From the Shionogi Institute for Medical Science, 2-5-1 Mishima,
Settsu-shi, Osaka 566, Japan
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INTRODUCTION |
Chemokines constitute a group of small, mostly basic,
heparin-binding cytokines with common structural features that mediate recruitment of leukocytes into sites of inflammation and immune responses. Chemokines are also considered to play roles in homeostatic recirculation and homing of lymphocytes (for review, see Refs. 1-4).
Based on the arrangement of the amino-terminal conserved cysteine
residues, chemokines are grouped into two major subfamilies, CXC and
CC. Molecules with a C or CX3C motif have also been described. CXC
chemokines with the Glu-Leu-Arg (ELR) motif immediately prior to the
CXC motif are potent chemoattractants for neutrophils, whereas those
without the ELR motif are mostly directed to lymphocytes. On the other
hand, CC chemokines are mainly directed to monocytes and also to
eosinophils, basophils, and/or lymphocytes with variable selectivity.
Notably, some recently identified CC chemokines are highly specific for
lymphocytes (4). The specific effects of chemokines are mediated by a
family of seven-transmembrane G-protein coupled receptors (1-4). In
humans, four CXC chemokine receptors (CXCR1 to 4), eight CC chemokine
receptors (CCR1 to 8),1 and
one CX3C chemokine receptor (CX3CR1) have been identified and defined
for their ligand specificities: CXCR1 for IL-8 (5); CXCR2 for IL-8 and
other CXC chemokines with the Glu-Leu-Arg motif (6-8); CXCR3 for IP-10
and Mig (9); CXCR4 for SDF-1/PBSF (10, 11); CCR1 for MIP-1
, RANTES,
and MCP-3 (12-15); CCR2 for MCP-1, MCP-3, and MCP-4 (15-18); CCR3 for
eotaxin, RANTES, MCP-2, MCP-3, MCP-4, and MPIF-2/eotaxin-2 (18-24);
CCR4 for TARC and MDC (25, 26); CCR5 for RANTES, MIP-1
, and MIP-1
(27, 28); CCR6 for LARC/MIP-3
/exodus (29-31); CCR7 for ELC/MIP-3
(32); CCR8 for I-309 (33, 34); and CX3CR1 for fractalkine/neurotactin (35).
Previously, we have described a novel human CC chemokine, termed
Secondary Lymphoid-tissue Chemokine
(SLC), which is mainly and constitutively expressed in the secondary
lymphoid tissues such as lymph nodes, appendix, and spleen (36).
Independently, the same chemokine has been reported with terms of
6Ckine and Exodus-2 (37, 38). We have demonstrated that recombinant
SLC, while not active on peripheral blood monocytes or neutrophils, is
a potent chemoattractant for peripheral blood lymphocytes and induces a
vigorous calcium mobilization in cultured normal T cells (36). Exodus-2
was also shown to be chemotactic for human T cells and B cells but not
for monocytes or neutrophils (38). Consistent with its
lymphocyte-specific activities, SLC fused with the secreted form of
alkaline phosphatase (SLC-SEAP) bound specifically to lymphocytes, and
its binding was fully displaced only by SLC among ten CC chemokines so
far tested (36). These findings suggest that lymphocytes express a
class of receptors highly specific for SLC. Notably, the SLC
gene (SCYA21) is mapped to chromosome 9p13 (36) where the
gene for another lymphocyte-specific CC chemokine ELC
(SCYA19) also exists (32), suggesting their divergence from
a common ancestral gene. We have shown that ELC is a high affinity
functional ligand for CCR7 that is expressed on T and B lymphocytes
(32). Here we demonstrate that SLC is also a high affinity functional
ligand for CCR7.
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EXPERIMENTAL PROCEDURES |
Cells--
A murine pre-B cell line L1.2 (39) was maintained in
RPMI 1640 supplemented with 10% fetal calf serum. L1.2 cells stably expressing transfected human CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, and
CCR7 were generated as described previously (25). Peripheral blood
mononuclear cells were isolated from EDTA-treated venous blood obtained
from healthy adult donors by using Ficoll-Paque (Pharmacia Biotech
Inc., Uppsala, Sweden). T cells were expanded by stimulation with
phytohemagglutinin (PHA) (Life Technologies, Inc.) for 2 days and
further cultivation with 200 units/ml interleukin 2 (IL-2) for a
week.
Chemokines--
Eotaxin, SLC, TARC, LARC, and MCP-1 were
produced by a baculovirus expression system and purified to essential
homogeneity as described previously (19, 36, 40, 41). MIP-1
and
RANTES were purchased from Peprotech (Rocky Hill, NJ). ELC was produced in Escherichia coli and purified as follows. To express a
fusion protein consisting of the amino-terminal (His)6 tag
and the enterokinase cleavage site (Asp-Asp-Asp-Asp-Lys) linked to
mature ELC, a DNA fragment termed EK-ELC-XhoI was generated
from pCRII-5'-RACE-ELC (32) by polymerase chain reaction using EK-ELC
primer (+5'-TCCGACGACGACGACAAGGGCACCAATGATGCTGAA) and
ELC-XhoI primer (
5'-ACGTCTCGAGTTAACTGCTGCGGCGCTTCAT).
Then, a DNA fragment termed NheI-EK-ELC-XhoI was
generated from EK-ELC-XhoI by polymerase chain reaction
using NheI-EK primer
(+5'-GCGCTAGCAGCAGCGGATCCGACGACGACGACAAG) and ELC-XhoI
primer. After digestion with NheI and XhoI, the
DNA fragment was cloned into an expression vector pRSET A to generate pRSET A-(His)6-EK-ELC. BL21(DE3)pLysS strain (Novagen) was
transformed with the expression vector and induced by
isopropyl-
-D-thiogalactopyranoside (Sigma) following the
standard protocols. The pellet was collected by centrifugation
and suspended by a lysis buffer (0.5% Sarkosyl, 20 mM
Tris-HCl, pH 8.0, and 150 mM NaCl). After sonication
and centrifugation, the supernatants were applied to a TARON
metal affinity resin column (CLONTECH). Eluted
fractions were analyzed by staining with Coomassie Blue after
SDS-polyacrylamide gel electrophoresis (PAGE). The fractions containing
the ELC-fusion protein were pooled, dialyzed against 0.5% acetic acid,
lyophilized, and dissolved in distilled water. The solution was applied
to a 1-ml HiTrap SP column (Pharmacia) equilibrated with 50 mM MES, pH 6.0, and eluted with a 25-ml linear gradient of
0.4-1.4 M NaCl in 50 mM MES, pH 6.0, at a rate
of 0.5 ml/min on a fast protein liquid chromatography (Pharmacia).
Fractions were analyzed by staining with Coomassie Blue after SDS-PAGE,
and fractions containing the ELC fusion protein were pooled, dialyzed
against 0.5% acetic acid, lyophilized, and dissolved in distilled
water. After digestion with enterokinase in 10 mM MES, pH
6.0, and 0.1% Tween 20 at 4 °C, the solution was injected into a
reverse-phase high performance liquid chromatography column (Cosmocil
5C4-AR-300, 4.6 × 250 mm) (Cosmo Bio, Tokyo, Japan) equilibrated
with 0.05% trifluoroacetic acid and eluted with a 0-60% linear
gradient of acetonitrile in 0.05% trifluoroacetic acid at a flow rate
of 1 ml/min. The peak fraction containing ELC was lyophilized
(Fig. 1A). The protein concentration was determined by a BCA kit (Pierce, Rockford, IL), and
the purity was analyzed by SDS-PAGE and silver staining (Fig. 1B). Endotoxin levels were always <4 pg/µg of ELC as
determined by the Limulus amoebocyte lysate assay (QCL-1000)
(BioWhittaker, Walkersville, MD).

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Fig. 1.
Purification of recombinant ELC produced by
E. coli. A, the elution profile of ELC from a
reverse-phase high performance liquid chromatography. ELC was eluted as
indicated by the arrow. B, analysis of purified ELC.
Purified ELC was analyzed by SDS-PAGE and silver staining. Positions of
size markers are shown on the right (kDa).
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Calcium Mobilization Assay--
Calcium mobilization in response
to chemokines was determined as described previously (36). In brief,
cells were loaded with 1 µM Fura 2-AM (Molecular Probe,
Eugene, OR) in RPMI 1640 supplemented with 1% fetal calf serum and 10 mM HEPES, pH 7.4, for 1 h at 37 °C in the dark.
Loaded cells were washed four times with phosphate-buffered saline
containing 1% fetal calf serum, 1 mM CaCl2,
and 1 mM MgCl2, and resuspended in the same
buffer at 2.5 × 106 cells/ml (L1.2 lines) or
1.25 × 106 cells/ml (cultured normal T cells). To
monitor intracellular calcium concentration, 2 ml of the cell
suspension in a quartz cuvette was placed in a spectrofluorimeter (LS
50B, Perkin-Elmer) and stimulated with each chemokine at 37 °C.
Emission fluorescence at 510 nm was monitored upon excitation at 340 (F340) and 380 nm (F380)
at every 200 ms. Data were expressed by the ratio of F340 to F380
(R340/380).
Binding Assay--
SLC and ELC fused with the secreted form of
placental alkaline phosphatase (SLC-SEAP and ELC-SEAP) were produced as
described previously (32, 36). For binding experiments, 2 × 105 L1.2 cells stably expressing CCR7 or cultured normal T
cells were incubated for 1 h at 16 °C with 1 nM of
SLC-SEAP or ELC-SEAP without or with increasing concentrations of
unlabeled competitors in 200 µl of RPMI 1640 containing 20 mM HEPES, pH 7.4, 1% bovine serum albumin (BSA), and
0.02% sodium azide. Cells were washed and lysed in 50 µl of 10 mM Tris-HCl, pH 8.0, and 1% Triton X-100. Cell lysates
were heated at 65 °C for 10 min to inactivate cellular phosphatases
and centrifuged to remove cell debris. AP activity in 10 µl of lysate
was determined by a chemiluminescence method as described previously
(32, 36). All assays were done in duplicate.
Chemotaxis Assay--
The chemotaxis assay was performed
essentially as described previously (36). In brief, parental L1.2 and
L1.2 stably expressing CCR7 were suspended in the assay buffer (RPMI
1640 supplemented with 10 mM HEPES, pH 7.4, 1% BSA). The
cells in 100 µl were placed in the upper compartments of transwell
chambers with 3 µm pore size (Costar), while 0.6 ml of the buffer
without or with chemokines were placed in the lower compartments. After
4 h at 37 °C, cells which migrated into lower chambers were
collected and counted on a FACStar Plus (Becton Dickinson, Mountain
View, CA). Results are expressed as percent input cells that migrated
through the filter. All assays were done in duplicate.
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RESULTS |
SLC Induces Calcium Mobilization in CCR7-Transfected L1.2
Cells--
To investigate the ability of SLC to induce signaling
through the known CC chemokine receptors, we examined calcium
mobilization in murine L1.2 cells stably expressing human CCR1, CCR2B,
CCR3, CCR4, CCR5, CCR6, and CCR7 upon stimulation with SLC. As shown in
Fig. 2, SLC did not induce significant
calcium flux in parental L1.2 or those expressing CCR1, CCR2B, CCR3,
CCR4, CCR5, or CCR6. We confirmed that L1.2 expressing each receptor
responded to an appropriate ligand with a vigorous calcium flux.
Strikingly, however, SLC induced a vigorous calcium flux in L1.2 cells
expressing CCR7, which is a known receptor for a CC chemokine ELC (32).
Thus, SLC is another functional ligand for CCR7. Furthermore, SLC fully desensitized CCR7-expressing L1.2 cells against subsequent stimulation with an equal amount of ELC. Conversely, ELC fully desensitized the
same cells against subsequent stimulation with an equal amount of SLC.
Thus, SLC and ELC are essentially equivalent in terms of calcium
mobilization via CCR7 expressed on transfected L1.2 cells.

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Fig. 2.
Calcium mobilization in CCR7-transfected L1.2
cells by SLC and ELC. Transfected L1.2 cells stably expressing
CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, or CCR7 were loaded with Fura 2-AM and stimulated with 10 nM each chemokine as indicated.
Intracellular concentrations of calcium were monitored by fluorescence
ratio R340/380. Representative results from
three separate experiments are shown.
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SLC Binds to CCR7--
We next compared the binding of SLC and ELC
to CCR7. As shown in Fig. 3, SLC-SEAP
bound specifically to CCR7-expressing L1.2 cells at high levels. The
binding was fully competed by unlabeled SLC and ELC with an
IC50 of 0.5 nM and 1.0 nM,
respectively. Conversely, the binding of ELC-SEAP to CCR7 was fully
competed by SLC and ELC with an IC50 of 8.2 and 6.1 nM, respectively. Thus, SLC and ELC are essentially
equivalent in terms of cross-competition in binding to CCR7
expressed on transfected L1.2 cells.

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Fig. 3.
Competition of SLC and ELC with SLC-SEAP and
ELC-SEAP for binding to CCR7. Transfected L1.2 cells (2 × 105 cells) stably expressing CCR7 were incubated with 1 nM SLC-SEAP or ELC-SEAP as indicated without or with
increasing concentrations of SLC (closed circle) and ELC
(open circle) at 16 °C for 1 h. After washing,
amounts of cell-bound SLC-SEAP or ELC-SEAP were determined
enzymatically. All assays were done in duplicate. Representative results from two separate experiments are shown.
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SLC Stimulates Chemotaxis in CCR7-Transfected L1.2 Cells--
We
next compared the ability of SLC and ELC to induce chemotaxis. As shown
in Fig. 4, both SLC and ELC induced
vigorous migration in CCR7-transfected L1.2 cells with a typical
bell-shaped dose-response curve and a maximal migration at 10 nM. The potency and efficiency of SLC and ELC in induction
of chemotaxis via CCR7 were very similar. Untransfected L1.2 cells
failed to migrate toward SLC or ELC (not shown). These results again
demonstrate that SLC and ELC are essentially equivalent as agonists for
CCR7 expressed on transfected L1.2 cells.

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Fig. 4.
Chemotaxis induction of CCR7 transfectants by
SLC and ELC. Parental L1.2 cells (closed squares) or
transfected L1.2 cells stably expressing CCR7 (circles) were
placed in upper wells and allowed to migrate toward SLC (closed
circles and closed squares) or ELC (open
circles) in the lower wells. Migrated cells were collected and
counted by flow cytometry. The assay was done in duplicate.
Representative results from two separate experiments are shown.
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SLC and ELC Share Receptors on Cultured Normal T Cells--
CCR7
is expressed on T and B lymphocytes (42-45). To test whether SLC and
ELC also share receptors on lymphocytes, we next examined induction of
calcium mobilization in cultured peripheral blood T cells that were
initially stimulated with PHA and subsequently expanded with IL-2. As
shown in Fig. 5, SLC and ELC at 10 nM induced vigorous calcium mobilization in cultured normal
T cells (A and B). In contrast to the results
with CCR7-transfected L1.2 cells (Fig. 2), however, SLC only partially
desensitized cultured normal T cells against subsequent stimulation
with an equal amount of ELC (Fig. 5A). On the other hand,
ELC fully desensitized T cells against subsequent stimulation with an
equal amount of SLC (Fig. 5B). As expected, SLC desensitized
T cells against subsequent stimulation with an equal amount of SLC
(Fig. 5C). SLC at 100 nM, however, fully
desensitized T cells against ELC at 10 nM (Fig. 5D).

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Fig. 5.
Calcium mobilization in cultured normal T
cells by SLC and ELC. Normal T cells expanded from peripheral
blood mononuclear cells by stimulation with PHA for 2 days and
subsequent cultivation with IL-2 for a week were loaded with Fura 2-AM
and stimulated with SLC or ELC as indicated by arrowheads.
Intracellular concentrations of calcium were monitored by fluorescence
ratio R340/380. Panels A,
B, and C, stimulated with SLC and ELC at 10 nM; panel D, stimulated with SLC at 100 nM and ELC at 10 nM. Representative results
from three separate experiments are shown.
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These results suggest that, even though SLC and ELC fully share a class
of receptors expressed on normal T cells, they may exhibit different
binding affinities. To test this possibility, SLC and ELC were compared
for their ability to compete with ELC-SEAP for binding to cultured
normal T cells. As shown in Fig. 6, both SLC and ELC were indeed capable of fully competing with ELC-SEAP for
binding to T cells. However, SLC and ELC exhibited an IC50 of 10 and 1.6 nM, respectively. Thus, even though SLC and
ELC fully share receptors expressed on cultured normal T cells, most probably CCR7, SLC appeared to bind to CCR7 expressed on these cells
with an affinity somehow lower than that of ELC in contrast to CCR7
expressed on transfected L1.2 cells (Fig. 3).

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Fig. 6.
Competition of SLC and ELC with ELC-SEAP for
binding to cultured normal T cells. Normal T cells expanded from
peripheral blood mononuclear cells by stimulation with PHA for 2 days
and subsequent cultivation with IL-2 for a week were incubated with 1 nM ELC-SEAP without or with increasing concentrations of
SLC (closed circles) or ELC (open circles) at
16 °C for 1 h. After washing, amounts of ELC-SEAP were
determined enzymatically. All assays were done in duplicate.
Representative results from two separate experiments are shown.
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DISCUSSION |
Previously, we have shown that ELC is a high affinity functional
ligand for CCR7 (32). ELC binds specifically to CCR7 with high affinity
and induces vigorous calcium mobilization and efficient chemotaxis in
CCR7-transfected human cells such as K562 and HEK293/EBNA-1 (32). In
the present study, we have demonstrated that SLC is also a high
affinity functional ligand for CCR7. SLC binds to CCR7 with high
affinity and induces calcium mobilization and chemotaxis in
CCR7-transfected murine L1.2 cells (Figs. 2-4). Quite remarkably, SLC
and ELC are almost equivalent as ligands for CCR7 when CCR7 is
expressed on L1.2 cells. This was revealed by full
cross-desensitization against each other in calcium mobilization via
CCR7 (Fig. 2), efficient cross competition in each binding to CCR7
(Fig. 3), and a very similar potency and efficiency in induction of
chemotaxis via CCR7 (Fig. 4). SLC, however, was much less efficient in
desensitizing cultured normal T cells against ELC (Fig. 5). Even though
SLC fully competed with ELC-SEAP for binding to cultured normal T cells, SLC appears to bind to these cells with an affinity lower than
that of ELC (Fig. 6). Collectively, the cell background may affect the
structure and/or function of CCR7 possibly through differential
coupling of G proteins (46). This may cause differences in binding
affinity and efficiency of cross-desensitization between SLC and ELC
depending on cell types.
SLC and ELC share only 32% amino acid identity (32, 36). SLC also has
a unique extension of about 30 amino acids with two extra cysteine
residues in its carboxyl terminus. As shown in
Fig. 7, however, certain amino acid
residues marked by closed circles are shared only by SLC and
ELC among the known human CC chemokines. Since the amino-terminal
region preceding the first conserved cysteine residue, the
amino-terminal loop region, the
-turn region containing the third
cysteine, and the residues preceding the fourth cysteine have been
shown to be important for the receptor specificity of IL-8 (1), the
residues shared only by SLC and ELC in such regions may be involved in
their specific binding to CCR7.

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Fig. 7.
Amino acid alignment of human SLC and
ELC. The residues highly conserved among the known human CC
chemokines are boxed. The residues shared only by SLC and
ELC among the known human CC chemokines are marked by closed
circles.
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In addition to sharing CCR7, SLC and ELC constitute a mini-cluster at
human chromosome 9p13 and have very similar patterns of
tissue-expression (32, 36). Thus, genetically and functionally, SLC and
ELC are highly related chemokines and possibly redundant to some
extent. In fact, a similar functional redundancy is also noted for
other sets of chemokines derived from mini-clusters. IP-10 and Mig form
a mini-cluster at human chromosome 4q21.21 (47). They are both highly
inducible by IFN-
(47) and share CXCR3 (9). TARC (40) and MDC (48)
form a mini-cluster at chromosome 16q13 (49). They are both expressed
mainly in the thymus (40, 48) and share CCR4 (26). Thus, the members of each mini-cluster, while relatively independent from the majority of
chemokines encoded by the traditional gene clusters at chromosome 4q12-q13 (CXC chemokines) (50) and 17q11.2 (CC chemokines) (51), obviously originate from a common ancestral gene and may have evolved
to serve overlapping if not identical functions in vivo.
Even though SLC and ELC are almost equivalent as ligands for CCR7,
there are some notable differences in their tissue patterns of
expression (32, 36). For example, ELC was found to be much more
strongly expressed in the thymus than SLC. Besides mostly overlapping
expression in various lymphoid tissues, SLC but not ELC was also
expressed in heart, pancreas, thyroid gland, etc. Such differences in
tissue distribution may indicate their differential roles in
vivo. Furthermore, as shown for IP-10 and Mig (47), the
inducibility of SLC and ELC may be different in certain in vivo situations. The carboxyl-terminal extension of SLC with two extra cysteine residues may also have unique biological functions in vivo through interaction with other molecules. Besides
CCR7, there may be still other receptors either for SLC or ELC.
Targeted disruption of the SLC and ELC genes may be helpful to
elucidate their respective physiological and pathological roles
in vivo.
CCR7 was originally identified through its strong up-regulation in an
EBV-negative Burkitt's lymphoma cell line upon infection with EBV
(thus originally termed EBI1 from Epstein-Barr virus-induced gene 1)
(42). EBI1/CCR7 was shown to be transactivated by EBV-encoded nuclear
antigen-2 (EBNA-2) (44), and also to be up-regulated in
CD4+ T cells upon infection with HHV6 and HHV7 (45).
EBI1/CCR7 is expressed at high levels in various lymphoid tissues and
on peripheral blood T and B lymphocytes (43). Circulating lymphocytes
emigrate from blood into interfollicular regions of the secondary
lymphoid tissues in a search for antigens presented on the reticular
network of cells. Homing of lymphocytes into specific secondary
lymphoid tissues is regulated by multi-step interactions between
circulating lymphocytes and high endothelial venules (HEV) via specific
combinations of adhesion molecules (52, 53). Several lines of evidence also indicate that G-protein-coupled receptors are essential for lymphocyte homing (54, 55). Chemokines, which elicit integrin activation and diapedesis via respective G-protein-coupled receptors, are thus likely to play important roles in directed migration of
various lymphocyte classes and subsets from blood into and within
lymphoid tissues, but their identity still remains mostly unknown. SLC
and ELC are constitutively expressed in various lymphoid tissues where
lymphocytes expressing CCR7 also exist abundantly (32, 36, 43). Thus,
SLC and ELC may be the ones that are involved in the homeostatic
lymphocyte recirculation and homing. They may also facilitate tissue
accumulation of lymphocytes in various immune responses. Furthermore,
they may affect tissue localization of lymphocytes infected by
lymphotropic herpesviruses such as EBV, HHV6, and HHV7. Thus, SLC, ELC,
and CCR7 may define new targets for drug development aiming at
controlling various immune responses and/or suppressing persistent
infections with certain lymphotropic herpesviruses.
We are grateful for Dr. Yorio Hinuma and Dr.
Masakazu Hatanaka for constant support and encouragement.