The chemokines RANTES (regulated on activation,
normal T cell expressed and secreted) and MIP (macrophage inflammatory
protein)-1
have been implicated in regulating T cell functions.
RANTES-induced T cell activation is apparently mediated via two
distinct signal transduction cascades: one linked to recruitment of
pertussis toxin-sensitive G proteins and the other linked to
protein-tyrosine kinase activation. In this report, we identified that
the transcription factors Stat1 and Stat3 (for signal transducers and
activators of transcription) are rapidly activated in T cells in
response to RANTES and MIP-1
. Nuclear extracts from MOLT-4 and
Jurkat T cells treated with RANTES or MIP-1
contain
tyrosine-phosphorylated Stat1:1 and Stat1:3 dimers that exhibit
DNA-binding activity. We demonstrated that RANTES and MIP-1
treatment of Jurkat cells resulted in transcriptional activation of a
Stat-inducible gene, c-fos, with kinetics consistent with
Stat activation by these chemokines. RANTES and MIP-1
mediate their
effects via shared chemokine receptors (CCRs): CCR1, CCR4, and CCR5.
Our data revealed a concordance between chemokine-induced Stat
activation and c-fos induction and CCR4 and CCR5
expression. These findings indicate that chemokine-mediated activation
of G-protein-coupled receptors leads to signal transduction that
invokes intracellular phosphorylation intermediates used by other
cytokine receptors.
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INTRODUCTION |
RANTES1
(regulated on activation, normal
T cell expressed and secreted) and MIP
(macrophage inflammatory protein)-1
are potent chemoattractant cytokines (chemokines) for T cells (1-3).
Accumulating evidence suggests that these CC or
-chemokines function
as regulators of inflammatory and immunoregulatory processes (4-15).
Chemokines mediate their shared and different biologic effects through
common receptors. Eight receptor cDNAs specific for the CC
chemokines have been cloned to date, whose nine gene products share a
seven transmembrane domain architecture linked to G-protein complexes (16-19). A number of studies have examined chemokine-induced signal transduction, yet defined signaling pathways have not been elaborated. Interleukin-8, the main chemotactic cytokine for neutrophils, will
stimulate serine/threonine protein kinases (20). Monocyte chemotactic
protein (MCP)-1 will activate p42/44 mitogen-activated protein kinases
(21). MCP-1-, MCP-2-, and MCP-3-induced monocyte chemotaxis can be
blocked by both serine/threonine and tyrosine kinase inhibitors (22).
MCP-1 and MCP-3 rapidly induce arachidonic acid in target monocytes
(23). RANTES, MIP-1
, and MCP-1, -2, and -3 have been shown to
promote Ca2+ mobilization in monocytes, eosinophils,
basophils, and T cells (7, 21, 24-27).
Many cytokines and growth factors mediate their effects via activation
of a common signal transduction pathway, the STAT pathway. Binding of
the ligand to its specific transmembrane receptor results in receptor
aggregation, which may involve single or multiple receptor chains.
Receptor aggregation leads to the catalytic activation of
receptor-associated cytoplasmic protein-tyrosine kinases, termed janus
kinases (JAK) and phosphorylation-activation of latent monomeric signal transducers and activators of
transcription, Stat proteins. Six Stat proteins have been
identified to date. Receptor-associated phosphorylated Stats then
dimerize via SH2-phosphotyrosyl interactions and translocate to the
nucleus, where they bind to specific promoter sequences, thereby
regulating gene expression (reviewed in Ref. 29). All Stat proteins,
with the possible exception of Stat2, differentially bind to more than
ten related DNA elements, which fit the consensus TTNNNNNAA (consensus
STAT recognition element). Conserved structural motifs within the
cytoplasmic domains among the cytokine receptors have been implicated
as Jak and STAT recognition sites (30, 31). Although Jak-STAT signaling
is likely a common feature of all cytokines, recent reports suggest
that STAT activation is not necessarily exclusively mediated via Jak
association with the cytoplasmic domains of receptors that constitute
the cytokine receptor superfamily. Angiotensin II binding to its
cognate seven transmembrane, G-protein-coupled receptor, activates
Stat1, Stat2, and Stat3 (32-34).
Apart from their potent chemotactic activities, there is accumulating
evidence that CC chemokines are also capable of stimulating T cells
in vitro (35-37). At µM concentrations,
RANTES-induced signaling in T cells is mediated by at least two
distinct signaling cascades: one associated with recruitment of G
proteins and the other to protein-tyrosine kinase activation (35). This
RANTES-induced tyrosine kinase activation has been functionally linked
to T cell proliferation, up-regulation of the IL-2 receptor, and the
production of cytokines. At these micromolar doses, RANTES will induce
the tyrosine kinase activity of the zeta-associated protein (ZAP)-70 and the focal adhesion kinase (FAK) pp125FAK (36). In the
presence of anti-CD3 monoclonal antibody, CC chemokines, at nanomolar
doses, exert costimulatory effects on T cells (37). This chemokine
enhancement of T cell activation, in combination with T cell
receptor-mediated signals, is also associated with proliferation and
IL-2 production (37). In this report, we investigated the potential
involvement of Stat proteins in chemokine-induced tyrosine
phosphorylation in T cells. In a similar manner to angiotensin II,
RANTES and MIP-1
can activate chemokine receptor (CCR)-mediated STAT
signaling in T cells. Our data indicate that both chemokines, at
nanomolar doses, induce the rapid tyrosine phosphorylation and
activation of Stat1 and Stat3. In addition, at nanomolar
concentrations, both chemokines induce the gene expression of the
Stat-inducible proto-oncogene c-fos.
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MATERIALS AND METHODS |
Cells, Chemokines, and Related Reagents--
MOLT-4 human
leukemic and Jurkat T cells were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum, 100 units/ml penicillin, and
100 mg/ml streptomycin. Cells were concentrated to 2 × 107 and treated with the appropriate chemokine for the
indicated time. Osteosarcoma cells stably expressing CCR4 (HOS-CCR4) or CCR5 (HOS-CCR5) were obtained from the National Institutes of Health
AIDS Research and Reference Reagent Program. Promyelocytic leukemic HL
60 cells expressing CCR1 were obtained from ATCC. Recombinant RANTES
(0.4 mg/ml) and MIP-1
(1.0 mg/ml) were a generous gift of Dr.
T. J. Schall (DNAX Research Inst., CA). Recombinant human
interferon (IFN)-
, IFN-Con1 (specific activity 3 × 109 units/mg of protein), was kindly provided by Amgen
Inc., CA.
Cell Extracts--
Nuclear extracts were prepared as described
previously (38). Briefly, cells were washed twice with ice-cold
phosphate-buffered saline that contained 1 mM
Na3VO4 and 5 mM NaF and once with
hypotonic buffer. Following incubation for 10 min in hypotonic buffer
at 108 cells/ml, supplemented with 0.2% Triton X-100,
cells were disrupted by repeated passage through a 25-gauge needle and
centrifuged at 12,000 × g for 20 s. The pellet
was incubated in high salt buffer at 2.5 × 108
cells/ml for 30 min and clarified by centrifugation at 12,000 × g for 20 min, and the supernatant was supplemented with
0.05% Triton X-100. Nuclear fractions that yielded 4.5-6.7 µg of
protein/106 cells, based on the Bradford method for protein
determination (Bio-Rad Labs., CA.) were aliquoted and stored at
70 °C. Hypotonic buffer contained 12 mM Hepes (pH
7.9), 4 mM Tris (pH 7.9), 0.6 mM EDTA, 10 mM KCl, 5 mM MgCl2, 1 mM Na3VO4, 1 mM
Na4P2O7, 1 mM NaF, 0.6 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 10 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin
A. The buffer that contained 300 mM KCl and 20% glycerol
constituted high salt buffer.
Oligonucleotides--
Double-stranded oligodeoxynucleotides,
representing the sis-inducing element (SIE) of the c-fos
promoter and a mutant IFN-stimulated response element (ISRE), were
synthesized. The sequences are: SIE, 5
-ATTTCCCGTAAATCCC-3
, and mutant
ISRE, 5
-CCTTCTGAGGCCACTAGAGCA-3
. These oligodeoxynucleotides were
synthesized with SalI-compatible linkers at the 5
terminus
(TCGAC). Gel-purified oligonucleotides were mixed with their respective
complements, heated to 65 ° for 15 min, and annealed at room
temperature for 18 h. Double-stranded elements were used directly
in competition experiments.
Reverse Transcription PCR--
Oligonucleotide primers were
designed to reverse transcribe and PCR amplify human CCR1, CCR4, and
CCR5 from 1 ng of poly(A)+ RNA. CCR primer sets were: CCR1,
sense primer 5
-ATCCTCTCTGGGTTTTATTACACA-3
, antisense primer
5
-GATGATCATGATGACAAAAATCAA-3
; CCR4, sense primer 5
-AAATGAAACCCACGGATATAGCAG-3
, antisense primer
5
-CCATGGTGGACTGCGTGAAG-3
; and CCR5, sense primer
5
-GCTGTGTTTGCTTTAAAAGCC-3
, antisense primer
5
-TTAGCCTCACAGCCCTATG-3
.
The reverse transcription-PCR conditions have been previously described
(39). Poly(A)+ RNA from promyelocytic HL60 leukemic cells
and osteosarcoma cells (HOS) transfected with cDNA for CCR4
(HOS-CCR4) and CCR5 (HOS-CCR5), were used as positive controls for
CCR1, CCR4, and CCR5, respectively.
Antibodies--
Monoclonal antibodies against Stat1 and Stat3
were purchased from Transduction Laboratories, KY. Anti-phosphotyrosine
(Tyr(P)) monoclonal antibody, clone 4G10 was purchased from Upstate
Biotechnology Inc., NY. For supershift studies, polyclonal antisera
against Stat1 and Stat2 were a gift from C. Schindler (Columbia
University College of Physicians & Surgeons, NY) and Stat3 antiserum
was a gift from D. Levy (NYU School of Medicine). Polyclonal antisera against CCR1 was a gift from R. Horuk, Berlex Biosciences, CA. Preimmune rabbit IgG (ICN Biomedicals, Inc., CA.) was used as a control
for immunoprecipitation.
Genomic DNA Affinity Chromatography (GDAC)--
GDAC has been
previously described (40). 50 mg of nuclear extract were incubated for
20 min with 25 µg of poly(dI-dC)·poly(dI-dC) (Pharmacia Biotech,
Uppsala, Sweden) in binding buffer. Where indicated, 600 ng of
double-stranded oligodeoxynucleotides were added. This mixture (200 ml)
was incubated for 2 h with 100 ml of bovine genomic DNA-cellulose
(Sigma) that was equilibrated in binding buffer. The DNA-cellulose was
then washed 3 times with 3 ml of wash buffer, and DNA-binding proteins
were eluted by incubation for 30 min in 200 µl of elution buffer.
Following centrifugation, the supernatant (high salt eluate) was
concentrated using Amicon-30 microconcentrators (Amicon, Inc., MA),
boiled with reducing SDS-PAGE sample buffer, and analyzed in Western
blotting experiments. Wash buffer contained 12% glycerol, 0.05%
Triton X-100, 12 mM Hepes (pH 7.9), 4 mM Tris
(pH 7.9), 0.6 mM EDTA, 60 mM KCl, 5 mM MgCl2, 1 mM
Na3VO4, 1 mM
Na4P2O7, 1 mM NaF, 250 mg/ml bovine serum albumin, 0.6 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The buffer that contained
1 mg/ml bovine serum albumin, 10 mg/ml aprotinin, 2 mg/ml leupeptin,
and 2 mg/ml pepstatin A constituted binding buffer. The buffer that
contained 360 mM KCl without bovine serum albumin
constituted elution buffer.
Mobility Shift Assay--
10 µg of nuclear extract from
untreated or chemokine-treated cells were analyzed using the
electrophoretic mobility shift assay (EMSA), by a modification of the
procedure described previously (41). Briefly, extracts were incubated
with or without double-stranded oligodeoxynucleotides corresponding to
the c-fos SIE or a mutant SIE, in the presence of 1.5 µg
of poly(dI-dC)·poly(dI-dC), in EMSA buffer for 30 min at room
temperature (final volume 30 ml). Protein-DNA complexes were resolved
on a 4.5% polyacrylamide gel using 0.5 × Tris-borate-EDTA as
running buffer. For supershift experiments, 1.0 µl of polyclonal
antisera to Stat1, Stat2, Stat3, anti-phosphotyrosine (4G10), or
preimmune sera were incubated with protein extracts for 30 min at
4 ° prior to the addition of DNA. EMSA buffer contained 13 mM Hepes (pH 7.9), 65 mM NaCl, 0.15 mM EDTA (pH 8.0), 0.06 mM EGTA (pH 8.0), 1.0 mM dithiothreitol, and 5% Ficoll.
RNA Purification, Gel Electrophoresis and Northern
Hybridization--
Poly(A)+ RNA extraction and Northern
hybridization procedures have been described elsewhere (42). A 1.3 kbp
v-fos cDNA insert in plasmid pFBH-1 was used.
 |
RESULTS AND DISCUSSION |
Chemokine-inducible Stat Activation--
RANTES and MIP-1
both
bind to the CC chemokine receptors designated CCR1, CCR4, and CCR5.
Target T cells for study were chosen initially based on CCR1 expression
determined by flow cytometric analysis of anti-CCR1 antibody binding to
native CCR1 on cells (Fig. 1). The lack
of availability of specific antibodies for the shared receptors CCR4
and CCR5 precluded identification of their cell surface expression. Our
analyses revealed that both the MOLT-4 and Jurkat T cell lines express
CCR1.

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Fig. 1.
CCR1 cell surface expression. Flow
cytometric analysis of CCR1 polyclonal antibody binding to native CCR1
on Jurkat cells. 0.5 × 106 cells were incubated with
fluorescence-activated cell sorter buffer (negative control) or CCR1
antisera for 45 min on ice, washed, and incubated with
biotin-SP-conjugated F(ab )2 rat anti-mouse IgG for an
additional 30-45 min. The cells were washed and then incubated with
R-phycoerythrin-conjugated streptavidin for an additional 30 min.
Immunofluorescence was analyzed with a Becton Dickinson FACScan.
Incubation with either medium alone or secondary and tertiary reagents
alone resulted in superimposable negative cytograms, represented as the
profile in panel A and positive cytogram (+CCR1 antisera)
represented in panel B. MOLT-4 CCR1 expression was likewise
confirmed (data not shown).
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Subsequently, we undertook studies to examine whether, in a similar
manner to angiotensin II, RANTES can activate Stats in MOLT-4 cells. We
employed a procedure that we have developed, GDAC, to assay for
chemokine-inducible Stat activation (40). GDAC does not require prior
knowledge of target DNA elements. Briefly, nuclear extracts from
RANTES-treated cells were mixed with genomic DNA bound to cellulose.
The mixture was allowed to equilibrate, following which DNA-binding
complexes were eluted in high salt buffer. Eluted fractions were
resolved by SDS-PAGE, and Stat proteins were detected using anti-Stat
immunoblots. Using this procedure, we identified that RANTES induces
Stat1- and Stat3-containing DNA-binding complexes in MOLT-4 cells
within 30 min (Fig. 2). Stat2, Stat5, and
Stat6 were not detected in the DNA-binding complexes.

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Fig. 2.
Identification of RANTES-inducible STAT
complexes by GDAC. Actively growing MOLT-4 cells were incubated
with or without RANTES (6 nM). Nuclear extracts were
prepared and analyzed for DNA-binding STAT complexes using GDAC.
Eluates from genomic DNA were resolved by SDS-PAGE (7%) and analyzed
by Western blotting. Blots were probed with antisera to Stat1, Stat2,
Stat3, Stat5, and Stat6. Lysates from whole cell extracts from
fibroblast (FL) and Jurkat (JL) cells that had
not undergone GDAC served as positive controls, as did a nucelar
extract from IFN-Con1-treated MOLT-4 cells that had
undergone GDAC. As indicated, a nuclear extract from untreated MOLT-4
cells that had undergone GDAC served as the negative control.
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Since both RANTES and MIP-1
bind to CCR1 (and CCR4 and CCR5), we
reasoned that MIP-1
might also invoke Stat1 and Stat3 activation in
MOLT-4 cells. All combinations of homo- and heterodimers of Stat1 and
Stat3 will bind to the high affinity c-fos SIE recognition element, m67 (38). Accordingly, we examined the kinetics of activation
of both RANTES- and possibly MIP-1
-inducible Stat1- and
Stat3-containing complexes in a standard mobility shift assay. MOLT-4
cells were treated with MIP-1
for varying times (15 min to 2 h),
then nuclear extracts were analyzed in a gel mobility shift assay. As
shown in Fig. 3, MIP-1
rapidly induced
SIE-binding activities in MOLT-4 cells, within 15 min, that were no
longer detectable 2 h after treatment. Similarly, RANTES and
MIP-1
rapidly induced SIE-binding activities in Jurkat cells, by 15 min, that were likewise no longer detectable after 2 h (Fig.
3).

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Fig. 3.
Chemokine-inducible SIE-binding activities in
T cells. MOLT-4 (A) and Jurkat (B) cells
were either left untreated ( ) or treated (+) with 2 nM
MIP-1 or 6 nM RANTES for varying times as indicated.
Nuclear extracts, prepared as descibed under "Materials and
Methods," were reacted with 30,000 cpm of a
32P-end-labeled sis-inducible element of the
c-fos promoter. Complexes were resolved using 4.5% native
PAGE and visualized by autoradiography. Mobility of
IFN-Con1-inducible Stat1:1 and Stat1:3 complexes are identified by arrows. FP, free probe.
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The apparent contradiction in kinetics of chemokine-induced Stat
activation observed using the two different approaches, GDAC and gel
mobility shift assay, may be attributable to the differences in DNA
recognition elements employed in the two procedures. Specifically, GDAC
allows for detection of STAT complexes that bind with relatively high
affinity to any number of different target genomic DNA
elements. Moreover, GDAC allows for DNA recognition by STAT complexes
in the context of any potential accessory factors that are also present in the nuclear extract and that may be involved in DNA-binding. By
contrast, gel mobility shift assays invoke the use of a specified DNA
target element, in this case the SIE, thereby restricting the scope of
STAT complex binding. The delay in chemokine-induced Stat activation
observed by GDAC compared with the gel shift assay, may reflect both
the low abundance of activated STAT complexes in the cell extracts and
the low abundance of SIE-like recognition elements in the genomic DNA
used for GDAC. With increased time, chemokine-induced STAT complexes
will accumulate, enhancing the likelihood of detection by GDAC. GDAC
identification of DNA-binding Stat-containing complexes in 2-h-induced
nuclear extracts may reflect STAT complexes that are induced with
slower kinetics and that recognize DNA elements distinct from the SIE
element employed in the gel shift assay. Moreover, these
Stat-containing complexes may be associated with DNA-binding adapter
proteins.
We confirmed the specificity of RANTES- and MIP-1
-induced
SIE-binding activities using unlabeled competitor SIE DNA and a nonspecific oligonucleotide element (a mutant interferon-stimulated response element) in the gel shift assays (Fig.
4). Specifically, both RANTES and
MIP-1
-inducible specific SIE-binding activities are present in
nuclear extracts from both MOLT-4 and Jurkat cells. Using anti-Stat
antibodies in a gel mobility supershift assay, we observed that
anti-Stat1 antibodies recognized both SIE-binding complexes in
chemokine-treated nuclear extracts (Fig.
5, A and B),
whereas anti-Stat3 antibodies only recognized one complex (Fig.
5A). We infer that the chemokine-induced SIE binding
activities correspond to the STAT complexes Stat1:1 and Stat1:3. The
mobilities of antibody-supershifted STAT-SIE complexes vary according
to the charge and size of the resultant complexes. Inclusion of
antisera to Stat2 resulted in the appearance of a slow migrating band
in both untreated (data not shown) and chemokine-treated cells. The mobilities of the chemokine-induced STAT-DNA complexes were, however, unaffected by anti-Stat2 antisera. We infer that Stat2 is not a
constituent of the chemokine-induced STAT complexes and that there are
constituents in the anti-Stat2 antisera that interact with DNA-binding
factors in cells to invoke a non-Stat-specific SIE-containing complex.
Additionally, the Stat1:1 and Stat1:3 complexes may be supershifted
with anti-phosphotyrosine antibody 4G10, confirming that, in common
with other cytokine-induced STAT complexes, the chemokine-induced STAT
complexes are phosphorylated on tyrosine residues (Fig.
5C).

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Fig. 4.
Specificity of chemokine-inducible
DNA-binding activities. PMA-stimulated (50 ng/ml for 16 h)
MOLT-4 (A) and Jurkat (B) cells were either left
untreated or treated with 2 nM MIP-1 or 6 nM
RANTES for 30 min. Nuclear extracts were prepared and reacted with
32P-SIE. Complexes were resolved by native gel
electrophoresis. For details refer to Fig. 3. Specific SIE-binding
complexes were displaced by 100-fold molar excess of unlabeled SIE, as
indicated. Inclusion of 100-fold molar excess of an unlabeled mutant
ISRE served as a negative control for displacement of SIE-binding
activities (mutant). Mobility of
IFN-Con1-inducible Stat1:1 and Stat1:3 complexes are
identified by arrows. FP, free probe.
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Fig. 5.
RANTES and MIP-1 activate Stat1:1 and
Stat1:3 STAT complexes in T cells. MOLT-4 (A) or Jurkat
(B) cells were either untreated or treated with RANTES (6 nM) or MIP-1 (2 nM) for 15 min. Nuclear
extracts were prepared, incubated with antisera to Stat1, Stat2, Stat3
or preimmune sera, and reacted with labeled SIE, and then the resultant
complexes were resolved by native gel electrophoresis. For details
refer to Fig. 3. Non-supershifted protein-DNA complexes are identified
as Stat1:1 and Stat1:3 by arrows. C, nuclear
extracts from RANTES-treated Jurkat cells were incubated with
anti-phosphotyrosine antisera (4G10) or nonspecific antisera
(anti-interferon receptor (IFNAR1) antibody, designated antiIFN / Rc), followed by addition of labeled SIE.
FP, free probe.
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Chemokine-inducible c-fos Gene Expression--
To address the
biological consequence of chemokine-inducible Stat activation, we
examined whether chemokine treatment leads to the transcriptional
regulation of a Stat-inducible gene. The promoter of the proto-oncogene
c-fos contains a Stat binding DNA element (43). Indeed, a
modified version of the SIE of the c-fos promoter was used
as the recognition element for Stat1- and Stat3-containing STAT
complexes in our gel shift assays.
Chemokine receptor expression is tightly regulated; we have observed
that both Jurkat and MOLT-4 cells transiently express chemokine
receptors. We have shown that PMA treatment for 16-18 h induces the
gene expression of chemokine receptors (data not shown), without
affecting Stat activation. Accordingly, PMA-treated Jurkat cells were
exposed to RANTES or MIP-1
in time course studies, then
poly(A)+ RNA extracted and probed for c-fos gene
expression by Northern hybridization. Our results, shown in Fig.
6A, reveal that both RANTES
and MIP-1
induce c-fos gene expression within 2 h,
which is absent by 6 h. The kinetics of c-fos induction
are consistent with the kinetics of chemokine-inducible Stat
activation. Interestingly, cyclohexamide treatment of cells 1 h
prior to chemokine treatment lead to a superinduction of
c-fos gene expression at 6 h. These data imply that
c-fos gene expression is under the control of a protein
synthesis-dependent pathway.

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Fig. 6.
Chemokine-inducible c-fos gene
expression correlates with CCR4 and CCR5 expression.
PMA-stimulated (50 ng/ml for 16 h) Jurkat cells were left
untreated or treated with 6 nM RANTES or 2 nM
MIP-1 for the times indicated. A, Northern analysis of 0.5 µg of poly(A)+ RNA with
32P-v-fos and 32P- -actin cDNA
probes. B, reverse transcription-PCR amplification of CCR1,
CCR4, and CCR5 from poly(A)+ RNA from cell extracts in
which chemokine-inducible c-fos gene expression was detected
(B1) or absent (B2).
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RANTES and MIP-1
mediate their effects by shared receptors: CCR1,
CCR4, and CCR5. We conducted experiments to determine the concordance
between chemokine-induced c-fos gene induction and expression of specific species of CCRs. The data in Fig. 6B
show a correlation between CCR4 and CCR5 gene expression and
chemokine-inducible c-fos gene expression.
Examination of the intracellular loops of CCR1 reveals a putative Stat3
recognition sequence, YRLQ (residues 311-314) (31). Close examination
of this conserved region within the carboxyl-terminal intracellular
loop of the different CCRs reveals a similar Stat recognition motif in
CCR4 (YILQ), as well as conserved tyrosine residues in the other
receptors. Moreover, a highly conserved motif in the second
intracellular loop of the eight CCRs contains a tyrosine residue in all
but CCR7: DRYI/LAI/VVH/Q. Interestingly, this
tyrosine-containing motif is present in the angiotensin II receptor
that is associated with ligand-induced Stat activation. Furthermore,
angiotensin II induces similar Stat complexes as RANTES and MIP-1
(33). Viewed together, these data suggest that chemokine-induced signal
transduction may be mediated, in part, via ligand activation of
intracellular domains of the CCR associated with Stat activation.
Antigen-independent activation of T cells by cytokines may be important
for recruiting effector T cells at the site of an immune response and
in maintaining the clonal size of memory T cells in the absence of
antigenic stimulation (28). Although there is evidence to suggest that
RANTES and MIP-1
can costimulate T cell activation at nanomolar
doses (37), the evidence to date for antigen-independent CC
chemokine-induced signaling in T cells, mediated by protein-tyrosine
kinase activation, is restricted to RANTES (35). In this study,
micromolar concentrations of RANTES induced T cell activation,
characterized by the up-regulation of the IL-2 receptor
chain, IL-2
and IL-5, and T cell proliferation. Our data indicate that, at
nanomolar concentrations, RANTES and MIP-1
will trigger
protein-tyrosine kinase activation, which in this instance is
associated with Stat activation. The biological consequence of this
Stat activation in terms of T cell functions remains to be elucidated.
It is likely that the protein-tyrosine kinases associated with
RANTES-induced T cell proliferation are distinct from those associated
with RANTES- and MIP-1
-induced Stat activation. Additionally, subtle
structural differences among the receptors that mediate
chemokine-induced protein-tyrosine kinase activation may define which
kinases are activated and hence which signaling pathways are invoked.
The existence of multiple signaling pathways associated with
tyrosine-phosphorylated intermediates suggests a complexity related to
regulation of T cell functions.
This report represents original findings with regard to chemokine
activation of Stat signaling pathways. Based on our GDAC studies, it is
intriguing to speculate that chemokine-induced Stat1- and
Stat3-containing DNA-binding complexes may accumulate in T cells and
bind to DNA elements that are distinct from the consensus STAT
recognition element, TTNNNNNAA. Such novel promoter elements might
provide the basis for grouping gene families, thereby identifying
potential gene targets of chemokine action. The immediate challenge,
however, is to determine the kinases that mediate Stat phosphorylation-activation in this system and the role of G-proteins in
Stat activation. These studies are currently in progress.