From the § ICOS Corporation, Bothell, Washington 98021 and Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan
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ABSTRACT |
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Macrophage-derived chemokine (MDC) is a recently
identified member of the CC chemokine family. MDC is not closely
related to other chemokines, sharing most similarity with thymus- and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with
little expression seen in other tissues. In addition, the genes for MDC
and TARC are encoded by human chromosome 16. To explore this
relationship in greater detail, we have more precisely localized the
MDC gene to chromosome 16q13, the same position reported for the TARC
gene. We have also examined the interaction of MDC with CC chemokine
receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a
fusion protein of MDC with secreted alkaline phosphatase, we observed
high affinity binding of MDC-secreted alkaline phosphatase to
CCR4-transfected L1.2 cells (Kd = 0.18 nM). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1, macrophage inflammatory protein-1
). MDC was tested for calcium mobilization in
L1.2 cells tranfected with seven different CC chemokine receptors. MDC
induced a calcium flux in CCR4-transfected cells, but other receptors
did not respond to MDC. TARC, which also induced calcium mobilization
in CCR4 transfectants, was unable to desensitize the response to MDC.
In contrast, MDC fully desensitized a subsequent response to TARC. Both
MDC and TARC functioned as chemoattractants for CCR4 transfectants,
confirming that MDC is also a functional ligand for CCR4. Since MDC and
TARC are both expressed in the thymus, one role for these chemokines
may be to attract CCR4-bearing thymocytes in the process of T cell
education and differentiation.
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INTRODUCTION |
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Chemokines are small secreted proteins that mediate recruitment of leukocytes to sites of inflammation (1, 2). The complexity and functions of the chemokine family have become increasingly diverse as more members have been identified and characterized. There are four subfamilies of chemokines based on the relative position of conserved cysteine residues (1, 3, 4). The largest subfamily consists of the CC chemokines, which generally induce migration of monocytes, T lymphocytes, and in some cases eosinophils, basophils, or mast cells. All chemokines mediate their activities through G protein-coupled receptors, which have a characteristic seven-transmembrane structure. These receptors are very selective and bind specific ligands with high affinity. Eight different CC chemokine receptors have been characterized to date. CCR1,1 CCR2, CCR3, and CCR5 each has ligand specificity for at least three CC chemokines (5-12), while single high affinity ligands have been identified that recognize CCR4, CCR6, CCR7, and CCR8 (13-17).
Macrophage-derived chemokine (MDC) is a novel CC chemokine synthesized by macrophages and dendritic cells (18). MDC shares only limited homology with other chemokines. It is most similar to thymus- and activation-regulated chemokine (TARC) (19), with 37% identity. As shown by tissue Northern blots, MDC also shares a very similar expression pattern with TARC, showing high levels in thymus and very low expression in other tissues. Most surprisingly, MDC and TARC are encoded by human chromosome 16 (18, 20), while other CC chemokines are closely linked on chromosome 17 (21).
TARC was recently found to be a highly specific ligand for CCR4 (13), and both are co-expressed in the thymus (19, 22). TARC is likely to be made by dendritic cells and may aid the recruitment, activation, and development of T cells that express CCR4. Because of the similarities between MDC and TARC, a study was undertaken to determine if MDC interacts with CCR4, the receptor for TARC. We show here that MDC is a ligand for CCR4 and in fact binds with a higher affinity than TARC.
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EXPERIMENTAL PROCEDURES |
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Chromosomal Localization-- A 20-kilobase pair genomic fragment containing the human MDC gene was labeled with digoxigenin by nick translation and used as a probe for fluorescence in situ hybridization of human chromosomes (Genome Systems, Inc., St. Louis, MO). The labeled probe was hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes. Reactions were carried out in the presence of sheared human DNA in 50% formamide, 10% dextran sulfate, 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate. Hybridization signals were detected by treating slides with fluoresceinated antidigoxigenin antibodies followed by counterstaining with 4,6-diamidino-2-phenylindole. Initial labeling implicated a group E chromosome. A genomic probe that specifically hybridizes to the short arm of chromosome 16 was used to demonstrate cohybridization of chromosome 16 with the MDC probe. A total of 80 metaphase cells were analyzed, with 61 exhibiting specific labeling.
Cell Culture-- The murine pre-B cell line L1.2 was kindly provided by Dr. Craig Gerard (Harvard Medical School, Boston, MA) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. L1.2 cells were stably transfected as described previously (13) by electroporation with 10 µg of linearized plasmid at 260 V, 960 microfarads using a Gene Pulser (Bio-Rad). HUT78 cells (ATCC) were maintained in Iscove's modified Dulbecco's medium with 15% fetal bovine serum.
Preparation of Recombinant Chemokines--
The mature sequences
of both MDC and TARC were chemically synthesized by Gryphon Sciences
(South San Francisco, CA) using t-butyl-oxycarbonyl
chemistries on a peptide synthesizer (model 430A; Applied Biosystems).
Lyophilized protein was dissolved at 10 mg/ml in 4 mM HCl
and immediately diluted to 0.1 mg/ml in phosphate-buffered saline plus
0.1% bovine serum albumin (BSA) for storage at 80 °C. Recombinant
MDC was expressed as a fusion protein with the secreted form of
placental alkaline phosphatase (SEAP) as described (13), in the
expression vector pcDNA3 (CLONTECH, Palo Alto
CA). Briefly, the coding region of MDC followed by the coding sequence
for a five-amino acid linker sequence (Ser-Arg-Ser-Ser-Gly) was fused in frame to the mature coding region of SEAP. The MDC-SEAP expression plasmid was transfected into COS cells by the DEAE-dextran method (23).
Twenty-four hours following transfection, the serum levels were reduced
from 10 to 1%. After 3-4 days, the culture supernatants were
collected, centrifuged, filtered through a 0.45-µm membrane, and
stored at 4 °C. The concentration of MDC-SEAP was determined by
comparison with the reported specific activity of secreted placental
alkaline phosphatase (24) and confirmed using known concentrations of
TARC-SEAP (13) as an internal reference standard.
Binding Assays--
For displacement and saturation experiments,
3 × 105 cells (or 4 × 105 cells for
HUT78) were incubated for 1 h at 16 °C in the presence of 0.5 nM MDC-SEAP in the presence or absence of various
concentrations of chemokines in 200 µl of binding buffer (RPMI 1640 medium containing 25 mM HEPES, pH 7.4, 1% BSA, and 0.02%
sodium azide). Following incubation, cells were washed four times in
binding buffer and lysed in 50 µl of 10 mM Tris-HCl, pH
8.0, and 1% Triton X-100. Samples were heated at 65 °C for 15 min
to inactivate cellular phosphatases, centrifuged, and stored at
20 °C until assayed. Alkaline phosphatase activity in 10 µl of
sample was determined by a chemiluminescence assay using the Great
Escape Detection kit (CLONTECH) according to the
manufacturer's instructions. The saturation binding curve was fitted
(table curve 228) using the Hill equation y = a(xc)/(xc + bc), where y is the amount
of ligand bound, a is the maximum amount of ligand bound,
x is the concentration of ligand, b is the
concentration of ligand at which 50% of receptor sites are occupied
(KD), and c is the Hill coefficient.
Binding competition curves were fitted (table curve 228) using a
three-parameter logistic model described by the equation
y = a/(1 + (x/b)c), where y is the
amount of labeled ligand bound, a is the maximum amount of
labeled ligand bound, x is the concentration of the competitive chemokine, b is the IC50, and
c is a parameter that determines the slope of the curve at
the IC50.
Calcium Mobilization-- Cells were suspended at 3 × 106 cells/ml in Hanks' balanced salt solution supplemented with 1 mg/ml BSA and 10 mM HEPES, pH 7.4. Cells were incubated with 1 µM fura-PE3-AM (Texas Fluorescence Labs) at room temperature for 1 h in the dark. After washing twice, cells were resuspended at 2.5 × 106 cells/ml. To measure intracellular calcium, cells in 2 ml were placed in a quartz cuvette in a Perkin-Elmer LS 50B spectrofluorimeter. Fluorescence was monitored at 340 nm (excitation wavelength 1), 380 nm (excitation wavelength 2), and 510 nm (emission wavelength) every 200 ms.
Chemotaxis Assay-- Cell migration was assayed using L1.2 cells transfected with CCR4 cDNA (13) or HUT78 cells. Approximately 106 cells resuspended in 0.1 ml of RPMI 1640 medium with 0.5% BSA were loaded in the upper wells of a transwell chamber (3-µm pore size, Costar). Test chemokines were added to the lower wells in a volume of 0.6 ml. After 4 h at 37 °C, cells in the lower chamber were collected and counted by fluorescence-activated cell sorting. Values are expressed as the percentage of input cells that migrate through the filter. Untransfected L1.2 cells were used as a control.
Northern Analysis-- The expression of CCR4 mRNA in T cells was examined by Northern analysis. Total RNA was extracted from the T cell line HUT78 using RNA Stat-60 (Tel-Test "B", Friendswood, TX). Peripheral blood lymphocytes (PBL) were obtained from human blood separated over Histopaque gradients. After separation, monocytes were removed by plastic adherence. Half of the cells were used immediately to obtain RNA ("unstimulated") and the other half were treated with IL-2 (50 units/ml, Boehringer Mannheim) for 10 days before extracting RNA. Ten µg of total RNA was loaded per lane, fractionated on 0.8% agarose-formaldehyde gel, and transferred to nitrocellulose as described (12). The blot was probed with a gel-purified polymerase chain reaction fragment containing the entire coding region of CCR4 (22) and washed as described (12).
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RESULTS |
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Chromosomal Localization-- To more precisely establish the chromosomal location of the human MDC gene, fluorescent in situ hybridization analysis was performed using a 20-kilobase pair human genomic clone as a probe. An initial hybridization experiment localized the gene to the q terminus of a group E chromosome. Subsequent co-hybridization experiments with a genomic probe specific for chromosome 16 implicated the q terminus of chromosome 16. The MDC probe hybridized to a region immediately adjacent to the heterochromatic/euchromatic boundary, corresponding to band 16q13. This localization is depicted in Fig. 1. Because the TARC gene is localized in this region (20), we next compared the receptor usage of MDC with TARC.
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Receptor Binding Assays--
Chemokines have previously been fused
to SEAP to generate probes for receptor binding studies (20, 25). We
utilized this strategy to prepare MDC as a fusion protein with SEAP. A
plasmid was prepared that directed the expression of a secreted fusion protein with MDC at the amino terminus followed by the alkaline phosphatase with His6 tag. The MDC-SEAP was used as a probe
to examine binding to CCR4-transfected L1.2 cells. As shown in Fig. 2A, the MDC-SEAP bound to
CCR4-expressing cells in a saturable manner. This binding was to a
single high affinity site with a Kd of 0.18 nM, as demonstrated by Scatchard analysis (Fig.
2B). Binding of MDC-SEAP was competitively inhibited with increasing concentrations of unlabeled MDC or TARC (Fig.
2C). The IC50 for MDC was 0.65 nM,
while the IC50 for TARC was 2.1 nM. This
suggests that both ligands recognize a common binding site on CCR4 and
that MDC has more than 3-fold higher affinity for CCR4. To examine
specificity of binding to CCR4, six additional chemokines were tested
for competition of MDC-SEAP binding. A 200-fold molar excess of each
chemokine was tested for competition with a constant quantity of
MDC-SEAP (0.5 nM), presented in Fig. 2D. The
chemokines MCP-1, MCP-3, MCP-4, RANTES, MIP-1, and MIP-1
did not
compete for binding of MDC-SEAP to CCR4. In contrast, both MDC and TARC
blocked binding to CCR4 transfectants.
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MDC Induces Calcium Flux in CCR4-transfected Cells-- TARC has previously been shown to signal through CCR4 by inducing calcium mobilization (13). To determine the ability of MDC to cause signaling through chemokine receptors, we examined calcium mobilization in L1.2 cells expressing CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, or CCR7 (Fig. 3). MDC did not cause calcium flux in L1.2 cells transfected with CCR1, CCR2B, CCR3, CCR5, CCR6, or CCR7, whereas each responded to its known cognate ligand. In contrast, L1.2 cells transfected with CCR4 produced a strong calcium flux when stimulated with 10 nM MDC. Similar to other G protein-coupled receptors, CCR4 was refractory to subsequent stimulation with the same concentration of MDC. MDC also completely desensitized CCR4 transfectants to subsequent TARC treatment when both were added at 10 nM. However, pretreatment with TARC did not desensitize the receptor to subsequent stimulation with MDC. The signal produced by initial TARC stimulation was of lower intensity than the primary MDC signal and the MDC signal secondary to TARC stimulation. These results further confirm that MDC is a ligand for CCR4.
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MDC Induces Chemotaxis in Cells with CCR4-- We next examined the ability of MDC to induce migration of CCR4-transfected L1.2 cells. As shown in Fig. 4A, both MDC and TARC induced migration of CCR4-transfected L1.2 cells. Both chemokines produced classic bell-shaped migration responses with maximal migration between 1 and 10 nM. The migration for MDC was significantly higher than that for TARC. Untransfected L1.2 cells failed to migrate when treated with MDC. These chemotaxis results confirm that both MDC and TARC are functional ligands for CCR4. The human T cell line HUT78 has previously been shown to express CCR4 mRNA and to respond to TARC (13). As shown in Fig. 4B, both MDC and TARC induced migration of this T cell line.
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DISCUSSION |
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As demonstrated by binding studies, calcium mobilization, and chemotaxis, MDC is a potent agonist for CCR4. The high affinity binding and concentrations of MDC required for signaling are similar to other chemokine receptor-ligand interactions. MDC and TARC both bind and signal through CCR4. In addition to this similar activity, both chemokines share similar in vivo expression patterns and are closely linked on human chromosome 16. MDC and TARC share these functional similarities despite sharing only 37% amino acid identity. Consequently, MDC and TARC can be considered to comprise a distinct class of CC chemokines with unique genetic localization, receptor utilization, expression pattern, and function.
Our experiments suggest that MDC is likely to act as a chemoattractant for CCR4-expressing cells including mature T lymphocytes. This prediction has been confirmed recently by other investigators. Chang and colleagues (26) have shown that MDC acts as a chemoattractant for activated T cells. Pal and colleagues (27) have demonstrated that MDC induces calcium mobilization in activated T cells and also acts as an human immunodeficiency virus-suppressive factor.
During T cell development, immature progenitor cells undergo differentiation and expansion leading to the establishment of the major T cell lineages and the elimination of potentially autoreactive T cells (28). These processes occur within the highly specialized microenvironment of the thymus. The signals regulating the directed movement of immature T cells within the thymus have yet to be determined. The abundant expression of MDC, TARC, and CCR4 in the thymus (with very little expression seen in other tissues (18, 19, 22)) suggests that they may play a role in T cell development. CCR4 is expressed on T cells (13, 22), while MDC and TARC are expressed by cells of the dendritic lineage, which form a major component of the thymic architecture (18, 19). MDC and TARC may function to attract or retain T cells in the thymus and thereby mediate their trafficking and education. To further elucidate the physiologic role of these molecules, responses on T cell subsets should be determined. The complexity of the cellular movements that need to occur during T cell development may explain the diversity of chemokine and chemokine receptor expression within the thymus. Other chemokines such as PARC (pulmonary and activation-regulated chemokine; Ref. 29)/DC-CK1 (30), SCM-1 (31)/lymphotactin (3), and TECK (thymus-expressed chemokine; Ref. 32) and receptors such as CCR8 (16, 17, 33) exhibit the greatest expression in the thymus. Determining the relationships and functions of these molecules may greatly help to understand the mechanisms of T cell development and regulation.
MDC may also play a role outside of the thymus in certain disease states. We have previously shown that MDC is expressed at high levels by cultured macrophages and dendritic cells (18). CCR4 is also expressed at high levels by activated T lymphocytes, principally of the CD4 subset (13). Thus, MDC may also play a role in the initiation and or triggering of the immune response by facilitating the interaction of T cells with antigen-presenting cells at sites of inflammation.
MDC was previously shown to stimulate migration of dendritic cells and IL-2 activated natural killer cells (18). It is not clear if this migration is mediated by CCR4, since it appears to be expressed primarily on T cells. MDC may be recognized by other chemokine receptors that are yet to be characterized. Further experiments are required to determine if TARC is also able to stimulate dendritic cell migration. By virtue of its ability to attract both T lymphocytes and dendritic cells, MDC may play a unique role in the initiation or amplification of antigen-specific immune responses.
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ACKNOWLEDGEMENTS |
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We thank Michael Siani (Gryphon Sciences, South San Francisco) for synthesis and chemical analysis of MDC and TARC; Dina Leviten and Marsalina Quiggle for DNA synthesis and sequencing; and Vicki Schweickart, Johnny Stine, and Larry Tjoelker for critical review of the manuscript.
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FOOTNOTES |
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* 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 may be addressed: 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan. Tel.: 81-6-382-2612; Fax: 81-6-382-2598.
To whom correspondence may be addressed: ICOS Corp., 22021 20th Avenue S.E., Bothell, WA 98021. Tel.: 425-485-1900; Fax:
425-485-0885.
1 The abbreviations used are: CCR, CC chemokine receptor; BSA, bovine serum albumin; IL-2, interleukin-2; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; PBL, peripheral blood lymphocytes; RANTES, regulated on activation normal T cell expressed and secreted; SEAP, secreted alkaline phosphatase; TARC, thymus- and activation-regulated chemokine.
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REFERENCES |
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