Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M406978200 on September 3, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48520-48534, November 19, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/47/48520    most recent
M406978200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barlic, J.
Right arrow Articles by Murphy, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barlic, J.
Right arrow Articles by Murphy, P. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Interleukin (IL)-15 and IL-2 Reciprocally Regulate Expression of the Chemokine Receptor CX3CR1 through Selective NFAT1- and NFAT2-dependent Mechanisms*

Jana Barlic{ddagger}, David H. McDermott{ddagger}, Maya N. Merrell{ddagger}, Jacqueline Gonzales§, Laura E. Via§, and Philip M. Murphy{ddagger}

From the {ddagger}Molecular Signaling Section, Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and §Tuberculosis Research Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852

Received for publication, June 22, 2004 , and in revised form, September 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2R{beta}{gamma}, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a {kappa}B-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the {kappa}B-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1–/– mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a {kappa}B-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte extravasation into inflamed tissue is coordinated by several families of differentially expressed chemoattractant and adhesive molecules. Fractalkine (or CX3CL1), a member of the chemokine family (1, 2), is particularly interesting because it can function as either an adhesion molecule (3–6) when expressed in a membrane-bound form on endothelial cells (7, 8) or as a soluble chemoattractant when cleaved at a membraneproximal region by the TNF-{alpha}1-converting enzyme (TACE/ADAM17) (9, 10) or ADAM10 (11). Both functions are governed by the distribution of CX3CR1, a specific seven transmembrane domain G protein-coupled receptor for fractalkine expressed on human peripheral blood CD8+ (12) and CD4+ perforin+ T cells (12, 13), CD56dim CD16+ NK cells, NK-T cells (12, 14), and CD14lowCD16high monocytes (15), as well as on mouse NK cells, NK-T cells, and monocytes (16, 17). Moreover, blood monocytes can be divided in the mouse into two subpopulations as follows: the short-lived CCR2+ Gr1+ cells and the longer lived CCR2– Gr1– cells, which have high and low levels of CX3CR1 expression, respectively (15, 17). Chemotactic signaling by CX3CR1 is G protein-dependent, whereas CX3CR1-dependent adhesion to CX3CL1-expressing cells is not, whether tested under static conditions or in the context of physiologic flow (4–6).

Studies of cx3cr1 gene knockout mice, a defective CX3CR1 allele in man, as well as CX3CR1 immunodepletion in rodents have implicated the receptor in several diseases, including crescentic glomerulonephritis in rats (18), cardiac allograft rejection in mice (19, 20), NK cell-dependent anti-tumor responses in mice (21, 22), and atherosclerosis in mouse (23, 24) and man (25, 26). These studies imply that the risk of acquiring certain immunologically mediated/inflammatory diseases may depend in part on the level of leukocyte CX3CR1 expression and that blocking or down-modulating the receptor with drugs or biologics may be an effective treatment strategy in specific settings. In this regard, several recombinant cytokines, including TGF-{beta}1 (27), IL-2, and IL-15 (28), have been reported to modulate CX3CR1 expression. Of these IL-2 and IL-15 are particularly interesting because work in mouse, both in vitro and in vivo, has indicated that they have opposite effects on CX3CR1 expression, with IL-2 increasing it and IL-15 decreasing it (28), despite the fact that both cytokines share the same receptor signaling chains, IL-2R{beta} and IL-2R{gamma}c (29). The same pattern has been observed with human peripheral blood lymphocytes and NK cells in vitro (30).2 IL-2 and IL-15 also have quite distinct immunological functions. IL-15 plays a central role in the development and maintenance of NK cells, NK-T cells, TCR{delta}+ lymphocytes, and memory T cells, as well as in the functional maturation of dendritic cells and macrophages (29, 32–34), whereas endogenous IL-2 is a key growth and death factor for antigen-activated T lymphocytes and a developmental factor for regulatory T cells (28, 35–37).

Negative regulation of CX3CR1 and potentially other chemokine receptors by IL-15 may be desirable in the setting of inflammatory disease but is undesirable in the setting of cell immunotherapy, for which IL-15 is being considered as an ex vivo NK cell growth factor for application in diseases such as cancer (33) and human immunodeficiency virus infection (38, 39). The loss of CX3CR1 and possibly other chemokine receptors by cells cultured in IL-15 (30) would cripple their ability to migrate in vivo to disease sites expressing their ligands. IL-2 has for many years been used ex vivo to expand NK cells from PBMCs or tumor infiltrating lymphocytes for adoptive immunotherapy of cancer (40), with limited success, possibly for a similar reason (41, 42).

How these {gamma}c cytokines stimulate distinct physiologic and pharmacologic responses through the same receptors is an interesting and unresolved question in immune cell signaling that could lead to new therapeutic targets (29, 32). Both cytokines are known to activate the AKT pathway, the Ras mitogen-activated protein kinase pathway, and Jak-dependent pathways leading to STAT-3 and STAT-5 transcription factor activation (29). However, they have opposing effects on the activity of nuclear factor of activated T cells (NFAT) (43), a transcription factor implicated in transactivation of numerous immunomodulatory genes in leukocytes, including the chemokine genes CXCL8 and CCL3 (44, 45). As with CX3CR1, IL-15 decreases NFAT activity, and IL-2 increases it (43), which suggested to us that these effects may be connected mechanistically. It is important to note that the NFAT family has five members (45). Most studies of NFAT1–4, the four immunologically relevant NFAT subtypes, have suggested that there is broad overlap and redundancy in their activities. NFAT1 and NFAT2 are the major subtypes expressed in T lymphocytes, and their relative abundance varies depending on the T cell subset in question. The specificity of IL-2 and IL-15 for activation of these various forms has not been investigated previously. Here we present pharmacologic, genetic, and biochemical evidence in vitro and in vivo that supports the hypothesis that IL-15 and IL-2 oppositely regulate CX3CR1 expression by differentially recruiting NFAT1 and NFAT2 specifically to an NFAT site in the CX3CR1 promoter.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human recombinant IL-2 and IL-15 were from Roche Diagnostics and PeproTech (Rocky Hill, NJ), respectively. Phycoerythrin-conjugated mAbs directed against human CD4, CD8, CD56, and CD14 and phycoerythrin-conjugated isotype-matched control mAbs were from Pharmingen. Mouse mAbs recognizing rat, mouse, and human NFAT1 and NFAT2 were from Affinity Bioreagents (Golden, CO). Rabbit polyclonal Ab specifically recognizing phosphorylated serine residues was from Abcam Inc. (Cambridge, MA). The human pro-monocytic cell line U937 and RPMI 1640 culture medium were purchased from American Type Culture Collection (Manassas, VA). Cyclosporin A (CsA) and the 16-amino acid peptide MAGPHPVIVITGPHEE, commonly known as "VIVIT," were from Calbiochem. Restriction endonucleases MluI and XhoI were obtained from Fermentas Inc. (Hanover, MD).

Plasmid Construction and Mutagenesis—A 1278-bp fragment from nt 39,247,004 to 39,248,282 (NCBI Human Genome, build 34 version 3) of chromosome 3p21 [PDB] .3 was amplified by PCR from human genomic DNA using the 5' primer 5'-CGACGCGTAGAAAACTGCTGGTGGACTTCTTCCA and the 3' primer 5'-CCTCGAGCCCACAGGTACCCAACTAGTCC. Cleavage sites for restriction enzymes MluI and XhoI were included in the primers (underlined sequences) to facilitate directional cloning into the corresponding sites of the promoterless firefly luciferase reporter plasmid pGL3-Basic (Promega, Madison, WI). The insert sequence of the resulting plasmid, named p1278-luc, was identical to the sequence found in NCBI, GenBankTM accession number AY016370 [GenBank] . This 1278-bp gene region is located 13,422 bp upstream of the start of the CX3CR1 open reading frame (ORF) and was selected for analysis because we conducted an in silico analysis of all the available 5'-expressed sequence tags and mRNA sequences, and we determined this to be the likely site of the main promoter used in leukocytes. In the course of this work, two groups published slightly different versions of the genomic organization of CX3CR1 (46, 47), both of which noted the location of a functional promoter within this 1278-bp region (Fig. 1). A library of pGL3 plasmids containing portions of the 1278-bp fragment truncated at the 5' end was created by PCR using the 5' primers 5'-CGACGCGTTAATGAAGGTGTATTGAAGGCCACA, 5'-CGACGCGTAGATATAGGGCAGGGCTTGAGGTGC, 5'-CGACGCGTCCCATGAGCTCTCCCAGCTTCCTGAA, and 5'-CGACGCGTCGCAGTCTTCCCTGAGGTTTAGATCT, paired with the same 3' primer used to clone the parent 1278-bp fragment. Each 5' primer contained a nested MluI site to facilitate cloning (underlined sequence). The resulting plasmids were named p1043-luc, p572-luc, p433-luc, and p287-luc according to the number of bp of the CX3CR1 promoter region that they contained. The 1278-bp sequence was analyzed by the TRANSFAC version 4.0 transcription factor data base (www.cbil.upenn.edu/cgi-bin/tess) for potential transcription factor binding sites. An NFAT site located between nt –298 and –291 of this gene region was scrambled (boldface/underlined sequence) using the mutagenesis primers: 5'-GCTCTCCCAGCTTCCTGAATCGCGCCCATTAATGAGAGCTGATGT and 5'-ACAGTCAGCTCATATGGGGCGCGATTCAGGAAGCTGGGAGAGC and the Quik-Change site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's instructions. The resulting mutated pGL3-luciferase plasmid was named p433*-luc.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1.
Identification of a strong constitutively active CX3CR1 promoter in human PBMCs. A, location of CX3CR1 gene region tested. The gene organization shown is based on two previous reports (46, 47). Note that exons 2–4 have been renamed relative to these two reports, because each group found one exon not found by the other. All mature transcripts reported to date consist of one of the first 4 exons spliced to exon 5. The most common transcript reported is the E3/E5 splice which is why we focused on the P2/P3 CX3CR1 genomic region. The gene regions tested in the bottom panel are indicated by the arrows and are named as indicated to the right of each arrow; the sequence landmarks are enumerated relative to the first nucleotide in the ORF, which is given number +1. P#, region reported to have promoter activity; boxes, exons; lines, introns; tsp, major transcriptional start point; shaded box, untranslated portions of exon 5. B, promoter activity of full-length and 5'-truncated portions of the 1278-bp CX3CR1 promoter region (p1278) in PBMCs and U937 cells. pGL3 plasmids with the indicated inserts upstream of the firefly luciferase gene were transfected into the cell type indicated above each panel, and constitutive luciferase activity was measured. Reporter expression is given as the relative light units (rlu) as defined under "Experimental Procedures." The results shown are pooled from three experiments each done in triplicate and are presented as the mean ± S.E.

 
Cell Transfection—A nucleofector, a cell line nucleofector kit V, and a Human T cell nucleofection kit were purchased from Amaxa Biosystems (Cologne, Germany). Heparinized whole blood from healthy human donors was separated at room temperature by centrifugation at 1600 rpm for 30 min using lymphocyte separation medium (Cellgro, Mediatech Inc., Kansas City, MO). The intermediate layer containing lymphocytes and monocytes was harvested, washed once with PBS (Cambrex Bioscience Inc., Walkersville, MD), and resuspended in an equal volume of nucleofection solution containing a proprietary supplement from the human T cell nucleofector kit. Cell line nucleofector kit V was used for transfection of the human promonocytic cell line U937. 1 x 106 U937 cells and 1 x 106 PBMCs were incubated with 1.5 µg of CX3CR1 promoter-firefly luciferase reporter plasmids or control plasmids (pGL3-luc, which contains the SV40 promoter was used as a positive control, and promoterless pGL3-Basic was used as a negative control) plus 0.25 µg of a Renilla luciferase-expressing plasmid in a total volume of 100 µl of the appropriately supplemented nucleofection solution, and were then nucleofected according to the instructions of the manufacturer. Following the procedure, the cells were immediately resuspended in 1 ml of RPMI 1640 prewarmed to 37 °C and supplemented with 15% fetal calf serum and 2 mM L-glutamine. 24 h after transfection, cell viability was examined, and the cells were incubated with various concentrations of cytokines and inhibitors at 37 °C for 12 h. All conditions were tested in triplicate.

Luciferase Assay—CX3CR1 promoter activity was measured by the dual-luciferase reporter assay system from Promega (Madison, WI), which has been designed to allow both firefly luciferase reporter and the transfection control Renilla luciferase reporter to yield linear assays with high sensitivities and no endogenous activity of either reporter in experimental host cells. Briefly, transfected cells were collected by centrifugation for 5 min at 1400 rpm, and supernatants were removed. Passive lysis buffer was added to the pellet which was lysed by passage 4 times through a 19-gauge needle. Cell lysates were cleared by a brief centrifugation at 5000 rpm, and supernatants were transferred to a fresh tube. A stabilized luminescent signal generated by firefly luciferase in 20 µl of cell lysate was detected immediately upon addition of 100 µl of luciferase assay reagent II and measured on a manual TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). After quantifying the firefly luciferase luminescence, this reaction was quenched, and the Renilla luciferase reaction was initiated upon addition of 100 µl of Stop and Glo Reagent, which produced a stabilized Renilla luciferase signal. Promoter activity is expressed as the adjusted luciferase intensity in relative light units (rlu), which is calculated as follows: firefly Luc x (Renilla Luc/Renilla Lucmax), where Luc denotes luciferase luminescence.

RNA Analysis—Total RNA was extracted from 1 x 106 human PBMCs after culture under various conditions for 12 h using the Qiagen RNeasy mini kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. Total RNA was also isolated from 0.5 x 106 mouse PBMCs and splenocytes using the same technique. Prior to reverse transcription, RNA was treated with RNase-free DNase I following the manufacturer's protocol. Total RNA (1 µg) was used for the synthesis of cDNA using the Superscript First Strand Synthesis System from Invitrogen in the presence of 40 units/µl recombinant RNasin (Promega, Madison, WI). Reverse transcription was performed for 120 min at 37 °C and stopped by heating the samples for 10 min at 72 °C. Unique primer sets for human {beta}-actin, human and mouse CX3CR1, and mouse {beta}2-microglobulin were designed, based on sequences deposited with the NCBI, and were synthesized by Invitrogen. PCR was performed using AmpliTaq Gold DNA polymerase with the GeneAmp PCR Gold buffer system (Applied Biosystems, Foster City, CA). PCR conditions for human {beta}-actin and CX3CR1 were as follows: 95 °C for 9 min, followed by 30 cycles of 1 min at 95 °C, 45 s at 57 °C, and 1.5 min at 72 °C, with a final extension at 72 °C for 10 min. PCR conditions for mouse {beta}2-microglobulin and mouse cx3cr1 were as follows: 95 °C for 9 min, followed by 35 cycles of 1 min at 95 °C, 45 s at 61 °C, and 1.5 min at 72 °C, with a final extension at 72 °C for 10 min. PCR conditions were optimized to allow for semi-quantitative comparison of the results. The plateau for amplification of human CX3CR1 mRNA was 35 cycles; however, maximum amplification of mouse cx3cr1 message was reached at 38 cycles. Expected sizes of the amplified PCR products were 500 bp for {beta}-actin, 310 bp for human CX3CR1, 220 bp for {beta}2-microglobulin, and 410 bp for mouse cx3cr1.

Chromatin Immunoprecipitation—Transcription factors regulated by IL-15 and IL-2 that interact with the CX3CR1 promoter in vivo were determined by using a kit from Active Motif (Carlsbad, CA) according to the methods recommended by the manufacturer. Stimulated PBMCs were fixed in 37% formaldehyde, disrupted by Dounce homogenization on ice in a lysis buffer containing protease inhibitors supplied by the manufacturer. Cellular DNA was sheared using a Branson Sonifier 450 sonicator (Pierce) with a 3-mm stepped microtip at 25% power, 10 pulses of 5 s each, and 30 s of rest on ice. Precleared chromatin, referred to as input DNA (500 ng), was immunoprecipitated by using 4 µg of mAbs directed against NFAT1 or NFAT2, an isotype control mouse IgG1, or 1.5 µg of polyclonal rabbit TFIIB Ab (Active Motif, Carlsbad, CA). RNA was removed from the immunoprecipitate by incubation with RNase A. DNA-binding proteins were removed by treatment with proteinase K, and the DNA was then purified by phenol/chloroform/isoamyl alcohol extraction (25:24:1; Sigma). To detect whether the 433-bp region of the CX3CR1 promoter coimmunoprecipitated with NFAT1 and NFAT2, DNA purified from immunoprecipitates was amplified by PCR using human CX3CR1 promoter-specific primers: 5'-ATGAGCTCTCCCAGCTTCCT and 5'-ATCTTTAGATGCTGCCACAGG. These primers amplify a 244-bp fragment containing the NFAT site of interest. PCR conditions were as follows: 95 °C for 3 min, followed by 30 cycles of 20 s at 94 °C, 30 s at 59 °C, and 30 s at 72 °C. The appropriate number of cycles was determined empirically by monitoring the amplification efficiency of the input and immunoprecipitated DNA at 25, 27, 30, 35, and 40 cycles. The plateau stage for amplification of immunoprecipitated DNA was 40 cycles and for the input DNA was 35 cycles. As recommended by the manufacturer, the positive control for chromatin immunoprecipitation analysis was detection of a 166-bp GAPDH promoter fragment that constitutively immunoprecipitates with TFIIB Ab. To determine the specificity of monoclonal NFAT Abs, we tested the ability of the 244-bp CX3CR1 promoter region to immunoprecipitate with mouse IgG1.

Protein Immunoprecipitations—Human PBMCs were isolated by centrifugation from freshly prepared leukopaks using lymphocyte isolation medium as described above. 107 cells were unstimulated or stimulated with 100 ng/ml IL-15 or 100 units/ml IL-2 for 12 h. As a positive control for NFAT activation, which requires serine dephosphorylation of the target protein, cells were treated for 2 h with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 10 ng/ml A23187 [GenBank] (Calbiochem). Costimulation of PBMCs with PMA and A23187 [GenBank] that lasted longer than 2 h resulted in cell death; therefore, 2-h costimulations were used as a control for stimulus-induced dephosphorylation of endogenously expressed NFATs. Following stimulation cells were collected by centrifugation at 1400 rpm for 10 min, washed twice in ice-cold TBS (pH 7.4), and lysed on ice by Dounce homogenization in lysis buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 2 mM Na3VO4, 0.1% SDS, 0.1% digitonin, 0.1% Nonidet P-40, 0.05% sodium deoxycholate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 tablet of complete protease inhibitors containing serine and cysteine protease blockers (Roche Applied Science). Cell lysates were sedimented for 5 min at 4000 rpm, supernatants transferred into fresh Eppendorf tubes, target proteins immunoprecipitated for at least 5 h with 6 µg of NFAT1 or NFAT2 mAbs or alternatively with 6 µg of mouse IgG1, which was used as an isotype control in immunoprecipitation of NFAT target proteins. The immune complexes were concentrated overnight at 4 °C by addition of 50 µl of protein G-agarose beads (Roche Applied Science). The immunoprecipitates were washed twice with ice-cold low salt wash buffer containing 10 mM Tris-HCl (pH 7.5), 0.1% Nonidet P-40, 0.05% sodium deoxycholate, and 1 tablet of complete protease inhibitors and eluted from the agarose beads by addition of 50 µl of 1x SDS-PAGE sample buffer (Bio-Rad). Immunoprecipitated proteins were separated on 8% SDS-PAGE gel, transferred onto nitrocellulose membranes, and membranes probed with NFAT1 (1:1500) or NFAT2 (1:1000) mAbs to determine the amount of immunoprecipitated target proteins. However, to investigate the amount of serine-phosphorylated NFAT1 and NFAT2, blots were stripped and re-probed with polyclonal rabbit anti-phosphoserine Ab at 1:1000 dilution.

Analysis of IL-2 and IL-15-mediated NFAT Activity in Vivo—All mice were bred and maintained under specific pathogen-free conditions, and all experiments were performed in compliance with the relevant laws and institutional guidelines that apply in the Animal Care and Use Committee protocol used in the Tuberculosis Research Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health. Seven-month-old female NFAT1–/– mice back-crossed to BALB/c for 7 generations and then mated with male NFAT1–/– (48, 49) and age-matched female NFAT1+/+ BALB/c mice were separated into 8 groups of 3 and injected in the tail vein with a single dose of recombinant human IL-15 (2 µg in 200 µl of PBS per mouse), or with recombinant human IL-2 (50,000 units in 200 µl of PBS per mouse), or with the same volume of sterile PBS. Total RNA was isolated from splenocytes, and PBMCs were isolated from animals sacrificed 24 h after injection. All tissues sampled from wild type and NFAT1–/– mice were evaluated for expression of mouse cx3cr1 by RT-PCR as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Strong Functional CX3CR1 Promoter in Human PBMCs—Sequence analysis of cloned cDNAs, 5'-RACE products, and human genomic DNA has shown that CX3CR1 is ~17 kb in length and consists of five exons and four introns on chromosome 3p21 [PDB] .3. The complete 1068-bp ORF, ~1946 bp of the 3'-untranslated region (UTR), and 9 bp of the 5'-UTR reside together on exon 5. The remaining 476 bp of the 5'-UTR reside on four other exons (Fig. 1A, top panel) which are differentially spliced to exon 5 to form five distinct mRNA forms. The major RNA species is the one composed solely of exons 3 and 5. Thus, the major transcriptional start point (tsp) is located 13,532 nt 5' from the start of the ORF (47). A potential TATA box can be found 28 bp upstream of exon 3. Two groups have analyzed the 5'-flanking region of CX3CR1 in model promoterassay systems based on expression of luciferase as a reporter and identified weak constitutive activity upstream of exon 4, moderate constitutive activity upstream of exon 1, and strong constitutive activity upstream of exon 3 (46, 47). Most interestingly, the promoter activity of the gene region upstream of exon 3 was greatly enhanced when it was extended 3' to include all of exon 3 (46). One significant limitation of these studies is that they were performed only in hematopoietic (THP-1, U87, HL-60, and Jurkat) and nonhematopoietic (HEK and HeLa) cell lines and not in primary cells. From the perspective of the present study, the effect of IL-2 and IL-15 on CX3CR1 expression in these cell lines is not known, and may not be physiologically relevant. Therefore, we attempted to develop a model system for promoter analysis of CX3CR1 in freshly isolated PBMCs from randomly selected healthy human donors.

We initially focused on a 1278-bp region of CX3CR1 (39,248,282 to 39,247,004 nt, NCBI Human Genome, build 34, version 3) which includes the 3'-most 849 bp of intron 1, all of exon 2 (97 bp), all of intron 2 (222 bp), all of exon 3 (78 bp), and 32 bp of intron 3 sequence immediately 3' relative to exon 3 (Fig. 1A, bottom panel). This fragment, named p1278, was cloned upstream of a luciferase reporter cassette and was introduced by nucleofection into primary PBMCs as described under "Experimental Procedures." Luciferase activity in PBMCs transfected with promoterless pGL3-Basic, a negative control plasmid (–), was very low and was increased by ~10–12-fold in cells transfected with pGL3-SV40, a positive control plasmid in which reporter expression was driven by the SV40 promoter (+). PBMCs transfected with p1278-luc exhibited intermediate luciferase activity ~6-fold over background and ~60% of the positive control, indicating that p1278 contains a strong CX3CR1 promoter (Fig. 1B). The relative activity was consistent in PBMCs from at least six independent healthy randomly selected donors. We next tested four fragments, named p1043, p572, p433, and p287, produced by serially truncating p1278 at the 5' end (Fig. 1A). All four fragments were highly active in the PBMC/luciferase reporter system. p572 and p1043 had activity similar to p1278, and when the fragment was further shortened to 433 bp, activity was consistently 3-fold higher, suggesting the removal of a negative element. Further truncation to 287 bp gave a fragment with activity equivalent to the positive control, double that of p1278, but ~20% reduced relative to p433 (Fig. 1B). Thus, p433 contains the strongest constitutive promoter activity of the series in PBMCs. Of note, we also tested this library of fragments in U937 cells, which most closely resemble the monocyte immunophenotype and express low levels of CX3CR1 (50), and we found that they were all active in the same rank order as in PBMCs but with markedly reduced absolute activity (Fig. 1B).

IL-15 and IL-2 Oppositely Regulate CX3CR1 Promoter Activity in Human PBMCs—We next tested whether IL-2 and IL-15 are able to modulate the constitutive activity of these five promoter regions. Freshly isolated PBMCs were transfected with the CX3CR1 promoter constructs and 24 h later were stimulated for an additional 12 h with 100 ng/ml IL-15 or 100 units/ml IL-2. Neither cytokine affected the level of luciferase activity in cells transfected with promoterless or SV40 control plasmids. In contrast, both cytokines modulated CX3CR1 promoter activity but in opposite directions (Fig. 2A). Specifically, IL-15 suppressed luciferase activity in cells transfected with any one of the five CX3CR1 promoter fragments. The suppressive effect was most prominent (3-fold) if reporter expression was driven by p433 and was the weakest for p287. In contrast, IL-2 up-regulated the reporter activity of p433, p572, p1043, and p1278 by 40–140%, respectively, but had no effect on p287 (Fig. 2A).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2.
IL-15 and IL-2 modulate CX3CR1 promoter reporter activity in human PBMCs. A–D, freshly isolated human PBMCs were transfected with the indicated luciferase reporter plasmids and 24 h later were stimulated for an additional 12 h with the indicated concentration of IL-2 or IL-15. Luciferase activity, expressed in rlu, was then measured in cell lysates. The results shown are pooled from three experiments each done in triplicate and are presented as the mean ± S.E. E, neither cytokine affects the relative proportion of CD4-, CD8-, CD14-, and CD56-positive cells in human PBMCs. Human PBMCs were stimulated for 12 h with IL-15 (100 ng/ml) or IL-2 (100 units/ml) or were unstimulated (–). Data presented are expressed as the % of total cells positive for CD4, CD8, CD56, or CD14. The data shown represent the mean ± S.E. of results from three different donors.

 
Because both cytokines rapidly and robustly altered the activity of p433, we selected this region for more detailed analysis. IL-15 treatment reduced p433 activity in a concentration-dependent manner (Fig. 2B). The threshold for inhibition was ~0.05 ng/ml, and the maximal reduction observed, 70% of base line, occurred at a concentration of 500 ng/ml (Fig. 2B). The IC50 was ~5 ng/ml. IL-2 up-regulation of p433 activity in PBMCs was also concentration-dependent, with a threshold between 0.5 and 5 units/ml. The maximal stimulation observed, 2.2-fold over unstimulated control, occurred at 1000 units/ml (Fig. 2C). The EC50 value for IL-2 in our assay was 30 units/ml. The reported EC50 for IL-2 determined by cytolytic activity of cytokine-stimulated peripheral blood lymphocytes from healthy donors is 20.2 ± 1.3 units/ml (51). As presented in Fig. 2D, the highest tested concentrations of IL-15 (500 ng/ml) and IL-2 (1000 units) had no effect on reporter activity in cells transfected with positive or negative control plasmids. Based on these results, we stimulated cells with 100 ng/ml of IL-15 or 100 units/ml of IL-2 for 12 h in all of the experiments that follow. Because both cytokines are known to induce proliferation of primary lymphocytes (32), we also checked whether the alterations in luciferase activity mediated by IL-15 and IL-2 were associated with differential changes in the distribution of cell subsets relative to the initial population of PBMCs. As shown in Fig. 2E, our stimulation protocols with IL-2 and IL-15 resulted in the same distribution of cell subsets that express CX3CR1, namely CD4-, CD8-, CD14-, and CD56-positive cells.

NFAT-directed Inhibitors Prevent IL-15- and IL-2-mediated Modulation of CX3CR1 Expression—We next investigated the signaling mechanisms responsible for the effects of IL-2 and IL-15 on CX3CR1 promoter activity. We hypothesized that the transcription factor NFAT may play a role because IL-15 and IL-2 have been reported to oppositely regulate NFAT in the human T cell line Jurkat (43). Moreover, we noted by computational analysis using the TRANSFAC version 4.0 transcriptional factor data base that p1278 has seven NFAT recognition sequences, all of them located within 750 bp upstream of the tsp. Of these only two fall within p433, at positions –64 and –298 relative to the tsp (Fig. 3). The putative –64 NFAT site, which was originally noted by Garin et al. (46), appears to be a core sequence (AGGAAAC) present in promoters of several immunoregulatory genes critically regulated by NFAT. It is the only one of the seven predicted NFAT sites in p1278 that falls within p287, which is the only fragment of p1278 that was insensitive to IL-2. This fragment also had little sensitivity to IL-15 (Fig. 2A). Thus, this site by itself was not a good candidate to mediate the effects of IL-2 and IL-15, and we decided to focus on the –298 NFAT site (AGGCTCCC), which has not been reported previously, and is the only NFAT site present in p433 but not in p287. This site can be described as a quasipalindromic {kappa}B-like element (45). Most interestingly, it is flanked by c-Jun/AP-1-like sequences 14 nucleotides upstream (TGAGCTCC) and 15 nucleotides downstream (TGCTGTGC), which are known to bind transcription factors able to interact with NFAT at functional NFAT sites (45).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 3.
The CX3CR1 promoter contains seven potential NFAT-binding sites. The p1278 sequence is shown with sites predicted to bind NFAT transcription factors. Numbering on the left corresponds to the positioning of the 1278-bp CX3CR1 region relative to the first nucleotide of the start codon. Potential NFAT, c-Jun, c-Fos, and AP-1 sites are numbered in parentheses relative to their position to the major tsp. AP-1/c-Jun sites are located flanking the –298 {kappa}B-like NFAT site. Potential AP-1, c-Jun, and c-Fos recognition sequences located upstream of the –631 {kappa}B-like NFAT site are also shown. Sites were identified by screening the TRANSFAC version 4.0 transcription factor data base (www.cbil.upenn.edu/cgi-bin/tess).

 
We first tested the putative functional role of NFAT pharmacologically, by culturing p433-transfected human PBMCs for 12 h in the presence of IL-15 (100 ng/ml) or IL-2 (100 units/ml) plus increasing concentrations of the NFAT inhibitors CsA and VIVIT. Neither CsA, which prevents activation of NFAT by inhibiting activity of the upstream Ca2+-dependent phosphatase calcineurin (52), nor VIVIT, which prevents activation of NFAT by hindering its physical association with calcineurin (53), altered the constitutive activity of positive and negative control plasmids or p433 at any dose tested (Fig. 4, A and C). In contrast, CsA in a dose-dependent fashion markedly reversed both the inhibitory effect of IL-15 and the stimulatory effect of IL-2 on p433 activity (Fig. 4, B and D). VIVIT was slightly less efficient than CsA in reversing IL-15 action (Fig. 4B) but was as effective as CsA in reversing IL-2 action (Fig. 4D).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 4.
NFAT-directed inhibitors CsA and VIVIT block IL-15 and IL-2 regulation of CX3CR1 minigene expression. Human PBMCs were transfected with the CX3CR1 p433-luc plasmid construct, and promoterless- or SV40-luc promoter reporter plasmids as indicated for 24 h and then were cultured with the indicated cytokines and/or NFAT inhibitors for 12 h. Luciferase activity, expressed in rlu, was then measured in cell lysates. The results shown are pooled from three experiments each done in triplicate and are presented as the mean ± S.E. IL-15 analysis, panels A and B; IL-2 analysis, panels C and D. Analysis of IL-2 and IL-15 used separate sets of donors, three donors for each analysis.

 
We next tested whether these NFAT-directed inhibitors could also reverse the effects of IL-2 and IL-15 on endogenous CX3CR1 gene expression in human PBMCs. Consistent with the p433-reporter system results, IL-15 decreased constitutive CX3CR1 expression by a factor of 3.4 (Fig. 5, A and B), whereas IL-2 up-regulated receptor mRNA 1.7-fold (Fig. 5, C and D). Consistent with the p433 experiment in Fig. 4, neither CsA nor VIVIT affected constitutive expression of endogenous CX3CR1 in PBMCs, suggesting that constitutive expression of CX3CR1 is NFAT-independent. Neither inhibitor was able to induce CX3CR1 expression in polymorphonuclear leukocytes where it is normally silent (Fig. 5). The mRNA levels of the housekeeping gene {beta}-actin were unaffected by cytokine and inhibitor exposure whether alone or in combination (Fig. 5, A and C). In striking contrast to these controls, both inhibitors, at concentrations that were maximally effective in the p433/PBMC model system, were able to prevent completely IL-15 inhibition and IL-2 stimulation of endogenous CX3CR1 expression in PBMCs (Fig. 5).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 5.
IL-15 and IL-2 reciprocally regulate expression of endogenous CX3CR1 in human PBMCs in an NFAT-dependent manner, pharmacologic analysis. A and C, human PBMCs were cultured for 12 h in the presence or absence of CsA (400 µM), VIVIT (400 µM), IL-15 (100 ng/ml), and IL-2 (100 units/ml), as indicated. Total RNA was isolated, and the expression of human CX3CR1 was determined by RT-PCR using primers specific for a 310-bp portion of the ORF (upper panel). Loading was assessed by amplification of a 500-bp portion of the {beta}-actin gene (lower panel). Each panel displays one experiment representative of three that were performed. Markers, 100-bp ladder. B and D, densitometry analysis of experiments illustrated in A and C, respectively. CX3CR1 PCR signals were quantitated from each experiment, adjusting for {beta}-actin. The results shown are pooled from three experiments using PBMCs from three independent donors, each of which was tested with IL-2 and IL-15, and are presented as the mean ± S.E.

 
The –298 NFAT Site Regulates CX3CR1 Promoter Activity— To evaluate these pharmacological results at the molecular level, we scrambled the –298 NFAT site in p433 (Fig. 6A). The resulting fragment, named p433*, retained constitutive promoter activity in PBMCs at a level equivalent to wild type p433. In contrast, the ability of IL-15 to suppress p433* activity was markedly decreased relative to its effects on wild type p433 activity (23 versus 77% inhibition, respectively) (Fig. 6B). Likewise, IL-2-stimulated reporter activity was decreased by 65% in cells expressing p433* compared with cells expressing wild type p433 (Fig. 6B).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 6.
IL-15 and IL-2 reciprocally regulate CX3CR1 promoter activity in an NFAT-dependent manner, genetic analysis. A, {kappa}B-like NFAT site mutagenesis. The p433 CX3CR1 promoter region was mutated at its predicted –298 {kappa}B-like NFAT-binding site as illustrated and was named p433*. E3 represents the third exon, and tsp determines positioning of the major transcriptional start point in human CX3CR1. B, functional analysis of p433*. Human PBMCs transfected with either wild type p433-luc or p433*-luc reporter plasmids were cultured for 12 h in the presence or absence of IL-15 (100 ng/ml) or IL-2 (100 units/ml). Luciferase activity was then measured in cell lysates. Data are presented as the mean ± S.E. rlu from three independent experiments carried out in triplicate using three independent donors.

 
NFAT1 and NFAT2 Differentially Interact with the CX3CR1 Promoter in Intact Human PBMCs—Although the data presented so far strengthen the hypothesis that IL-2 and IL-15 regulate CX3CR1 promoter activity in an NFAT-dependent fashion, they do not directly demonstrate NFAT interaction with the endogenous CX3CR1 –298-NFAT binding site nor do they explain how IL-2 and IL-15 exert opposite effects on CX3CR1 promoter activity. To address these issues we performed chromatin immunoprecipitation assays by using NFAT1 and NFAT2 mAbs and human CX3CR1 promoter-specific primers, which were designed to amplify a genomic region –74 to –318 relative to the tsp, on human PBMCs cultured in the presence or absence of IL-2 or IL-15. The 244-bp target fragment did not coimmunoprecipitate with NFAT1 in unstimulated PBMCs (Fig. 7, A and B). However, in IL-15-stimulated PBMCs NFAT1 efficiently coimmunoprecipitated with the target in a time-dependent fashion (Fig. 7, A, top panel, and B, black bars). In contrast, IL-15 very inefficiently recruited NFAT2 to the same target DNA, such that a very weak PCR signal could only be detected after 12 h of stimulation (Fig. 7, A, middle panel, and B, white bars). In contrast to IL-15, IL-2 induced very poor binding of NFAT1 to this genomic region (Fig. 7, C, top panel, and D, black bars). Instead, IL-2 very efficiently and rapidly induced a complex with NFAT2. Results of PCR amplification presented in Fig. 7C (middle panel) show that the target region of the CX3CR1 promoter was not detectable in NFAT2 immunoprecipitates from unstimulated PBMCs, whereas maximum complex formation occurred 8 h after cytokine addition and persisted for at least 12 h (Fig. 7, C, middle panel, and D, white bars). Binding of NFAT1 and NFAT2 with the endogenous proximal CX3CR1 promoter was specific because no PCR product was obtained when isotype control (mouse IgG1) (Fig. 7, A and C, bottom panels) or control TFIIB mAb were used in immunoprecipitations (Fig. 7E, top panel) or, alternatively, when chromatin immunoprecipitated using NFAT2 mAb (Fig. 7E, middle panel) or NFAT1 mAb (Fig. 7E, bottom panel) was amplified with primers specific for unrelated NFAT-independent genes, such as GAPDH (Fig. 7E). The absence of NFAT proteins on the target DNA in unstimulated PBMCs (Fig. 7, A–D) is consistent with the lack of an effect of CsA and VIVIT on constitutive CX3CR1 expression in leukocytes (Fig. 5).




View larger version (121K):
[in this window]
[in a new window]
  		FIG. 7.
IL-2 and IL-15 differentially regulate NFAT1 and NFAT2 binding to the CX3CR1 promoter in intact PBMCs. A and C, chromatin immunoprecipitation of NFAT1 and NFAT2. Human PBMCs were cultured for the indicated time in the presence of IL-15 (100 ng/ml) or IL-2 (100 units/ml) in A and C, respectively. Cells were then analyzed by chromatin immunoprecipitation (IP) using 4 µg of mAbs to mouse NFAT1 (top panels) or NFAT2 (middle panels) or with an equivalent amount of isotype matched control Ab, mouse IgG1 (bottom panels). Immunoprecipitated DNA was analyzed by PCR using human CX3CR1 promoter-specific primers that amplify a 244-bp region containing the {kappa}B-like NFAT site and no others. Each panel displays one experiment representative of 3–4 that were performed. Input DNA represents DNA purified from chromatin that has not been immunoprecipitated. M, 100-bp ladder markers. B and D, densitometry analysis of experiments illustrated in A and C, respectively. The results shown are pooled from 3 to 4 experiments using PBMCs from 3 to 4 independent donors and are presented as the mean ± S.E. E, NFAT1 and NFAT2 mAb specificity controls. The TATA box region of the promoter of the GAPDH gene was detected by PCR in chromatin immunoprecipitated by TFIIB mAb from human PBMCs, but the amount was not altered by treatment of the cells with either IL-15 or IL-2 (top two panels). The TATA-box containing GAPDH promoter was not detected by PCR in immunoprecipitates using NFAT1 or NFAT2 mAb from cells treated with either IL-2 or IL-15 (bottom four panels). Results shown in E are representative of two separate experiments.

 
Binding of NFATs to DNA target sequences implies that the proteins have been activated by dephosphorylation of serine residues in the regulatory domain (45). To address this directly in the case of IL-2- and IL-15-mediated activation of NFATs, we immunoprecipitated NFAT1 and NFAT2 from unstimulated and cytokine-stimulated PBMCs, and we determined the amounts of phosphorylated serines in target proteins. As shown in Fig. 8, IL-15 stimulation decreased steady state NFAT1 serine phosphorylation by 58%, and the effect of IL-15 was comparable with the change in NFAT1 phosphorylation status induced by the positive control, PMA plus A23187 [GenBank] . Consistent with the chromatin immunoprecipitation results, IL-2 stimulated a weaker reduction (~ 20%) in NFAT1 serine phosphorylation. Cytokine stimulations and PMA plus A23187 [GenBank] costimulations up-regulated NFAT2 expression 3.2-fold compared with levels of NFAT2 expressed in resting PBMCs (Fig. 8C). Moreover, both cytokines reduced the total amount of steady state serine-phosphorylated NFAT2 compared with the amount of serine-phosphorylated NFAT2 in unstimulated PBMCs (Fig. 8D). However, IL-2 decreased serine phosphorylation of NFAT2 by 80% which was equivalent to the impact of PMA and A23187 [GenBank] costimulation on NFAT2 dephosphorylation, whereas IL-15 reduced serine phosphorylation of NFAT2 to a lesser extent, ~ 53% respectively (Fig. 8, C and D). This differential activity is consistent with the differential chromatin immunoprecipitation results described previously.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 8.
IL-15 and IL-2 preferentially activate NFAT1 and NFAT2, respectively. A and C, NFAT1 and NFAT2 were immunoprecipitated from 107 human PBMCs that were either unstimulated (2nd lane) or treated for 12 h with 100 ng/ml of IL-15 (3rd lane) or 100 units/ml of IL-2 (4th lane) or, alternatively, were stimulated for 2 h with 10 ng/ml of PMA + 10 ng/ml of A23187 [GenBank] (5th lane), and the amounts of total NFAT1 and NFAT2 (top panels) and serine-phosphorylated NFAT1 or NFAT2 (P-Ser NFAT1/P-Ser NFAT2; bottom panels) were determined by sequential Western blot (WB) analysis with NFAT1, NFAT2, and phosphoserine-directed Abs as indicated. Immunoprecipitation (IP) of proteins by mouse IgG1 (1st lane) was used as a negative (isotype Ab) control in immunoprecipitation of NFAT target proteins. B and D, quantitation of the results shown in A and C, respectively. Densitometry was performed on each Ser-P (P-Ser) NFAT1/NFAT2 protein band adjusting each for the corresponding NFAT1/NFAT2 signal.

 
IL-15-induced Down-regulation of CX3CR1 Is NFAT1-dependent in Vivo—The proximal 325 bp of the CX3CR1 promoter that associates with NFAT1 and NFAT2 shows 57% sequence identity with the corresponding region of the mouse genome. Furthermore, this region in the mouse contains two putative {kappa}B-like NFAT sites that are positioned 60 and 278 nucleotides upstream of the predicted tsp (Fig. 9). Therefore, we sought to confirm that NFATs are differential transcription factors in the regulation of CX3CR1 expression in vivo by examining IL-15- and IL-2-mediated regulation of mouse cx3cr1 in NFAT1–/– mice. NFAT1–/– and NFAT1+/+ BALB/c mice were injected with a single dose of human IL-15 (2 µg per mouse) or IL-2 (50,000 units per mouse), and expression of mouse CX3CR1 mRNA was analyzed in PBMCs and splenocytes 24 h later. We were able to detect equivalent amounts of CX3CR1 and {beta}2-microglobulin mRNA in spleen and PBMCs from noninjected control NFAT1–/– and NFAT1+/+ BALB/c mice (Fig. 10, top panels). The exogenous {gamma}c cytokines did not alter {beta}2-microglobulin expression; however, IL-15 abolished and IL-2 markedly increased CX3CR1 mRNA in both PBMCs and splenocytes (Fig. 10) from NFAT1+/+ BALB/c mice, which is consistent with our previous report (28). In comparison, IL-15 was less effective in down-regulating cx3cr1 expression in both PBMCs (100 versus 63% compared with PBS-injected mice) and splenocytes (62 versus 24% compared with PBS-injected animals) from NFAT1–/– mice (Fig. 10). Consistent with the hypothesis that IL-2 stimulation of cx3cr1 expression is dependent on NFAT2 and not NFAT1, as suggested by our results using human PBMCs, we did not observe any difference in IL-2-stimulated CX3CR1 mRNA expression in either PBMCs or splenocytes between wild type and NFAT1–/– mice injected with IL-2 (Fig. 10). NFAT2–/– mice die during embryogenesis and therefore cannot be used to test our hypothesis further in vivo (54).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 9.
Sequence alignment of the proximal CX3CR1 promoter region in human and mouse. Alignment of sequence upstream of the tsp in human CX3CR1 with the corresponding mouse genomic sequence (mouse chromosome 9 genomic contig, GenBankTM accession number NT039482.2) is shown with sites predicted to bind NFAT transcription factors. Numbering on the left corresponds to the position relative to the tsp in human and mouse. Potential NFAT sites are numbered relative to their position to the tsp. Potential NFAT recognition sequences were identified by screening the TRANSFAC version 4.0 transcription factor data base (www.cbil.upenn.edu/cgi-bin/tess), and the alignment was conducted by using the GCG LITE Gap-Global Alignment of Two Sequences program (molbio.info.nih.gov/molbio/gcglite). Dots indicate gaps inserted by the program to achieve the optimal alignment, and vertical lines indicate identical bases in both sequences.

 



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 10.
IL-15 modulates mouse CX3CR1 expression in an NFAT1-dependent manner in vivo. A and C, RNA from PBMCs and splenocytes, harvested 24 h after intravenous injection of NFAT1–/– or NFAT1+/+ BALB/c mice with 2 µg of IL-15 or 50,000 units of IL-2 per mouse, was analyzed by RT-PCR using mouse cx3cr1 or {beta}2-microglobulin ORF-specific primers (top and bottom panels, respectively). Each lane in A represents PBMC PCR products from a different mouse injected with the substance indicated at the top. C, the splenocyte PCR products are from the same mice as in A arranged in the same order. B and D, quantitation of the results shown in A and C, respectively. Densitometry was performed on each CX3CR1 PCR product, adjusting each for the corresponding {beta}2-microglobulin signal. The results are presented as the mean ± S.E. of values from three mice per condition. Sk. mus, skeletal muscle RNA was used as a negative control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that the {gamma}c cytokines IL-15 and IL-2 oppositely regulate expression of the chemokine receptor CX3CR1 in primary leukocytes through a related transcriptional mechanism involving the major immunoregulatory transcription factor family NFAT. This work provides novel insight into the biochemical mechanism underlying our unexpected observation in the mouse (28), confirmed by ourselves and others in human (30),2 that IL-2 increases, whereas IL-15 silences, constitutive CX3CR1 expression.

Implication of NFAT in this pathway rests on the following evidence drawn from a series of pharmacologic, biochemical, and genetic experiments. First, the 1168-bp region upstream of the main human CX3CR1 tsp contains a functional promoter and seven predicted NFAT-binding sites. This is the first demonstration that this region functions in primary leukocytes as a promoter, which extends results for this region by using model promoter assays in U937, THP-1, and Jurkat cell lines (46, 47). We have also identified for the first time what we have named the –298 NFAT site, which is located in the proximal CX3CR1 promoter. Second, two pharmacologic inhibitors of NFAT, CsA, which blocks the essential NFAT activator calcineurin (52), and VIVIT, a targeted NFAT inhibitor (53), both inhibited IL-2- and IL-15-regulated expression of both endogenous CX3CR1 and CX3CR1 promoter-luciferase minigene reporter activity in human PBMCs. Third, direct mutagenesis of the predicted –298 NFAT site in the CX3CR1 promoter markedly reduced the ability of IL-2 and IL-15 to regulate CX3CR1 promoterreporter activity in human PBMCs. Thus, NFAT appears capable of both activating and silencing the same gene, depending on the cell stimulus.

Further mechanistic insight into this paradox came from chromatin immunoprecipitation experiments in which we found that stimulation of human PBMCs with IL-2 and IL-15 caused selective physical recruitment of NFAT1 and NFAT2, respectively, to the proximal promoter region containing the –298 NFAT site. DNA-binding sites for NFAT are classified into two major groups, classic NFAT sites and {kappa}B-like sites (45). The –298 NFAT site we have analyzed is a {kappa}B-like site. Such sites have been found in promoters and enhancers for many chemokine and cytokine genes (45). The {kappa}3 element in the TNF-{alpha} promoter, which behaves as a strong CsA-sensitive element in stimulated T cells, is the best studied example of a functional nonpalindromic {kappa}B-like NFAT site (45, 55–57). Consistent with our CX3CR1 chromatin immunoprecipitation results, electromobility shift analysis has shown that NFAT1 binds with a much higher affinity than NFAT2 to this site (55–57). With regard to function, IL-2 and IL-15 have also been reported to increase and decrease NFAT activity, respectively, in the human T cell line Jurkat; however, the role of specific NFAT subtypes has not been defined (43).

Taken together these results suggest a model in which negative regulation of CX3CR1 expression by IL-15 is mediated by NFAT1, whereas positive regulation of the same gene by IL-2 is mediated by NFAT2. We obtained direct evidence for this model and for its relevance in vivo from experiments in the mouse showing that intravenous IL-15 injection was able to suppress CX3CR1 mRNA in both PBMCs and splenocytes from NFAT1+/+ BALB/c mice but not in cells from NFAT1–/– mice. Moreover, consistent with our chromatin immunoprecipitation results showing selectivity of IL-2 for recruitment of NFAT2 over NFAT1, IL-2 injection up-regulated CX3CR1 to a similar level in leukocytes from NFAT1–/– mice and wild type BALB/c animals. It is important to note that mice unconditionally deficient in NFAT2 due to gene knockout are not viable in vivo (54) so that other approaches will have to be developed to test the role of this protein in IL-2 and IL-15 regulation of CX3CR1 in vivo. It is also important to note that additional mechanisms, possibly including other NFAT subtypes, evidently cooperate with NFAT to modulate constitutive CX3CR1 expression when leukocytes are stimulated with exogenous IL-2 and IL-15 because neither pharmacologic inhibitors, nor scrambling the –298 NFAT site, nor in vivo NFAT1 gene disruption succeeded in completely reversing the effects of IL-2 or IL-15. We have not yet defined a functional NFAT1-binding site in mouse cx3cr1 as we have in human; however, the mouse and human genes contain at least two stretches of conserved nonrepetitive, noncoding sequence upstream of the ORF, one of which is located in the human CX3CR1 proximal promoter we tested in vitro (47). This homologous genomic region in mouse also contains two potential NFAT {kappa}B-like sites located at positions –60 and –278 relative to the predicted tsp.

Our model departs from the classic NFAT signaling pathway, which is initiated by antigen receptor, Fc receptor, or G protein-coupled receptor triggering of sustained elevation of intracellular calcium ion in leukocytes, a critical step toward activation of calcineurin through a calmodulin-dependent mechanism. In the classic pathway, calcineurin promotes NFAT serine dephosphorylation, exposure of the nuclear translocation sequence, nuclear import, and activation (45, 58). Cytokines do not classically induce sustained calcium flux, although IL-2 and IL-15 have been reported to induce transient calcium flux (59–62), and we have confirmed this (data not shown). We have also observed that IL-15 stimulation results in preferential dephosphorylation of serines in NFAT1, whereas IL-2 treatment induces prominent NFAT2 dephosphorylation. Because both cytokines reduce both serine phosphorylation of NFAT1 and NFAT2 and localization of these proteins with chromatin, we conclude that they activate NFATs, albeit to different degrees. Whether IL-2 and IL-15 can also activate NFAT by a calcium-independent mechanism is an important issue for future study.

NFATs are involved in transcriptional activation of numerous homeostatic and immunomodulatory cytokines including IL-1 to IL-6, IL-9 to IL-13, granulocyte-macrophage colony-stimulating factor, interferon-{alpha}, -{beta}, and -{gamma}, TNF-{alpha}, TGF-{beta}, CXCL8, and CCL3, as well as apoptotic factors (Fas, Fas ligand, Bcl-x, and Bcl-2), and surface receptors such as CD40 ligand, CD69, CTLA-4, and IL-2R{alpha} (45). However, there are also reports of possible negative regulation, for example in the case of IL-13 (63), TGF-{beta} (64), cyclin-dependent kinase 4 (CDK4) expression (65), and now CX3CR1. In addition, a recent microarray analysis of CsA-treated T cells has revealed sets of both up-regulated and down-regulated genes (66, 67), suggesting that NFAT may inhibit the expression of certain genes; however, known calcineurin-independent side effects of CsA preclude strong conclusions from these results. Most analysis of NFAT function has been restricted to inducible genes (45, 58). In contrast, CX3CR1 is constitutively expressed on subsets of resting leukocytes, as described in the Introduction.

Classically, NFAT subtypes have been thought to have redundant functions (58). However, experiments with knockout mice have shown that this is an overly simplistic model. NFAT2–/– mice die in utero because of their inability to form cardiac valves and septum (54), but NFAT2–/– T cells, when evaluated by RAG-2–/– blastocyst complementation, show only mildly impaired proliferation and a selective decrease in IL-4 production (68). In contrast, mice lacking NFAT1 develop normally and are immunocompetent with normal production of IL-2, IL-4, TNF-{alpha}, and interferon-{gamma} by stimulated spleen cells but have moderate splenomegaly with relatively normal splenic architecture and distribution of splenocyte subsets (69). Moreover, when the total number of splenic mononuclear cells was estimated, no difference in numbers of CD8+, CD4+, CD3+, and CD11b+ cells were observed between 6- and 14-week-old BALB/c and NFAT1–/– mice or in the leukocyte immunophenotypes present in spleen and peripheral blood between 6- and 8-week-old BALB/c and NFAT1–/– animals. However, NFAT1–/– mice do have increased intrapleural eosinophils and serum IgE in a model of allergic inflammation and increased susceptibility to Leishmania major, with increased production of IL-4 and other Th2-type cytokines in both challenges (69, 70). These results suggest that under certain conditions NFAT1 and NFAT2 may suppress and activate IL-4 gene transcription, respectively.

Additional work will be needed to explain the specificity of IL-2 for NFAT2 and IL-15 for NFAT1, and whether these factors are directly responsible for activation and suppression of CX3CR1. Of note, these NFAT subtypes differ dramatically in distribution among leukocyte subsets and in expression level. NFAT1 is expressed in thymus, spleen, T cells, B cells, NK cells, eosinophils, monocytes, and macrophages and represents 80–90% of total NFAT in resting immune cells (45, 58); NFAT2 is expressed less widely and, at least in some cell types (e.g. human NK cells), is not expressed unless cells are stimulated with immune complexes or undergo ligand-induced activation (31).

The reported differences in affinity between NFAT1 and NFAT2 for {kappa}B-like NFAT sites could have important effects on their ability to form specific transcription complexes. Assembly of transcription complexes on promoters and enhancers can occur as simple independent binding of different proteins to adjacent binding sites or at composite DNA elements, such as the ternary complexes of NFAT with AP-1, GATA, and ATF-2/Jun (45). The human TNF-{alpha} promoter {kappa}3 element is adjacent to an ATF-2/Jun site that binds dimers required for maximal function of the {kappa}3 site in reporter assays, although no stabilizing protein-protein interactions between NFAT1 and ATF-2/Jun bound to the contiguous sites have been described (55, 56). Additional work will be needed to define whether such complexes are differentially assembled at the CX3CR1 –298 NFAT site by NFAT1 and NFAT2. This site is flanked by a potential ATF-2/Jun at position –320 and at –278 by a potential AP-1 element.

To date our experiments have focused on the effects of exogenous recombinant human IL-2 and IL-15. The NFAT-dependent effects we have described suggest that prolonged exposure to IL-15 will cripple immune cell products in their ability to traffic in vivo to disease sites where the CX3CR1 ligand CX3CL1/fractalkine makes up a high percentage of the total resident chemoattractant activity. Before our initial report on IL-2 and IL-15 regulation of CX3CR1, Hanna and co-workers (30) have published a survey in which IL-15 was found to markedly down-regulate surface expression of multiple chemokine receptors on human peripheral blood CD16– NK cells, including CXCR1, CXCR2, CCR1, CCR2, CCR3, CCR5, CCR8, and CX3CR1. Thus, the NFAT-dependent mechanism we have identified may be more generally relevant to chemokine receptors and could conceivably modulate expression of other classes of immunoregulatory genes.

In conclusion, our data are consistent with a model in which NFAT1 and NFAT2 proteins are differentially regulated in primary leukocytes by IL-2 and IL-15, respectively, and bind to the same –298 NFAT DNA element in the CX3CR1 promoter, whereupon NFAT2 acts to increase and NFAT1 to reduce CX3CR1 promoter activity and gene transcription. These findings are clinically relevant in the setting of adoptive immunotherapy of diseases such as cancer, where recombinant IL-2 is used and recombinant IL-15 is being considered for use to expand killer cells from PBMCs or tumor-infiltrating lymphocytes (33, 38, 42). The findings may also be relevant during the normal immune response, in which the balance of NFAT1 and NFAT2 in cells could fine-tune expression of CX3CR1, and potentially other immunoregulatory genes, to promote appropriate cellular immune responses while avoiding pathologic overreactions. Finally, our data raise interesting new biochemical questions for future research regarding how IL-2 and IL-15 are able to differentially activate NFAT1 and NFAT2, and how these NFATs, acting at a common promoter binding site, are able to have opposite effects on transcriptional activity of the same gene.


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

To whom correspondence should be addressed: Bldg. 10, Rm. 11N113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8616; Fax: 301-402-4369; E-mail: pmm{at}nih.gov.

1 The abbreviations used are: TNF-{alpha}, tumor necrosis factor-{alpha}; IL, interleukin; PBMCs, peripheral blood mononuclear cells; NFAT, nuclear factor of activated T cells; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcription; ORF, open reading frame; Ab, antibody; mAb, monoclonal Ab; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; TGF, transforming growth factor; nt, nucleotide; CsA, cyclosporin A; rlu, relative light units; PBS, phosphate-buffered saline. Back

2 Sechler, J. M., Barlic, J., Grivel, J.-C., and Murphy, P. M. (2005) Cell. Immunol., in press. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, J. A., Vath, J., Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrez-Ramos, J. C., and Gearing, D. (1997) Nature 387, 611–617[CrossRef][Medline] [Order article via Infotrieve]
  2. Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. R., Zlotnik, A., and Schall, T. J. (1997) Nature 385, 640–644[CrossRef][Medline] [Order article via Infotrieve]
  3. Ancuta, P., Rao, R., Moses, A., Mehle, A., Shaw, S. K., Luscinskas, F. W., and Gabuzda, D. (2003) J. Exp. Med. 197, 1701–1707[Abstract/Free Full Text]
  4. Fong, A. M., Robinson, L. A., Steeber, D. A., Tedder, T. F., Yoshie, O., Imai, T., and Patel, D. D. (1998) J. Exp. Med. 188, 1413–1419[Abstract/Free Full Text]
  5. Haskell, C. A., Cleary, M. D., and Charo, I. F. (1999) J. Biol. Chem. 274, 10053–10058[Abstract/Free Full Text]
  6. Haskell, C. A., Cleary, M. D., and Charo, I. F. (2000) J. Biol. Chem. 275, 34183–34189[Abstract/Free Full Text]
  7. Imaizumi, T., Matsumiya, T., Fujimoto, K., Okamoto, K., Cui, X., Ohtaki, U., Yoshida, H., and Satoh, K. (2000) Tohoku J. Exp. Med. 192, 127–139[CrossRef][Medline] [Order article via Infotrieve]
  8. Yoshida, H., Imaizumi, T., Fujimoto, K., Matsuo, N., Kimura, K., Cui, X., Matsumiya, T., Tanji, K., Shibata, T., Tamo, W., Kumagai, M., and Satoh, K. (2001) Neurosci. Lett. 303, 132–136[CrossRef][Medline] [Order article via Infotrieve]
  9. Tsou, C. L., Haskell, C. A., and Charo, I. F. (2001) J. Biol. Chem. 276, 44622–44626[Abstract/Free Full Text]
  10. Garton, K. J., Gough, P. J., Blobel, C. P., Murphy, G., Greaves, D. R., Dempsey, P. J., and Raines, E. W. (2001) J. Biol. Chem. 276, 37993–38001[Abstract/Free Full Text]
  11. Hundhausen, C., Misztela, D., Berkhout, T. A., Broadway, N., Saftig, P., Reiss, K., Hartmann, D., Fahrenholz, F., Postina, R., Matthews, V., Kallen, K. J., Rose-John, S., and Ludwig, A. (2003) Blood 102, 1186–1195[Abstract/Free Full Text]
  12. Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T. J., and Yoshie, O. (1997) Cell 91, 521–530[CrossRef][Medline] [Order article via Infotrieve]
  13. Nishimura, M., Umehara, H., Nakayama, T., Yoneda, O., Hieshima, K., Kakizaki, M., Dohmae, N., Yoshie, O., and Imai, T. (2002) J. Immunol. 168, 6173–6180[Abstract/Free Full Text]
  14. Campbell, J. J., Qin, S., Unutmaz, D., Soler, D., Murphy, K. E., Hodge, M. R., Wu, L., and Butcher, E. C. (2001) J. Immunol. 166, 6477–6482[Abstract/Free Full Text]
  15. Geissmann, F., Jung, S., and Littman, D. R. (2003) Immunity 19, 71–82[CrossRef][Medline] [Order article via Infotrieve]
  16. Combadiere, C., Gao, J., Tiffany, H. L., and Murphy, P. M. (1998) Biochem. Biophys. Res. Commun. 253, 728–732[CrossRef][Medline] [Order article via Infotrieve]
  17. Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A., and Littman, D. R. (2000) Mol. Cell. Biol. 20, 4106–4114[Abstract/Free Full Text]
  18. Feng, L., Chen, S., Garcia, G. E., Xia, Y., Siani, M. A., Botti, P., Wilson, C. B., Harrison, J. K., and Bacon, K. B. (1999) Kidney Int. 56, 612–620[CrossRef][Medline] [Order article via Infotrieve]
  19. Haskell, C. A., Ribeiro, S., and Horuk, R. (2002) Curr. Opin. Investig. Drugs 3, 399–405[Medline] [Order article via Infotrieve]
  20. Haskell, C. A., Hancock, W. W., Salant, D. J., Gao, W., Csizmadia, V., Peters, W., Faia, K., Fituri, O., Rottman, J. B., and Charo, I. F. (2001) J. Clin. Investig. 108, 679–688[CrossRef][Medline] [Order article via Infotrieve]
  21. Robinson, L. A., Nataraj, C., Thomas, D. W., Cosby, J. M., Griffiths, R., Bautch, V. L., Patel, D. D., and Coffman, T. M. (2003) Cell. Immunol. 225, 122–130[CrossRef][Medline] [Order article via Infotrieve]
  22. Lavergne, E., Combadiere, B., Bonduelle, O., Iga, M., Gao, J. L., Maho, M., Boissonnas, A., Murphy, P. M., Debre, P., and Combadiere, C. (2003) Cancer Res. 63, 7468–7474[Abstract/Free Full Text]
  23. Combadiere, C., Potteaux, S., Gao, J. L., Esposito, B., Casanova, S., Lee, E. J., Debre, P., Tedgui, A., Murphy, P. M., and Mallat, Z. (2003) Circulation 107, 1009–1016[Abstract/Free Full Text]
  24. Lesnik, P., Haskell, C. A., and Charo, I. F. (2003) J. Clin. Investig. 111, 333–340[CrossRef][Medline] [Order article via Infotrieve]
  25. McDermott, D. H., Halcox, J. P., Schenke, W. H., Waclawiw, M. A., Merrell, M. N., Epstein, N., Quyyumi, A. A., and Murphy, P. M. (2001) Circ. Res. 89, 401–407[Abstract/Free Full Text]
  26. McDermott, D. H., Fong, A. M., Yang, Q., Sechler, J. M., Cupples, L. A., Merrell, M. N., Wilson, P. W., D'Agostino, R. B., O'Donnell, C. J., Patel, D. D., and Murphy, P. M. (2003) J. Clin. Investig. 111, 1241–1250[CrossRef][Medline] [Order article via Infotrieve]
  27. Chen, S., Luo, D., Streit, W. J., and Harrison, J. K. (2002) J. Neuroimmunol. 133, 46–55[CrossRef][Medline] [Order article via Infotrieve]
  28. Barlic, J., Sechler, J. M., and Murphy, P. M. (2003) Blood 102, 3494–3503[Abstract/Free Full Text]
  29. Waldmann, T. A. (2002) J. Clin. Immunol. 22, 51–56[CrossRef][Medline] [Order article via Infotrieve]
  30. Hanna, J., Wald, O., Goldman-Wohl, D., Prus, D., Markel, G., Gazit, R., Katz, G., Haimov-Kochman, R., Fujii, N., Yagel, S., Peled, A., and Mandelboim, O. (2003) Blood 102, 1569–1577[Abstract/Free Full Text]
  31. Aramburu, J., Azzoni, L., Rao, A., and Perussia, B. (1995) J. Exp. Med. 182, 801–810[Abstract/Free Full Text]
  32. Waldmann, T. (2002) Arthritis Res. 4, Suppl. 3, 161–167
  33. Fehniger, T. A., Cooper, M. A., and Caligiuri, M. A. (2002) Cytokine Growth Factor Rev. 13, 169–183[CrossRef][Medline] [Order article via Infotrieve]
  34. Ohteki, T. (2002) Curr. Mol. Med. 2, 371–380[CrossRef][Medline] [Order article via Infotrieve]
  35. Malek, T. R. (2003) J. Leukocyte Biol. 74, 961–965[Abstract/Free Full Text]
  36. Schluns, K. S., and Lefrancois, L. (2003) Nat. Rev. Immunol. 3, 269–279[CrossRef][Medline] [Order article via Infotrieve]
  37. Hurlstone, A., and Clevers, H. (2002) EMBO J. 21, 2303–2311[CrossRef][Medline] [Order article via Infotrieve]
  38. Forcina, G., D'Ettorre, G., Mastroianni, C. M., Carnevalini, M., Scorzolini, L., Ceccarelli, G., D'Agostino, C., Lichtner, M., Massetti, A. P., and Vullo, V. (2004) Cytokine 25, 283–290[CrossRef][Medline] [Order article via Infotrieve]
  39. Mastroianni, C. M., d'Ettorre, G., Forcina, G., and Vullo, V. (2004) Trends Immunol. 25, 121–125[CrossRef][Medline] [Order article via Infotrieve]
  40. Fehniger, T. A., and Caligiuri, M. A. (2001) Int. Rev. Immunol. 20, 503–534[Medline] [Order article via Infotrieve]
  41. Kershaw, M. H., Wang, G., Westwood, J. A., Pachynski, R. K., Tiffany, H. L., Marincola, F. M., Wang, E., Young, H. A., Murphy, P. M., and Hwu, P. (2002) Hum. Gene Ther. 13, 1971–1980[CrossRef][Medline] [Order article via Infotrieve]
  42. Dudley, M. E., Wunderlich, J. R., Robbins, P. F., Yang, J. C., Hwu, P., Schwartzentruber, D. J., Topalian, S. L., Sherry, R., Restifo, N. P., Hubicki, A. M., Robinson, M. R., Raffeld, M., Duray, P., Seipp, C. A., Rogers-Freezer, L., Morton, K. E., Mavroukakis, S. A., White, D. E., and Rosenberg, S. A. (2002) Science 298, 850–854[Abstract/Free Full Text]
  43. Eicher, D. M. (2003) Cell. Immunol. 223, 133–142[CrossRef][Medline] [Order article via Infotrieve]
  44. Masuda, E. S., Imamura, R., Amasaki, Y., Arai, K., and Arai, N. (1998) Cell. Signal. 10, 599–611[CrossRef][Medline] [Order article via Infotrieve]
  45. Rao, A., Luo, C., and Hogan, P. G. (1997) Annu. Rev. Immunol. 15, 707–747[CrossRef][Medline] [Order article via Infotrieve]
  46. Garin, A., Pellet, P., Deterre, P., Debre, P., and Combadiere, C. (2002) Biochem. J. 368, 753–760[CrossRef][Medline] [Order article via Infotrieve]
  47. DeVries, M. E., Cao, H., Wang, J., Xu, L., Kelvin, A. A., Ran, L., Chau, L. A., Madrenas, J., Hegele, R. A., and Kelvin, D. J. (2003) J. Biol. Chem. 278, 11985–11994[Abstract/Free Full Text]
  48. Tsytsykova, A. V., and Goldfeld, A. E. (2000) J. Exp. Med. 192, 581–586[Abstract/Free Full Text]
  49. Hodge, M. R., Ranger, A. M., Charles de la Brousse, F., Hoey, T., Grusby, M. J., and Glimcher, L. H. (1996) Immunity 4, 397–405[CrossRef][Medline] [Order article via Infotrieve]
  50. Raport, C. J., Schweickart, V. L., Eddy, R. L., Jr., Shows, T. B., and Gray, P. W. (1995) Gene (Amst.) 163, 295–299[CrossRef][Medline] [Order article via Infotrieve]
  51. Seidel, M. G., Freissmuth, M., Pehamberger, H., and Micksche, M. (1998) Naunyn-Schmiedeberg's Arch. Pharmacol. 358, 382–389[CrossRef][Medline] [Order article via Infotrieve]
  52. Kiani, A., Rao, A., and Aramburu, J. (2000) Immunity 12, 359–372[CrossRef][Medline] [Order article via Infotrieve]
  53. Aramburu, J., Yaffe, M. B., Lopez-Rodriguez, C., Cantley, L. C., Hogan, P. G., and Rao, A. (1999) Science 285, 2129–2133[Abstract/Free Full Text]
  54. Ranger, A. M., Grusby, M. J., Hodge, M. R., Gravallese, E. M., de la Brousse, F. C., Hoey, T., Mickanin, C., Baldwin, H. S., and Glimcher, L. H. (1998) Nature 392, 186–190[CrossRef][Medline] [Order article via Infotrieve]
  55. Tsai, E. Y., Jain, J., Pesavento, P. A., Rao, A., and Goldfeld, A. E. (1996) Mol. Cell. Biol. 16, 459–467[Abstract]
  56. Tsai, E. Y., Yie, J., Thanos, D., and Goldfeld, A. E. (1996) Mol. Cell. Biol. 16, 5232–5244[Abstract]
  57. Goldfeld, A. E., McCaffrey, P. G., Strominger, J. L., and Rao, A. (1993) J. Exp. Med. 178, 1365–1379[Abstract/Free Full Text]
  58. Crabtree, G. R., and Olson, E. N. (2002) Cell 109, (suppl.) 67–79[CrossRef]
  59. Utsunomiya, N., Tsuboi, M., and Nakanishi, M. (1986) Biochemistry 25, 2582–2584[CrossRef][Medline] [Order article via Infotrieve]
  60. Gearing, A. J., Wadhwa, M., and Perris, A. D. (1985) Immunol. Lett. 10, 297–302[CrossRef][Medline] [Order article via Infotrieve]
  61. Cao, C. M., Xia, Q., Bruce, I. C., Zhang, X., Fu, C., and Chen, J. Z. (2003) J. Pharmacol. Exp. Ther. 306, 572–580[Abstract/Free Full Text]
  62. Bulanova, E., Budagian, V., Pohl, T., Krause, H., Durkop, H., Paus, R., and Bulfone-Paus, S. (2001) J. Immunol. 167, 6292–6302[Abstract/Free Full Text]
  63. van der Pouw Kraan, T. C., Boeije, L. C., Troon, J. T., Rutschmann, S. K., Wijdenes, J., and Aarden, L. A. (1996) J. Immunol. 156, 1818–1823[Abstract]
  64. Ahuja, S. S., Shrivastav, S., Danielpour, D., Balow, J. E., and Boumpas, D. T. (1995) Transplantation 60, 718–723[Medline] [Order article via Infotrieve]
  65. Baksh, S., Widlund, H. R., Frazer-Abel, A. A., Du, J., Fosmire, S., Fisher, D. E., DeCaprio, J. A., Modiano, J. F., and Burakoff, S. J. (2002) Mol. Cell 10, 1071–1081[CrossRef][Medline] [Order article via Infotrieve]
  66. Cristillo, A. D., and Bierer, B. E. (2002) J. Biol. Chem. 277, 4465–4476[Abstract/Free Full Text]
  67. Cristillo, A. D., Macri, M. J., and Bierer, B. E. (2003) Blood 101, 216–225[Abstract/Free Full Text]
  68. Yoshida, H., Nishina, H., Takimoto, H., Marengere, L. E., Wakeham, A. C., Bouchard, D., Kong, Y. Y., Ohteki, T., Shahinian, A., Bachmann, M., Ohashi, P. S., Penninger, J. M., Crabtree, G. R., and Mak, T. W. (1998) Immunity 8, 115–124[CrossRef][Medline] [Order article via Infotrieve]
  69. Xanthoudakis, S., Viola, J. P., Shaw, K. T., Luo, C., Wallace, J. D., Bozza, P. T., Luk, D. C., Curran, T., and Rao, A. (1996) Science 272, 892–895[Abstract]
  70. Kiani, A., Viola, J. P., Lichtman, A. H., and Rao, A. (1997) Immunity 7, 849–860[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S.-J. Lee, S. Namkoong, Y.-M. Kim, C.-K. Kim, H. Lee, K.-S. Ha, H.-T. Chung, Y.-G. Kwon, and Y.-M. Kim
Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways
Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2836 - H2846.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
X. Ding, N. K. Saxena, S. Lin, A. Xu, S. Srinivasan, and F. A. Anania
The Roles of Leptin and Adiponectin: A Novel Paradigm in Adipocytokine Regulation of Liver Fibrosis and Stellate Cell Biology
Am. J. Pathol., June 1, 2005; 166(6): 1655 - 1669.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/47/48520    most recent
M406978200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barlic, J.
Right arrow Articles by Murphy, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barlic, J.
Right arrow Articles by Murphy, P. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement