(Received for publication, April 27, 1995)
From the
Lymphocyte chemoattractant factor (LCF) is a polypeptide
cytokine which induces both cell motility and activation of T
lymphocytes. These LCF-induced events demonstrate an absolute
requirement for the cell surface expression of CD4. Because many
CD4-mediated T lymphocyte activation events have been demonstrated to
require the association of the src-related tyrosine kinase
p56
Accumulation of T cells in tissue at sites of antigen deposition
requires the mobilization, adhesion, transendothelial migration,
activation, and proliferation of lymphocytes(1) . Locally
produced chemotactic cytokines contribute the major driving force to
directed lymphocyte migration during this process via their interaction
with cell surface receptors(2) . In addition to their role as
chemotactic factors, many cytokines have growth factor activity which
may also play a role in the phenotype of the accumulated
cells(3) . Despite the fact that a large number of growth
factor like-cytokines have chemotactic activity for lymphocytes,
relatively little is known about the mechanism by which they transduce
the motile signal. This study addresses the mechanism by which the
chemotactic cytokine lymphocyte chemoattractant factor functions.
LCF
Along these lines, the CD4 ligand gp120
induces rises in [Ca
In the present investigations
we examine LCF-induced p56
Figure 1:
A, in vitro kinase assay of
LCF-induced wild type CD4-associated p56 kinase activity. 5
Figure 2:
In vitro kinase assay of
LCF-induced mutant CD4-associated p56 kinase activity. 5
Figure 3:
LCF-induced murine T cell migration. 10
Figure 4:
Anti-T cell receptor antibody-induced T
cell migration. Murine T cell migration was assayed as in Fig. 3using 2C11 antibody.
Figure 5:
Effect of herbimycin A on in vitro kinase assay of LCF-induced CD4 associated p56 kinase activity.
Kinase activity was assayed as in Fig. 1, following 18 h of
incubation with 3 µg/ml herbimycin A. Lane 1,
precipitation with Protein A alone. CD4 immunoprecipitation from T cell
hydridomas were either untreated (lane 2), treated with
cross-linked Leu 3A (lane 3), or treated with LCF (lane
4).
Figure 6:
Effect of tyrosine kinase inhibitor
herbimycin A on LCF-induced T cell migration. Murine CS430 T cell
migration was assayed as in Fig. 3following an 18 h incubation
with 3 µg/ml herbimycin A.
Figure 7:
Anti-T cell receptor antibody-induced T
cell migration. Murine T cell migration was assayed as in Fig. 6using 2C11 antibody.
Figure 8:
LCF-induced murine T cell migration. 10
CD4 is required as an essential component of the cell surface
signaling complex engaged during LCF-induced motility. The evidence for
this relationship rests with CD4 transfection experiments in which we
have previously shown that the LCF-induced rises in
[Ca
In conjunction with previous work,
For the motile response, one of the principle events is
the induction of cellular shape changes through the reorganization of
the cytoskeletal structures(2) . Along these lines, the second
messengers generated by phospholipase C
Our data also
suggest an explanation for the observations that the CD4 ligands HIV-1
gp120 and anti-CD4 antibodies appear to require cross-linking to induce
some, but not all CD4 related activation pathways and cellular
functions. The lymphocyte chemoattractant factor is a 14-kDa protein
which exists as a bioactive tetramer of 56 kDa in supernatants of
conconavalin A or histamine-stimulated peripheral blood mononuclear
cells(39, 40) . We subsequently demonstrated that the
cDNA for LCF codes for a 14kDa protein, which autopolymerizes to 56
kDa. There is an absolute requirement for a polymerized form of LCF for
the biologic activities(8) . Thus, cross-linking of other
ligands such as anti-receptor (CD4) antibody
However,
the relationship of CD4 ligands to CD4 must be more complex than a
simple cross-linking phenomena as noted for a number of other
receptors(41) . Fab of anti-CD4 antibodies do not induce
motility; however, intact dimeric antibody induces comparable migration
as seen with LCF or gp120. In addition, further cross-linking of
divalent anti-CD4 antibody does not further augment the motile
response. Intact antibody also down-modulates subsequent activation via
CD3, but unlike LCF it does not induce IL-2R nor major
histocompatability complex Class II molecules(5) . In addition,
intact antibody does not induce any of the traditional intracellular
signal transduction pathways including phosphatidyl inositol turnover,
rises in [Ca
We propose, therefore, that the src homology domains of lck,
independent of the activation of the kinase, act to recruit
intracellular molecules which initiate changes in cytoskeletal
structure essential for LCF-induced T lymphocyte migration. Therefore,
the induction of p56
with the cytoplasmic domain of CD4, we
examined the role of p56
in LCF-induced
lymphocyte migration in a murine T cell hybridoma line expressing
transfected human CD4. LCF induces the catalytic activity of CD4
associated p56
at chemoattractant concentrations
of cytokine. Hybridoma cells that express CD4 with cytoplasmic point
mutations which uncouple the CD4-lck association lack both lck
enzymatic activity and chemotactic responses to LCF. The enzymatic
activity of lck however does not appear to be required for CD4-mediated
migratory signal. First, the protein tyrosine kinase inhibitor
herbimycin A blocked LCF-induced p56
activation
but had no effect on the LCF-induced motile response. Second, T cell
hybridomas expressing a chimeric receptor combining the extracellular
domain of human CD4 and murine p56
which lacked
the kinase domain had a normal LCF-induced motile response. We conclude
from these observations that CD4-lck coupling is essential for
LCF-induced T lymphocyte migration but the motile response is
independent of the enzymatic activity of CD4-associated
p56
.
(
)is a CD4
immunocyte-specific chemotactic cytokine of CD8
T cell origin(4, 5, 6) . In addition to
the activation of cell migration, LCF induces rises in
[Ca
]
and inositol
trisphosphate (IP
)(7) . The relationship between
these messengers and the induction of motility, however, is yet to be
defined. The initiation of all these responses, however, is absolutely
dependent upon the cell surface expression of
CD4(7, 8) .
]
,
IP
generation and motile responses in T cells(9) .
In addition, HIV-1 gp120 induces CD4-associated activation of
p56
(10) , a member of the src-tyrosine
kinase family which is tightly associated with
CD4(11, 12) . From these observations, we investigated
the possible requirement for p56
in CD4-mediated
lymphocyte migration induced by LCF.
activity in a murine
T cell hybridoma line which has been transfected with human CD4. Our
experiments demonstrate that LCF induces CD4-associated p56
kinase activity in these cell lines. In order to establish a
relationship between the LCF-induced enzymatic activity of the kinase
and lymphocyte motility, hybridomas expressing CD4 cytoplasmic domain
mutants were also studied. The mutations result in the disruption of
the physical coupling of CD4 with p56
(13) . Neither the CD4-associated kinase activity nor the
migration induced by LCF occurs in cells expressing CD4 mutations
incapable of binding p56
. The relationship
between p56
catalytic activity and lymphocyte
migration was examined in cells treated with the tyrosine-kinase
specific inhibitor, herbimycin A. At concentrations that completely
abolished the enzymatic activity, the LCF-induced motile response was
unaffected. Finally, the removal of the kinase domain from CD4-lck
chimeras expressed in this hybridoma line resulted in migratory
responses comparable to full-length kinase controls. These data suggest
that the physical association of p56
with CD4,
and not the enzymatic activity of the kinase, is the essential
component for the LCF-mediated lymphocyte motile response.
Murine T Cell Hybridoma Cell Lines
All
CD4-expressing murine T cell hybridomas were the generous gift of Dr.
Steven J. Burakoff (Dana Farber Cancer Institute Boston,
MA)(14) . Briefly, The murine T cell line, By155.16 was
infected with the MNC retroviral vector which contains a neomycin
resistance gene, a cytomegalovirus promoter, and the gene for human
CD4. The MNC-CD4 transfectants were selected (G418) and assessed for
CD4 surface expression. All cell lines examined had comparable
expression of CD4. In addition to wild type CD4, By155.16 was
transfected with CD4 containing cysteine to serine point mutations at
positions 420, 422, and 430 (MNC-CS420, CS422,
CS430)(13, 15) . Chimeric molecules containing the
extracellular domain of CD4 directly ligated to p56 were also expressed in murine cells. All cells were grown in
RPMI 1640 medium (Sigma) containing 200 units/ml of penicillin and 200
mcg/ml streptomycin, 2 mM glutamine, 20 mM HEPES, pH
7.4, and 10% fetal bovine serum.
Reagents
Recombinant LCF was produced as described
previously(8) . Anti-human CD4 antibody Leu 3A was purchased
from Becton Dickinson (San Jose, CA), Anti-murine CD3 antibody
2C11 was the generous gift of Dr. Anne Rothstein of the Boston
University School of Medicine. Rabbit anti-mouse antibodies used as a
cross-linking reagent for Leu 3A was purchased from Fisher (Pittsburgh,
PA). The protein tyrosine kinase inhibitor herbimycin A was the
generous gift of Dr. Y. Uehara (National Institute of Health Tokyo,
Japan). The p56
antibody used in Western blot
analysis was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Chemotaxis
Cell migration was performed as
described previously(4, 5) . Migration was assessed by
a modification of a Boyden chamber technique, by using a
microchemotaxis chamber (Neuroprobe, Cabin John, MD). T lymphocytes (10
10
/ml) were loaded in the upper well of the
chamber, with 30 µl of various concentrations of antibody as
chemoattractant placed in the lower well. The two wells were separated
by a nitrocellulose filter paper with a pore size of 8 µm.
Migration chambers were incubated for 3 h, after which the filters were
fixed in ethanol, stained, and migration was assessed by counting of
the number of cells that migrated beyond 50 µm, by light
microscopy. Cells treated with herbimycin A for both chemotaxis in
vitro kinase analysis were incubated with 3 µg/ml
(Me
SO) for 18 h, at 37 °C in 5% CO
and were
assayed for viability by trypan blue exclusion. Cells were >90%
viable. All migration was expressed as percentage values of cell
migration in control buffer and statistics calculated by the
Student's t test. Data are the mean value ± the
standard deviation of three or more experiments.
In Vitro Kinase Assay
5 10
murine T cells/sample were removed from culture and suspended in
ice-cold serum-free RPMI 1640 medium. Agonist was added to each sample
on ice. The samples were then placed in a 37 °C heat block for 2
min. The reaction was stopped with the addition of ice cold wash buffer
(150 mM NaCl, 50 mM Tris, pH 7.6) and the cells were
pelleted. The supernatants were removed, and the pellets were
solubilized in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50
mM Tris, pH 7.6) containing protease inhibitors for 20 min at
4 °C. The samples were then centrifuged for 10 min at 11,000
g and the supernatants transferred to fresh Eppendorff
tubes. Precipitating antibody and Protein A-Sepharose beads were then
added with the samples incubated for 1 h at 4 °C. The beads were
washed and 50 µl of ice-cold kinase buffer (20 mM Tris, pH
7.4, 10 mM MnCl
) was added to each sample. The
kinase reaction was started with the addition of 10 µCi/sample of
[
-
P]ATP and an exogenous substrate,
acid-denatured enolase. The reaction was stopped with the addition of 4
SDS-Laemmli sample buffer. The samples were boiled and
subjected to 10% SDS-PAGE. The gels were dried, subjected
autoradiography, and quantitated by densitometry. Under these
conditions the incorporation of
P label was linear for the
duration of agonist exposure.
Western Blot Analysis
Treated immunoprecipitates
were separated on 10% SDS-PAGE gels and electrophoretically transferred
to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The
filters were blocked in a solution of 5% non-fat milk in
phosphate-buffered saline (PBS: 125 mM NaCl, 8 mM NaHPO
, 2 mM NaH
PO
, 5 mM KCl, pH 7.4),
washed, and incubated in p56
antiserum (1:500)
in PBS with 0.05% Tween 20 for 2 h. Washed filters were then incubated
in goat anti-rabbit-horseradish peroxidase. Following a 1-min exposure
to Lumi-GLO
substrate (Kirkegaard and Perry Laboratories,
Gaithersburg, MD) filters were subjected to chemiluminescense
autoradiography.
LCF Induction of p56
Murine T cell hybridomas expressing human
CD4 were examined for their ability to induce the catalytic activity of
p56 in Murine T
Cell Hybridoma Cells
in response to chemotactically active
(10
M) concentrations of LCF. In addition
to the wild type CD4 hybridomas, cells expressing CD4 with selected
cysteine to serine mutations were also examined. Alterations in
cysteines 420 and 422 have previously been shown to disrupt the
lck-binding site on CD4 (15) , while the mutation of cysteine
430, which lies outside of this domain, has no effect on the
association of p56
with CD4 (15) . Cells
lines were selected for equal expression of CD4 and exposed to LCF. CD4
immunoprecipitates from treated cells were then assayed for kinase
activity by the addition of radioactive [
P]ATP
and the exogenous phosphorylation substrate, enolase.
Immunoprecipitates from untreated wild type (data not shown) and CS430
murine hybridoma cells, the cysteine to serine control transfectant, (Fig. 1A, lane 2) demonstrated a background
level of kinase and enolase phosphorylation, consistent with previous
reports(16, 17) . As described
previously(16, 18) ,
(
)a
dramatic increase in the autophosphorylation of lck protein was seen
following treatment of cells with cross-linked CD4 antibody (Fig. 1A, lane 3). A lower, yet detectable,
increase in enolase phosphorylation was also observed following
antibody stimulation (Fig. 1B). LCF-induced
autophosphorylation of lck was also observed although less pronounced (Fig. 1A, lane 4). Enolase phosphorylation
following exposure of cells to LCF, however, was substantially
increased (Fig. 1B). It is at present unclear what
mechanism might regulate the differential incorporation of
P in respective substrates with these two agonists. The
association of p56
protein with CD4 was,
however, not affected by these treatments as comparable levels of lck
protein were immunoprecipitated for each condition (Fig. 1C). LCF-induced kinase activity was observable
at 30 s, reached a maximum at 2 min, and began to diminish at 5 min
(data not shown). The mutants CS420 and CS422, as anticipated,
demonstrated no receptor associated kinase activity in either control
or LCF-treated cells (Fig. 2).
10
CS430 murine T cells were incubated with LCF, detergent
solubilized, and immunoprecipitated with anti-receptor antibody. The
immunoprecipitates were assayed for kinase activity and separated by
SDS-PAGE. The gels were dried and subjected to autoradiography for
12-18 h. lck represents autophosphorylated p56 protein, en denotes acid denatured enolase. Lane 1,
precipitation with Protein A alone. CD4 immunoprecipitation from T cell
hydridomas were either untreated (lane 2), treated with
cross-linked Leu 3A (lane 3), or treated with LCF (lane
4). B, densitometric analysis of LCF-induced kinase
activity. Incorporation of labeled phosphate into both CD4-associated
lck protein (Top) and enolase (Bottom) was
quantitated by densitometry using the Molecular Dynamics Computing
Densitometer. C, p56 Western blot of LCF-treated murine T cell
hybridomas kinase activity. 5
10
CS430 murine T
cells were incubated with LCF, detergent solubilized, and
immunoprecipitated with anti-receptor antibody. The immunoprecipitates
were assayed separated by SDS-PAGE transferred to nitrocellulose. The
filters were first blocked in 5% milk in phosphate-buffered saline,
then incubated in p56 antiserum followed by goat
anti-rabbit-horseradish peroxidase, and finally subjected to
chemiluminescense autoradiography. Arrow indicates p56
protein. Lane 1, precipitation with Protein A alone. CD4
immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3),
or treated with LCF (lane 4).
10
CS420 murine T cells were treated as in Fig. 1. Lane 1, precipitation with Protein A alone. CD4
immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3),
or treated with LCF (lane 4).
Murine T Cell Hybridomas Migrate in Response to
LCF
We next investigated these murine T cell hybridomas for
their ability to migrate in response to LCF. Cells which express either
wild type CD4 (open square) or CS430 CD4 (closed
diamond) exhibited dose dependent migration following exposure of
cells to micromolar-picomolar LCF concentrations (Fig. 3). The
hybridoma cells, which express either CS420 (open diamond) or
CS422 (closed square) CD4, failed to respond to any dose of
LCF (Fig. 3). These results suggested that the binding of the
tyrosine kinase was essential to CD4-mediated LCF-induced T cell
migration. In order to confirm the CD4 specificity of the migration
inactive clones, the cells were examined for their ability to migrate
in response anti-CD3 antibody. We have previously shown (8) that the parental and mock transfected hybridoma lines
migrate in response to CD3 antibody. Fig. 4shows that all four
of the murine hybridoma lines expressing different forms, but equal
amounts, of CD4 migrated in a similar dose-dependent manner following
exposure to anti-CD3 antibody. These experiments demonstrate that the
impaired motility observed in the CS420 and CS422 clones in response to
LCF was restricted to alterations in CD4 and suggests that the CD4
signaling pathway induced during lymphocyte migration is independent of
the signal transduction pathway utilized for anti-CD3-induced motility.
10
/ml murine T cells were incubated in a modified
Boyden chamber in the presence of 1 pM to 0.1 µM of LCF for 3 h. The results are expressed as a percentage of the
migration of untreated T cells.
, MNC-CD4 T cells;
,
CS430 T cells;
, CS420 T cells;
, CS422 T cells. Asterisks denote a significant difference in migration from
control at p = 0.05.
, MNC-CD4 T cells;
,
CS430 T cells;
, CS420 T cells;
, CS422 T
cells.
Effect of Tyrosine Kinase Inhibitor, Herbimycin A, on LCF
Induction of Murine T Cell Hybridomas Migration and p56
A number of groups have demonstrated
that functions associated with lck interaction with CD4 are independent
of the kinase activity of lck(10, 20, 21) .
Along these lines we have recently demonstrated that divalent,
uncross-linked anti-CD4 antibodies, incapable of inducing lck kinase
activity do induce motile responses dependent on physical association
of CD4 with p56 Kinase Activity
.
LCF induction of
the kinase enzymatic activity, however, led us to determine whether
LCF-induced lymphocyte migration requires the enzymatic activity of
p56
activity. In these experiments CS430
hybridoma cells were exposed to the kinase inhibitor herbimycin A.
Herbimycin A has a bimodal effect on tyrosine kinases, first directly
decreasing the activity of the kinase(22, 23, 24) and then acting to increase the degradation rate of the
steady state levels of the protein(25) . At 18 h, a time when
there is complete inhibition of enzymatic activity (Fig. 5) but
minimal protein degradation(23) , the motility induced by LCF (Fig. 6) and CD3 antibodies (Fig. 7) was identical to
that observed in untreated cells ( Fig. 3and Fig. 4). To
further understand the role of lck enzymatic activity in CD4-mediated
migration, we used two T cell hybridoma cell lines expressing two
different CD4-lck chimeras.
, with treatment;
,
without.
, MNC-CD4 T cells;
,
CS430 T cells;
, CS420 T cells;
, CS422 T
cells.
Chimeric CD4-lck T Cell Hybridoma
Migration
Chimeric CD4-lck containing either full-length lck
protein or kinase domain-deleted lck protein were expressed in the
By155.16 T cell line. Cells were selected for stable and equivalent
surface expression of these constructs and examined for their
LCF-induced migratory response. Fig. 8shows that hybridoma
cells expressing full-length CD4-lck responded to LCF in a
dose-dependent manner similar to that of the CD4 wild type control.
Cells expressing CD4-lck which lacks the catalytic domain of the kinase
demonstrated comparable motility. Thus T cells in which the kinase
activity was eliminated either by herbimycin A treatment or
construction of kinase deficient chimeric molecules have normal motile
responses to LCF. These data indicate that while LCF induces
CD4-associated lck kinase activity and that induced migration requires
lck association with CD4, LCF-induced cell migration is independent of
the catalytic activity of the kinase.
10
/ml murine T cells were incubated in a modified
Boyden chamber in the presence of 10 pM to 0.1 µM of LCF for 3 h. The results are expressed as a percentage of the
migration of untreated T cells.
, By155.16 parent line T cells;
, MNC-CD4 T cells;
, full-length CD4-lck chimeric T cells;
, kinase-deleted CD4-lck chimeric T
cells.
]
and inositol
trisphosphate (IP
) are CD4 dependent(7) , as is the
motile response (8, Fig. 3). Further, LCF-induced chemotactic
responses in monocytes (5) and eosinophils (6) are also
directly dependent upon the expression of cell surface CD4, and Fab
fragments of anti-CD4 monoclonal antibody inhibit all LCF-induced
functions on these
cells(5, 6, 7, 8) . The data
presented in this paper extend these observations to show that LCF
induces CD4-associated p56
kinase activity and
therefore provides further evidence for a direct signaling role of CD4
in LCF-induced functions.
the physical association between the src family tyrosine kinase
p56
and CD4 is essential for LCF-induced
lymphocyte migration. The activation of T cell motile response mediated
via CD4 is independent of the catalytic activity of the
receptor-coupled kinase. These findings are consistent with recent
reports which have identified a kinase independent role for
p56
during T cell activation (10, 20, 21) and our own recent analysis
demonstrating that uncross-linked anti-CD4 induced migration is
independent of the enzymatic activity of
p56
.
While kinase activity is not
required for the induction of T cell migration, p56
binding of other intracellular proteins (e.g. GTPase p32, c-raf, phosphatidylinositol 3-kinase,
phospholipase C
) as an adaptor molecule, may explain the
requirement for lck-CD4 association in the motile
response(26, 27, 28, 29) . Binding
to the src homology domains (30) SH2 and SH3 of p56
appear most likely to provide a recruitment site for these
molecules to the CD4-lck complex. The ability of cells, expressing
CD4-lck chimeras which lack the catalytic domain, to migrate in
response to LCF strongly suggests the involvement of SH2/SH3 binding
intermediates in the signal transduction mechanism of lymphocyte
motility.
, IP
, and
[Ca
]
are both released in
response to LCF (7) and have been reported to regulate
actin-binding proteins (31) and changes in the
cytoskeleton(32) , as well as the activation of their down
stream effector enzyme protein kinase C(33) . The link to the
cytoskeleton could be the lipid kinase, phosphatidylinositol-3 kinase
which associates with src kinases, by binding to SH3
domains(34, 35) and has recently been shown to play a
role in cell shape change(36) . Alternatively, there might be a
direct link between actin-binding proteins, which have been
demonstrated to contain SH3 domains(37, 38) to the
CD4-coupled p56
complex.
or multimeric
gp120(9) , might mimic the natural state of LCF.
]
, nor
p56
enzymatic activity(19) .
The inability of uncross-linked anti-CD4 antibodies to induce
p56
kinase activity
but their
ability to induce motile responses highlights the dissociation between
a proposed ``adaptor'' function for lck which appears to be
essential for communication with motility-dependent cytoskeletal
structures and functions associated with lck kinase activity. More
extensive aggregation of surface CD4 by LCF, cross-linked anti-CD4
antibodies, and gp120 appear to have the ability to induce cell
activation and the intracellular signal transduction systems noted
above.
kinase activity by LCF and
cross-linked antibodies suggests a role for the catalytic activity in
LCF-induced cell activation events other than motility.
]
, intracellular calcium;
IP
, inositol trisphosphate; gp120, glycoprotein 120; PAGE,
polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.