymethylesther (Molecular Probes) for 30 minutes at 37°C, washed, and kept on ice until use. Cells then were preincubated and the fluorescence followed at 340/380 nm dual excitation and 510 nm emission wavelengths using a Hitachi F-4500 spectrofluorimeter. At the end of each run, 0.2% Triton X-100 was added to determine the fluorescence of Fura-2 saturated with Ca2+, followed by addition of 10 mM EDTA (ethylenediaminetetraacetic acid) to obtain the same values in the absence of any free Ca2+. The intracellular Ca2+ concentration then was calculated as described by Rebres et al.39 To determine the release of Ca2+ from intracellular stores only, 5 mM EGTA (ethylene glycol tetraacetic acid) was added to the cells prior to stimulation. In these runs, fluorescence values corresponding to saturating Ca2+ concentrations were derived from parallel runs in the absence of EGTA treatment.Activation of the ERK and p38 MAP kinases was determined by immunoblotting, using phospho-specific anti-ERK and anti-p38-MAP kinase antibodies (both from Cell Signaling Technologies, Beverly, MA) as described.27 Non-phospho-specific control antibodies were obtained from Santa Cruz Biotechnologies. Polymerization of cellular actin was measured by a phalloidin binding assay. Cells were stimulated with the appropriate agonists for 30 seconds and then fixed in 10% formalin for 20 minutes at 4°C. Cells then were lysed with 0.2% Triton X-100, stained with 0.2 µM rhodamine-phalloidine (Molecular Probes) for 30 minutes on ice, washed, and the fluorescence of the cells determined by flow cytometry using a Becton Dickinson FACScan (San Jose, CA). Mast cell experiments Bone marrow cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 mM nonessential amino acids, 50 µM 2-merkaptoethanol, and 8% conditioned medium of interleukin-3 (IL-3) gene-transfected cells (bone marrow-derived mast cell [BMMC] medium). More than 95% of the trypan blue-excluding viable cells were mast cells after 4 weeks of culture. No discernible differences in morphology were detected between the wild-type and syk / genotypes, and surface expression of
FC -receptor (FCR) I and c-kit at similar
levels was confirmed by flow cytometry using a FACS-Calibur apparatus
and CELLQuest software (Becton Dickinson). In stimulation experiments,
BMMCs were cultured with BMMC medium without IL-3 for 3 hours followed
by one wash in Tyrode buffer (112 mM NaCl, 2.7 mM KCl, 0.4 mM
NaH2PO4, 1.6 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES (pH 7.5), 0.05% gelatin, 0.1%
glucose). 2 × 107 cells/mL in Tyrode buffer were
stimulated by 10 mM adenosine (Sigma-Aldrich, St Louis, MO)
for the indicated intervals. For stimulation of the
FCRI, BMMCs were sensitized by an overnight incubation
with 0.5 µg/mL anti-dinitrophenol (DNP) IgE, washed once in
Tyrode buffer, resuspended in Tyrode buffer to
2 × 107 cells/mL, and stimulated by polyvalent antigen,
DNP conjugates of human serum albumin (DNP-HSA at 100 ng/mL) for the
indicated time intervals.
Cells were lysed in ice-cold 1% NP-40-containing lysis buffer (20 mM Tris [tris(hydroxymethyl)aminomethane]-HCl [pH 8.0] 0.15 M NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin,10 mg/mL leupeptin, 25 mM p-nitrophenyl p-guanidinobenzoate, 1 mM pepstatin, and 0.1% sodium azide) immediately after stimulation. Lysates were centrifuged in an Eppendorf microcentrifuge at 4°C for 10 minutes. Protein concentrations of cleared lysates were measured using Dc protein assay reagents (Bio-Rad Laboratories, Hercules, CA). The lysates were directly analyzed by SDS-PAGE. Proteins separated by SDS-PAGE were electrophoretically transferred to polyvinylidene difluoride membranes (NEN Life Science Products, Boston, MA). Membranes were blocked then incubated consecutively with primary antibody (see next paragraph) and horseradish peroxidase-conjugated secondary antibody, and immunoreactive proteins were visualized by enhanced chemiluminescence reagents (NEN, Life Science Products). Anti-phospho-MAP kinase, anti-phospho-p38 MAP kinase, and anti-phospho-Akt (Ser473) were purchased from New England Biolabs (Beverly, MA). Anti-Akt1 (C-20), anti-ERK (C-16), and anti-p38 MAP kinase (C-20) were from Santa Cruz Biotechnologies. Presentation of data All presented data are representative of 3 or more independent experiments. Error bars represent SD of triplicate or quadruplicate readings.
syk / mice, we have generated bone marrow
chimeras with a syk/ hematopoietic system by
transplanting syk/ fetal liver cells into
lethally irradiated wild-type recipients.12 No Syk
immunoreactivity could be observed in lysates of neutrophils purified
from these syk/ bone marrow chimeras (Figure
1A).
In a number of cases, deleting the gene of one protein results in the
overexpression of another member of the same protein family and
concomitant functional compensation for the lack of the deleted gene
product. To exclude this possibility, we tested the presence of ZAP-70,
the only other known Syk-related protein, in
syk The function of Src-family kinases is closely related to that of
Syk/ZAP-70 in multiple biologic systems, raising the possibility of a
functional compensation by these kinases for the absence of Syk. As
shown in Figure 1B, syk Syk is required for the integrin-dependent, but not the integrin-independent, component of the neutrophil respiratory burst We recently have reported that Syk is required for a number of integrin-dependent functions of neutrophils.12 One of the several assay systems used assessed the activation of neutrophils by multiple proinflammatory stimuli while adherent to the extracellular matrix protein fibrinogen. syk/ neutrophils
were significantly reduced in their capacity to release superoxide
under these conditions (Mócsai et al12 and Figure 2). Interestingly, while
syk/ neutrophils were completely defective
in responses to tumor necrosis factor (TNF) (and several other
stimuli) in this assay system, their responses to fMLP were only
partially decreased (compare Figures 2A-B; see Mócsai et
al12). We hypothesized that this difference is due to the
differential requirement for integrins in the TNF- and fMLP-induced
responses. Since Mg2+ ions are essential for the binding of
integrins to their natural ligands, testing the requirement for
Mg2+ salts is a simple assay for the involvement of
integrins in any biologic process. Importantly, removal of
Mg2+ from the assay medium nearly completely abolished the
responses of fibrinogen-plated wild-type neutrophils to TNF (Figure 2A) but only partially decreased those to fMLP stimulation (Figure 2B).
Furthermore, the Mg2+-dependent (integrin-mediated)
response to either TNF or fMLP had a longer (10 minutes) lag phase,
while the Mg2+, and thus integrin-independent, phase after
fMLP stimulation was rapid and practically completed within the first
5-10 minutes. Thus, fMLP stimulation of neutrophils on fibrinogen leads
to a rapid, integrin-independent activation, followed by a slower, integrin-dependent activation. Since the response of
syk/ neutrophils is similar to that of
wild-type cells in the absence of Mg2+ (Figure 2B)
and withdrawal of Mg2+ had no further effect on
syk/ cells (not shown), Syk seems to be
involved in the integrin-dependent, but not in the
integrin-independent, component of the fMLP-induced respiratory burst.
The above results prompted us to analyze GPCR signaling mechanisms in
more detail in syk Syk is not required for the fMLP-induced respiratory burst Using cells maintained in suspension as described above, no major defect could be observed in respiratory burst of fMLP-stimulated syk/ neutrophils (Figure
3A). Both the kinetics of the superoxide production and the fMLP dose-response curve were normal or even slightly augmented in the Syk-deficient cells.
Under physiologic conditions, the cortical actin cytoskeleton of
neutrophils prevents the release of superoxide and of other antibacterial agents into the extracellular space and thus ensures that
these rather harmful intermediates are specifically targeted to the
phagosome. Disrupting the cortical actin network with cytochalasins converts the plasma membrane into a surface resembling that of the
phagosome and thus allows the release of antimicrobial agents into the
extracellular space. Through this effect, cytochalasin B (CB) leads to
a strong increase in fMLP-induced respiratory burst both in
human40 and in murine neutrophils (compare Figures 3A-B).
Even in the presence of CB, the responses of
syk Normal fMLP-induced degranulation in
syk / neutrophils.
The primary granules contain the most toxic components of neutrophils
and thus, under physiologic conditions, are strictly directed to the
phagosome. However, pretreatment of the cells with CB leads to the
exocytosis of a significant fraction of primary granules into the
extracellular space. This is shown by the fact that fMLP-stimulated
wild-type neutrophils release elastase only in the presence and not in
the absence of CB (Figure 4A). The syk
As shown in Figure 4B, optimal release of the secondary granule marker
lactoferrin also requires preincubation with CB. However, a fraction of
lactoferrin also can be released in the absence of CB, allowing us to
study the mechanisms underlying granule exocytosis under more
physiologic conditions. Again, syk Migration of neutrophils toward fMLP is not affected by the
syk
The overall transmigration of syk Syk is not required for intracellular signaling events triggered by fMLP To directly compare the intracellular signaling reactions induced by fMLP, we examined Ca2+ mobilization, activation of ERK and p38 MAP kinase, as well as polymerization of cellular actin in wild-type and Syk-deficient neutrophils.fMLP initiates a biphasic rise in intracellular Ca2+
concentration in neutrophils (Figure 6A).
The first phase of this response is caused mainly by the release of
Ca2+ from intracellular stores, while the second phase is
attributed to the opening of plasma membrane Ca2+ channels
supposedly responding to the emptying of intracellular stores
(store-operated Ca2+ influx). As shown in Figure 6A, no
difference between wild-type and syk
fMLP triggers a rapid activation of both the ERK and the p38 MAP kinase
pathways (Figure 6B). While the functional importance of the dramatic
increase in the activation of ERK is still unclear, p38 MAPK has been
implicated in a number of antimicrobial processes, including the
fMLP-mediated degranulation of the cells.27 As shown in
Figure 6B, activation of ERK and p38 MAPK in fMLP-stimulated syk A number of the functional responses of neutrophils to fMLP stimulation
rely on changes of cell shape and the rapid reorganization of the actin
cytoskeleton. This is reflected in the transient increase in the amount
of F-actin within the first minutes after fMLP stimulation. As shown in
Figure 6C, no difference could be observed between wild-type and
syk Taken together, these results suggest that multiple fMLP-induced intracellular signaling events proceed normally in the absence of Syk. Normal responses to LTB4 and C5a in
syk As shown in Figure 7A, no defect was
observed in the maximal migratory response of
syk
Figure 7B shows the Ca2+ signal initiated by
LTB4 and C5a. Although the overall shape of these
Ca2+ response curves was rather different from that induced
by fMLP, syk Taken together, these results suggest that Syk does not play a major role in the responses of neutrophils initiated by LTB4 or C5a. Normal responses to chemokines in
syk  , which acts through
the CXC chemokine receptor CXCR2 (unlike humans, mice don't express
the related receptor, CXCR1).47 Murine neutrophils also
respond, though to a significantly lesser extent, to the CC chemokine
MIP-1 (CCL3) through its cognate receptor CCR1.
Both MIP-1
Figure 8B shows migration of wild-type and
syk Both MIP-1 Thus, similar to responses initiated by fMLP, LTB4, and
C5a, those triggered by the chemokines MIP-1 Normal GPCR signaling in syk receptors by IgE,8
but its role in mast cell responses to GPCR agonists is presently
unclear (see "Introduction"). To directly test this latter
question, we have examined the responses of bone marrow-derived
syk/ mast cells to GPCR ligation. As shown
in Figure 9A, activation of the
phosphatidylinositol-(3,4,5)P3-dependent kinase Akt or of the ERK and
p38 MAP kinases occurred normally in syk/
mast cells stimulated by the GPCR agonist adenosine,48
indicating that Syk is not required for GPCR signaling in these
cells. In contrast, activation of the same kinases by IgE-mediated
cross-linking of FC receptors is completely
abolished by the syk/ mutation (Figure 9B),
confirming the critical role for Syk in FC receptor
signaling.8 These data demonstrate that, similar to
syk/ neutrophils,
syk/ mast cells also respond normally to
ligands that use G-protein-coupled receptors.
In this report we have shown that syk We previously have reported that Syk is essential for the responses of
neutrophils to engagement of cell surface integrins.12 Under most in vivo conditions, activation of neutrophils by classical chemoattractants or chemokines occurs while the cells are adherent, through their integrins, to endothelial cells or extracellular matrix
proteins. Thus, despite a normal GPCR signaling,
syk It should be noted that syk Our results, which suggest that Syk is dispensable for all
tested GPCR-mediated responses, are in contrast with previous reports suggesting that Syk is involved in GPCR signaling in a number of
different cell types and assay systems.18,19,21-24 This
apparent contradiction may stem from the fact that those reports used
long-propagated cell lines, transfection of nonendogenous surface
receptors, as well as piceatannol, an inhibitor of limited specificity.
In contrast, our results are based on stimulation, through their
endogenous GPCRs, of primary cells that are genetically deficient of
the syk gene. In the case of the neutrophil assays, these
cells have even developed in vivo and were used within hours after
freshly isolating them from the mice. We feel that the possible number of misleading artefacts are considerably less in our assay systems than
in those described in references 18, 19, and 21-24. In accordance with
our data, thrombin-induced release of 5-HT and AA was not affected by
the syk This study and most previous reports deal with the possible role of Syk in signaling from receptors coupled to the Gi family of heterotrimeric G proteins. It is presently unclear whether Syk is involved in signaling from other classes of GPCR. However, hematopoietic cells, which express the highest levels of Syk and are the most functionally dependent on it, predominantly use GPCR coupled to Gi. While this fact underscores the possible importance of Syk in signaling from other classes of heterotrimeric G proteins, it also makes it rather difficult to study the role of Syk in non-Gi pathways in any physiologically relevant assay system. Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) was first isolated as a naturally occurring plant antitumor agent49 and later shown to act as an inhibitor of degranulation of mast cells.50 It was soon recognized as an inhibitor of tyrosine kinases,51 and its major target was later suggested to be Syk.52 However, its specificity toward Syk was seriously questioned by discrepancies between piceatannol-treated platelets and platelets genetically deficient of syk, as well as by the fact that both Src-family kinases and FAK were inhibited by moderate concentrations of the drug.29 Another recent report provided further doubts about the selectivity of piceatannol toward Syk.30 The latest evidence supporting the nonspecific nature of the drug comes from the apparent contradiction between the results presented in this paper and those obtained by using piceatannol to study the role of Syk in GPCR signaling,18,19,21-24,27 in particular our own previous observation that piceatannol inhibits fMLP-induced degranulation and activation of the p38 MAP kinase in human neutrophils.27 Taken together, these results indicate that piceatannol should not be considered a specific inhibitor of the Syk tyrosine kinase. The syk
We thank Victor Tybulewicz for the syk+/
Submitted August 5, 2002; accepted January 10, 2003.
Prepublished online as Blood First Edition Paper, January 16, 2003; DOI 10.1182/blood- 2002-07-2346.
Supported by National Institutes of Health grants DK58066 (C.A.L.), AI33617 (T.K.), and AI38348 (T.K.). A.M. was a recipient of a Bolyai postdoctoral fellowship from the Hungarian Academy of Sciences. C.A.L. was a Scholar of the Leukemia and Lymphoma Society.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Clifford A. Lowell, Department of Laboratory Medicine, Box 0134, University of California, San Francisco, 505 Parnassus Ave, Rm HSE-590, San Francisco, CA 94143-0134; e-mail: clowell{at}cgl.ucsf.edu.
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J. Schymeinsky, A. Sindrilaru, D. Frommhold, M. Sperandio, R. Gerstl, C. Then, A. Mocsai, K. Scharffetter-Kochanek, and B. Walzog The Vav binding site of the non-receptor tyrosine kinase Syk at Tyr 348 is critical for beta2 integrin (CD11/CD18)-mediated neutrophil migration Blood, December 1, 2006; 108(12): 3919 - 3927. [Abstract] [Full Text] [PDF]
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A. Utomo, X. Cullere, M. Glogauer, W. Swat, and T. N. Mayadas Vav Proteins in Neutrophils Are Required for Fc{gamma}R-Mediated Signaling to Rac GTPases and Nicotinamide Adenine Dinucleotide Phosphate Oxidase Component p40(phox) J. Immunol., November 1, 2006; 177(9): 6388 - 6397. [Abstract] [Full Text] [PDF]
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S. A. Francis, X. Shen, J. B. Young, P. Kaul, and D. J. Lerner Rho GEF Lsc is required for normal polarization, migration, and adhesion of formyl-peptide-stimulated neutrophils Blood, February 15, 2006; 107(4): 1627 - 1635. [Abstract] [Full Text] [PDF]
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J.-C. Gevrey, B. M. Isaac, and D. Cox Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine) J. Immunol., September 15, 2005; 175(6): 3737 - 3745. [Abstract] [Full Text] [PDF]
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J. Kitaura, T. Kinoshita, M. Matsumoto, S. Chung, Y. Kawakami, M. Leitges, D. Wu, C. A. Lowell, and T. Kawakami IgE- and IgE+Ag-mediated mast cell migration in an autocrine/paracrine fashion Blood, April 15, 2005; 105(8): 3222 - 3229. [Abstract] [Full Text] [PDF]
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S. Pereira, H. Zhang, T. Takai, and C. A. Lowell The Inhibitory Receptor PIR-B Negatively Regulates Neutrophil and Macrophage Integrin Signaling J. Immunol., November 1, 2004; 173(9): 5757 - 5765. [Abstract] [Full Text] [PDF]
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Z. Jakus, G. Berton, E. Ligeti, C. A. Lowell, and A. Mocsai Responses of Neutrophils to Anti-Integrin Antibodies Depends on Costimulation through Low Affinity Fc{gamma}Rs: Full Activation Requires Both Integrin and Nonintegrin Signals J. Immunol., August 1, 2004; 173(3): 2068 - 2077. [Abstract] [Full Text] [PDF]
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 M. A. M. Gakidis, X. Cullere, T. Olson, J. L. Wilsbacher, B. Zhang, S. L. Moores, K. Ley, W. Swat, T. Mayadas, and J. S. Brugge Vav GEFs are required for {beta}2 integrin-dependent functions of neutrophils J. Cell Biol., July 19, 2004; 166(2): 273 - 282. [Abstract] [Full Text] [PDF]
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Y. Liu, S. K. Shaw, S. Ma, L. Yang, F. W. Luscinskas, and C. A. Parkos Regulation of Leukocyte Transmigration: Cell Surface Interactions and Signaling Events J. Immunol., January 1, 2004; 172(1): 7 - 13. [Full Text] [PDF]
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D. Pradip, X. Peng, and D. L. Durden Rac2 Specificity in Macrophage Integrin Signaling: POTENTIAL ROLE FOR Syk KINASE J. Biol. Chem., October 24, 2003; 278(43): 41661 - 41669. [Abstract] [Full Text] [PDF]
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 T. Willeke, J. Schymeinsky, P. Prange, S. Zahler, and B. Walzog A role for Syk-kinase in the control of the binding cycle of the {beta}2 integrins (CD11/CD18) in human polymorphonuclear neutrophils J. Leukoc. Biol., August 1, 2003; 74(2): 260 - 269. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
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