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Department of Physiology, Semmelweis University of Medicine, Budapest, Hungary;
Joint Research Organization of the Hungarian Academy of Sciences and Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Semmelweis University of Medicine, Budapest, Hungary;
Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy; and
§
Department of Laboratory Medicine, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The receptor for the bacterial tripeptide fMLP, the most widely studied chemoattractant of neutrophils, activates a pertussis toxin-sensitive, heterotrimeric G protein belonging to the Gi/o family. One of the most characteristic features of fMLP signal transduction is the rapid induction of tyrosine phosphorylation of several intracellular proteins, including tyrosine kinases (2, 3), different members of the mitogen-activated protein kinase (MAPK)3 family (4, 5, 6), adaptor proteins (3), and components of the small G protein network (7). Despite clear evidence of phosphorylation (and activation) of these proteins, little is known about their role in triggering the fMLP-induced effector responses.
Using broad specificity tyrosine kinase inhibitors, we and others suggested that tyrosine kinases might play a role in the fMLP-induced release of primary and secondary granules (Ref. 8 and references therein). In the last few years great progress has been made in understanding the roles of the different tyrosine kinases in the regulation of neutrophil functions (9). Activation of the Src-related kinase Lyn in response to fMLP stimulation has been reported by several authors (2, 3, 9, 10). Fgr, another member of the Src kinase family, has been shown to be associated with the secondary granules and to translocate to the plasma membrane upon fMLP stimulation of the cells (11). Similarly, Hck, the third Src family member present in neutrophils, is associated with primary granules and translocates to the phagosome during phagocytosis of serum-opsonized zymosan (12). Our pharmacological and gene knockout experiments performed on murine neutrophils showed that the fMLP-induced exocytosis of secondary granules is mediated by Src-related tyrosine kinases (13). However, these reports did not provide any clue to identification of possible downstream targets of tyrosine kinases. In addition, they did not address in detail the distinctive role of tyrosine phosphorylation in regulating exocytosis of the different granule populations and of secretory vesicles.
In the present report we analyzed the fMLP-induced exocytosis of primary and secondary granules as well as secretory vesicles in response to fMLP using inhibitors with reported selectivity for different tyrosine kinases or tyrosine-phosphorylated proteins and studying mice deficient in Src family kinases expressed in neutrophils.
This study followed two major directions of investigation. First, to obtain information on the molecular identity of the kinases participating in the fMLP-induced degranulation response, we focused on 1) the ERK and p38 MAPK proteins, members of the MAP kinase family shown to become rapidly phosphorylated on adjacent Tyr and Thr residues and thereby activated upon chemoattractant stimulation (4, 5, 6, 14, 15); 2) the Src family of tyrosine kinases, the members of which have been implicated in several aspects of the degranulation process (see above); and 3) the Syk tyrosine kinase, the function of which is closely related to that of Src family kinases in several cellular systems (16, 17, 18, 19). Our second objective was to compare the involvement of the above pathways in the fMLP-induced release of three exocytic compartments of human neutrophils: primary granules, secondary granules, and secretory vesicles. Previous reports indicated differences in calcium sensitivity (20) and inhibition by wortmannin (21) of fMLP-induced exocytosis among these three granule populations. All these differences warranted a comparative study on the role of tyrosine kinases in the release of the three exocytic compartments.
In the present paper we provide evidence that p38 MAPK, activated by Src family kinases has a central role in eliciting the fMLP-induced release of primary and secondary granules, but not in that of secretory vesicles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dextran T-500 and Ficoll-Paque were obtained from Pharmacia (Uppsala, Sweden). fMLP, cytochalasin B (CB), and genistein were obtained from Sigma (St. Louis, MO). PD98059, PP1, and piceatannol were obtained from Calbiochem (San Diego, CA). SB203580 was provided by SmithKline Beecham (King of Prussia, PA). Human lactoferrin (Lfr), human serum albumin (HSA), and goat anti-HSA Abs were obtained from Sigma. Rabbit anti-human Lfr Abs were either purchased from Sigma or provided by Dr. Katalin Német (National Institute of Hematology and Blood Transfusion, Budapest, Hungary). Where necessary, Abs were labeled with HRP (22).
Unless otherwise stated, incubation of neutrophils was performed in HBSS (containing 0.5 mM CaCl2 and 1 mM MgCl2) supplemented with 20 mM Na-HEPES (pH 7.4) and 0.1% BSA (HBSS-HB). All incubation media were prepared using sterile and endotoxin-free water.
Preparation of human neutrophils
Venous blood was drawn from healthy volunteers. After dextran sedimentation at room temperature, neutrophils were obtained by centrifugation at 4°C through Ficoll-Paque followed by hypotonic lysis of erythrocytes (8, 13). Unless otherwise stated, cells were then resuspended in ice-cold HBSS-HB and kept on ice until use. The preparations usually contained >98% polymorphonuclear cells; the viability, as determined by the erythrosin B dye exclusion test, was >98%.
Degranulation of human neutrophils
Human neutrophils at 106/ml were incubated in HBSS-HB with or without the indicated inhibitors and/or 10 µM cytochalasin B for 15 min on ice and for an additional 15 min at 37°C. The cells were then stimulated for 10 min with 100 nM fMLP. The reaction was stopped by cooling, and the suspension was centrifuged for 10 min at 2000 x g at 4°C. The extent of degranulation was determined by measuring the concentrations of the different granule markers in the supernatant. Addition of up to 0.2% DMSO (the maximum solvent concentration added with the inhibitors) had no influence on any of the exocytic responses tested (not shown).
The activity of the primary granule marker ß-glucuronidase (ß-GU) was determined by a fluorometric assay described previously (8). In some experiments the total cellular ß-GU activity was also measured, using suspensions treated with 0.02% cetyl-trimethyl-ammonium bromide as detergent. The concentrations of the secondary granule marker Lfr (23) and HSA, a marker of secretory vesicles (24), were determined by ELISA. Maxisorp F96 plates (Nunc, Naperville, IL) were covered with the relevant unlabeled Abs and blocked by PBS supplemented with 0.5% BSA and 0.1% Tween 20. Plates were then incubated with diluted supernatants or known concentrations of Lfr or HSA, followed by treatment with peroxidase-labeled anti-human Lfr or anti-HSA Abs, and developed with o-phenylenediamine. The OD of the samples was read using a Labsystems (Helsinki, Finland) iEMS microplate reader.
For determination of adhesion-dependence of the exocytic response, the
effect of withdrawal of Mg2+ on the release of
secondary granules was tested (13, 25). Cells were
resuspended in HBSS-HB without CaCl2,
MgCl2, and BSA, supplemented with 5 mM Na-EDTA.
After incubation for 10 min at room temperature, cells were pelleted
and resuspended in ice-cold HBSS-HB with or without 1 mM
MgCl2. The fMLP-stimulated release of Lfr was
then determined as described above. Simultaneously, cells were plated
on tissue culture plates precoated with human fibrinogen
(13) and stimulated with 20 ng/ml human TNF-
(PeproTech, Rocky Hill, NJ) for 30 min at 37°C. Spreading of the
cells was assessed visually under a phase contrast microscope.
Immunoblotting of human cell lysates and kinase assays
Cells at 2.5 x 107/ml were pretreated exactly as described for degranulation experiments, followed by a 2-min stimulation with 100 nM fMLP at 37°C. Samples were then immersed in liquid N2 to stop the reaction and were lysed by adding a Triton-based lysis buffer with protease and phosphatase inhibitors (8) from a 5x concentrated stock solution followed by a 10-min incubation on ice and spinning down at 16,000 x g for 1 min at 4°C. The Triton-soluble supernatant was used in further determinations.
For immunoblot experiments, the Triton-soluble lysate was mixed with sample buffer, boiled for 10 min, run on 12% SDS-PAGE, and blotted onto nitrocellulose sheets. Blots were processed using phospho-specific rabbit polyclonal anti-ERK or anti-p38 MAPK Abs (both from New England Biolabs, Beverly, MA) followed by a peroxidase-labeled anti-rabbit Ab (Amersham, Aylesbury, U.K.). Blots were developed using Amersham’s enhanced chemiluminescence system and exposed to x-ray film, and signal intensities were quantified by a Pharmacia-LKB (Uppsala, Sweden) Ultroscan XL laser densitometer. For control blots, non-phospho-specific rabbit polyclonal anti-ERK1 and anti-ERK2 (used in combination; both from Santa Cruz Biotechnology, Santa Cruz, CA) or anti-p38 MAPK (Upstate Biotechnology, Lake Placid, NY) with peroxidase-labeled anti-rabbit Abs (Amersham) were used. It should be noted that the slight difference in the migrations of ERK1 and ERK2 could only be resolved when the acrylamide/bisacrylamide ratio of the SDS-PAGE gel was increased to 37.5:1. Even when such gels were used, we never observed any duplication of the anti-p38 MAPK immunoreactive band.
The activity of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase
downstream of p38 MAPK, was measured by an in vitro kinase assay
(26) using recombinant heat shock protein 27 (Hsp27), a
substrate of MAPKAPK2. Triton-soluble lysates were supplemented with
kinase buffer (15 mM Na-HEPES (pH 7.4), 10 mM
MgCl2, 1 mM Na-EGTA, 2 mM DTT, 50 mM NaCl, 10 mM
NaF, 5 µM sodium pyrophosphate, 20 mM ß-glycerophosphate, 2 µM
okadaic acid, 0.2 mM [
-32P]ATP (
200
µCi/µmol), and 20 µg/ml human Hsp27 (Stressgen, Victoria,
Canada)) from a 2x concentrated stock solution and incubated for 20
min at room temperature. The reaction was stopped by adding sample
buffer, followed by boiling for 10 min. Samples were then run on a 12%
SDS-PAGE, dried, and exposed to x-ray film or quantitated using a
Bio-Rad (Hercules, CA) GS-525 PhosphorImager. To determine the
incorporation of radioactivity into endogenous cellular proteins,
parallel measurements without Hsp27 were also performed. Radioactivity
in these samples was then subtracted from values obtained from
Hsp27-containing tubes.
For the in vitro ERK kinase activity measurements (27),
100 µl Triton-soluble cell lysates were incubated for 3 h with 3
µg of rabbit polyclonal anti-ERK2 Abs (provided by Dr. Jackie R.
Vandenheede, Catholic University of Leuven, Leuven, Belgium).
Immunocomplexes were captured with protein A-TSK gel and were washed
three times with PBS containing 1% Nonidet P-40, and twice with kinase
buffer containing 20 mM MOPS (pH 7.6), 2 mM EGTA, 20 mM
MgCl2, 1 mM DTT, 1 mM
Na3VO4, and 0.1% Triton
X-100. The washed beads were resuspended in 30 µl of kinase reaction
mixture containing kinase buffer supplemented with 40 µM ATP, 5 µCi
of [-32P]ATP, and 50 µg myelin basic
protein as kinase substrate. After incubation at 30°C for 15 min, the
reaction was terminated by addition of sample buffer and heating at
100°C for 3 min. The proteins were separated on 11% SDS-PAGE, dried,
and exposed to x-ray film. The myelin basic protein band was then cut
out, and the incorporated radioactivity was quantified using a Wallac
(Turku, Finland) 1409 liquid scintillation counter.
Determination of Tyr phosphorylation of Syk
Tyr phosphorylation of Syk was determined by immunoprecipitation followed by immunoblotting with anti-phosphotyrosine (DFP) Abs. To prevent the degradation of Syk, these experiments were performed on neutrophils pretreated with 0.2 µl/ml di-isopropyl fluorophosphate at the end of the cell preparation. Cells were stimulated with 100 nM fMLP or by hyperosmotic shock (addition of an extra 120 mM NaCl; Ref. 28) for 2 min. The reaction was stopped by the addition of an equal volume of ice-cold PBS, followed by lysing the cells with a 5x concentrated lysis buffer. Triton-soluble fractions were then prepared as described above and precleared by incubating for 60 min with protein G-Sepharose beads (Pharmacia). The precleared lysates were incubated for 60 min with 5 µg/sample mouse monoclonal anti-human Syk Abs (Santa Cruz Biotechnology) and for a further 60 min with protein G-Sepharose beads. The beads were washed three times with PBS containing 0.1% Triton X-100 and 1 mM Na3VO4 and were boiled for 10 min in sample buffer. An aliquot of the supernatant was run on an 8% SDS-PAGE and immunoblotted using mouse monoclonal anti-phosphotyrosine Abs (clone 4G10 from Upstate Biotechnology). The Tyr phosphorylation of Syk was quantitated by densitometric analysis. The amounts of Syk immunoprecipitated from the different samples were compared by immunoblotting aliquots of the immunoprecipitates with the precipitating Abs.
Experiments on murine neutrophils
The hck-/-fgr-/-lyn-/- triple knockout mice deficient in all three Src family kinases present in neutrophils have been described previously (29). Preparation and stimulation of wild-type (C57BL/6) or hck-/-fgr-/-lyn-/- bone marrow neutrophils as well as determination of the release of the secondary granule marker Lfr were performed as previously described (13), using 1 µM fMLP as stimulus. Exocytosis of mouse serum albumin (MSA) was measured by ELISA, using rabbit anti-MSA Abs (unlabeled or peroxidase labeled for capturing and developing, respectively; both from Nordic Immunology, Tilburg, The Netherlands). The assay was calibrated using purified MSA (Sigma). Phosphorylation of murine p38 MAPK was determined as described for human neutrophils, except that 1 µM fMLP was used to induce a well measurable phosphorylation of the protein.
Presentation of data and statistical analysis
Degranulation assays were performed in triplicate or quadruplicate. The concentration of granule markers in the supernatants was expressed as a percentage of values of fMLP-stimulated samples of human cells without kinase inhibitors or of wild-type murine cells, in the presence (primary and secondary granules) or the absence (secretory vesicles) of CB. The 100% values for human cells corresponded to 29 ± 16% of the total cellular ß-GU content in the case of primary granules (CB present; n = 8), 3.4 ± 1.3 µg Lfr/106 cells in the case of secondary granules (CB present; n = 19), and 189 ± 77 ng HSA/106 cells in the case of secretory vesicles (CB absent; n = 26). Further information on presentation of results of degranulation experiments is provided at the beginning of Results. Data for phosphorylation and kinase activity are also expressed as a percentage of the fMLP-stimulated values without kinase inhibitors, except for the Syk phosphorylation experiments, where data are expressed as the fold increase over values obtained from nonstimulated samples.
Because of the floating zero point of the fluorometer used, the results of the ß-GU measurements reflect the differences between but not the absolute values of the ß-GU activity of the samples. For this reason, only fMLP-stimulated values of ß-GU (i.e., after subtraction of values in nonstimulated samples) are provided. None of the inhibitors used had any considerable effect on ß-GU activity in supernatants of nonstimulated cells (not shown).
For statistical analysis throughout this paper, nonstimulated values
were subtracted from fMLP-stimulated ones, and the obtained data were
expressed as a percentage of similarly calculated values in the absence
of kinase inhibitors or the
hck-/-fgr-/-lyn-/-
mutation. The mean ± SD of these values from the indicated number
of experiments are provided. The difference in these normalized values
from 100% was then analyzed using Student’s paired two-population
t test. Values of p < 0.05 at
n 
3 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report we investigated the signaling mechanisms leading to
exocytosis of three different intracellular compartments of
neutrophils: the primary and secondary granules as well as the
secretory vesicles. The microfilament disrupting agent CB has long been
known to enhance certain responses of neutrophils, including the
release of primary and secondary granules (30). To
determine optimal conditions for studying the exocytic activity of
neutrophils we first investigated the effect of CB on the release of
the three exocytic compartments tested. In accordance with previous
findings (30), while fMLP induced a substantial release of
the primary granule marker ß-GU from human neutrophils pretreated
with 10 µM CB, no exocytosis of ß-GU was observed in the absence of
CB (not shown). CB strongly increased fMLP-induced release of the
secondary granule marker Lfr, but exocytosis of Lfr from CB-untreated
neutrophils could also be easily observed (for example, see Fig. 1
B). In contrast, the
exocytosis of HSA, a constituent of the secretory vesicles, was maximal
even in the absence of CB, and no further increase was observed after
pretreating the cells with CB (not shown). On the basis of these
findings, only degranulation of primary granules from CB-pretreated
cells and release of secretory vesicles from CB-untreated cells are
shown throughout this paper (Figs. 1, 3, 4, 5, and 7). However, data on
Lfr release are provided in both the presence and the absence of CB.
Comparing the effects of kinase inhibitors on Lfr release in the
presence and the absence of CB allowed us to speculate
about whether the drugs act on fMLP signaling or on the
cytoskeletal changes induced by CB.
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As shown in Fig. 1
, 100 µM genistein, a broad specificity
tyrosine kinase inhibitor, caused a strong inhibition of the
fMLP-induced exocytosis of primary (67 ± 12% inhibition) and
secondary (71 ± 14% inhibition) granules from CB-treated
neutrophils. The fact that genistein also decreased the release of the
secondary granule marker Lfr from CB-untreated neutrophils (77 ±
17% inhibition) suggests that the drug affects a signaling pathway
initiated by fMLP itself, instead of acting on the cytoskeletal changes
induced by CB. On the other hand, the release of HSA, a marker of
secretory vesicles, was only moderately influenced by 100 µM
genistein (29 ± 22% inhibition). Taken together, these data
suggest that tyrosine kinases are strongly involved in the fMLP-induced
degranulation of primary and secondary granules, but to only a lesser
extent in that of secretory vesicles.
Exocytosis of the secondary granules can be induced in an
integrin-dependent (adherent) manner, and we have recently shown that
this adherent exocytosis is mediated by tyrosine kinases
(13). To exclude the possibility that adhesive (e.g.,
intercellular) interactions were also involved in the fMLP-induced
exocytosis by suspended neutrophils, we exploited the evidence that
integrin function requires the presence of Mg2+
ions in the extracellular medium (13, 25). Withdrawal of
Mg2+ ions from the incubation medium had no
effect on the fMLP-induced release of Lfr (CB absent; 102 ± 26%
remaining exocytosis; n = 4), while it prevented the
TNF--induced spreading of neutrophils over fibrinogen-coated surface
(not shown), a response known to involve integrin activation
(25). Thus, in the assay conditions that we employed, fMLP
triggered the exocytosis of secondary granules in an
integrin-independent manner.
One of the earliest events of fMLP signal transduction is the tyrosine
phosphorylation of several intracellular proteins, including ERK (ERK1
and ERK2) and p38 MAPK proteins. As shown in Fig. 2, phosphorylation of ERK (90 ± 7%
inhibition) and p38 MAPK (94 ± 9% inhibition) proteins was
blocked by 100 µM genistein, raising the possibility that the
inhibition of ERKs and/or that of p38 MAPK might mediate the effect of
genistein on the degranulation response. To test this possibility, we
next investigated the effect of pharmacological inhibition of the ERK
and p38 MAPK pathways in fMLP-induced degranulation of neutrophils.
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Despite evidence of the rapid activation of the ERK cascade in
response to fMLP stimulation (4, 14, 15) and the intensive
search for its function in the fMLP-induced responses (8, 31, 32), no clear data supporting such a role has been presented. To
determine the involvement of the ERK cascade in the exocytic activity
of neutrophils, we used PD98059, an inhibitor of MEK1 and MEK2, the
kinases responsible for phosphorylation of the ERK1 and ERK2 proteins
(33). As shown in Fig. 3, 50
µM PD98059 had no considerable effect on the release of ß-GU
(96 ± 19% remaining degranulation), Lfr (109 ± 37 and
89 ± 9% remaining degranulation in the absence and the presence
of CB, respectively), or HSA (91 ± 24% remaining activity).
Under identical conditions, 50 µM PD98059 strongly inhibited
fMLP-induced activation of the ERK cascade as determined using an in
vitro ERK2 kinase assay (Fig. 3D; 88 ± 2% inhibition)
or by immunoblotting using anti-phospho-ERK Abs (not shown). These
data suggest that the ERK pathway, although clearly activated by fMLP,
plays no role in triggering the exocytic responses of neutrophils.
In contrast to the ERK pathway, the p38 MAPK cascade has been
implicated in fMLP-induced superoxide production and chemotaxis
(6, 26) as well as in several responses triggered by TNF
or LPS stimulation (26, 34) in neutrophils. We tested
the role of p38 MAPK in the fMLP-induced exocytic activity of
neutrophils using SB203580, an inhibitor of this kinase (35, 36). As shown in Fig. 4, SB203580
decreased the exocytosis of primary and secondary granules from
CB-pretreated neutrophils, reaching 48 ± 13 and 51 ± 19%
inhibition, respectively, at a 100 µM inhibitor concentration. The
release of Lfr from CB-untreated neutrophils was also reduced by the
drug (62 ± 5% inhibition at 100 µM SB203580). However, 100
µM SB203580 had no effect (102 ± 27% remaining exocytosis) on
the release of the secretory vesicle marker HSA. The effect of
SB203580 on p38 MAPK activity was determined using an in vitro kinase
assay for MAPKAPK2, a known in vivo substrate of p38 MAPK
(5). Fig. 4D shows that SB203580 blocked the
activation of MAPKAPK2 in a concentration-dependent manner, reaching
85 ± 1% inhibition at 100 µM. Taken together, these data
indicate that the p38 MAPK pathway is involved in the fMLP-induced
release of primary and secondary granules, but not in that of secretory
vesicles. It should be noted, however, that while a major part of the
inhibition of secondary granule release and MAPKAPK2 activation was
attained by 10 µM SB203580, a moderately higher concentration of the
drug was required to prevent the exocytosis of primary granules. This
observation points to a possible difference between the signaling of
the two granule populations.
It should also be noted that in addition to the classical p38 MAPK
(also known as p38 or SAPK2a), a novel isoform of p38 MAPK, p38
(or SAPK4) has been shown to be present in neutrophils (37, 38). Although LPS stimulation of neutrophils activates only
p38, treatment of the cells with
H2O2 activates both p38
and p38 (37). At present it is not known which of the
two isoforms becomes activated in response to fMLP stimulation. In
contrast to p38, p38 is not sensitive to SB203580 (39, 40) and does not phosphorylate MAPKAPK2 (39, 40, 41).
Thus, the above experiments provide information only on the role of the
classical isoform, p38.
The role of Src family kinases in neutrophil degranulation and activation of p38 MAPK
In the last few years great progress has been made in understanding the role of the different tyrosine kinases in the regulation of neutrophil functions (9). Several reports implicated the Src kinase family in different aspects of the fMLP-induced degranulation process (2, 3, 9, 10, 11, 12). These findings together with the effect of genistein on the degranulation response prompted us to investigate the role of Src-related kinases in the exocytic activity of the different granules and secretory vesicles of neutrophils.
As shown in Fig. 5, PP1, a selective
inhibitor of Src family kinases (42, 43) diminished the
exocytosis of primary and secondary granules from CB-pretreated human
neutrophils (48 ± 12 and 58 ± 11% inhibition,
respectively, at 30 µM PP1). PP1 also prevented the release of Lfr
from CB-untreated neutrophils (83 ± 11% inhibition at 30 µM
PP1), but had no considerable effect on the release of the secretory
vesicle marker HSA (92 ± 26% remaining activity at 30 µM
PP1).
To confirm that the effect of PP1 on granule release was in fact due to
its action on Src family kinases, we investigated the fMLP-induced
release of Lfr from neutrophils prepared from mice deficient in all
three Src family kinases (Hck, Fgr, and Lyn) present in neutrophils
(29). As shown in Fig. 5D, exocytosis of Lfr
was strongly decreased in
hck-/-fgr-/-lyn-/-
triple mutant neutrophils compared with wild-type cells (97 ± 2
and 119 ± 14% inhibition in the presence and the absence of CB,
respectively). This finding was in accordance with our previous
observation that in the presence of CB, fMLP-induced release of Lfr was
significantly decreased in
hck-/-fgr-/-
double-mutant or PP1-treated wild-type murine neutrophils
(13) and confirmed that Src family kinases are in fact
involved in the exocytic activity of neutrophils. The more pronounced
effect of the
hck-/-fgr-/-lyn-/-
mutation compared with that of PP1 probably reflects the complete
absence of any Src-related kinase activity in the
hck-/-fgr-/-lyn-/-
neutrophils, while some residual activity might be present in the
PP1-treated cells.
Unfortunately, we were unable to study the fMLP-induced exocytosis of
primary granules in the
hck-/-fgr-/-lyn-/-
mice because murine neutrophils do not release their intracellular
ß-GU enzymes under conditions inducing strong release of ß-GU from
human or of Lfr from murine cells (13). Surprisingly, we
could not observe consistent release of MSA from wild-type or mutant
cells in response to 1 µM fMLP, i.e., under conditions causing
maximal release of HSA from human neutrophils (data not shown). While
this fact did not allow us to study fMLP-induced release of MSA from
hck-/-fgr-/-lyn-/-
neutrophils, we did consistently observe that, similar to the effect of
PP1 on HSA release (Fig. 5C), the unstimulated release of
MSA was significantly lower in the triple mutant than in the wild-type
cells (data not shown).
Several reports have implicated Src family kinases in the activation of the ERK pathway by G proteins (44, 45, 46), but very little is known of their role in the activation of p38 MAPK. This fact together with our observation that both the p38 MAPK pathway and Src family kinases seem to be involved in degranulation of neutrophils prompted us to examine the possible involvement of Src family kinases in the fMLP-induced activation of p38 MAPK.
As shown in Fig. 6A, 30 µM
PP1 exerted a strong (67 ± 17%) inhibition on the fMLP-induced
phosphorylation of p38 MAPK in human neutrophils. Similarly,
phosphorylation of p38 MAPK was strongly decreased in
hck-/-fgr-/-lyn-/-
triple-mutant murine neutrophils (Fig. 6B; 67 ± 23%
decrease in phosphorylation). These observations suggest that Src
family kinases play a major role in linking the
Gi-coupled fMLP receptor to the p38 MAPK
pathway.
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suggest that
the fMLP-induced release of primary and secondary granules is mediated
by p38 MAPK activated by a Src family kinase-dependent mechanism. Piceatannol inhibits both degranulation and activation of p38 MAPK
Besides Src family tyrosine kinases, Syk, a member of the
Syk/ZAP-70 kinase family is also present in neutrophils
(47). Activation of Syk by agonists of neutrophils
(19, 47, 48) and the closely related functions of the Src
and Syk/ZAP-70 kinase families in several cell types
(16, 17, 18, 19) prompted us to investigate the role of Syk in the
signaling mechanisms initiated by fMLP in neutrophils. As shown in Fig. 7, piceatannol, a reportedly selective
inhibitor of Syk (49), exerted a strong inhibition of the
fMLP-induced release of primary granules (69 ± 8% inhibition at
100 µM). The drug also prevented the exocytosis of secondary granules
in both the presence and the absence of CB (71 ± 6 and 83 ±
4% inhibition, respectively, at 100 µM), but exerted a statistically
nonsignificant effect on the release of secretory vesicles (49 ±
30% inhibition at 100 µM). Furthermore, as shown in Fig. 7D, piceatannol prevented the activation of p38 MAPK
triggered by fMLP (74 ± 21% inhibition). These data resembled
the effects of genistein and PP1 as well as the responses observed in
hck-/-fgr-/-lyn-/-
neutrophils and raised the possibility that Syk, similar to its role in
several other cellular systems, participates in a signaling pathway
closely related to the Src family of tyrosine kinases.
Next we investigated the possible activation of Syk by fMLP in
neutrophils. These experiments had two purposes. First, we planned to
reveal whether Syk, similar to the immunoreceptor signaling pathways
(16, 17, 18), is activated by an Src family-dependent
mechanism in fMLP-treated neutrophils. Second, since a recent report
raised the possibility of a nonspecific effect of piceatannol
(50), we aimed to determine whether the effect of the drug
on the degranulation and p38 MAPK activation correlated with that on
the activity of Syk. Because the activity of Syk is closely related to
its tyrosine phosphorylation, we investigated the tyrosine
phosphorylation status of Syk in fMLP-stimulated neutrophils.
Unexpectedly, the tyrosine phosphorylation of Syk precipitated from
lysates of neutrophils stimulated for 2 min by 100 nM fMLP did not
differ from that of unstimulated cells (94 ± 6% remaining
phosphorylation; Fig. 7E), while a strong (2.7 ±
1.8-fold) increase in the Tyr phosphorylation of Syk was observed in
neutrophils subjected to a hyperosmotic shock (addition of an extra 120
mM NaCl for 2 min) (28), showing that our method was
capable of determining changes in the tyrosine phosphorylation status
of Syk. We did not observe any increase in Syk Tyr phosphorylation when
the cells were treated for a shorter (30 to 60 s) period, stimulated
with 1 µM fMLP, preincubated with 10 µM CB or when pretreatment
with DFP was omitted (data not shown). We did not observe any
fMLP-induced increase in Syk activity when the immunoprecipitates were
subjected to an in vitro kinase assay (data not shown). Although these
data do not exclude the involvement of Syk in the signaling mechanisms
initiated by fMLP in neutrophils, they point to the need for more
specific approaches to define the role of this enzyme in the
fMLP-induced neutrophil responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 4, 5, and 7). In fact, tyrosine kinase activity does
not seem to play a decisive role in the release of the secretory
vesicles upon chemotactic stimulation.
The use of inhibitors acting on different tyrosine
phosphorylation-dependent signaling pathways as well as the study of
neutrophils genetically deficient in Src family kinases revealed that
several types of kinases are involved in the exocytic process of
primary and secondary granules. The following observations indicate
that phosphorylation and consequent activation of p38 MAPK represents a
key element: 1) the rapid activation of p38 MAPK, following the ligand
binding to fMLP receptors, precedes or coincides with the degranulation
response (5); 2) the decrease in degranulation in response
to treatment with genistein, PP1, and piceatannol or in the
hck-/-fgr-/-lyn-/-
neutrophils (Figs. 1, 5, and 7) correlates with a similar decrease in
the phosphorylation of p38 MAPK (Figs. 2, 6, and 7); and, most
importantly, 3) direct inhibition of p38 MAPK activity by SB203580
impairs the degranulation response (Fig. 4). To our knowledge this is
the first demonstration of the involvement of p38 MAPK in exocytosis
from neutrophils. However, it should be noted that the effective
inhibition of p38 MAPK by SB203580 results in only 50% inhibition
of the release of granule contents. This finding points to the
existence of a signaling pathway that does not involve the p38 MAPK
cascade (or proceeds through p38, the p38 MAPK isoform of
neutrophils not sensitive to SB203580). The fact that genistein had a
more profound effect on degranulation than SB203580 (Figs. 1 and 4),
while both drugs strongly inhibited the p38 MAPK pathway (Figs. 2 and 4), suggests that some of the pathways bypassing the SB203580-sensitive
p38 MAPK also require tyrosine kinase activity.
It should be noted that SB203580 has recently been reported to affect
the Raf protein kinase. Paradoxically, while in vitro treatment with
SB203580 inhibited the activity of Raf, the in vivo treatment of the
cells led to an activation of the kinase (51, 52).
Nevertheless, the inhibitor did not influence the signaling pathways
known to be upstream or downstream of Raf (51, 52). These
uncertainties together with the fact that the ERK cascade, the main
downstream effector of Raf, does not seem to be involved in the
degranulation process (Fig. 3), suggest that the effect of SB203580 on
the release of primary and secondary granules is not mediated by its
influence on Raf.
Inhibition of the ERK cascade by PD98059 had no effect on the
fMLP-induced exocytosis of secretory vesicles, and in accordance with
previous findings (8, 31, 32), it did not affect the
release of primary or secondary granule marker proteins either (Fig. 3). It should be noted that despite its immediate and profound
activation, clear evidence for a functional role of ERKs in any of the
fMLP-induced responses of neutrophils is still lacking.
fMLP has also been shown to activate Src family kinases in neutrophils
(2, 3, 9, 10). These kinases seem to participate in the
signaling of the exocytic process, because application of the Src
family-selective inhibitor PP1 or the genetic deficiency of the Src
family kinases expressed in neutrophils significantly decreased the
fMLP-induced granule release (Fig. 5). This is in accord with our
previous findings suggesting a role for Src family kinases in the
fMLP-induced degranulation of the secondary granules in murine
neutrophils (13).
The observation that both PP1 and the genetic deficiency of Src family
kinases prevented activation of p38 MAPK (Fig. 6) places the site of
action of Src-related kinases between the receptor and p38 MAPK. These
data together with those presented in Figs. 4 and 5 suggest that the
exocytosis of primary and secondary granules proceeds through p38 MAPK
activated by a Src family kinase-dependent mechanism.
In addition to providing information on neutrophil functions, some of
the experiments presented in this paper may be relevant to G protein
signal transduction in other cell types as well. In the last years
great effort has been made to elucidate the mechanisms linking G
protein-coupled receptors to the MAP kinase cascades (for review, see
Ref. 53). It has been shown that nonreceptor tyrosine
kinases activated by G proteins impinge on the Ras-Raf-ERK pathway
through tyrosine phosphorylation of Shc and recruitment of Grb2 and Sos
to the plasma membrane. The tyrosine kinases implicated in the G
protein-mediated activation of ERK include members of the Src family as
well as Syk, Btk, Csk, and Pyk2 (44, 45, 46, 54). On the other
hand, despite several observations showing activation of the p38 MAPK
pathway by G protein-coupled receptors, very little is known about the
role of tyrosine kinases in this process. To our knowledge, the only
relevant information on this topic was provided by Nagao et al.
(55), who described the involvement of Src family kinases
in the activation of p38 MAPK by a constitutively active form of
G11. Our finding that
Src-related kinases participate in the activation of the p38 MAPK by
fMLP in neutrophils (Fig. 6) raises the possibility that members of the
Src family have a more general role in linking G protein-coupled
receptors to the p38 MAPK pathway in different cell types of the
organism.
In this study we also tried to gain more information about the role of
the Syk tyrosine kinase in the fMLP signaling of neutrophils.
Piceatannol, a reportedly selective inhibitor of Syk, prevented the
fMLP-induced release of primary and secondary granules as well as the
activation of p38 MAPK (Fig. 7). The effect of the drug on granule
release is in accordance with previous reports showing that Syk plays a
major role in degranulation of mast cells and basophilic leukemia cells
mediated by FC
-receptor activation (49, 56). Furthermore, the pattern of inhibition of neutrophil
responses by piceatannol resembles the effect of PP1 and that of Src
kinase deficiency, raising the possibility that Syk might participate
in the same pathway linking fMLP receptors to the degranulation
response via Src family kinases and p38 MAPK.
Surprisingly, the concentration of piceatannol effectively inhibiting
the fMLP-induced degranulation of CB-treated neutrophils (30–100 µM)
is an order of magnitude higher than that recently described to inhibit
spreading and H2O2 release
of adherent neutrophils (3–10 µM) (57). Interestingly,
a similar difference between adhesion-dependent responses and
degranulation in suspension exists in the case of genistein and PP1 as
well. Although concentrations of genistein as low as 2–10 µM are
sufficient to block the adhesion-dependent superoxide release of
neutrophils (58), 25–100 µM of the drug is required to
block granule release in suspension (Fig. 1 and Ref. 8).
PP1 (10 µM) almost completely abolishes the adhesion-dependent Lfr
release of human and murine neutrophils (13), whereas
20–30 µM was required to cause a partial decrease in the
degranulation of suspended cells (Fig. 5) (13). In light
of the fact that degranulation in suspension is defective
in both
hck-/-fgr-/-
(13) and
hck-/-fgr-/-lyn-/-
neutrophils (Fig. 5D), it is highly unlikely that the
inhibition of degranulation in suspension by the latter two compounds
was due to a nonspecific effect. Furthermore, the effective
concentration of piceatannol in inhibiting the granule release in
suspension falls within the range (50–100 µM; i.e., 12–24
µg/ml) required for inhibiting several cellular responses as well as
the in vivo activation of Syk or ZAP-70 in other cell types
(59, 60, 61, 62). Thus, the above-mentioned
differences can reflect the extreme sensitivity of the
adhesion-dependent neutrophil responses to tyrosine kinase inhibitors,
rather than the nonspecific action of all three compounds. This high
sensitivity of the adhesion-dependent functions might be related to
tyrosine kinases involved in pathways (e.g., integrin signaling or
the cytoskeletal rearrangement required for cell spreading) used by
adhesion-dependent, but not by fMLP-induced, activation
mechanisms.
However, while initial studies reported piceatannol to be highly
selective toward Syk (49), a recent observation showed the
inhibition of other tyrosine kinases by high concentrations of the drug
(50). Although Src family kinases are not or are only
minimally sensitive to piceatannol (49, 50, 59, 60), the
compound inhibited focal adhesion kinase (Fak) at moderately high
concentrations (50). The fact that neutrophils contain
both Fak (57, 63, 64) and Pyk2 (28, 65, 66),
a kinase closely related to Fak, raises the possibility that, in
addition to Syk, piceatannol might affect other kinases present
in neutrophils. Our finding that despite the effects of piceatannol on
degranulation and p38 MAPK activation, no increase in the tyrosine
phosphorylation of Syk in fMLP-stimulated neutrophils could be
observed (Fig. 7E) supports the possibility of a
nonspecific action of the drug. All these data point to the need for
more specific approaches to determine whether Syk is in fact involved
in fMLP signaling in neutrophils (e.g., by having a permissive effect
or being activated only in a minor compartment of the cell) or whether
piceatannol exerts its effect by inhibiting other cellular targets
(e.g., members of the Fak kinase family). Solving this question is
hampered by the fact that deficiency of the syk gene leads
to perinatal lethality in mice (67, 68), making studies of
syk-/- neutrophils difficult to
attain.
Taken together, our observations based on pharmacological and gene knockout approaches suggest that the binding of fMLP to its Gi-coupled seven-transmembrane receptor on the surface of neutrophils leads to the phosphorylation of p38 MAPK by a mechanism involving Src family kinases. p38 MAPK, in turn, plays a role in dictating, by an as yet unidentified mechanism, the release of both primary and secondary granules of the cells. The possible involvement of Syk in the above processes requires further consideration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Attila Mócsai, Department of Physiology, Semmelweis University of Medicine, P.O. Box 259, 1444 Budapest, Hungary.
3 Abbreviations used in this paper: MAPK or MAP kinase, mitogen-activated protein kinase; CB, cytochalasin B; DFP, di-isopropyl fluorophosphate; ERK, extracellular signal-regulated kinase; Fak, focal adhesion kinase; ß-GU, ß-glucuronidase; HSA, human serum albumin; Hsp27, heat shock protein 27; Lfr, lactoferrin; MAPKAPK2, MAPK-activated protein kinase 2; MBP, myelin basic protein; MSA, mouse serum albumin.
Received for publication August 11, 1999. Accepted for publication February 11, 2000.
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References |
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Q. Wang, G. R. Pfeiffer II, and W. A. Gaarde Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells J. Biol. Chem., November 28, 2003; 278(48): 47731 - 47743. [Abstract] [Full Text] [PDF]
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L. J. Crossley Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1 J. Leukoc. Biol., October 1, 2003; 74(4): 583 - 592. [Abstract] [Full Text] [PDF]
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N. G. Shenoy, G. J. Gleich, and L. L. Thomas Eosinophil Major Basic Protein Stimulates Neutrophil Superoxide Production by a Class IA Phosphoinositide 3-Kinase and Protein Kinase C-{zeta}-Dependent Pathway J. Immunol., October 1, 2003; 171(7): 3734 - 3741. [Abstract] [Full Text] [PDF]
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I. Komuro, Y. Yokota, S. Yasuda, A. Iwamoto, and K. S. Kagawa CSF-induced and HIV-1-mediated Distinct Regulation of Hck and C/EBP{beta} Represent a Heterogeneous Susceptibility of Monocyte-derived Macrophages to M-tropic HIV-1 Infection J. Exp. Med., August 4, 2003; 198(3): 443 - 453. [Abstract] [Full Text] [PDF]
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S. Pereira and C. Lowell The Lyn Tyrosine Kinase Negatively Regulates Neutrophil Integrin Signaling J. Immunol., August 1, 2003; 171(3): 1319 - 1327. [Abstract] [Full Text] [PDF]
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G. S. Laszlo and N. M. Nathanson Src Family Kinase-independent Signal Transduction and Gene Induction by Leukemia Inhibitory Factor J. Biol. Chem., July 18, 2003; 278(30): 27750 - 27757. [Abstract] [Full Text] [PDF]
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T. Pedron, R. Girard, and R. Chaby TLR4-dependent Lipopolysaccharide-induced Shedding of Tumor Necrosis Factor Receptors in Mouse Bone Marrow Granulocytes J. Biol. Chem., May 30, 2003; 278(23): 20555 - 20564. [Abstract] [Full Text] [PDF]
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A. Mocsai, H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C. A. Lowell G-protein-coupled receptor signaling in Syk-deficient neutrophils and mast cells Blood, May 15, 2003; 101(10): 4155 - 4163. [Abstract] [Full Text] [PDF]
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B. Zhong, K. Jiang, D. L. Gilvary, P. K. Epling-Burnette, C. Ritchey, J. Liu, R. J. Jackson, E. Hong-Geller, and S. Wei Human neutrophils utilize a Rac/Cdc42-dependent MAPK pathway to direct intracellular granule mobilization toward ingested microbial pathogens Blood, April 15, 2003; 101(8): 3240 - 3248. [Abstract] [Full Text] [PDF]
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J. S. Park, J. Arcaroli, H.-K. Yum, H. Yang, H. Wang, K.-Y. Yang, K.-H. Choe, D. Strassheim, T. M. Pitts, K. J. Tracey, et al. Activation of gene expression in human neutrophils by high mobility group box 1 protein Am J Physiol Cell Physiol, April 1, 2003; 284(4): C870 - C879. [Abstract] [Full Text] [PDF]
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C. Stambe, R. C. Atkins, G. H. Tesch, A. M. Kapoun, P. A. Hill, G. F. Schreiner, and D. J. Nikolic-Paterson Blockade of p38{alpha} MAPK Ameliorates Acute Inflammatory Renal Injury in Rat Anti-GBM Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2003; 14(2): 338 - 351. [Abstract] [Full Text] [PDF]
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K. Parthasarathi, H. Ichimura, S. Quadri, A. Issekutz, and J. Bhattacharya Mitochondrial Reactive Oxygen Species Regulate Spatial Profile of Proinflammatory Responses in Lung Venular Capillaries J. Immunol., December 15, 2002; 169(12): 7078 - 7086. [Abstract] [Full Text] [PDF]
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 L. W. de Rossi, N. A. Horn, W. Buhre, F. Gass, G. Hutschenreuter, and R. Rossaint The Effect of Isoflurane on Neutrophil Selectin and {beta}2-Integrin Activation In Vitro Anesth. Analg., September 1, 2002; 95(3): 583 - 587. [Abstract] [Full Text] [PDF]
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S. R. Yan, W. Al-Hertani, D. Byers, and R. Bortolussi Lipopolysaccharide-Binding Protein- and CD14-Dependent Activation of Mitogen-Activated Protein Kinase p38 by Lipopolysaccharide in Human Neutrophils Is Associated with Priming of Respiratory Burst Infect. Immun., August 1, 2002; 70(8): 4068 - 4074. [Abstract] [Full Text] [PDF]
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M. P. Scott, F. Zappacosta, E. Y. Kim, R. S. Annan, and W. T. Miller Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1 J. Biol. Chem., July 26, 2002; 277(31): 28238 - 28246. [Abstract] [Full Text] [PDF]
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L. Vicentini, P. Mazzi, E. Caveggion, S. Continolo, L. Fumagalli, J. A. Lapinet-Vera, C. A. Lowell, and G. Berton Fgr Deficiency Results in Defective Eosinophil Recruitment to the Lung During Allergic Airway Inflammation J. Immunol., June 15, 2002; 168(12): 6446 - 6454. [Abstract] [Full Text] [PDF]
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G. Lachance, S. Levasseur, and P. H. Naccache Chemotactic Factor-induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils. IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASES J. Biol. Chem., June 7, 2002; 277(24): 21537 - 21541. [Abstract] [Full Text] [PDF]
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M. Takami, V. Terry, and L. Petruzzelli Signaling Pathways Involved in IL-8-Dependent Activation of Adhesion Through Mac-1 J. Immunol., May 1, 2002; 168(9): 4559 - 4566. [Abstract] [Full Text] [PDF]
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J. Branger, B. van den Blink, S. Weijer, J. Madwed, C. L. Bos, A. Gupta, C.-L. Yong, S. H. Polmar, D. P. Olszyna, C. E. Hack, et al. Anti-Inflammatory Effects of a p38 Mitogen-Activated Protein Kinase Inhibitor During Human Endotoxemia J. Immunol., April 15, 2002; 168(8): 4070 - 4077. [Abstract] [Full Text] [PDF]
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K. Kaldi, J. Szeberenyi, B. K. Rada, P. Kovacs, M. Geiszt, A. Mocsai, and E. Ligeti Contribution of phopholipase D and a brefeldin A-sensitive ARF to chemoattractant-induced superoxide production and secretion of human neutrophils J. Leukoc. Biol., April 1, 2002; 71(4): 695 - 700. [Abstract] [Full Text] [PDF]
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C. Rubel, G. C. Fernandez, F. A. Rosa, S. Gomez, M. B. Bompadre, O. A. Coso, M. A. Isturiz, and M. S. Palermo Soluble Fibrinogen Modulates Neutrophil Functionality Through the Activation of an Extracellular Signal-Regulated Kinase-Dependent Pathway J. Immunol., April 1, 2002; 168(7): 3527 - 3535. [Abstract] [Full Text] [PDF]
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P. J. Mansfield, V. Hinkovska-Galcheva, S. S. Carey, J. A. Shayman, and L. A. Boxer Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D Blood, February 15, 2002; 99(4): 1434 - 1441. [Abstract] [Full Text] [PDF]
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H. Yoshitake, Y. Takeda, T. Nitto, and F. Sendo Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin J. Leukoc. Biol., February 1, 2002; 71(2): 205 - 211. [Abstract] [Full Text] [PDF]
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 T. Sato, L. Koike, Y. Miyata, M. Hirata, Y. Mimaki, Y. Sashida, M. Yano, and A. Ito Inhibition of Activator Protein-1 Binding Activity and Phosphatidylinositol 3-Kinase Pathway by Nobiletin, a Polymethoxy Flavonoid, Results in Augmentation of Tissue Inhibitor of Metalloproteinases-1 Production and Suppression of Production of Matrix Metalloproteinases-1 and -9 in Human Fibrosarcoma HT-1080 Cells Cancer Res., February 1, 2002; 62(4): 1025 - 1029. [Abstract] [Full Text] [PDF]
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E. Nijhuis, J.-W. J Lammers, L. Koenderman, and P. J. Coffer Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning J. Leukoc. Biol., January 1, 2002; 71(1): 115 - 124. [Abstract] [Full Text] [PDF]
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R. Liu, K. Aupperle, and R. Terkeltaub Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells J. Leukoc. Biol., December 1, 2001; 70(6): 961 - 968. [Abstract] [Full Text] [PDF]
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 W. Kang, H. Saito, K. Fukatsu, A. Hidemura, H. Koyama, T. Sakamoto, and K. Maekawa Effects of Tyrosine Kinase Signaling Inhibition on Survival After Cecal Ligation and Puncture in Diet-Restricted Mice JPEN J Parenter Enteral Nutr, November 1, 2001; 25(6): 291 - 298. [Abstract] [PDF]
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P. Desaulniers, M. Fernandes, C. Gilbert, S. G. Bourgoin, and P. H. Naccache Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals J. Leukoc. Biol., October 1, 2001; 70(4): 659 - 668. [Abstract] [Full Text] [PDF]
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M. O. Hannigan, L. Zhan, Y. Ai, A. Kotlyarov, M. Gaestel, and C.-K. Huang Abnormal Migration Phenotype of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-/- Neutrophils in Zigmond Chambers Containing Formyl-Methionyl-Leucyl-Phenylalanine Gradients J. Immunol., October 1, 2001; 167(7): 3953 - 3961. [Abstract] [Full Text] [PDF]
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X. Zhu, H. Sano, K. P. Kim, A. Sano, E. Boetticher, N. M. Munoz, W. Cho, and A. R. Leff Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils J. Immunol., July 1, 2001; 167(1): 461 - 468. [Abstract] [Full Text] [PDF]
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Z. Korade-Mirnics and S. J. Corey Src kinase-mediated signaling in leukocytes J. Leukoc. Biol., November 1, 2000; 68(5): 603 - 613. [Abstract] [Full Text]
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R. A. Ward, M. Nakamura, and K. R. McLeish Priming of the Neutrophil Respiratory Burst Involves p38 Mitogen-activated Protein Kinase-dependent Exocytosis of Flavocytochrome b558-containing Granules J. Biol. Chem., November 17, 2000; 275(47): 36713 - 36719. [Abstract] [Full Text] [PDF]
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P. Kumar, S. Hosaka, and A. E. Koch Soluble E-selectin Induces Monocyte Chemotaxis through Src Family Tyrosine Kinases J. Biol. Chem., June 8, 2001; 276(24): 21039 - 21045. [Abstract] [Full Text] [PDF]
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