From the
Intracellular adhesion molecule-2 (ICAM-2), one of the ligands
of CD11a/CD18 (LFA-1), is mainly expressed on endothelial and
hematopoietic cells. The biological significance of ICAM-2 has remained
unclear. Previous findings have shown that a peptide from ICAM-2,
spanning residues 21-42 from the first immunoglobulin domain,
enhances natural killer (NK) cell cytotoxicity and induces T cell
aggregation. We have now studied the effect of the same ICAM-2 peptide
on NK cell migration in the Boyden chamber assay. The peptide
significantly increased NK cell migration up to 215 ± 21%, as
compared to migration of control cells (100%), and the induction was
inhibited by anti-CD11a monoclonal antibodies. The ICAM-2 peptide also
induced polymerization of F-actin at the leading edge of migratory NK
cells. Cross-linking of CD11a/CD18 receptors with anti-CD11a or
anti-CD18 monoclonal antibodies and secondary antibodies resulted in
receptor recycling, increased migration, and actin polymerization, but
led to slight inhibition of cytotoxicity. The ICAM-2 peptide did not
induce such a receptor recycling. Phosphotyrosine immunoblotting
experiments showed that the ICAM-2 peptide increased the
phosphorylation of 150- and 35-kDa proteins. During cross-linking with
antibodies, only the 150-kDa protein showed increased phosphorylation.
The results show that depending on the type of CD11a/CD18 receptor
ligation different kinds of signals are transduced in NK cells. These
signals may either trigger only locomotion, or both locomotion and
cytotoxicity. Based on these findings, a major function for ICAM-2 on
endothelium may be triggering of migration of adhering leukocytes.
NK
Many factors that attract leukocytes at
low concentrations also increase cytotoxicity at higher concentrations
(6) . This is biologically meaningful because the highest
concentration of a factor exists in the actual lesion. When confronting
the target cell, the NK cell stops its migration, adheres to the target
mostly via
Adhesion receptors play a pivotal role in inflammatory responses. To
be able to locomote, a cell must attach to other cells or to the
extracellular matrix, and cytotoxicity requires a tight and transient
adhesion of the effector cell to the target. CD11a/CD18 (LFA-1) is the
major integrin of lymphocytes. It is a heterodimer and composed of
noncovalently bound
Ligands for the CD11a/CD18 integrin are ICAM-1, ICAM-2, and ICAM-3.
ICAM-1 differs from ICAM-2 in distribution and inducibility. Apart from
mere adhesion very little is known about the biological roles of ICAMs.
ICAM-1 is widely distributed, and its expression can be up-regulated by
various cytokines. ICAM-2 is mainly present on vascular endothelium and
lymphohematopoietic cells
(29, 30, 31) , and it
is generally constitutively expressed
(29) although its
expression may be increased in lymphoid malignancies
(32) . A
costimulatory cross-talk between TCR and ICAM-1
(33) or ICAM-2
(34) has been demonstrated. The ICAM-1 and ICAM-2 expression on
target cells contributes to LAK cell killing sensitivity
(35) but not to endogenous NK cell killing
(36) .
We
have previously shown that a soluble 22-amino-acid long peptide from
the first immunoglobulin domain of ICAM-2, but not from the homologous
region of ICAM-1 activates T cell aggregation and NK cytotoxicity
(37) . In the present work we show that the same peptide induces
actin polymerization, redistribution of actin, and increased migration
of endogenous NK cells. In addition, we show that although receptor
cross-linking with CD11a- or CD18-specific mAbs resulted in actin
polymerization and increased migration, it did not affect cytotoxicity.
The results indicate that CD11a/CD18 alone is capable of signal
transduction and that the response to outside signals depends on ligand
quality. Furthermore, our results suggest a biological role for ICAM-2
in activation of cell migration.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Because the ICAM-2 peptide is known to increase the cytotoxicity of
NK cells
(37) (Fig. 4), we also studied the effect of
CD11a/CD18 cross-linking on cytotoxicity. Cross-linking (IOT16, 7E4, or
IB4) slightly inhibited cytotoxicity during the 4-h assay, as seen in
Fig. 4. The cytotoxicity did not increase even if the cells were
preincubated for 4 h with primary and secondary mAbs before the
experiments. CD18 has a role as an adhesion receptor in the binding and
migration of NK cells
(23, 48) . Therefore, when the IB4
epitope on CD18 antigens was covered with IB4 mAbs without
cross-linking, both migration
(23) and cytotoxicity
(48) of endogenous NK cells were inhibited (Fig. 4).
Control peptide or control mAb had no effect. Surprisingly, when the
7E4-binding site on CD18 was covered, without cross-linking, migration
was inhibited but not cytotoxicity (not shown).
The effect of the ICAM-2 peptide and CD11a/CD18
cross-linking on NK cells in the presence of inhibitors was then
studied. The inhibitors had similar effect on activated cells and on
uninduced cells; moreover, migration induced by the ICAM-2 peptide or
by CD11a/CD18 cross-linking were similarly dependent on the inhibitors
(Table I).
Earlier studies have indicated that the CD11a/CD18 integrin
binds to the first immunoglobulin domain of ICAM-1
(50) . The
6D5 mAb to ICAM-2 reacted with its first immunoglobulin domain and
inhibited leukocyte adhesion to ICAM-2-transfected COS-1 cells
indicating that this domain is most important in CD11a/CD18 binding
(51) . Subsequent studies showed that a synthetic 22-amino-acid
long peptide, covering residues 21-42 of the first immunoglobulin
domain, inhibited binding of endothelial cells to purified CD11a/CD18
and to B lymphoblastoid cells
(43) . The same soluble ICAM-2
peptide induced CD11a/CD18-dependent T cell aggregation, showing that
this ligand is able to increase the avidity of CD11a/CD18
(37) .
Furthermore, NK cell binding and cytotoxicity to K562 were increased by
this peptide, suggesting that ICAM-2 also triggers signal transduction
(37) .
In this study we investigated the effect of the ICAM-2
peptide on NK cell motility. Our results show that this peptide, but
not ICAM-1 nor ICAM-3 peptides made from the homologous regions,
significantly increased endogenous NK cell migration and that the
migration was inhibited by anti-CD11a mAb. A comparable increase of
migration was achieved when the CD11a or CD18 polypeptides were
cross-linked with specific antibodies. Both the ICAM-2 peptide and
antibody cross-linking increased actin polymerization and cell
spreading, showing that they can induce changes in the
actin-cytoskeleton.
Compounds that increase cell migration are
usually soluble chemotactic or chemokinetic factors. The ICAM-2 peptide
that we used in our experiments increased NK cell migration both as
soluble and when substrate-bound (not shown) forms. It is well known
that soluble ICAM-1 exists in human blood, and it is increased in
various diseases
(52, 53, 54, 55) . In
addition, Makgoba
(56) has recently found that soluble ICAM-2
also circulates in blood and is increased in older people. Yet the role
of these soluble ICAMs is unknown, but it has been suggested that they
may down-regulate inflammation
(55) . However, we cannot exclude
the possibility that the soluble peptide would also attach to the
substrate, and lymphocytes would recognize it in solid phase-coupled
form only. Such substrate-bound haptotactic or haptokinetic factors can
increase cell migration
(57) . Unsoluble attractants are
especially necessary on endothelium where soluble factors would quickly
be diluted in circulating blood
(16) . Because ICAM-2 is
predominantly expressed on endothelium
(29) our results suggest
that ICAM-2 could be involved in the activation of cell migration and
extravasation.
When an attractant molecule binds to the neutrophil
surface, it is rapidly internalized, and the receptor-ligand complex
dissociates in the cytoplasm
(58) . Free receptors recycle in
3-10 min back to the cell surface
(59) , probably to the
leading edge of the cell where they can bind to another attractant
molecule. Higher migratory activity is detectable in a few minutes
after the attractant binding
(60) . In our experiments the first
signs of activation of human NK cells by the peptide, i.e. clustering or spreading of the cells, were seen after 30 min to 1
h of stimulation, but only after 4 h of incubation could the migration
increment be measured. Also, upon activation by cross-linking, the
kinetics was about the same: 1.5-2 h after the cross-linking the
first free receptors had reappeared, but only after 4 h of incubation
it was seen that migration had significantly increased. Probably the
increase of migration already takes place earlier, but the sensitivity
of the Boyden chamber method is not sufficient for its detection.
Usually the role of chemotactic factors, besides stimulating
locomotion, is to increase cell adhesion. In neutrophils, chemotactic
factors cause the exposition of cytoplasmic adhesion receptor storage
on cell surface. After activation, the amount of adhesion receptors can
increase in a few minutes up to 10-fold
(61) . In our
experiments, the ICAM-2 peptide that caused increased migration of NK
cells, either in a chemotactic or chemokinetic way, was itself the
ligand of the adhesion receptor and did not increase the amount of
CD11a/CD18 receptors on cell surface. Therefore, our results suggest
that the peptide stimulates locomotion through direct signaling and
up-regulated avidity of CD11a/CD18.
When migratory activity is
plotted against attractant concentration, a bell-shaped curve is
typical for a chemotactic factor. This is logical because at higher
concentrations of a chemoattractant, near foci of inflammation, cells
should slow down the migration and finally stop and adhere on the
target cell
(60) . We did not see any bell-shaped curve with the
ICAM-2 peptide. This may be biologically meaningful since ICAM-2 is
expressed by endothelium, the site that in inflammation is the starting
place rather than the final goal for migrating leukocytes.
Surprisingly, 7E4 mAb did not inhibit NK cell cytotoxicity, but it
inhibited migration, whereas IB4 mAb inhibited both cytotoxicity and
migration
(22, 23) . These results suggest that IB4 and
7E4 bind to different epitopes and that the epitope responsible for the
binding to a target cell differs from the epitope which is responsible
for the migration. 7E4 mAb has been shown to inhibit binding of
endothelial cells to purified CD11a/CD18
(29) and aggregation
of phorbol 12-myristate 13-acetate-stimulated neutrophils
(41) .
IB4 mAb has also been shown to abrogate the binding of various cells to
endothelium and their transmigration
(62, 63, 64, 65) . Both mAbs, which
covered the epitope responsible for migration, stimulated migration
when a secondary antibody was used as a cross-linking Ab. By
cross-linking the receptors were internalized, and they reappeared at
the surface of the cell as free and probably high avidity receptors.
Several other mAbs, when cross-linked with secondary antibodies, have
also been shown to cause both avidity increase in the CD11a/CD18
receptor and signal transduction
(28, 66, 67) .
The data suggest that the activation seen after cross-linking by
antibodies is epitope-independent.
The region of CD11a/CD18 that
binds ICAM-2 seems to be different from the region binding of IOT16
(anti-CD11a), 7E4, and IB4 (anti-CD18). The presence of ICAM-2 peptide
did not inhibit the binding of the mAbs, and the binding of radioactive
ICAM-2 peptide to purified CD11a/CD18 is not inhibited by 7E4
(43) . Furthermore, the different kinds of functional activities
caused by the peptide and the mAbs suggest that their binding sites are
different: the ICAM-2 peptide induced both migration and cytotoxicity,
but the cross-linking of CD11a/CD18 with mAb induced only migration.
Furthermore, we found as others have shown earlier
(28) , that
PI
(4, 5) P
Kanner
et al. (66) have described that CD18 on T cells is
linked to the tyrosine kinase-phosphatidylinositol pathway that
stimulates tyrosine phosphorylation and activation of PLC-
In order
to examine signal transduction caused by the ICAM-2 peptide or by
antibody cross-linking, we used specific protein kinase and phosphatase
inhibitors. We first explored random migration and endogenous
cytotoxicity. Random migration is known to be mediated mainly by CD11b
(23) , and endogenous NK cell cytotoxicity by CD11a, CD11b, and
CD11c/CD18 complexes
(24) . Both of these activities are most
probably dependent on transient and repeated changes between a high and
a low avidity state of the integrin receptors. In migration the
adhesion at the leading edge and the dissociation at the trailing edge
of a cell is of pivotal importance, enabling a cell to crawl on
substratum
(69) . In cytotoxicity, cell needs to detach from a
target cell after killing. Hedman and Lundgren
(70) have shown
that staurosporin, the serine/threonine kinase inhibitor, is able to
induce a high avidity state of CD11a/CD18, and okadaic acid, the
serine/threonine phosphatase inhibitor, inhibits the formation of a
high avidity state of CD11a/CD18 on B cells. On the other hand, in the
work of Miron et al. (71) , staurosporin decreased the
fibronectin receptor avidity and okadaic acid delayed the formation of
high avidity state of the fibronectin receptor on T cells. Both these
inhibitors decreased random migration in our experiments. However, we
have no information whether the inhibitory effect is due to changes in
the avidity of integrin receptors. In our experiments okadaic acid
enhanced endogenous NK cell cytotoxicity at 1 n
M-1 µ
M concentrations, but others
(72, 73) have described
with longer okadaic acid exposure times that low concentrations
(<100 n
M) of the drug increases the cytotoxicity of NK or T
cells and high concentrations (>200 n
M) decreases it.
Staurosporin also blocked the ICAM-2-activated migration and
cytotoxicity, but okadaic acid had no significant effect on these
functions. Altogether, in our experiment the random migration and
endogenous cytotoxicity of NK cells were dependent on the
serine/threonine phosphorylation/dephosphorylation, but increased
migration and cytotoxicity after the ICAM-2 activation were dependent
on serene/threonine phosphorylation but not on dephosphorylation.
To
conclude, we have demonstrated that an ICAM-2-derived peptide is a
potent inducer of NK cell migration. The same peptide has earlier been
shown to stimulate T cell aggregation and NK cell activity
(37) . These data suggest that ICAM-2 is involved in lymphocyte
activation, and thus the ICAM-2-
Enriched NK cells were
preincubated with the inhibitors for 30 min and then activated. After 4
h the migration of CD56+/CD3- NK cells was calculated or the
cytotoxicity of NK cells against K562 cells was measured. The values
are expressed as percent of migration and cytotoxicity without the
inhibitor. Mean ± S.E. of three to six experiments.
(
)
activity represents a form of early
defense mechanisms
(1, 2) . While normally found mostly
in blood and spleen, NK cells accumulate to sites of microbial
infections, tumors
(1, 2) , and rejecting allografts
(3, 4) . The trafficking of NK cells to strategic sites
involves the ability to respond either to soluble or surface-bound
signals that stimulate cell migration. When a lymphocyte is triggered
to locomote, its initial carbohydrate-mediated loose binding on
endothelium is followed by firm integrin-mediated adhesion
(5) .
Thereafter, the lymphocyte spreads on endothelium, and subsequent
transmigration is associated with the polymerization of actin to the
leading edge of a cell, as a result of receptor-mediated signal
transduction
(6) .
-integrins, and the cytolytic mechanism is
triggered: the Golgi apparatus, the centrioles, granules, and
polymerized actin reorient toward the contact area
(2, 7, 8) . In addition, cytoplasmic calcium
increase, inositol turnover, and protein kinase activation are
associated with the function of the cytotoxic machinery
(2) .
- (CD18) and
-subunits
(CD11a). In resting circulating lymphocytes, CD11a/CD18 is in a
nonadhesive conformation, and in order to become adhesive it requires
triggering by an external signal. Certain anti-CD11a/CD18 mAbs
(9, 10) , divalent cations
(11, 12) , or
a newly found lipid (IMF-1)
(13) , can directly induce an
adhesive conformation to this receptor. The avidity increase of
CD11a/CD18 is also induced by signaling through certain chemotactic
receptors
(14, 15, 16) through CD3
cross-linking
(17) and through the E-selectin ligand
(18) . In stimulated NK cells, the high avidity state CD11a/CD18
receptors participate in many adhesion-dependent functions: binding to
endothelial cells
(19, 20) , migration
(21, 22, 23) , and cytotoxicity
(24) . In
addition to binding, CD11a/CD18 may also function as a triggering
receptor in the activation of migration and cytotoxicity. Cross-linking
of TCR
(25, 26, 27) on resting T cells results
in both a high avidity state of CD11a/CD18 and rapid cell
proliferation, when CD11a/CD18 is coligated. In addition, cross-linking
of CD11a itself has been shown to be capable of signal transduction
through calcium increase and inositol hydrolysis
(28) .
Cell Purification
Buffy coats from healthy blood
donors were obtained from the Finnish Red Cross Blood Transfusion
Service. NK and T cells were isolated by Ficoll-Isopaque (Pharmacia
Fine Chemicals AB, Uppsala, Sweden) gradient centrifugation, filtration
through nylon wool columns, and by four-step Percoll (Pharmacia)
gradient centrifugation in RPMI 1640 (Life Technologies, Inc.)
supplemented with 0.29 mg/ml glutamine (Life Technologies, Inc.), 100
IU/ml penicillin, 10 µg/ml streptomycin, and 5% heat-inactivated
fetal calf serum (Life Technologies, Inc.), as described previously
(38) , with minor modifications. Purified lymphocytes were
phenotyped by flow cytometry. Lymphocytes in the NK cell fraction
consisted of 54.1 ± 9.6% of CD56+CD3-, 44.2 ± 11.7%
of CD3+, and 1.3 ± 1.1% of CD14+ cells ( n = 39) (mean ± S.D.). Highly purified NK cells were
used in immunofluorescence microscopy, immunoblotting, and
phosphatidylinositol turnover experiments. They were obtained as
described by Timonen et al. (39) : NK fraction cells
from the Percoll gradients were incubated at 29 °C for 45 min with
sheep red blood cells at a ratio of 1:150. The sheep red blood cells
with the attached CD3+ cells were removed by a subsequent
Ficoll-Isopaque gradient centrifugation. The resulting cell populations
contained 90.1 ± 4.8% CD56+CD3-, 4.2 ± 1.8%
CD3+, and 0.3 ± 0.4% CD14+ lymphocytes.
Antibodies and Phenotypic Analysis
The mAbs used
in migration, cytotoxicity, and phosphatidylinositol (IP) assays were:
anti-CD11a (IOT16, IgG(40) , Serotec, Bicester,
Oxon, UK), anti-CD18 (7E4
(41) purified IgG
, or
IB4, IgG2a, courtesy of Dr. Samuel Wright, Rockefeller University, New
York), anti-CD16 (3G8
(42) purified IgG
from
supernatant of hybridoma cell line), and F(ab`)
fragments
of goat-anti-mouse-IgG (Coulter Immunology, Hialeah, FL) as secondary
cross-linking antibody. The mAbs used in phenotyping were: anti-CD16,
anti-CD56, anti-CD3, and anti-CD14 (anti-Leu11b, anti-Leu19, anti-Leu4,
and LeuM3, respectively, all from Becton-Dickinson, Mountain View, CA).
Normal mouse IgG, including all subclasses (Coulter Immunology), was
used as a control. Phenotypic analysis was carried out using standard
procedures
(23) , and the cells were analyzed using a FACScan
(Becton-Dickinson) flow cytometer.
Peptides
A 22 amino peptide (residues 21-42)
from the first immunoglobulin domain of ICAM-2
(GSLEVNCSTTCNQPEVGGLETSL), and a control peptide containing the same
amino acids as the ICAM-2 peptide, but in random order, except for the
two conserved cysteines (EVGTGSCNLECVSTNPLSTGTEQ) were used
(43) . Additionally, ICAM-1
(GSVLVTCSTSCDQPKLLGIETPL)
and ICAM-3 (GSLFVNCSTDCPSSEKIALETSL)
peptides made from the homologous regions were used. The peptides were
synthesized by t-Boc chemistry on an Applied Biosystems 430A peptide
synthesizer and purified to >98% purity by high performance liquid
chromatography. The quality of the purified peptides was verified by
amino acid sequence analysis and mass spectrometry
(43) .
Cell Activation
The soluble ICAM-2 peptide or the
soluble control peptide (0-100 µg/10 10
cells/ml) was added to lymphocyte suspensions 30 min before
experiments and left in the supernatants during the assay. Activating
mAbs (anti-CD18 (7E4 or IB4), anti-CD11a (IOT16), or anti-CD16 (3G8))
were added (10 µg/1
10
cells/100 µl) at 4
°C 30 min before experiments and left in the supernatants during
the assay. In cross-linking experiments, the cells were first incubated
with a primary mouse mAb for 30 min at 4 °C, washed, and
F(ab`)
fragments of goat-anti-mouse-IgG were added,
incubated for 30 min at 4 °C, and left in the supernatants during
the assay.
Migration Assay
The Boyden chamber migration assay
described by Axelsson et al. (44) was used.
Polycarbonate filters with pores of 3-µm diameter (Nuclepore
Corporation, Pleansanton, NY) were used between the lower and upper
compartments of the Boyden chambers. The lower compartments were filled
with 400 µl of RPMI 1640 supplemented with 0.5% human AB serum and
0.5% human serum albumin (both from Finnish Red Cross Blood Transfusion
Service). The Percoll fractionated lymphocytes were suspended in the
same medium at 10 10
/ml and activated as described
above. 200 µl of lymphocyte suspensions were added to the upper
compartments. As a background control to monitor passive relocation of
cells to the lower compartment of the chamber, the divalent cation
chelator EDTA (10 m
M), which inhibits migration was used
(23) . After incubation for indicated hours at 37 °C, the
amount of migratory cells in the lower chambers was counted. NK or T
cell frequencies were determined by phenotyping, the amounts of
migrated NK cells were calculated, and the mean values in triplicate
chambers were calculated. The cell number of the EDTA-treated cells
(<5% of input) in the lower chamber was subtracted from the
experimental cell numbers. In some experiments the cells were treated
with various concentrations of genistein (Life Technologies, Inc.),
staurosporin (Sigma), or okadaic acid (Sigma) for 30 min at 37 °C
before the assays and left in the supernatants during the assay.
Genistein and staurosporin were dissolved in Me
SO and
okadaic acid in ethanol. Me
SO and ethanol at their final
concentrations used had no effect on the parameters determined. The
results are expressed as the mean (
± S.E.) values of
repeated experiments.
Cytotoxicity Assays
1 10
K562
target cells
(45) (American Type Culture Collection, Rockville,
MD) were labeled with 20 µCi of sodium [
Cr]
(Radiochemical Centre, Amersham, United Kingdom). Various
concentrations of effector cells were added in 100 µl to 100 µl
of target cells 1
10
/ml, to produce effector/target
ratios 20:1, 10:1, 5:1, and 2.5:1. After a 4-h incubation at 37 °C,
100 µl of the supernatant from each well was counted in a gamma
counter (Wallac, Turku, Finland). The percentage of radioactivity
released was calculated as follows:
Fluorescence Microscopy
Highly purified NK cells
were used. For visualization of filamentous F-actin, activated NK cells
were fixed in 3.5% paraformaldehyde for 10 min, washed with
phosphate-buffered saline, and permeabilized with 0.1% Triton X-100 for
5 min. The cells were then incubated with tetrametylrhodamine
isothiocyanate (TRITC)-conjugated phallacidin (Molecular Probes Inc.
Pitchford, TX) which binds to F-actin. In cross-linking experiments,
unspecific Fc binding was first blocked with 20% human AB serum. The
cells were incubated with the primary mAb (anti-CD18 (7E4) or
anti-CD11a (IOT16)) for 30 min at 4 °C, washed, and further
incubated with F(ab`)fragments of goat-anti-mouse-IgG for
30 min at 4 °C, then further incubated at 37 °C for various
times (0-3 h), washed, and stained with the FITC-labeled
rabbit-anti-goat-IgG (Cappel, Organon Teknika Corp., Durham, NC) for 30
min at 4 °C. When indicated, the cells were permeabilized with 0.1%
Triton X-100 for 5 min and then stained with anti-CD11a (N225, N226,
and N217 obtained through the Fourth International Leukocyte Workshop),
anti-CD18 (7E4), and the secondary FITC-labeled goat-anti-mouse-IgG for
the detection of cytoplasmic CD11a/CD18. After washing, the cells were
and fixed in 3.5% paraformaldehyde.
SDS-polyacrylmide Gel Electrophoresis and
Immunoblotting
Highly purified NK cells were used. 10
10
NK cells were lysed in 50 m
M HEPES, pH 7.2, 1%
Triton X-100, 0.1% SDS, 10 m
M EDTA, 100 m
M NaH
PO
, 10 m
M NaF, 1 m
M Na
VO
, and 10 µg/ml aprotinin and
leupeptin. The soluble proteins were mixed in Laemmli buffer, separated
in 8% polyacrylamide gels under reducing conditions
(46) , and
subsequently transferred to nitrocellulose filters. The filters were
blocked with 4% bovine serum albumin overnight, incubated with
mouse-anti-phosphotyrosine (ascites fluid 1:1000) (Sigma) for 1 h, and
exposed to peroxidase-conjugated rabbit-anti-mouse IgG (Dako A/S,
Glostrup, Denmark) for 30 min. Bound antibody was visualized by ECL
(Amersham) according to the manufacturer's instructions.
Phosphatidylinositol Experiments
The procedure
described by Stand Carpén
(47) was used.
Activated highly purified NK cells were incubated (2.5 10
cells/ml) in inositol-free culture medium (RPMI 1640 sine
inositol, Pharmacy of Helsinki University, Finland), containing 0.5% of
human serum albumin, for 24 h together with
[
H]inositol 35 µCi/ml (NET 906, DuPont NEN).
After washing, the cells were suspended (20
10
cells/ml) in inositol-free culture medium containing 0.5% human
serum albumin and 10 m
M LiCl in order to inhibit the activity
of inositol phosphate phosphatase. The cells were then activated with
indicated agents for 3 min at 37 °C. The inositol phosphate
metabolism was terminated by the addition of methanol/chloroform (2:1)
and thereafter by chloroform/water (1:1) to the cell suspensions. The
water-soluble inositol phosphates were separated and fractionated on
MonoQ anion-exchange (fast protein liquid chromatography) system
(Pharmacia, Uppsala, Sweden) with a continuous gradient of 0-0.1
M ammonium formate. One-ml fractions were collected, and the
radioactivity was counted with a
-counter (LKB-Wallac, Bromma,
Sweden).
Statistical Methods
The statistical significance
of the results in cell migration assays was evaluated by the paired
t test (* = p < 0.001, = 0.001 <
p < 0.01, * = 0.01 < p < 0.05).
Effect of the ICAM-2 Peptide on NK Cell
Migration
To extend the formerly described experiment, where the
ICAM-2 peptide was shown to stimulate NK cell binding and cytotoxicity
(37) , the effect of increasing concentrations of the soluble
ICAM-2 peptide or control peptide on the migratory capacity of NK cells
was examined. Freshly isolated and ICAM-2 peptide-treated peripheral
blood lymphocytes were seeded on the top of filters dividing Boyden
chambers into two compartments. The ability of NK cells to migrate
through the filters into the lower compartments of the chamber during 4
h was determined. As shown in Fig. 1, the NK cell migration was
significantly increased by the ICAM-2 peptide. The migration was
dependent on the concentration of the peptide, and the plateau response
was reached at 75 µg/ml. The increase of migration was inhibited by
anti-CD11a (IOT16 10 µg/1 10
cell/100 µl)
mAb (not shown) although the random migration was not dependent on
CD11a. ICAM-1, ICAM-3, and control peptides had no effect on the
migration (not shown). In kinetic experiments, significantly increased
NK cell migration was first detectable after a 4-h treatment with the
ICAM-2 peptide (Fig. 2).
The Effect of the ICAM-2 Peptide on F-actin Distribution
and the Spreading of NK Cells
Cell movement involves
polymerization of globular actin into F-actin and its distribution
toward the leading edge. We therefore wanted to examine the effect of
the ICAM-2 peptide on actin polymerization in highly purified NK cells.
Prior to stimulation the cells were round (Fig. 3 A), and
F-actin visualized by TRITC-phallacidin was homogeneously distributed
in the cytoplasm. After stimulation, all NK cells spread on the
substratum and obtained the morphology of migratory cells with a
leading edge and a trailing edge. F-actin staining in these cells
became more abundant than in control cells, and often F-actin was
localized in the leading edges of the cells (Fig. 3 B).
Figure 3:
Analysis of the distribution of F-actin in
NK cells. Highly purified CD56+/CD3- NK cells were activated by a
control peptide ( A), ICAM-2 peptide (7.5 µg/1
10
cell/100 µl) ( B), or by cross-linking of
the CD11a (IOT16 mAbs followed by F(ab)
fragments of
goat-anti-mouse IgG Ab) ( C) or CD18 (7E4 mAbs 10 µg/1
10
cell/100 µl and the secondary Ab)
( D). After fixation F-actin was visualized by
rhodamine-phallacidin.
Antibody-induced cross-linking of CD11a and CD18 caused similar cell
spreading and F-actin polarization as the ICAM-2 peptide did
(Fig. 3, C and D). If CD11a/CD18 was not
cross-linked with secondary antibody, no effect was seen (not shown).
Effect of Cross-linked CD11a/CD18 on Migration and
Cytotoxicity
It is apparent that the induction of NK cell
migration by the ICAM-2 peptide involves ligation of the CD11a/CD18
integrin. We wanted to find out whether cross-linking of CD11a or CD18
by specific mAbs would have a similar effect. As seen in Fig. 4,
cross-linking of CD11a with IOT16 mAb and secondary Ab increased the
migration as strongly as the soluble ICAM-2 peptide. The cross-linking
of CD18 with 7E4 or IB4 mAb and a secondary Ab also increased NK cell
migration, but less than the cross-linking of the -chains.
Figure 4:
Effects of ICAM-2
peptide and antibody cross-linking on NK cell migration and
cytotoxicity. Enriched NK cells were activated by ICAM-2 peptide (7.5
µg/1
10
cell/100 µl) or by cross-linking of
CD11a or CD18 receptors (IOT16 or IB4 mAbs 10 µg/1
10
cell/100 µl and a secondary Ab). After a 4-h incubation at 37
°C, the migration of CD56+/CD3- NK cells was calculated or the
cytotoxicity of NK cells against K562 cells was measured. The results
are expressed as percent increase compared to untreated control cells.
The average control migration was 120-200
10
migrated cells/4 h incubation and control cytotoxicity 500-2000
cpm/4 h incubation. Mean values from four to eight experiments ±
S.E. *** = p < 0.001, ** = 0.001 < p < 0.01, * = 0.01< p <
0.05.
Because
cross-linking of CD18 with the mAbs (IB4 and 7E4), which cover the
epitopes responsible for migration, increased migration we examined the
effect of cross-linking on possible recycling of the CD11a/CD18
integrin and the re-expression of free integrin during the 3-h
incubation. When the cells were treated with anti-CD11a (IOT16) or
anti-CD18 (7E4) mAbs, the fluorescence intensity of the surface CD11a
or CD18 molecules did not change during the 3-h incubation at 37
°C. If molecules were cross-linked with the secondary Ab, the
intensity of the surface CD11a or CD18 decreased significantly (Fig. 5
shows CD18 expression). Immunofluorescence microscopy revealed that
CD11a or CD18 started to patch immediately after the cross-linking, and
after 3 h of incubation the receptors were completely capped in 98% of
the cells. If the cells were permeabilized at this time point,
restaining visualized the internalized CD11a and CD18 molecules, and
surface restaining of CD11a or CD18 revealed the re-expression of free
adhesion molecules, as seen in Fig. 5 F. The ICAM-2 peptide did
not induce such a receptor recycling (not shown).
Role of Protein Phosphatases and Kinases on Stimulated
Migration and Cytotoxicity
Experiments were performed to analyze
possible differences between the two stimulation mechanisms, which in
one case leads to increased migration and cytotoxicity (ICAM-2
peptide), and in the other to an increased migration but not to
increased cytotoxicity (CD11a/CD18 cross-linking). We first studied
whether different protein kinases and phosphatases participate in
random uninduced migration and endogenous cytotoxicity of NK cells.
Both the tyrosine protein kinase inhibitor genistein and the
serine/threonine protein kinase inhibitor staurosporin decreased the
random migration and cytotoxicity in a dose-dependent fashion (Fig. 6).
Staurosporin abrogated the activities completely, whereas genistein
inhibited migration by 49% and cytotoxicity by 59%. Okadaic acid is a
specific protein phosphatase 2A inhibitor when used at lower
concentrations than 10 n
M, and a protein phosphatase 1
inhibitor when used at 10 n
M-1 µ
M (49) .
It had no effect on migration at concentrations lower than 400
n
M, but at a concentration of 1 µ
M it decreased
migration by 52%. In contrast, all concentrations of okadaic acid
tested (from 150 n
M to 1 µ
M) increased endogenous
cytotoxicity.
Effect of the ICAM-2 Peptide and CD11a/CD18 Cross-linking
on Tyrosine Phosphorylation
The results indicated that protein
phosphorylation, among other effects, is required for migration and
cytotoxicity. The possibility that activation of NK cells by the ICAM-2
peptide or CD11a/CD18 cross-linking by mAbs could lead to different
patterns of protein phosphorylation was studied next. The
detergent-soluble proteins of the ICAM-2 peptide-activated or
CD11a/CD18 cross-linking-activated highly purified NK cells were probed
with anti-phosphotyrosine antibodies. As seen in Fig. 7 A, the
ICAM-2 peptide, which activated both migration and cytotoxicity,
increased phosphorylation of two cellular proteins, of 150 and 35 kDa.
The phosphorylation was detected most clearly after 8 and 15 min of
activation. The control peptide did not cause any phosphorylation.
Cross-linking with mAbs resulted in phosphorylation of the 150 kDa, but
not the 35-kDa protein (Fig. 7 B).
Effect of the ICAM-2 Peptide and CD11a/CD18 Cross-linking
on Phosphatidylinositol Turnover
Caincrease,
a consequence of the hydrolysis of plasma membrane phosphoinositides,
is involved in both cytotoxicity and migration. The effect of the
ICAM-2 peptide and CD11a/CD18 cross-linking on the NK cell inositol
turnover was therefore investigated. Fig. 8 shows that the ICAM-2
peptide increased only marginally the phosphatidylinositol
monophosphate (IP1) accumulation. Cross-linking of CD11a/CD18 increased
the IP1 accumulation significantly, although not as much as the
positive control, a mAb to CD16. No increase was observed when NK cells
were incubated with anti-CD11a or anti-CD18 mAbs without secondary
antibodies.
is hydrolyzed when CD11a/CD18 is
cross-linked with the primary and secondary mAbs, but we found only
very little PI
(4, 5) P
hydrolysis after
activation by the ICAM-2 peptide. The tyrosine phosphorylation profile
of NK cells was different after the two kinds of stimulation. These
results together support the interpretation that the ICAM-2 peptide and
the antibody cross-linking stimulate NK cells differently, and
therefore probably affect different regions of CD11a/CD18.
1
(66) . PLC-
1 has a mass of 150 kDa and may correspond to
the 150-kDa protein, which is observed when NK cells were activated
through either CD11a/CD18 cross-linking or by the ICAM-2 peptide. In
addition, we detected a phosphorylated 35-kDa protein in the ICAM-2
peptide-activated cells, but not in cells activated by cross-linking. A
35-kDa protein has previously been shown to coimmunoprecipitate with
PLC-
1
(68) with the ligation of TCR or the ligation of TCR
and CD18. As suggested by Kanner et al. (66) , our data
also suggest the possibility that the PLC-
1 protein complex may be
differently regulated depending on the type of triggering.
2-integrin pathway has a function
more complex than mere adhesion in lymphocyte physiology. Being
abundant on endothelial and lymphohematopoietic cells, ICAM-2 may be
especially relevant in the induction of migration as a result of
lymphocyte contact with endothelium or other regulatory cells of the
lymphoreticular system.
Table:
The
effect of protein kinase and phosphatase inhibitors on stimulated
migration and cytotoxicity of NK cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.