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*
Institute of General Pathology, University of Verona, Verona, Italy;
Department of Physiology and Laboratory of Cellular and Molecular Physiology, Semmelweis University of Medicine, Budapest, Hungary; 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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and this
response was inhibited by PP1, a Src family tyrosine kinase inhibitor.
This drug had no effect on lactoferrin secretion induced by PMA, an
adhesion-independent agonist of PMN degranulation. However, PP1 blocked
secretion in PMN plated on plain tissue culture plastic, a surface
inducing PMN spreading in the absence of any stimulus. Double knockout
hck-/-fgr-/-
PMN adherent to collagen or fibrinogen failed to release lactoferrin in
response to TNF- but responded to PMA as wild-type PMN.
Degranulation induced by spreading over tissue culture plastic was also
defective in
hck-/-fgr-/-
PMN. Defective adhesion-dependent degranulation required the absence of
both kinases, because single knockout
fgr-/- or
hck-/- PMN responded as wild-type cells.
Analysis of lactoferrin secretion in
hck-/-fgr-/-
or PP1-treated, suspended PMN showed that Src kinases are not
implicated in degranulation dependent on activation of protein kinase C
or increase in intracellular free Ca2+ but may play a role
in the response to FMLP of cytochalasin B-treated PMN. These findings
identify a role for Src family kinases in a signaling pathway leading
to granule-plasma membrane fusion and suggest that Fgr and Hck would be
targets for pharmacological control of adhesion-dependent degranulation
in the inflammatory site. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the last few years, a great progress has been made in the understanding of signaling from adhesion receptors (14, 15). Adhesion and spreading of PMN are tightly coupled to an increase in tyrosine phosphorylation of several proteins, and tyrosine kinase inhibitors block adhesion-dependent generation of ROI by PMN (7, 8). The Src family tyrosine kinases Fgr, Hck, and Lyn, as well as p72syk, have been recently implicated in signaling from adhesion receptors (16, 17, 18, 19).
Signals involved in triggering degranulation by adherent PMN are poorly understood. The evidence that protein tyrosine phosphorylation (7, 16) and the Src family kinases Fgr and Hck (18) play a critical role in ROI generation prompted us to investigate whether adhesion-dependent degranulation is also regulated by the same signaling pathways. In this report, we show that adhesion-dependent release of lactoferrin is blocked by a newly described tyrosine kinase inhibitor the effect of which has been reported to be selective for Src family tyrosine kinases (20). Investigations with PMN isolated from mice with the double deficiency of Fgr and Hck allowed us to demonstrate that these kinases play an essential role in signaling for adhesion-dependent degranulation. These results provide the first direct evidence that Src family kinases function in a signaling pathway leading to granule-plasma membrane fusion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human PMN were isolated from buffy coats of healthy volunteers as previously described (9, 16). Wild-type (C57BL/6), p59/61hck, or p58c-fgr-deficient single knockout mice and double knockout mice deficient in both p59/61hck and p58c-fgr were described previously (21). Bone marrow of 6–12-wk-old mice of both sexes were used in this study. Murine PMN were isolated essentially as described (18) except that a simplified three-layer gradient was utilized to separate neutrophils from other bone marrow cells (i.e., bone marrow cells suspended in Ca2+-/Mg2+-free HBSS supplemented with 0.1% BSA were layered on the top of a 62/81% two-layer Percoll (Pharmacia, Uppsala, Sweden) gradient, and after centrifugation, PMN were harvested from the 62/81% interface). At the end of the preparation, PMN were suspended in ice-cold HBSS containing 0.5 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose (HBSS) and kept in ice until use. For experiments in which the Mg2+ dependence of the PMN response was investigated, cells were isolated as above described but resuspended in Ca2+/Mg2+-free HBSS supplemented with 5 mM EDTA and, after 10 min of incubation, washed and suspended in HBSS either with or without 1 mM Mg2+ (17).
Coating of tissue culture plastic plates with proteins
Flat bottom polystyrene tissue culture plates with 96 wells (Greiner, Frickenhausen, Germany) were left untreated or were covered with rat collagen, human fibrinogen, or FCS as described (18). After coating, the plates were washed once with PBS and once with HBSS.
Cell stimulation
Human and murine PMN were washed at 4°C and resuspended in
ice-cold HBSS at 1 x 106 or 2 x 106
cells/ml, respectively. For adhesion assays, cell suspensions (100
µl/well), added or not with 10 µM PP1 (Calbiochem-Novabiochem Int.,
La Jolla, CA) were dispensed in protein-coated plates and prewarmed for
10 min at 37°C before addition of 20 ng/ml human or murine TNF-
(TNF, Peprotech, London, U.K.), 100 ng/ml PMA (Sigma, St. Louis, MO),
or diluent alone. Since the TNF stock solution was stored in the
presence of BSA, all the other samples were supplemented with the same
concentration of BSA. After incubation at 37°C for the indicated
times, samples were transferred to ice-cold polypropylene tubes and
centrifuged at 8000 x g for 1 min at 4°C.
Supernatants were used for the determination of granule marker release
into the extracellular space. For suspension assays, 100-µl aliquots
of a 2 x 106/ml cell suspension were dispensed in
polypropylene tubes and either preincubated or not with 10 µM
cytochalasin B (CB) and/or PP1 for 10 min at 37°C. PMN were
stimulated with 1 µM FMLP (Sigma), 1 µM ionomycin, or 100 ng/ml
PMA, and after 10 min samples were transferred to ice-cold
polypropylene tubes and processed as described above for adhesion
assays.
Determination of lactoferrin release
Release of the specific granule marker lactoferrin was measured
by an ELISA using anti-human lactoferrin Abs (Sigma). Supernatants
of human or murine PMN were diluted 4- or 20-fold, respectively, in 50
mM CO32-/HCO3 buffer (pH 9.6). A
total of 100 µl of the diluted supernatants or of known
concentrations of human lactoferrin were added to Nunc Maxisorp F96
(Nunc, Roskilde, Denmark) immunoplate wells and incubated overnight at
4°C. All subsequent steps were conducted at room temperature and
separated by several washings. Nonspecific binding sites were blocked
with PBS supplemented with 0.5% BSA and 0.5% Tween 20 (blocking
solution). Plates were then treated with affinity-purified rabbit
anti-human lactoferrin (Sigma; dilution, 1/500) followed by
peroxidase-conjugated anti-rabbit Ab (Amersham, Little Chalfont,
U.K.; dilution, 1/5000), both dissolved in blocking solution. Color was
developed by the
o-phenylenediamine/H2O2 system.
Absorbance of the wells were read at 490 nm with a Packard Argus 300
microplate reader, and the lactoferrin concentration was calculated
using the human lactoferrin calibration curve. With both human and
murine samples, no signal was detected if the anti-lactoferrin Ab
was substituted with an irrelevant rabbit antiserum. The reactivity of
the anti-human lactoferrin Ab with murine lactoferrin was
determined by Western blotting. Murine PMN suspended at
108/ml in HBSS were incubated for 15 min at 37°C in the
presence or the absence of 100 ng/ml PMA. Cells were then spun down and
the supernatant solubilized in 4x concentrated SDS-PAGE sample buffer
and boiled for 10 min. Samples were run on a 10% SDS-PAGE,
electroblotted onto nitrocellulose sheets (Hybond C, Amersham),
blocked, and probed with anti-human lactoferrin primary and
peroxidase-labeled secondary Abs exactly as described for ELISA
measurements. Signal was developed using an Amersham enhanced
chemiluminescence reagent. The anti-human lactoferrin Ab recognized
one single band at 
78 kDa in supernatants of murine PMN (not
shown). The m.w. of this band and the fact that its intensity increased
in the supernatants of PMA-stimulated PMN confirmed this protein to be
lactoferrin.
Measurement of ß-glucuronidase release
Release of the primary granule marker ß-glucuronidase from the same supernatants used for lactoferrin measurements was conducted essentially as described (22).
Expression of the data
All the experiments were performed in duplicates or triplicates. Since data obtained with mouse samples could not be exactly quantified using standard curves done with human lactoferrin, lactoferrin release from murine PMN is expressed as fold increase compared with the amount detected in supernatants of unstimulated PMN (see figure legends).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human adherent PMN can be stimulated to mobilize
lactoferrin-containing specific granules in response to
granulocyte-macrophage-CSF, FMLP, or the Ca2+ ionophore
A23187 (5). As shown in Fig. 1
A, TNF is also an effective
agonist of lactoferrin secretion in adherent human PMN. As previously
reported with other stimuli (5), TNF-induced degranulation by adherent
PMN is delayed in its onset and prolonged up to 60 min of incubation
(Fig. 1A). In accord with previous studies (4, 23, 24, 25), we
could not detect release of the primary granule marker
ß-glucuronidase in response to TNF (data not shown). Additionally, we
also found that the response to TNF depended on adhesion (25, 26),
since suspended PMN failed to release lactoferrin following TNF
stimulation while such cells did respond to PMA or ionomycin (data not
shown; see Fig. 6 for data with murine PMN).
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A, 10 µM PP1 completely inhibited lactoferrin
secretion in response to TNF. Importantly, PP1 inhibited only the
adhesion-dependent response; in fact, PMA-induced degranulation was
unaffected by the drug (Fig. 1B).
Previous evidence established a strict link between adhesion-dependent
stimulation of PMN functions and cell spreading (5, 6, 7, 8). In line with
this evidence, we observed that PP1 completely blocked PMN spreading on
fibrinogen in response to TNF, but not PMA (Fig. 2). In addition, generation of superoxide
anion, which is also stimulated as PMN are induced to spread by TNF,
was blocked by PP1 (L. Fumagalli and G. Berton, unpublished
observation). It is unlikely that PP1 acts by inhibiting TNF receptor
signaling because it also blocked lactoferrin secretion that occurred
when PMN were plated on plain tissue culture plastic, a surface that
can rapidly induce PMN spreading, in the absence of any stimulus (Fig. 1C).
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D). As seen with human cells, murine PMN
released lactoferrin when incubated on collagen in the presence of TNF
or on plain tissue culture plastic in the absence of any stimulus (see
also Fig. 3). PP1 inhibited murine PMN
lactoferrin secretion in both conditions (Fig. 1D). The high
degranulation response induced by PMA in mouse PMN was unaffected by
PP1 (see legend to Fig. 1D). As found with human PMN (Fig. 2), PP1, in parallel with its inhibition of degranulation also
completely blocked murine PMN spreading in response to TNF, but not PMA
(data not shown).
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Previous studies demonstrated that adhesion-dependent generation
of superoxide anion is defective in
hck-/-fgr-/- PMN
(18). Having established that adherence and spreading induce
lactoferrin release in murine PMN and that the Src family selective
inhibitor PP1 blocks this response in both human and murine PMN (Fig. 1), we addressed whether deficiency of Fgr and Hck results in a
defective adhesion-dependent degranulation. Wild-type PMN released
lactoferrin in response to TNF if incubated on collagen or, albeit to a
lower extent, on fibrinogen, while they responded poorly if incubated
on FCS (Fig. 3). In addition, wild-type PMN released lactoferrin when
incubated on plain tissue culture plastic even in the absence of TNF.
Comparable results were previously obtained by assaying superoxide
anion release by wild-type PMN (18). Lactoferrin release in response to
TNF by hck-/-fgr-/-
double knockout PMN was defective on all the surfaces tested.
Importantly, the spontaneous lactoferrin release by PMN incubated on
tissue culture plastic was also totally defective in
hck-/-fgr-/- PMN.
As lactoferrin release in response to TNF was optimal when PMN were
adherent to collagen we investigated degranulation by collagen-adherent
PMN in more detail. As shown in Fig. 4A, lactoferrin release by
wild-type PMN adherent to collagen and stimulated with TNF was delayed
and maximal at about 30 min. We did not detect any release of the
primary granule marker ß-glucuronidase under these conditions.
hck-/-fgr-/- double
knockout PMN did not release lactoferrin in response to TNF at any of
the time points tested. However, PMA induced release of lactoferrin to
a comparable extent in wild-type and double knockout PMN (Fig. 4B). Light microscopy observation confirmed previous
findings (18) that
hck-/-fgr-/- PMN did
not spread in response to TNF but responded to PMA as well as wild-type
cells.
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, double knockout, but not single
knockout fgr-/- or
hck-/-, PMN were totally defective in their
capability to release lactoferrin in response to TNF when plated on
collagen. In addition, degranulation induced by spreading on tissue
culture plastic was comparable in wild-type and single knockout PMN,
but totally defective in the double knockout cells.
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The findings described in
Figs. 3–5 implicate Fgr and Hck in
signaling for adhesion-dependent degranulation. However, the defective
response of
hck-/-fgr-/- PMN
could be also due to the fact that Hck and Fgr play a more general role
in signaling for degranulation and/or granule-plasma membrane fusion.
As a first approach to demonstrate that Hck and Fgr are required for
signaling from integrins, we addressed whether TNF-induced lactoferrin
release by murine PMN is indeed dependent on adhesion. To this purpose,
we exploited previous findings demonstrating that PMN adhesion in
response to TNF is absolutely dependent on the presence of
Mg2+ in the incubation medium (17). As shown in Fig. 6, in experiments in which lactoferrin
release by murine PMN was investigated in suspension assays, TNF was
unable to induce a response whether Mg2+ was included or
not in the assay medium. However, wild-type, but not
hck-/-fgr-/- PMN,
released lactoferrin in response to TNF if incubated on collagen, and
this response was strictly dependent on the presence of
Mg2+. Since Mg2+ is required for recognition of
extracellular matrix proteins by integrins, the data demonstrate that
impairment of integrin-mediated adhesion in wild-type cells produced
the same defect in degranulation seen in mutant PMN under normal
culture conditions. This supports the model that the defect we found in
hck-/-fgr-/-
PMN concerns an adhesion-dependent cell response.
To test directly whether
hck-/-fgr-/- PMN were
able to release lactoferrin in assays not requiring adhesion, we
analyzed degranulation occurring in suspended PMN in response to
different stimuli. As shown in Fig. 7A), lactoferrin release in
standard suspension assays in response to PMA or the Ca2+
ionophore ionomycin is comparable in wild-type and
hck-/-fgr-/- PMN.
Hence, Hck and Fgr do not regulate granule-plasma membrane fusion and
are dispensable for lactoferrin secretion dependent on protein kinase C
(PKC) and Ca2+. However, we found that
hck-/-fgr-/- PMN were
indeed defective in the response to FMLP (Fig. 7B). This
finding suggests a role of Src family kinases in signaling by FMLP and
is concordant with the evidence that FMLP activates Src family kinases
in PMN (Refs. 27–29, see Discussion). It must be, however,
noted that FMLP-induced lactoferrin secretion required disruption of
the actin-based cytoskeleton with CB, while PMA- and ionomycin-induced
degranulation occurred also in the absence of this drug (Fig. 7). At
present, we do not know whether Hck and Fgr are implicated in signaling
by FMLP and/or in facilitation of FMLP signaling by cytoskeletal
rearrangements induced by CB.
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A, PP1 inhibited the
FMLP + CB-induced degranulation, albeit at a higher concentration than
that required to block adhesion-dependent degranulation (see Fig. 1).
However, lactoferrin release induced by PMA or ionomycin was unaffected
by PP1 (Fig. 8B). We conclude that Src family kinase
inhibition by PP1 affects PMN degranulation in the same fashion as the
knockout of hck and fgr, i.e., inhibits the
response to FMLP + CB but does not modify the PKC- and
Ca2+-induced response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown for ROI generation (18), adhesion-dependent degranulation requires cell stimulation with a cytokine, such as TNF (Refs. 4 and 23–26, and this article), or other agonists (4, 5). It is unlikely that Fgr and Hck are primarily involved in agonist signaling. In fact, stimulus-independent degranulation induced by spreading of PMN on tissue culture plastic was blocked by PP1 and was also defective in hck-/-fgr-/- PMN. Additionally, TNF-induced up-regulation of CD11b/c is normal in hck-/-fgr-/- PMN (18). The evidence that PP1 blocks human and murine PMN spreading and that hck-/-fgr-/- fail to spread (18) suggests a strict link between PMN spreading and degranulation. Thus, it is most likely that signaling from adhesion receptors requires Src family tyrosine kinases to initiate cytoskeletal rearrangements leading to cell spreading and subsequent degranulation.
Analyzing degranulation in suspension assays, we found that
hck-/-fgr-/- PMN and
PMN treated with the Src kinase inhibitor PP1 are defective in the
response induced by FMLP in the presence of CB. These findings suggest
that signaling by the FMLP receptor also involves Src family kinases.
Indeed, several reports have implicated Src family kinases in signaling
by FMLP in PMN (27, 28, 29), as well as by heterotrimeric GTP-binding
protein-coupled receptors in other cell types (reviewed in 32 .
The data reported in Fig. 7 do suggest that Hck and Fgr play a critical
role in FMLP signaling in PMN. Indeed, we have also found that
activation of the respiratory burst by FMLP in CB-treated
hck-/-fgr-/- PMN is
defective and that FMLP activates the Src family kinases Hck and Fgr in
CB-treated human PMN (L. Fumagalli, C. A. Lowell, and G. Berton,
manuscript in preparation). Activation of PMN responses by FMLP in
murine PMN is absolutely dependent on the disassembly of the
actin-based cytoskeleton by cytochalasins. In a previous study (18) and
the present one, we invariably found that
hck-/-fgr-/- PMN
display alterations in the organization of the actin cytoskeleton,
which may contribute to their inability to spread over integrin ligands
or even plain tissue culture plastic. Hence we cannot exclude that
impairment of the response to FMLP + CB results from cytoskeletal
alterations present in double mutant PMN.
A clear hierarchy of mobilization of different granule compartments, i.e., secretory vesicles > gelatinase granules > specific granule (lactoferrin-containing) > azurophil granules, has been demonstrated in human PMN (33). To our knowledge, it is not known whether specific granules of murine PMN can be divided into gelatinase-containing and lactoferrin-containing subsets. Since hck-/-fgr-/- PMN are not defective in the capability to up-regulate CD11b/CD11c (18), which are also stored in secretory vesicles (33), it is tempting to speculate that Fgr- and Hck-regulated adhesion selectively controls mobilization of distinct granule subsets.
Although PMN spreading can occur independently of degranulation (5), previous studies (5) and those described in this report suggest that degranulation by adherent PMN requires cell spreading. PP1 blocked spreading of both human and murine PMN in response to TNF or FMLP, but not PMA, and hck-/-fgr-/- PMN were unable to spread on different surfaces (18). As with degranulation, ROI generation was also shown to be strictly correlated with spreading (18). Thus, it appears that loss of Src family kinase activity, either through the use of PP1 or in the knockouts, primarily affects PMN spreading which is in turn required for ROI generation and degranulation.
To some extent, it is surprising that degranulation proceeds in
coincidence with the extensive process of actin polymerization that
accompanies cell spreading. In fact, the block of actin polymerization
by cytochalasins has been used since the early 1970s to enhance PMN
degranulation (see Ref. 34 for a review of early studies).
Additionally, at least with murine PMN, we could not detect any
significant release of lactoferrin in FMLP-stimulated cells assayed in
suspension if cells were not pretreated with CB (Fig. 7). Cytoskeleton
rearrangement is a highly dynamic process (35). Thus, localized
rearrangements of the actin cytoskeleton may provide a driving force
for membrane fusion between adjacent granules and plasma membrane. In
the absence of Hck and Fgr activity, the signals to initiate the
appropriate cytoskeletal changes do not occur. Interestingly, members
of the Rho subfamily of GTP-binding proteins that play a central role
in actin polymerization have been described to regulate degranulation
in mast cells (36, 37, 38).
PMN degranulation is thought to play an important role in tissue damage and development of inflammation (12, 13, 33). Importantly, we recently found that hck-/-fgr-/- mice are markedly resistant to the lethal effect of high dose lipopolysaccharide injection, and this correlates with reduction in liver and renal damage (39). Reduced production of reactive oxygen intermediate (18) and release of granule constituents (this article) by PMN adherent to the vascular wall of inflamed tissues may explain resistance of hck-/-fgr-/- mice to endotoxic shock. Implication of the Src family tyrosine kinases Fgr and Hck as regulators of adhesion-dependent PMN degranulation suggests that they would be new targets for pharmacological control of the inflammatory process.
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Footnotes |
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2 Recipient of a short term fellowship from the European Molecular Biology Organization.
3 Address correspondence and reprint requests to Dr. Giorgio Berton, Istituto di Patologia Generale, Università di Verona, Strada Le Grazie, 37134 Verona, Italy. E-mail address:
4 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; ROI, reactive oxygen intermediates; TNF, TNF-; PKC, protein kinase C; CB, cytochalasin B.
Received for publication March 26, 1998. Accepted for publication October 6, 1998.
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 S. Chakrabarti, J. M. Zee, and K. D. Patel Regulation of matrix metalloproteinase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for tertiary granule release J. Leukoc. Biol., January 1, 2006; 79(1): 214 - 222. [Abstract] [Full Text] [PDF]
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 G. Lominadze, D. W. Powell, G. C. Luerman, A. J. Link, R. A. Ward, and K. R. McLeish Proteomic Analysis of Human Neutrophil Granules Mol. Cell. Proteomics, October 1, 2005; 4(10): 1503 - 1521. [Abstract] [Full Text] [PDF]
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E. Gaudreault, C. Thompson, J. Stankova, and M. Rola-Pleszczynski Involvement of BLT1 Endocytosis and Yes Kinase Activation in Leukotriene B4-Induced Neutrophil Degranulation J. Immunol., March 15, 2005; 174(6): 3617 - 3625. [Abstract] [Full Text] [PDF]
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 P. Scapini, A. Carletto, B. Nardelli, F. Calzetti, V. Roschke, F. Merigo, N. Tamassia, S. Pieropan, D. Biasi, A. Sbarbati, et al. Proinflammatory mediators elicit secretion of the intracellular B-lymphocyte stimulator pool (BLyS) that is stored in activated neutrophils: implications for inflammatory diseases Blood, January 15, 2005; 105(2): 830 - 837. [Abstract] [Full Text] [PDF]
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 D. Lau, H. Mollnau, J. P. Eiserich, B. A. Freeman, A. Daiber, U. M. Gehling, J. Brummer, V. Rudolph, T. Munzel, T. Heitzer, et al. Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins PNAS, January 11, 2005; 102(2): 431 - 436. [Abstract] [Full Text] [PDF]
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 R. A. Clemens, S. A. Newbrough, E. Y. Chung, S. Gheith, A. L. Singer, G. A. Koretzky, and E. J. Peterson PRAM-1 Is Required for Optimal Integrin-Dependent Neutrophil Function Mol. Cell. Biol., December 15, 2004; 24(24): 10923 - 10932. [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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W. S. Choi, T. Hiragun, J. H. Lee, Y. M. Kim, H.-P. Kim, A. Chahdi, E. Her, J. W. Han, and M. A. Beaven Activation of RBL-2H3 Mast Cells Is Dependent on Tyrosine Phosphorylation of Phospholipase D2 by Fyn and Fgr Mol. Cell. Biol., August 15, 2004; 24(16): 6980 - 6992. [Abstract] [Full Text] [PDF]
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C. Ryckman, C. Gilbert, R. de Medicis, A. Lussier, K. Vandal, and P. A. Tessier Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils J. Leukoc. Biol., August 1, 2004; 76(2): 433 - 440. [Abstract] [Full Text] [PDF]
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D. Abdel-Latif, M. Steward, D. L. Macdonald, G. A. Francis, M. C. Dinauer, and P. Lacy Rac2 is critical for neutrophil primary granule exocytosis Blood, August 1, 2004; 104(3): 832 - 839. [Abstract] [Full Text] [PDF]
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M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
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P. Scapini, M. Morini, C. Tecchio, S. Minghelli, E. Di Carlo, E. Tanghetti, A. Albini, C. Lowell, G. Berton, D. M. Noonan, et al. CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A J. Immunol., April 15, 2004; 172(8): 5034 - 5040. [Abstract] [Full Text] [PDF]
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 A Talmor-Cohen, R Tomashov-Matar, E Eliyahu, R Shapiro, and R Shalgi Are Src family kinases involved in cell cycle resumption in rat eggs? Reproduction, April 1, 2004; 127(4): 455 - 463. [Abstract] [Full Text] [PDF]
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R. Boxio, C. Bossenmeyer-Pourie, N. Steinckwich, C. Dournon, and O. Nusse Mouse bone marrow contains large numbers of functionally competent neutrophils J. Leukoc. Biol., April 1, 2004; 75(4): 604 - 611. [Abstract] [Full Text] [PDF]
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B. Kasper, E. Brandt, S. Bulfone-Paus, and F. Petersen Platelet factor 4 (PF-4)-induced neutrophil adhesion is controlled by src-kinases, whereas PF-4-mediated exocytosis requires the additional activation of p38 MAP kinase and phosphatidylinositol 3-kinase Blood, March 1, 2004; 103(5): 1602 - 1610. [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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N. Yokoyama and W. T. Miller Biochemical Properties of the Cdc42-associated Tyrosine Kinase ACK1: SUBSTRATE SPECIFICITY, AUTOPHOSPHORYLATION, AND INTERACTION WITH Hck J. Biol. Chem., November 28, 2003; 278(48): 47713 - 47723. [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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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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J. Kim, Y. Ogata, and R. A. Feldman Fes Tyrosine Kinase Promotes Survival and Terminal Granulocyte Differentiation of Factor-dependent Myeloid Progenitors (32D) and Activates Lineage-specific Transcription Factors J. Biol. Chem., April 18, 2003; 278(17): 14978 - 14984. [Abstract] [Full Text] [PDF]
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L. Tatton, G. M. Morley, R. Chopra, and A. Khwaja The Src-selective Kinase Inhibitor PP1 Also Inhibits Kit and Bcr-Abl Tyrosine Kinases J. Biol. Chem., February 7, 2003; 278(7): 4847 - 4853. [Abstract] [Full Text] [PDF]
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M. Ernst, M. Inglese, G. M. Scholz, K. W. Harder, F. J. Clay, S. Bozinovski, P. Waring, R. Darwiche, T. Kay, P. Sly, et al. Constitutive Activation of the Src Family Kinase Hck Results in Spontaneous Pulmonary Inflammation and an Enhanced Innate Immune Response J. Exp. Med., September 2, 2002; 196(5): 589 - 604. [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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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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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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 J. H. Lee, N. Kaminski, G. Dolganov, G. Grunig, L. Koth, C. Solomon, D. J. Erle, and D. Sheppard Interleukin-13 Induces Dramatically Different Transcriptional Programs in Three Human Airway Cell Types Am. J. Respir. Cell Mol. Biol., October 1, 2001; 25(4): 474 - 485. [Abstract] [Full Text] [PDF]
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P. Piccardoni, R. Sideri, S. Manarini, A. Piccoli, N. Martelli, G. de Gaetano, C. Cerletti, and V. Evangelista Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin Blood, July 1, 2001; 98(1): 108 - 116. [Abstract] [Full Text] [PDF]
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 C. L. Smith, P. Mittaud, E. D. Prescott, C. Fuhrer, and S. J. Burden Src, Fyn, and Yes Are Not Required for Neuromuscular Synapse Formation But Are Necessary for Stabilization of Agrin-Induced Clusters of Acetylcholine Receptors J. Neurosci., May 1, 2001; 21(9): 3151 - 3160. [Abstract] [Full Text] [PDF]
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S. Pereira, M. Zhou, A. Mocsai, and C. Lowell Resting Murine Neutrophils Express Functional {{alpha}}4 Integrins that Signal Through Src Family Kinases J. Immunol., March 15, 2001; 166(6): 4115 - 4123. [Abstract] [Full Text] [PDF]
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C. Rubel, G. C. Fernandez, G. Dran, M. B. Bompadre, M. A. Isturiz, and M. S. Palermo Fibrinogen Promotes Neutrophil Activation and Delays Apoptosis J. Immunol., February 1, 2001; 166(3): 2002 - 2010. [Abstract] [Full Text] [PDF]
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S. Verploegen, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase Blood, November 1, 2000; 96(9): 3215 - 3223. [Abstract] [Full Text] [PDF]
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R. Menegazzi, S. Busetto, R. Cramer, P. Dri, and P. Patriarca Role of Intracellular Chloride in the Reversible Activation of Neutrophil {beta}2 Integrins: A Lesson from TNF Stimulation J. Immunol., October 15, 2000; 165(8): 4606 - 4614. [Abstract] [Full Text] [PDF]
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A. Mocsai, Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, and E. Ligeti Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases J. Immunol., April 15, 2000; 164(8): 4321 - 4331. [Abstract] [Full Text] [PDF]
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 P. Suen, D Ilic, E Caveggion, G Berton, C. Damsky, and C. Lowell Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages J. Cell Sci., January 11, 1999; 112(22): 4067 - 4078. [Abstract] [PDF]
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S. Carreno, M.-E. Gouze, S. Schaak, L. J. Emorine, and I. Maridonneau-Parini Lack of Palmitoylation Redirects p59Hck from the Plasma Membrane to p61Hck-positive Lysosomes J. Biol. Chem., November 10, 2000; 275(46): 36223 - 36229. [Abstract] [Full Text] [PDF]
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S. Sergeant, K. A. Waite, J. Heravi, and L. C. McPhail Phosphatidic Acid Regulates Tyrosine Phosphorylating Activity in Human Neutrophils. ENHANCEMENT OF Fgr ACTIVITY J. Biol. Chem., February 9, 2001; 276(7): 4737 - 4746. [Abstract] [Full Text] [PDF]
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P. Peyron, I. Maridonneau-Parini, and T. Stegmann Fusion of Human Neutrophil Phagosomes with Lysosomes in Vitro. INVOLVEMENT OF TYROSINE KINASES OF THE Src FAMILY AND INHIBITION BY MYCOBACTERIA J. Biol. Chem., September 14, 2001; 276(38): 35512 - 35517. [Abstract] [Full Text] [PDF]
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