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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2767-2777
Signaling Through CD43 Induces Natural Killer Cell
Activation, Chemokine Release, and PYK-2 Activation
By
Marta Nieto,
José Luis Rodríguez-Fernández,
Francisco Navarro,
David Sancho,
José M.R. Frade,
Mario Mellado,
Carlos Martínez-A,
Carlos Cabañas, and
Francisco Sánchez-Madrid
From the Servicio de Inmunología, Hospital de la Princesa,
Universidad Autónoma de Madrid, Madrid, Spain; the Departamento
de Bioquímica y Biología Molecular, Facultad de
Medicina, Universidad Complutense, Madrid, Spain; and the Department of
Immunology and Oncology, Centro Nacional de Biotecnología,
CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco,
Madrid, Spain.
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ABSTRACT |
Natural killer (NK) cell activation is the result of a balance
between positive and negative signals triggered by specific membrane
receptors. We report here the activation of NK cells induced through
the transmembrane glycoprotein CD43 (leukosialin, sialophorin).
Engagement of CD43 by specific antibodies stimulated the secretion of
the chemokines RANTES, macrophage inflammatory protein
(MIP)-1 , and MIP-1 , which was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, signaling through CD43 increased the cytotoxic activity of
NK cells and stimulated an increase in the tyrosine kinase activity in
antiphosphotyrosine immune complexes of NK cell lysates. PYK-2 was
identified among the tyrosine kinase proteins that become activated.
Hence, PYK-2 activation was observed after 20 minutes of CD43
stimulation, reached a maximum after 45 to 60 minutes, and decreased to
almost basal levels after 120 minutes of treatment. Together, these
results demonstrate the role of CD43 as an activation molecule able to
transduce positive activation signals in NK cells, including the
regulation of chemokine synthesis, killing activity, and tyrosine
kinase activation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
AS ESSENTIAL COMPONENTS of the
innate immune system, natural killer (NK) cells kill cellular targets
and secrete cytokines that modulate acquired immunity.1 NK
cell activity is tightly regulated by a counter-balance of signals that
activate and inhibit their effector function.2-4 Inhibitory
receptors, specific for major histocompatibility complex (MHC) class I,
have been widely characterized and are known to mediate their effects
through cytoplasmic sequences termed immuno- receptor tyrosine-based
inhibitory motifs (ITIMs). In contrast, receptor-ligand interactions
responsible for activation of NK cells remain less characterized.
Nevertheless, it is largely known that the activation process involves
soluble factors such as the  / interferons, cytokines (eg, tumor
necrosis factor- [TNF-], interleukin-2 [IL-2], and IL-15),
and chemokines, as well as activation signals transduced through
certain membrane receptors. Receptors that have been reported to
mediate positive signals in NK cells include the low-affinity receptor
for IgG or CD16 (Fc RIII), CD2, CD28, and the CD40
ligand.2,5 Other receptors, such as CD69 and CD44, have
been implicated in NK cell activation based on the ability of
monoclonal antibodies (MoAbs) against these molecules to induce lysis
of Fc receptor-bearing target cells.6,7 Activation
receptors are thought to mediate positive signals in NK cells through
protein kinase activation, whereas NK cell receptor inhibitory signals
involve recruitment of protein tyrosine phosphatases (ie, SHP). In this
regard, it has been reported that ligation of CD16 and CD2 receptors
triggers activation and recruitment of tyrosine kinases of the Src
family.2,8
CD43 (leukosialin, sialophorin) is a transmembrane glycoprotein,
selectively expressed by hematopoietic cells, that bears a heavily
O-glycosylated extracellular N-terminal region.9 CD43
belongs to the growing family of cell-associated mucins, from which at
least 4 members, GlyCAM, CD34, MadCAM, and PSGL-1, appear to be
physiological ligands of selectins.10 However, it has been
proposed that the extended structure of mucins and their negative
charge may provide a repulsive barrier around the cell that limits
nonspecific adhesion phenomena. In the case of CD43, its role as an
adhesion receptor remains controversial, and different reports have
provided evidence supporting both its adhesive and antiadhesive
activity. Hence, this molecule has been described as a counter-receptor
for intercellular adhesion molecule-1 (ICAM-1), MHC class I molecules,
C1q, E-selectin, galectin-1, and for a putative endothelial cell
ligand.11-17 In contrast, several reports indicate that,
due to its stiffened and large negatively charged structure, CD43
prevents the interaction of surface receptors with their ligands,
acting as an antiadhesive molecule that negatively regulates T-cell
adhesion and homing.18-21 Both adhesive and antiadhesive properties of CD43 have been recently integrated in a model that proposes a dual functionality of CD43 in regulating cell interactions depending on different immune events.10 This is further
supported by recent data showing the impairment of
CD43 leukocytes to emigrate in response to
chemoattractants.22 Nevertheless, it is now clear that CD43
plays a role in the activation of T lymphocytes, monocytes, B cells,
and cytotoxic lymphocytes.23-27 According to its role as an
accessory molecule, engagement of CD43 with specific MoAbs regulates
integrin-mediated T-cell adhesion to endothelial and extracellular
matrix proteins and promotes cell aggregation in monocytes and T
lymphocytes, thus demonstrating that CD43 regulates integrin
function.28-31
Despite its well-established role as a stimulatory molecule, the
signals triggered by CD43 receptor are poorly characterized. In this
regard, it has been reported that CD43 is able to transduce signals
that lead to intracellular Ca2+ mobilization and activation
of protein kinase C (PKC). PKC, in turn,
hyperphosphorylates the cytoplasmic domain of CD43.32,33 Furthermore, it has also been reported that CD43 regulates tyrosine phosphorylation, including phosphorylation of Vav and mitogen-activated protein kinase, and that activation through CD43 induces its
association to the tyrosine kinase Fyn in T
lymphocytes.23,34,35
PYK-2 (proline-rich tyrosine kinase 2), also called RAFTK (related
adhesion focal tyrosine kinase), CAK (cell adhesion kinase ), and
CadTK (cell adhesion dependent tyrosine kinase), is a member of the FAK
nonreceptor tyrosine kinase family and is expressed by different cell
types, including brain, platelets, and other hematopoietic cells. PYK-2
shares significant sequence homology with FAK (60% identity in the
central catalytic domain and 40% identity in both the C and N termini)
and, like FAK, does not contain SH2 or SH3 domains, but presents
several sites for binding of SH2/SH3-containing signaling proteins.
PYK-2 is rapidly phosphorylated in response to stimuli that elevate
calcium or activate PKC.36-40
We studied here the role of CD43 as an activation molecule able to
transduce positive activation signals in NK cells, including the
regulation of the tyrosine kinase PYK-2.
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MATERIALS AND METHODS |
Cells.
IL-2-cultured NK cells were obtained essentially as
described.41 In brief, peripheral blood T lymphocytes (PBL)
were cultured with irradiated (5 Gy) RPMI 8866 lymphoblastoid cells for
6 to 9 days in RPMI 1640 supplemented with 10% fetal calf serum (FCS; complete medium), followed by a negative selection step using an
anti-CD3 MoAb plus rabbit complement (Behring, Marburg, Germany). The
CD3 cells (<5% CD3+) were cultured
with 50 IU/mL of recombinant human IL-2 (rhIL-2) until
use. These cell populations are hereafter referred to as NK cells.
After each purification process, the resulting population was
characterized by flow cytometry analysis. We routinely obtained a cell
population with a proportion of CD56+ and CD16+
cells greater than 95% and with less than 5% of CD3+,
CD19+, or CD14+ contaminating cells.
Antibodies and reagents.
The anti-CD43 MoAb TP1/36 (IgG1), HP2/21 (IgM), the nonactivatory
anti-CD44 MoAb HP2/9 (IgG1), and the anti-CCR5 MoAb CCR5-01(IgM) have
been previously described.42,43 The anti-CD56 K218 (IgG1) was kindly provided by Dr A. Moretta (Istituto Nazionale per la Ricerca
sul Cancro e Centro Biotecnologie Avanzate, University of Genova,
Genova, Italy). F(ab')2 fragments were obtained by pepsin digestion of purified antibody.41 PYK-2 antipeptide
polyclonal antibodies C-19 and N-19 were from Santa Cruz Biotechnology
Inc (Santa Cruz, CA). The anti-Tyr(P) PY20 MoAb was from
Transduction Laboratories (Lexington, KY). [32P]ATP
(4,000 Ci/mmol) was from ICN (Irvine, CA). Protein
A-agarose and protein G-agarose were from Boehringer Mannheim
(Mannheim, Germany). ECL reagents were from Amersham
(Buckinghamshire, UK). Tyrosine kinase inhibitor genistein
was from Calbiochem-Novabiochem Ltd (Nottingham, UK). All other
reagents used were of the purest grade available.
Preparation of antibody-coated dishes.
Plates were precoated overnight at 4°C with 2.5 µg/mL of antibody
in adhesion buffer (20 mmol/L Tris/HCl, 150 mmol/L NaCl, pH 8.2),
blocked with 1% bovine serum albumin (BSA) in adhesion buffer for 1 hour at room temperature, and then washed twice with phosphate-buffered
saline (PBS).
Chemokine quantification.
NK cells (106) were incubated in polypropylene tubes, to
prevent signaling through integrins, in a final volume of 1 mL complete medium in the presence of 5 µg/mL of different MoAbs for different times at 37°C, 5% CO2. Cells were then centrifuged and
the chemokines present in the supernatant were quantified. Human RANTES
was measured using the Cytoscreen immunoassay kit (Biosource
International, Inc, Camarillo, CA), and human macrophage inflammatory
protein (MIP)-1 and MIP-1 were measured using the
Quantikine immunoassay kit (R&D Systems, Minneapolis, MN), following
the manufacturer's instructions.
Redirected lysis assays.
Redirected lysis assays were performed as described.41 NK
cells were in triplicate tested in a 4-hour 51Cr release
assay against the murine P815 (FcR+) mastocytoma cell
line at different E/T ratios. MoAb were preincubated for 15 minutes
with the target cells before NK cell addition. The percentage of
specific lysis was calculated as described previously.41 Data are expressed as the arithmetic mean of triplicates. In each case,
spontaneous release was always less than 10% of the maximum lysis.
Preparation of cell lysates/immunoprecipitations.
IL-2-activated NK cells were washed twice with RPMI. Experiments were
initiated by adding the cells (10 × 106 cells, unless
otherwise stated) to 60-mm cultured dishes precoated with the relevant
antibodies. Cells were incubated in ice for 15 minutes and then were
incubated at 37°C for the indicated times. The incubation was
stopped by solubilizing the cells in 1 mL of ice-cold lysis buffer (10 mmol/L Tris/HCl, pH 7.65, 5 mmol/L EDTA, 150 mmol/L NaCl, 30 mmol/L
sodium pyrophosphate, 50 mmol/L NaF, 2 mmol/L sodium orthovanadate, 1%
Triton X-100, 50 µg/mL aprotinin, 50 µg/mL leupeptin, 5 µg/mL
pepstatin, and 1 mmol/L phenylmethylsulfonyl fluoride). Cell lysates
were clarified by centrifugation at 14,000 rpm for 10 minutes and the
pellets were discarded. After centrifugation, supernatants were
transferred to fresh tubes and proteins were immunoprecipitated at
4°C overnight with protein G-agarose-linked MoAbs directed against
Tyr(P) proteins (PY20 MoAb) or protein G-agarose-linked goat
polyclonal anti-Pyk-2 (C-19) antibody. Immunoprecipitates were washed
3 times with lysis buffer and either used for in vitro kinase reaction
(see below) or extracted in 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (200 mmol/L Tris/HCl, pH 6.8, 0.1 mmol/L sodium orthovanadate, 1 mmol/L
EDTA, 6% SDS, 2 mmol/L EDTA, 4% 2-mercaptoethanol, and 10% glycerol)
by boiling 5 minutes, were fractionated by SDS-PAGE (7.5%), and were
further analyzed.
In vitro kinase reactions.
Reactions were performed as described.44 Briefly,
immunoprecipitates were washed and pelleted (2,500 rpm 10 minutes at
4°C) 3 times in lysis buffer and twice with kinase buffer (20 mmol/L HEPES, 3 mmol/L MnCl2, pH 7.35). Pellets were
dissolved in 40 µL of kinase buffer and reactions were initiated by
adding 10 µCi of [32P] ATP. The reactions were
performed at 30°C for 15 minutes and were stopped on ice by adding
10 mmol/L EDTA. After the in vitro kinase reactions, the pellets were
washed in lysis buffer containing 10 mmol/L EDTA, extracted for 5 minutes at 95°C in 2× SDS-PAGE sample buffer, and analyzed by
SDS-PAGE. After fixing and drying of the gels, autoradiography was
performed at  80°C. Autoradiograms were analyzed using an
AGFA Studio ScanIIsi scanner (Montiel, Belgium) and bands
were quantified using the Bio-Rad Molecular Analyst Software (Bio-Rad,
Hercules, CA).
Western blotting.
Cell lysis and immunoprecipitations were performed as described above.
After SDS-PAGE, proteins were transferred to Immobilon membranes using
a Bio-Rad SD Transblot. Membranes were blocked using 3% nonfat dried
milk in PBS, pH 7.2, and incubated for 2 hours at 22°C with the
polyclonal antibody anti-Pyk-2 (C-19 or N-19) or with the anti-Tyr (P)
PY20 MoAb, all diluted 1:500 in PBS containing 3% nonfat dried milk.
After incubating membranes with horseradish peroxidase-conjugated
secondary antibodies, immunoreactive bands were visualized using ECL reagents.
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RESULTS |
Increased secretion of chemokines and NK cell cytotoxicity by
engagement of CD43.
Upon activation, NK cells are able to secrete cytokines that modulate
NK cytotoxic response and other immune events.45,46 Different chemokines, including RANTES, MIP-1, MIP-1, and
lymphotactin, are secreted by NK cells.43,47-49 These
chemokines trigger the directional migration or chemotaxis of NK cells,
regulate cell polarization and redistribution of adhesion molecules,
stimulate intracellular Ca2+ mobilization and cytolytic
granule release, and augment NK-mediated cytotoxicity.50-53
To investigate the possible role of CD43 as a triggering molecule in NK
cells, we assayed the effect on chemokine secretion by NK cells of
anti-CD43 MoAbs that have previously been described to stimulate
integrin-mediated adhesion, cell polarization, and homotypic
aggregation in T lymphocytes.28,54 IL-2-activated NK cells
constitutively secrete MIP-1, MIP-1, and RANTES
(Fig 1A through C).
Engagement of CD43 with TP1/36 MoAb significantly increases the
production of these chemokines by NK cells. The increased release of
chemokines in NK cell supernatants was clearly detectable after 4 hours
of incubation with the anti-CD43 TP1/36 and persisted after 8 hours of
incubation (Fig 1A). Treatment of NK cells with cycloheximide abolished
the secretion of chemokines induced by anti-CD43, thus indicating the
requirement of protein synthesis for this process (data not shown).
Engagement of CD43 with the anti-CD43 MoAb (HP2/21) of a different
isotype also increased chemokine production by NK cells (Fig 1B).
Furthermore, the incubation of NK cells on TP1/36
Fab'2 fragment-coated dishes also stimulated the
secretion of chemokines (Fig 1C). In contrast, control isotype-matched MoAbs against the highly expressed membrane adhesion molecule CD44 or
the chemokine receptor anti-CCR5 did not affect cytokine production,
ruling out the possibility of an Fc-receptor mediated effect (Fig 1B
and C).
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| Fig 1.
Induction of chemokine release in NK cells by engagement
of CD43. (A) Kinetics of the chemokine production induced by the
pro-activatory anti-CD43 TP1/36 MoAb in NK cells. IL-2-activated NK
cells were incubated in complete medium for different periods of time
in the absence or presence of anti-CD43 TP1/36 MoAb (5 µg/mL). Cell
supernatants were then assayed for different chemokines as described in
Materials and Methods. This is a representative experiment of 2 independent experiments. (B) Secretion of RANTES and MIP-1 by NK
cells stimulated with different MoAb. IL-2-activated NK cells were
incubated in complete medium for 4 hours in the absence or the presence
of the following MoAbs (5 µg/mL): anti-CD44 (HP2/9, IgG1); anti-CD43
(TP1/36 and HP2/21, IgG1 and IgM, respectively); and anti-CCR5 (IgM).
Chemokines present in cell supernatants were then measured as described
in Materials and Methods. In the absence of NK cells, the presence of
chemokines was undetectable in complete medium (medium). The arithmetic
mean ± SE of 6 independent experiments performed with cells from 6 different donors are shown. (C) The divalent F(ab')2
fragments of anti-CD43 MoAb induce RANTES and MIP-1 production in NK
cells. IL-2-activated NK cells were allowed to adhere on dishes
precoated with saturating concentrations of different MoAbs for 4 hours
in complete medium. Chemokines present in cell supernatants were then
measured. Chemokines were undetectable in complete medium (medium). The
arithmetic mean ± SE of 3 experiments performed with cells from
different donors are shown.
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We next assessed the possible effect of the anti-CD43 MoAb TP1/36 on
the cytotoxic activity of NK cells. IL-2-activated NK cells treated
with the anti-CD43 MoAb showed a significant increase of cytotoxic
activity. Similarly to the chemokine production response, the increase
in NK cell-mediated cytotoxicity triggered by anti-CD43 varied among
donors (Fig 2). Control isotype-matched
MoAb did not modify the cytotoxic activity of effector NK cells (Fig
2). These data further indicate that CD43 functions as a stimulatory molecule in NK cells.
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| Fig 2.
Enhancement of NK cell-mediated cytotoxicity by
engagement of CD43. Effect of the proactivatory anti-CD43
TP1/36 MoAb and the control isotype-matched anti-CD56 K218 MoAb in the
cytolytic activity of IL-2-cultured NK cells against P815 target
cells. NK killing was determined in a 4-hour 51Cr release
assay, as described in Materials and Methods. The arithmetic mean ± SE of 3 different experiments corresponding to 3 independent donors is
shown. The differences were significant according to Student's
t-test (P < .05).
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CD43-mediated signaling involves the activation of protein tyrosine
kinases.
Positive regulation of NK cell activity involves protein kinase
activation.2-4 To examine the possible involvement of
protein tyrosine kinases in the signals triggered by CD43, NK cells
were pretreated with the tyrosine kinase inhibitor
genistein55 before stimulation with anti-CD43 MoAb, and
then chemokines released were quantitated in cell supernatants. As
shown in Fig 3, CD43-induced production of
RANTES and MIP-1 was abolished by genistein, suggesting that
genistein-sensitive tyrosine kinases are required for signaling through
CD43 in NK cells.
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| Fig 3.
Effect of genistein on CD43-induced chemokine production
and NK cell cytotoxicity. (A and B) IL-2-activated NK cells were
incubated for 1 hour in ( ) complete medium or complete medium in the
presence of ( ) 30 µmol/L genistein or an equivalent amount of
( ) disolvent dimethyl sulfoxide (DMSO). Anti-CD43
TP1/36 or anti-CD44 HP2/9 MoAbs were then added and cells were
incubated for additional 4 hours. Chemokines present in cell
supernatants were measured as described above. The arithmetic mean ± SE of 3 experiments performed with cells from different donors is
shown.
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Engagement of CD43 induces activation of the PYK-2 tyrosine-kinase in
NK cells.
We next examined the effect of anti-CD43 TP1/36 MoAb on the tyrosine
kinase activities present in NK cells. Cells plated on dishes coated
with the anti-CD43 MoAb TP1/36 were allowed to attach for different
times and then lysed. Tyrosine-phosphorylated proteins were
immunoprecipitated from the cell lysates with the MoAb PY20, and the
resulting immunocomplexes were incubated with
[32P]--ATP and then analyzed by SDS-PAGE. As shown in
Fig 4A, we observed a time-dependent
phosphorylation of proteins in the molecular weight (Mr)
of 110 to 80 kD and in the 48 to 83 kD range (Fig 4A, left panel).
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| Fig 4.
Time-course of CD43-induced tyrosine kinase activity and
presence of PYK-2 in anti-Tyr (P) immunoprecipitates. (A) NK cells were
allowed to adhere for the indicated times on dishes precoated with the
anti-CD43 TP1/36 MoAb. The cells were then lysed and the extracts were
immunoprecipitated (IP) with the PY20 anti-Tyr(P) MoAb [IP: Tyr (P)],
and kinase reactions performed as described in Materials and Methods
(left panel). After the kinase reaction was performed, the major Tyr
(P)-labeled bands in the cells stimulated for 60 minutes were eluted
from the PY20 immunocomplex by boiling the pellet in 100 µL of a
solution containing 10 mmol/L Tris, pH 7.4, and 1% SDS. Denaturated
Tyr(P) proteins were then reimmunoprecipitated (r-IP) with the C-19
anti-PYK-2 antibody to confirm the presence of PYK-2 [IP: Tyr(P);
r-IP: PYK-2] or with nonimmune serum control [IP: Tyr (P); r-IP: IgG;
right panel]. After SDS-PAGE (7.5%) of immunoprecipitates, gels were
subjected to autoradiography. The position of the major phosphorylated
bands in the gel is indicated with arrowheads. PYK-2 position is
indicated by an arrow. Molecular weight markers (in kilodaltons) are
shown on the left side of the figure. (B) Cells were lysed and the
extracts were incubated with C-19 antibody to immunoprecipitate PYK-2.
Tyrosine phosphorylation levels of PYK-2 were determined by Western
blot analysis with the PY20 anti-Tyr (P) MoAb.
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PYK-2 has been recently identified as a 115-kD tyrosine kinase
homologous to FAK that is expressed in different cell types, including
NK cells.36-38,56 A protein band displaying an Mr equal to
that of PYK-2 was among the phosphorylated proteins present in the
lysates stimulated with the anti-CD43 TP1/36 MoAb. To
determine whether PYK-2 was a component of the Mr 110- to 180-kD bands
in the PY20 immune complexes, parallel cultures of NK cells were treated with the anti-CD43 MoAb for 45 minutes, in vitro kinase analysis was performed, and [32P]-labeled
phosphotyrosinated proteins were eluted from the complexes by
denaturation and then reimmunoprecipitated with the anti-PYK-2 polyclonal antibody. The results shown in Fig 4A (right panel) demonstrate that PYK-2 is a constituent of the Mr 110- to 180-kD Tyr
(P) bands induced after engagement of CD43. We confirmed by Western
blotting that CD43 induces tyrosine phosphorylation of PYK-2. Lysates
obtained from cells that were plated on dishes coated with the
anti-CD43 TP1/36 MoAb for different times were immunoprecipitated with
the C-19 anti-PYK-2 antibody and tyrosine phosphorylation of PYK-2 was
determined by immunoblotting using the MoAb PY20. As shown in Fig 4B,
tyrosine phosphorylation of PYK-2 increased gradually, reaching a
maximum after 60 minutes of CD43 stimulation.
To confirm directly that CD43 stimulates PYK-2 activity, NK cells were
incubated for 60 minutes on dishes coated with the anti-CD43 MoAb
TP1/36. Cells were then lysed and immunoprecipitated with the C-19
anti-PYK-2 polyclonal antibody. PYK-2 immunoprecipitates were
incubated with [32P]--ATP and analyzed by SDS-PAGE. As
shown in Fig 5A, CD43 induced an increase
in the PYK-2 autokinase activity. F(ab')2 of
anti-CD43 TP1/36 MoAb also induced a similar activation PYK-2 (Fig 5C). The increase in PYK-2 phosphorylation of activity was not observed when
NK cells were plated on dishes precoated with the nonstimulating isotype control anti-CD44 MoAb HP2/9 or when NK cells were incubated with conditioned medium obtained from cells plated on TP1/36
MoAb-coated dishes for 60 minutes, ruling out an autocrine loop in the
activation of PYK-2 (Fig 5A and data not shown). Immunoblotting with
anti-PYK-2 C-19 antibody immunoprecipitates performed in parallel
verified that similar amounts of PYK-2 were recovered after stimulation with anti-CD43 MoAbs or its F(ab')2 fragments (Fig 5A
and C; IP: C-19; WB: C-19). Densitometric scanning showed that
anti-CD43 MoAb TP1/36 induced a 4- ± 0.5-fold increase (n = 5) in
the phosphorylation level of PYK-2 (Fig 5B).
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| Fig 5.
CD43-induced stimulation of PYK-2 tyrosine kinase
activity. (A) NK cells were allowed to adhere for 60 minutes, on dishes
precoated with BSA, with the HP 2/9 anti-CD44 MoAb, TP 1/36 anti-CD43
MoAb, or TP1/36 F(ab')2 fragments. Cells were then
lysed and the extracts were incubated with C-19 antibody to
immunoprecipitate PYK-2 (C-19) or with nonimmune serum (NIS) control
and in vitro kinase reactions, performed as described in Materials and
Methods (IVK, top). PYK-2 levels were determined by immunoprecipitation
with the C-19 anti-PYK-2 antibody and Western blot analysis with C-19
anti-PYK-2 antibody (IP: C-19; WB: C-19; bottom). The position of
PYK-2 is indicated with an arrow. (B) Quantification by densitometric
scanning of the effect of stimulation with BSA, anti-CD44, and
anti-CD43 on PYK-2 activity. PYK-2 was immunoprecipitated with the C-19
antibody and in vitro kinase reactions performed as described in
Materials and Methods. Values are the mean± SEM of 5 independent
experiments and are expressed as fold-stimulation above control. (C) NK
cells were allowed to adhere for 60 minutes on dishes precoated with
BSA or TP1/36 F(ab')2 fragments. Cells were then
lysed and processed as described above.
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Kinetic studies of CD43-induced activation of PYK-2 showed that the
increase in the autokinase activity of PYK-2 reached a maximum after 45 to 60 minutes, returning to basal levels after 180 minutes. When
soluble anti-CD43 MoAb was added directly to the cell suspension, it
was also able to induce activation of PYK-2
(Fig 6A and B). Taken together, these
results show that engagement of CD43 increases the phosphotyrosine
kinase activity of PYK-2.
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| Fig 6.
Time-course of CD43-stimulated PYK-2 activity in NK cells.
(A) NK cells were either plated on dishes coated with BSA
(BSA), HP 2/9 anti-CD44 MoAb (-CD44), and anti-CD43 MoAb TP1/36
(-CD43) or plated on dishes coated with BSA and stimulated with
soluble anti-CD43 MoAb (soluble -CD43). The cells were allowed to
adhere for the indicated times and lysed. Lysates were incubated with
the C-19 antibody to immunoprecipitate PYK-2 and activities in the
resulting immunoprecipitates were measured by in vitro kinase
reactions, as described in Materials and Methods. A representative
experiment of 3 is shown. (B) Quantification by densitometric scanning
of the effect of stimulation with anti-CD43 on PYK-2 activity. PYK-2
was immunoprecipitated with the C-19 antibody and in vitro kinase
reactions performed as described in Materials and Methods. (s) -CD43
indicates soluble antibody. Values are the mean ± SE of 3 independent
experiments and are expressed as fold-stimulation above control.
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| |
DISCUSSION |
We provide evidence here suggesting that CD43 positively regulates NK
cell activity through phosphorylation signals. In addition, we have
investigated the involvement of the protein tyrosine kinase PYK-2 in
this signaling pathway. Despite the fact that a definitively confirmed
ligand for CD43 is still lacking and its controversial function as an
adhesion molecule, our data further support the role of this receptor
as an accessory molecule in lymphocytes. In this regard, we have
explored different aspects of the activation events triggered by CD43
in NK cells. Our results show that engagement of CD43 induces the
augmented secretion of chemokines. This effect appeared to be
independent of homotypic cell aggregation triggered by the anti-CD43
MoAb,28 as demonstrated by the results obtained with
antibody-coated dishes, in which cells adhere and do not aggregate.
This fact allows us to rule out the possibility of an activation effect
as a consequence of cell-cell interactions.
Chemokines have been shown to be major factors regulating the directed
migration of leukocytes, including NK cells, in inflammation and
immunity.57-59 In addition, it has been described that
chemokines stimulate Ca2+ mobilization and cytolytic
granule release, promote cytotoxic activity, and regulate the
adhesiveness of NK cells.51,52 We have previously reported
that chemokine secretion by NK cells is dramatically increased by
contact with target cells and that the release of these soluble factors
induces the chemotaxis of additional NK cells. This suggests that
chemokines may function by amplifying the immune response at sites of
NK cell activation.43 It is therefore conceivable that the
chemokines RANTES, MIP1-, and MIP-1, released upon activation
through CD43, may increase the cytotoxic activity of NK cells at the
targeted tissue and may direct the migration of other neighboring NK
cells towards the target cells. In addition, these chemokines may
contribute to modulate the function of other leukocytes. In this
regard, it is worth mentioning that, in addition to its cytotoxic
activity, growing evidence points to the role of NK cells as regulators of the immune response.46
The role of CD43 as a positive modulator of NK cell function was
further reinforced by our finding that engagement of this receptor
increases the cytotoxic activity of NK cells. Furthermore, CD43 induced
phosphorylation and activation of tyrosine kinase proteins that are
thought, in general, to transduce the positive signals that trigger the
cytotoxic process. We have analyzed in detail some of the molecular
components of the phosphorylation pathways triggered through CD43.
PYK-2 was among the tyrosine kinases that became activated. PYK-2 is a
member of the FAK nonreceptor tyrosine kinase family expressed by
different cell types, including brain cells, platelets, and other
hematopoietic cells. The cascade of signaling events that trigger PYK-2
activation includes various stimuli that elevate intracellular calcium
or induce PKC activation.36,37 Hence, a possible linkage
between CD43 engagement and PYK-2 activation could be established,
because it has been reported that ligation of CD43 induces PKC
activation.33 Nevertheless, it should be taken into account
that PYK-2 tyrosine phosphorylation can be mediated via different
pathways in various cell sytems. The Src tyrosine kinase Fyn has been
reported to selectively phosphorylate PYK-2 in T cells. This
phosphorylation does not depend on intracellular Ca2+.60 Fyn, which associates with the
cytoplasmic tail of CD43,34 could be another candidate for
the molecule responsible for the phosphorylation and activation of
PYK-2 triggered through CD43.
The precise role of PYK-2 in the activation of NK cells through CD43
currently remains unknown. The homology sequence between FAK and PYK-2
seems to correspond to the functions that these two PTKs perform in the
cell, although the exact role of PYK-2 may differ depending on the cell
type.38 In this regard, studies using
FAK mutant mice have shown that expression of PYK-2
is induced in fibroblasts from these mice and that this expression
counterbalances FAK signaling functions triggered by 1
integrins.61 This suggested that the expression of both
proteins is regulated in a mutually exclusive fashion and that they
might undertake similar functions in the cell. Nevertheless, as occurs
with FAK, PYK-2 is best known for its functional association to 1,
2, and 3 integrins and its activation upon interaction of these
adhesion receptors with their ligands.56,62,63 Recently, it
has been described that NK cells express PYK-2, but not FAK, and that
outside-in signaling triggered by 1 integrin receptors stimulates
PYK-2 activation.56 Moreover, the constitutive association
of PYK-2 to paxillin, a cytoskeletal protein that is also associated to
pp125FAK, and to other proteins present in focal adhesions
such as vinculin has been recently reported.56,64 Integrins
are two-way signaling receptors, and it is therefore feasible that
PYK-2, as occurs with different second-messengers and mediators
involved in the outside-in integrin signaling, may also participate in
transducing signals from inside the cell to the exterior. The
possibility that CD43-mediated activation of PYK-2 may regulate
integrin affinity must be taken into account, because CD43 has been
reported to regulate 1 and 2 integrin-mediated lymphocyte
adhesion,28 a phenomenon that may lead to an increase in
the cytotoxic activity of NK cells.
Nevertheless, it is also feasible that PYK-2 may function independently
of integrin receptors and participates in other different signaling
events during CD43-induced activation. Hence, PYK-2 phosphorylation has
been involved in other processes, such as the signal events triggered
by the interaction of CCR5 with either the chemokine RANTES or the HIV
envelope protein gp120, the stress signals, or cytokine
signaling.65-67 Interestingly, different reports have shown
that engagement of the T-cell receptor triggers PYK-2 activation, which
is selectively phosphorylated by Fyn, and the association of PYK-2 to
Lck.60,68,69
Our results add novel elements to the range of protein kinases that
participate in the positive regulation of NK cell activity. It is clear
that the function of NK cells is modulated by the interplay between
negative and positive signals within the cell. Membrane receptors as
well as the signaling pathways involved in the activation of NK cells
have not been studied to the same extent as the inhibitory receptors
that provide specific recognition.2-4 The CD16 molecule
functions as a prototype activating receptor in NK cells, and the
signaling through this molecule has been extensively investigated
(reviewed in Lanier2 and Yokoyama4). Engagement
of CD16 triggers recruitment of tyrosine kinase of the src-family and
tyrosine phosphorylation of residues contained within the
immunoreceptor tyrosine-based motifs (ITAMs) in the cytoplasmic domains
of CD16-associated zeta and gamma chains, followed by activation of
ZAP-70, activation of phospholipase C, and the MAP kinase activation
pathway. As occurs with CD43, ligation of CD16 stimulates cytokine
production.70 These findings, together with the fact that
CD43 has been reported to be associated to the Src family tyrosine
kinase Fyn,34 suggest that the activation pathway triggered
by these two receptors, as well as by other positive modulators of NK
cell activity, may share more common elements. Therefore, it is likely
that CD16 receptor triggers PYK-2 phosphorylation. However, this issue
requires further investigation. It is therefore feasible that signals
triggered by positive regulators of NK cell-mediated cytotoxicity may
converge in a common signaling pathway. This activatory cascade of
signals may be induced by different receptors depending on the type of
NK cell cytotoxicity (ie, antibody-dependent cellular cytotoxicity or
MHC-recognition regulated killing). Triggering of this cascade by CD43,
as well as by other costimulatory receptors, such as CD16, may serve to augment the primary activation of NK cells through specific MHC receptors.
| |
ACKNOWLEDGMENT |
The authors thank Dr Miguel López-Botet for critical reading of
the manuscript.
| |
FOOTNOTES |
Submitted January 25, 1999; accepted June 9, 1999.
Supported by Grants No. SAF 99/0034 and 2FD97-0680-C02-02 from the
Ministerio de Educación y Cultura, Grants No. 07/44/96 (to
F.S.-M.) and 08.1/0015/97 (to C.C.) from the Comunidad Autónoma de Madrid, a grant from "Fundación Cientifica de la
Asociación Española contra el Cáncer" (to F.S.-M.
and C.C.), Grant No. SAF 98/0080 (to C.C.), and by fellowships from the
Fondo de Investigaciones Sanitarias BAE 97/5089 (to M.N.). J.L.R.-F.
was supported by a "Contrato de Reincorporación" associated
to Grants No. PB94-0231 and SAF98/0080, awarded by the "Ministerio
Español de Educación y Cultura." The Department of
Immunology and Oncology was founded and is supported by the CSIC,
Pharmacia, and Upjohn.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Francisco Sánchez-Madrid, PhD,
Servicio de Inmunología, Hospital de la Princesa, Universidad
Autónoma de Madrid, Diego de León 62, E-28006, Madrid,
Spain; e-mail: fsmadrid/princesa{at}hup.es.
| |
REFERENCES |
1.
Trinchieri G:
Biology of natural killer cells.
Adv Immunol
47:187, 1989[Medline]
[Order article via Infotrieve]
2.
Lanier L:
NK cell receptors.
Annu Rev Immunol
16:359, 1998[Medline]
[Order article via Infotrieve]
3.
Moretta A, Moretta L:
HLA class I specific inhibitory receptors.
Curr Opin Immunol
9:694, 1997[Medline]
[Order article via Infotrieve]
4.
Yokoyama WM:
Natural killer receptors.
Curr Opin Immunol
10:298, 1998[Medline]
[Order article via Infotrieve]
5.
Carbone E, Ruggiero G, Terrazzano G, Palomba C, Manzo C, Fontana S, Spits H, Karre K, Zappacosta S:
A new mechanism of NK cell cytotoxicity activation: The CD40-CD40 ligand interaction.
J Exp Med
185:2053, 1997[Abstract/Free Full Text]
6.
Moretta A, Poggi A, Pende D, Tripodi G, Orengo AM, Pella N, Augugliaro R, Bottino C, Ciccone E, Moretta L:
CD69 mediated pathway of lymphocyte activation: Anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolitic T lymphocytes expressing T cell receptor /.
J Exp Med
174:1393, 1991[Abstract/Free Full Text]
7.
Galandrini R, De Maria R, Piccoli M, Frati L, Santoni A:
CD44 triggering enhances human NK cell cytotoxic functions.
J Immunol
153:4399, 1994[Abstract]
8.
Leibson PJ:
Signal transduction during natural killer cell activation: Inside the mind of a killer.
Immunity
6:655, 1997[Medline]
[Order article via Infotrieve]
9.
Remold-O'Donnell E, Rosen F:
Proteolytic fragmentation of sialophorin (CD43). Localization of the activation-inducing site and examination of the role of sialic acid.
J Immunol
145:3372, 1990[Abstract]
10.
Ostberg JR, Barth RK, Frelinger JG:
The roman god Janus: A paradigm for the function of CD43.
Immunol Today
19:546, 1998[Medline]
[Order article via Infotrieve]
11.
Rosenstein Y, Park JK, Hahn WC, Rosen FS, Bierer BE, Burakoff SJ:
CD43, a molecule defective in Wiskott-Aldrich syndrome, binds ICAM-1.
Nature
354:233, 1991[Medline]
[Order article via Infotrieve]
12.
Stökl J, Madjic O, Khol P, Rick WF, Menzel JE, Knapp W:
Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation.
J Exp Med
184:1769, 1996[Abstract/Free Full Text]
13.
Guan EN, Burgess WH, Robinson SL, Goodman EB, McTigue KJ, Tenner AJ:
Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis.
J Biol Cell
266:20345, 1991
14.
Sawada R, Tsuboi S, Fukuda M:
Differential E-selectin-dependent adhesion efficiency in sublines of a human colon cancer exhibiting distinct metastatic potentials.
J Biol Chem
269:1425, 1994[Abstract/Free Full Text]
15.
Zhang K, Baeckstrom D, Brevinge H, Hansson GC:
Comparison of sialyl-Lewis a-carrying CD43 and MUC1 mucins secreted from a colon carcinoma cell line for E-selectin binding and inhibition of leukocyte adhesion.
Tumour Biol
18:175, 1997[Medline]
[Order article via Infotrieve]
16.
Baum LG, Pang M, Perillo NL, Wu T, Delegeane A, Uittenbogaart CH, Fukuda M, Seilhamer JJ:
Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells.
J Exp Med
181:877, 1995[Abstract/Free Full Text]
17.
McEvoy LM, Sun H, Frelinger JG, Butcher EC:
Anti-CD43 inhibition of T cell homing.
J Exp Med
185:1493, 1997[Abstract/Free Full Text]
18.
Ardman B, Sikorski MA, Staunton DE:
CD43 interferes with T lymphocyte adhesion.
Proc Natl Acad Sci USA
89:5001, 1992[Abstract/Free Full Text]
19.
McFarland TA, Ardman B, Manjunath N, Fabry JA, Lieberman J:
CD43 diminishes susceptibility to T lymphocyte-mediated cytolysis.
J Immunol
154:1097, 1995[Abstract]
20.
Manjunath N, Johnson RS, Staunton DE, Pasqualini R, Ardman B:
Targeted disruption of CD43 gene enhances T lymphocyte adhesion.
J Immunol
151:1528, 1993[Abstract]
21.
Stockton BM, Cheng G, Manjunath N, Ardman B, von Adrian UH:
Negative regulation of T cell homing by CD43.
Immunity
8:373, 1998[Medline]
[Order article via Infotrieve]
22.
Woodman RC, Johnston B, Hickey MJ, Teoh D, Reinhardt P, Poon BY, Kubes P:
The functional paradox of CD43 in leukocyte recruitment: A study using CD43 deficient mice.
J Exp Med
188:2181, 1998[Abstract/Free Full Text]
23.
Manjunath N, Ardman B:
CD43 regulates tyrosine phosphorylation of a 93-kD protein in T lymphocytes.
Blood
86:4194, 1995[Abstract/Free Full Text]
24.
Mentzer SJ, Remold-O'Donell E, Crimmins MAV, Bierer BE, Rosen FS, Burakoff SJ:
Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation.
J Exp Med
165:1383, 1987[Abstract/Free Full Text]
25.
Sperling AI, Jonathan M, Green R, Mosley RL, Smith PL, DiPaolo RJ, Klein JR, Bluestone JA, Thompson CB:
CD43 is a murine T cell costimulatory receptor that functions independently of CD28.
J Exp Med
182:139, 1995[Abstract/Free Full Text]
26.
Axelsson B, Youseffi-Etemad R, Hammarström S, Perlmann P:
Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43.
J Immunol
141:2912, 1988[Abstract]
27.
Vargas-Cortes M, Axelsson B, Larsson A, Berzins T, Perlmann P:
Enhancement of human spontaneous cell-mediated cytotoxicity by a monoclonal antibody against the large syaloglycoprotein (CD43) on peripheral blood lymphocytes.
Scan J Immunol
27:661, 1988[Medline]
[Order article via Infotrieve]
28.
Sánchez-Mateos P, Campanero MR, del Pozo MA, Sánchez-Madrid F:
Regulatory role of CD43 leukosialin on integrin-mediated T-cell adhesion to endothelial and extracellular matrix ligands and its polar redistribution to cellular uropod.
Blood
86:2228, 1995[Abstract/Free Full Text]
29.
Nong Y-H, Remold-O'Donell E, LeBien TW, Remold HG:
A monoclonal antibody to sialophorin (CD43) induces homotypic adhesion and activation of human monocytes.
J Exp Med
170:259, 1989[Abstract/Free Full Text]
30.
Kuijpers TW, Hoogerwerf M, Kuijpers KC, Schwartz BR, Harlan JM:
Cross-linking of sialophorin (CD43) induces neutrophil aggregation in a CD18-dependent and a CD18-independent way.
J Immunol
149:998, 1992[Abstract]
31.
Youseffi-Etemad R, Axelsson B:
Parallel pattern of expression of CD43 and LFA-1 on the CD45RA+ (naive) and CD45R0+ (memory) subset of human CD4+ and CD8+ cells. Correlation with the aggregative response of the cells to CD43 monoclonal antibodies.
Immunology
87:439, 1996[Medline]
[Order article via Infotrieve]
32.
Piller V, Piller F, Fukuda M:
Phosphorylation of the major leukocyte surface sialoglycoprotein, leukosialin, is increased by phorbol 1-myristate 13 acetate.
J Biol Chem
264:18824, 1989[Abstract/Free Full Text]
33.
Wong RCK, Remold-O'Donell E, Vercelli D, Sancho J, Terhorst C, Rosen F, Geha R, Chatila T:
Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C.
J Immunol
144:1455, 1990[Abstract]
34.
Pedraza-Alva G, Mérida LB, Burakoff SJ, Rosenstein Y:
CD43-specific activation of T cells induces association of CD43 to Fyn-kinase.
J Biol Chem
271:27564, 1996[Abstract/Free Full Text]
35.
Pedraza-Alva G, Mérida LB, Burakoff SJ, Rosenstein Y:
T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation.
J Biol Chem
273:14218, 1998[Abstract/Free Full Text]
36.
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J:
Protein tyrosine kinase PYK-2 involved in Ca2+ induced regulation of ion channel and MAP kinase functions.
Nature
376:737, 1995[Medline]
[Order article via Infotrieve]
37.
Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li S, Jiang S, Pasztor LM, White RA, Groopman JE, Avraham H:
Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain.
J Biol Chem
270:27742, 1995[Abstract/Free Full Text]
38.
Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K, Sasaki T:
Cloning and characterization of cell adhesion kinase p, a novel protein tyrosine kinase of the focal adhesion kinase subfamily.
J Biol Chem
270:21206, 1995[Abstract/Free Full Text]
39.
Earp HS, Huckle WR, Dawson TL, Li X, Graves LM, Dy R:
Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells.
J Biol Chem
270:28440, 1995[Abstract/Free Full Text]
40.
Herzog H, Nicholl J, Hort YJ, Sutherland GR, Shine J:
Molecular cloning and assignment of FAK-2, a novel human focal adhesion kinase to 8p11.2-p22 by nonisotopic in situ hybridization.
Genomics
32:484, 1996[Medline]
[Order article via Infotrieve]
41.
Aramburu J, Balboa MA, Ramírez A, Silva A, Acevedo A, Sánchez-Madrid F, de Landázuri MO, López-Botet M:
A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-/ + T lymphocytes. Inhibition of IL-2 dependent proliferation by anti-Kp43 monoclonal antibody.
J Immunol
147:714, 1990[Abstract]
42.
Campanero MR, Pulido R, Alonso JL, Pivel JP, Pimentel-Muiños FX, Fresno M, Sánchez-Madrid F:
Down regulation by tumor necrosis factor alpha of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism.
Eur J Immunol
21:3045, 1991[Medline]
[Order article via Infotrieve]
43.
Nieto M, Navarro F, Perez-Villar JJ, del Pozo MA, González-Amaro R, Mellado M, Frade JMR, Martínez-A C, López-Botet M, Sánchez-Madrid F:
Roles of chemokines and receptor polarization in NK-target cell interactions.
J Immunol
161:3330, 1998[Abstract/Free Full Text]
44.
Rodríguez-Fernández JL, Rozengurt E:
Bombesin, vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine induce focal adhesion kinase activation on intact Swiss 3T3 cells.
J Biol Chem
273:19321, 1998[Abstract/Free Full Text]
45.
Cassatella MA, Anegón I, Cuturi MC, Griskey P, Trinchieri G, Perussia B:
FcR (CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in FcR (CD16)-induced transcription and expression of lymphokine genes.
J Exp Med
169:549, 1989[Abstract/Free Full Text]
46.
Horwitz DA, Gray JD, Ohtsuka K, Hirokawa M, Takahashi T:
The immunoregulatory effects of NK cells: the role of TGF-beta and implications for autoimmunity.
Immunol Today
18:538, 1997[Medline]
[Order article via Infotrieve]
47.
Somersalo K, Carpén O, Saksela E:
Stimulated natural killer cells secrete factors with chemotactic activity, including NAP-1/IL-8, which supports VLA-4 and VLA-5-mediated migration of T lymphocytes.
Eur J Immunol
24:2957, 1994[Medline]
[Order article via Infotrieve]
48.
Bluman EM, Bartynski KJ, Avalos BR, Caligiuri MA:
Human natural killer cells produce abundant macrophage inflammatory protein-1 alpha in response to monocyte-derived cytokines.
J Clin Invest
12:2722, 1996
49.
Hedrick JA, Saylor V, Figueroa D, Mizoue L, Xu Y, Menon S, Abrams J, Handel T, Zlotnik A:
Lymphotactin is produced by NK cells and attracts both NK cells and T cells in vivo.
J Immunol
158:1533, 1997[Abstract]
50.
Maghazachi AA, Al-Aoukaty A, Schall TJ:
C-C chemokines induce the chemotaxis of NK and IL-2-activated NK cells.
J Immunol
153:4969, 1994[Abstract]
51.
Taub DD, Sayers TJ, Carter CRD, Ortaldo JR:
and chemokines induce NK cell migration and enhance NK-mediated cytolysis.
J Immunol
155:3877, 1995[Abstract]
52.
Loetscher P, Seitz M, Clark-Lewis, Baggiolini M, Moser B:
Activation of NK cells by CC chemokines.
J Immunol
156:322, 1996[Abstract]
53.
Allavena P, Bianchi G, Zhou D, van Damme J, Jîlek P, Sozzani S, Mantovani A:
Induction of natural killer cell migration by monocyte chemotactic protein-1,-2 and -3.
Eur J Immunol
24:3233, 1994[Medline]
[Order article via Infotrieve]
54.
Serrador JM, Nieto M, Alonso-Lebrero JL, del Pozo MA, Calvo J, Furthmayr H, Schwartz-Albiez R, Lozano F, Gónzalez-Amaro R, Sánchez-Mateos P, Sánchez-Madrid F:
CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts.
Blood
91:4632, 1998[Abstract/Free Full Text]
55.
Akiyama SK, Yamada SS, Yamada KM, LaFlame SE:
Transmembrane signal transduction by integrin cytoplasmic domain expressed in single-subunit chimeras.
J Biol Chem
269:15961, 1994[Abstract/Free Full Text]
56.
Gismondi A, Bisogno L, Mainiero F, Palmieri G, Piccoli M, Frati L, Santoni A:
Proline-rich tyrosine kinase-2 activation by 1 integrin fibronectin receptor cross-linking and association with paxillin in human natural killer cells.
J Immunol
159:4729, 1998[Abstract]
57.
Baggiolini M, Dewald B, Moser B:
Human chemokines: An update.
Annu Rev Immunol
15:675, 1997[Medline]
[Order article via Infotrieve]
58.
Luster AD:
Chemokines-chemotactic cytokines that mediate inflammation.
N Engl J Med
338:436, 1998[Free Full Text]
59.
Ward SG, Bacon K, Westwick J:
Chemokines and T lymphocytes: More than an attraction.
Immunity
9:1, 1998[Medline]
[Order article via Infotrieve]
60.
Qian D, Lev S, van Oers NSC, Dikic I, Schlessinger J, Weiss A:
Tyrosine phosphorylation of PYK-2 is selectively regulated by Fyn during TCR signaling.
J Exp Med
185:1253, 1997[Abstract/Free Full Text]
61.
Sieg DJ, Ilic D, Jones KC, Damsky CH, Hunter T, Schlaepfer DD:
Pyk2 and src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but pyk2 does not fully function to enhance FAK-cell migration.
EMBO J
15:5933, 1998
62.
Astier A, Avraham H, Manie S, Groopman J, Canty T, Avraham S, Freedman AS:
The related adhesion focal tyrosine kinase is tyrosine phosphorylated after 1-integrin stimulation in B cells and binds to p130cas.
J Cell Biol
272:228, 1997
63.
Ma EA, Lou O, Berg NN, Ostergaard HI:
Cytotoxic T lymphocytes express a 3 integrin which can induce the phosphorylation of focal adhesion kinase and the related PYK-2.
Eur J Immunol
27:329, 1997[Medline]
[Order article via Infotrieve]
64.
Schaller MD, Parsons JT:
Focal adhesion kinase and associated proteins.
Curr Opin Cell Biol
6:705, 1994[Medline]
[Order article via Infotrieve]
65.
Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, Thompson DA, Schlessinger J, Littman DR:
Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.
J Exp Med
186:1793, 1997[Abstract/Free Full Text]
66.
Tokiwa G, Dikic I, Lev S, Schlessinger J:
Activation of PYK2 by stress signals and coupling with JNK signaling pathway.
Science
273:792, 1996[Abstract]
67.
Hatch WC, Ganju RK, Hiregowdara D, Avraham S, Groopman JE:
The related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated and participates in colony stimulating factor-1/macrophage colony-stimulating factor signaling in monocyte-macrophages.
Blood
91:3967, 1998[Abstract/Free Full Text]
68.
Ganju RK, Hatch WC, Avraham H, Ona MA, Druker B, Avraham S, Groopman JE:
RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature lymphocytes.
J Exp Med
185:1055, 1997[Abstract/Free Full Text]
69.
Berg NN, Ostergaard HL:
T cell receptor engagement induces tyrosine phosphorylation of FAK and PYK-2 and their association with Lck.
J Immunol
159:1753, 1997[Abstract]
70.
Daeron M:
Fc receptor biology.
Annu Rev Immunol
15:203, 1997[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION