RANTES and MIP-1alpha Activate Stats in T Cells*

Mark WongDagger and Eleanor N. FishDagger §

From the Departments of § Medical Genetics & Microbiology and Dagger  Immunology, University of Toronto, Toronto, Ontario M5S 3E2, Canada

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MIP (macrophage inflammatory protein)-1alpha 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-1alpha . Nuclear extracts from MOLT-4 and Jurkat T cells treated with RANTES or MIP-1alpha contain tyrosine-phosphorylated Stat1:1 and Stat1:3 dimers that exhibit DNA-binding activity. We demonstrated that RANTES and MIP-1alpha 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-1alpha 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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

RANTES1 (regulated on activation, normal T cell expressed and secreted) and MIP (macrophage inflammatory protein)-1alpha are potent chemoattractant cytokines (chemokines) for T cells (1-3). Accumulating evidence suggests that these CC or beta -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-1alpha , 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-1alpha 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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-1alpha (1.0 mg/ml) were a generous gift of Dr. T. J. Schall (DNAX Research Inst., CA). Recombinant human interferon (IFN)-alpha , 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
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Chemokine-inducible Stat Activation-- RANTES and MIP-1alpha 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).

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.

Since both RANTES and MIP-1alpha bind to CCR1 (and CCR4 and CCR5), we reasoned that MIP-1alpha 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-1alpha -inducible Stat1- and Stat3-containing complexes in a standard mobility shift assay. MOLT-4 cells were treated with MIP-1alpha 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-1alpha 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-1alpha 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-1alpha 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.

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-1alpha -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-1alpha -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-1alpha 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-1alpha 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-1alpha (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 antiIFNalpha /beta Rc), followed by addition of labeled SIE. FP, free probe.

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-1alpha 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-1alpha 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-1alpha for the times indicated. A, Northern analysis of 0.5 µg of poly(A)+ RNA with 32P-v-fos and 32P-beta -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).

RANTES and MIP-1alpha 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-1alpha (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-1alpha 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 alpha  chain, IL-2 and IL-5, and T cell proliferation. Our data indicate that, at nanomolar concentrations, RANTES and MIP-1alpha 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-1alpha -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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medical Genetics & Microbiology, University of Toronto, Rm. 73, FitzGerald Bldg., 150 College St., Toronto, Ontario M5S 3E2, Canada. Tel.: 416-978-2755; Fax: 416-978-4761; E-mail: en.fish{at}utoronto.ca.

1 The abbreviations used are: RANTES, regulated on activation, normal T cell expressed and secreted; MIP-1alpha , macrophage inflammatory protein; CC, chemoattractant cytokines; CCR, chemokine receptor; IL, interleukin; JAK, janus kinase; SIE, sis-inducing element; ISRE, IFN-stimulated response element; PCR, polymerase chain reaction; HOS, human osteosarcoma; PAGE, polyacrylamide gel electrophoresis; GDAC, genomic DNA affinity chromatography.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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