(Received for publication, April 4, 1997, and in revised form, May 21, 1997)
From the By searching the Expressed Sequence Tag (EST)
data base, we identified partial cDNA sequences potentially
encoding a novel human CC chemokine. We determined the entire cDNA
sequence which encodes a highly basic polypeptide of 134 amino acids
total with a putative signal peptide of 23 amino acids. The predicted
mature protein of 111 amino acids has the four canonical cysteine
residues and shows 21-33% identity to other human CC chemokines, but
has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed
strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and
purified to homogeneity, was a highly efficient chemoattractant for
certain human T cell lines and a highly potent one for freshly isolated
peripheral blood lymphocytes and cultured normal T cells expanded by
phytohemagglutinin and interleukin 2. Unlike most other CC chemokines,
however, this novel chemokine was not chemotactic for monocytes or
neutrophils, suggesting that it is specific for lymphocytes. From these
results, we designated this novel CC chemokine as SLC from
secondary lymphoid-tissue
chemokine. SLC fused with the secreted form of alkaline
phosphatase (SLC-SEAP) was used to characterize the SLC receptor.
Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC
(IC50, 0.12 nM) but not by any other CC
chemokine so far tested, suggesting that resting lymphocytes express a
class of receptors highly specific for SLC. By using somatic cell
hybrids, radiation hybrids, and selected yeast and bacterial artificial
chromosome clones, we mapped the SLC gene
(SCYA21) at chromosome 9p13 and between chromosomal
markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another
novel CC chemokine termed ELC from EBI1-ligand
chemokine (SCYA19) also exists. Collectively,
SLC is a novel CC chemokine specific for lymphocytes and, together with
ELC, constitutes a new group of chemokines localized at chromosome
9p13.
It is now known that structurally related polypeptides,
collectively called chemokines, play important roles in inflammatory and immunological responses primarily by virtue of their ability to
recruit selective subsets of leukocytes (1, 2). Some chemokines may
also be involved in constitutive migration and homing of lymphocytes
(3, 4). Furthermore, some chemokines have been shown to have other
biological activities such as suppression of hematopoiesis (5-7),
stimulation of angiogenesis (8), suppression of angiogenesis (9, 10),
suppression of apoptosis (11), and suppression of infection of human
immunodeficiency viruses (12-14). The chemokines are grouped into two
major subfamilies from the arrangement of the amino-terminal two of the
four canonical cysteine residues. One amino acid separates the two
cysteines in the CXC chemokines, whereas the two cysteines
are adjacent in the CC chemokines. The CXC chemokine genes
are clustered at chromosome 4q12-q21, whereas the CC chemokine genes at
chromosome 17q11.2. Most CXC chemokines are primarily
chemotactic for neutrophils, whereas most CC chemokines are chemotactic
for monocytes. Furthermore, most CXC and CC chemokines share
receptors with some other members of the respective subfamilies (1, 2).
Thus, the functional redundancy may be more or less the norm of the
chemokine system. In this context, three novel CC chemokines,
TARC,1 LARC, and ELC, that we have recently
identified, are quite unique among the known CC chemokines because they
are all functionally specific for lymphocytes, each interacts with a
class of receptors not shared by any other CC chemokines so far tested,
and their genes are distinctly mapped at chromosomes 16q13, 2q33-q37,
and 9p13, respectively (15-20). Thus, these chemokines may constitute a new category of CC chemokines, each playing a specific role in the
immune system. Furthermore, besides CXC and CC chemokines, new molecules related to the chemokine superfamily have been
identified. Lymphotactin/SCM-1 is a cytokine which carries only the
second and the fourth of the four canonical cysteine residues and seems to act specifically on lymphocytes (21, 22). Fractalkine is a
transmembrane molecule that carries a chemokine-like domain with a
distinct CX3C motif on top of an extended
mucin-like stalk. A soluble form of fractalkine was shown to be
efficiently chemotactic for monocytes and T cells (23).
The data base of expressed sequence tags (EST) consists of partial
single pass cDNA sequences from various tissues (24). Analysis of
the EST data base is becoming a powerful approach to look for new
members of gene families. Recently, we have identified a number of
novel human CC chemokines by initially probing the EST data base with
nucleotide and amino acid sequences of known CC chemokines (16, 18,
25). Here we report another novel human CC chemokine termed SLC from
secondary lymphoid-tissue chemokine which was also initially identified in the EST data base. SLC is
expressed strongly in lymphoid tissues such as lymph nodes, Appendix,
and spleen and is specifically chemotactic for lymphocytes. Freshly
isolated peripheral blood lymphocytes possess a class of receptors
binding SLC with high affinity and specificity. The SLC gene
(SCYA21) is mapped to chromosome 9p13 where the gene for
another novel CC chemokine ELC (SCYA19) also exists
(18).
Human T cell lines, Hut78 and Hut102, and a human
monocytic cell line, THP-1, were cultured in RPMI 1640 supplemented
with 10% fetal calf serum (FCS). 293/EBNA-1 cells were purchased from Invitrogen (San Diego, CA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Sf9 insect cells were maintained at 27 °C in Grace insect medium (Life Technologies, Inc.)
supplemented with 10% FCS. High FiveTM cells were
purchased from Invitrogen and maintained at 27 °C in EX-CELL 400 medium (JRH Bioscience, Lenexa, KS). Peripheral blood mononuclear cells
(PBMC) were isolated from EDTA-treated venous blood obtained from
healthy adult donors by using Ficoll-Paque (Pharmacia Biotech, Uppsala,
Sweden). Granulocytes were isolated from the pellet of Ficoll-Paque
gradient by dextran sedimentation and hypotonic lysis of residual
erythrocytes. Peripheral blood lymphocytes (PBL) (>97% lymphocytes)
were negatively selected by MiniMACS (Milteny Biotec, Bergisch,
Germany) after staining with fluorescein isothiocyanate-conjugated
anti-CD14. In some experiments, T cells were expanded from PBMC by
stimulation with phytohemagglutinin (PHA) (Life Technologies, Inc.) for
2 days and cultivation for another week with a supplement of 400 units/ml IL-2 (Shionogi, Osaka, Japan) (>99% CD3+ T
cells). In some experiments, cells were preincubated with 500 ng/ml
pertussis toxin (PT) (Sigma) at 37 °C for 90 min before assays.
Production and purification of recombinant TARC,
LARC, and eotaxin were described previously (15, 16, 26). MIP-1 The EST data base (24), a division of
GenBankTM, was searched with nucleotide sequences and amino
acid sequences of various CC chemokines as queries using the data base
search and analysis service Search Launcher (27) available on the World
Wide Web. The program used was Basic Local Alignment Search Tool
(BLAST) (28).
By using
primers based on an EST sequence (GenBankTM accession
number W84422), cDNA clones were obtained by the method of rapid
amplification of cDNA ends (RACE) (29). In brief, PCR was performed
with 0.5 ng of human fetal lung cDNA commercially available for
RACE-PCR (CLONTECH, Palo Alto, CA) in 10 µl of
reaction mixture containing 0.2 mM each dNTP, 10 pmol each
of primers (5 The 0.5-kb
BamHI-XbaI fragment of the SLC cDNA
containing the entire coding region was labeled with 32P
using Prime-It II kit (Stratagene, La Jolla, CA) at a specific activity
of 3.1 × 109 cpm/µg. The probe hybridized with a
single band in human genomic DNA digested with EcoRI,
HindIII, or BamHI at high stringency conditions.
Northern blot filters containing human poly(A)+ RNA from
various tissues (2 µg/lane) were purchased from
CLONTECH. Filters were hybridized with the
32P-labeled probe at 42 °C for 16 h in a solution
consisting of 50% formamide, 5 × SSPE, 10 × Denhardt's
solution, 2% SDS, and 100 µg/ml denatured salmon sperm DNA, washed
twice at 42 °C for 30 min in 0.1 × SSC and 0.1% SDS, and
exposed to x-ray films (Eastman Kodak, New Haven, CT) at Recombinant SLC was
produced by using Bac-to-Bac baculovirus expression system (Life
Technologies, Inc.) following the protocol recommended by the
manufacturer. Briefly, the BamHI-XbaI fragment of
the SLC cDNA containing the entire coding region was subcloned into
BamHI-XbaI sites of pFastBac 1 baculovirus
transfer vector in the downstream of the polyhedrin promoter. The
resulting recombinant plasmid termed pFastBac-NCC8.1 was transfected
into Escherichia coli DH10Bac containing shuttle vector
"bacmid" with a mini-attTn7 target site and the helper
plasmid. The mini-Tn7 element on the pFastBac 1 plasmid transposes to
the mini-attTn7 target site on the bacmid in the presence of
transposition proteins provided by the helper plasmid. Insertion of the
mini-Tn7 into the mini-attTn7 target site disrupts the
coding sequence for the LacZ Migration of cells was assessed in a
48-well chemotaxis chamber (Neuroprobe, Cabin John, MD) as described
previously (15). In brief, Hut78, Hut102, THP-1, PBMC, PBL, and
cultured normal T cells expanded by PHA and IL-2 were suspended in RPMI
1640 containing 1% bovine serum albumin and 20 mM HEPES,
pH 7.2, at 2 × 106/ml (Hut78, Hut102, THP-1, PBMC) or
at 4 × 106/ml (PBL, cultured T cells). Granulocytes
were suspended in Hank's balanced salt solution containing 1% bovine
serum albumin at 1 × 106/ml. The lower wells were
filled with 30 µl of assay buffer without or with chemokines, while
the upper wells were filled with 50 µl of cell suspension. The lower
and upper wells were separated by a polycarbonate filter (Neuroprobe)
with 5-µm pore size. Polyvinylpyrrolidone-free filters were used for
assays with neutrophils, PBL, cultured T cells, and T-cell lines,
whereas polyvinylpyrrolidone-treated filters were used for assays with
monocytes (PBMC) and THP-1. In assays using PBL and cultured T cells,
the surface of filters facing the lower wells was precoated with 5 µg/ml fibronectin for 2 h at room temperature and extensively
washed with distilled water. Similarly, in assays using T cell lines,
the surface of filters facing the lower wells was precoated with 5 µg/ml collagen type IV for 2 h at room temperature and
extensively washed with distilled water. Assays were carried out at
37 °C for 1 h with neutrophils, 2 h with PBMC (monocytes)
and THP-1, or 4 h with PBL, cultured normal T cells, and
established T cell lines (Hut78 and Hut102). Filters were washed,
fixed, and stained with Diff-Quick (Harleco, Gibbstown, NJ). The number
of migrated cells in five randomly selected high power (× 400) fields
was counted. All assays were done in triplicate.
Cells were incubated at 37 °C
for 1 h with 2 µM Fura-2/AM (Molecular Probe,
Eugene, OR) at 1 × 106/ml in RPMI 1640 containing 1%
FCS and 20 mM HEPES, pH 7.2, in the dark. Cells were washed
three times and resuspended at 1.25 × 106/ml in
phosphate-buffered saline containing 1% FCS, 50 mM
CaCl2, and 50 mM MgCl2. Two ml of
cell suspension was placed in a cuvette and set into a luminescence
spectrometer (LS50B, Perkin-Elmer, Norwalk, CT) with constant stirring.
Emission fluorescent intensity at the wavelength of 520 nm was measured
upon excitation at 340 nm and 380 nm with a time resolution of 2.5 or 5 points/s. Data were presented by the ratio of
F340 divided by F380
(R340/380). After each measurement,
R340/380 at the presence and absence of excess
calcium was determined by the sequential addition of 10 µl of 10%
Triton X-100 and 120 µl of 0.5 M EGTA.
This was carried out essentially as
described previously (16). In brief, to produce a soluble fusion
protein of SLC with the secreted form of placental alkaline phosphatase
(SEAP) with the carboxyl-terminal histidine tag, the SLC cDNA was
amplified by PCR using the 5 DNA samples were prepared from a panel of human × rodent somatic cell hybrids containing human monochromosomes (National
Institute of General Medical Science Mapping Panel No. 2, version 2, Coriell Cell Repositories, Camden, NJ), from 93 radiation hybrids of
the GeneBridge 4 Mapping Panel (30) (Research Genetics, Huntsville, AL), and from 83 radiation hybrids of the Stanford Human Genome Center
G3 RH panel (31) (Research Genetics, Huntsville, AL). To determine the
SLC gene locus, these DNA samples were analyzed by PCR using
SLC primers (+5 To find new members of the human CC
chemokine family, we searched the EST data base (24) with amino acid
sequences and nucleotide sequences of various CC chemokines as probes.
We have found a number of EST sequences derived from a single species
of cDNA encoding a novel CC chemokine (GenBankTM
accession numbers W17274, W67885, W84375, W84422, T25128, W67812,
AA027314, AA027315, AA149456, and AA151607) (Fig. 1). To
determine the full-length cDNA, we carried out the 5
The deduced polypeptide sequence contains a hydrophobic amino-terminal
region characteristic of a signal peptide with a putative cleavage site
between Gly-23 and Ser-24 (Fig. 2A) (34). The calculated
molecular weight and pI of the predicted mature protein are 12,237 and
10.72, respectively. The polypeptide contains no putative
N-glycosylation site. As shown in Fig. 2B, the
mature protein (indicated as SLC, see below) shows 21-33% identities with other CC chemokines and retains four properly placed cysteine residues as well as certain amino acid residues highly conserved among
the CC chemokines such as Pro-41, Tyr-48, Phe-64, Trp-82, and Val-83.
However, the polypeptide has a carboxyl-terminal extension of about 30 amino acids in comparison with other CC chemokines. This region, which
is highly rich in basic amino acids and contains two cysteine residues,
has no homologous sequences in the GenBankTM data base.
We examined constitutive
expression of this novel CC chemokine in various human tissues by
Northern blot analysis (Fig. 3). Consistent with the
size of the cDNA plus poly(A) tail, a single species of transcripts
of about 0.9 kb was detected strongly in lymph nodes, intermediately in
spleen, small intestine, thyroid gland, and trachea, and weakly in
heart, pancreas, thymus, and colon. When the mRNA expression in
various lymphoid tissues was examined further, the transcripts were
present at high levels in lymph nodes and Appendix, at intermediate
levels in spleen, and at low levels in thymus. The expression was very
low in bone marrow and fetal liver and virtually negative in peripheral
blood leukocytes. From this pattern of tissue expression, we designated this novel CC chemokine as SLC from secondary
lymphoid-tissue chemokine.
To obtain the SLC
protein, High FiveTM insect cells (Invitrogen) were
infected with a recombinant baculovirus encoding SLC under the control
of the polyhedrin promoter. Recombinant SLC was purified from pooled
culture supernatants by cation-exchange FPLC and reverse-phase high
performance liquid chromatography. SLC was eluted from the reverse-phase column as a single major peak at the acetonitrile concentration of 28% (Fig. 4A). The purified
SLC migrated as a single band of about 15 kDa on SDS-polyacrylamide gel
electrophoresis (Fig. 4B). Amino acid sequence analysis
demonstrated that the amino terminus of mature SLC started at Ser-24 as
predicted (data not shown). The final yield of SLC was ~0.8 µg/ml
starting culture supernatants.
Previously, we showed that two
human T cell lines, Hut78 and Hut102, responded well to a
T-cell-directed CC chemokine TARC in the chemotaxis assay (15). Because
SLC was expressed most abundantly in lymph nodes (Fig. 3), SLC might be
chemotactic for T cells. Therefore, we first tested the chemotactic
activity of SLC using Hut78 and Hut102. As shown in Fig.
5A, SLC induced vigorous cell migration in
both types of cells with a maximal chemotactic index (the ratio of the
number of migrated cells toward a chemotactic factor divided by the
number of cells migrated toward control medium) of >100 at 10-100
nM. A checkerboard-type analysis revealed that the
migration of the responder cells toward SLC was chemotactic and not
chemokinetic (Fig. 5B). Furthermore, PT completely inhibited the migration of the cells toward SLC (Fig. 5C), suggesting
that the SLC receptor is coupled with a trimeric G-protein of the
Gi class.
We next tested chemotactic activity of SLC on peripheral blood
leukocyte subsets (Fig. 6). Freshly isolated PBL showed
a vigorous migration toward SLC with a maximal chemotactic index of
3.7 ± 0.5 (mean ± S.E., n = 3) at 0.1 nM. On the other hand, SLC induced no significant migration
in neutrophils, monocytes, or a monocytic cell line THP-1 within a dose
range from 0.01-100 nM. These cells, however, vigorously
responded to respective positive controls, MCP-1 and IL-8. SLC also
induced vigorous chemotactic responses in cultured normal T cells
expanded with PHA + IL-2 (>99% CD3+ T cells) with a
maximal chemotactic index of 4.5 ± 0.4 (n = 3) at
0.1 nM.
We next examined induction
of calcium flux by SLC. Cells were loaded with Fura-2, a calcium
indicator, and the intracellular calcium concentration (presented by
R340/380) was measured before and after addition
of SLC. Fresh PBL did not show detectable levels of calcium
mobilization in response to SLC (not shown). On the other hand,
cultured normal T cells expanded with PHA + IL-2 responded vigorously
to SLC with an EC50 of 1 nM (Fig.
7A). SLC, but not RANTES or MCP-1,
desensitized cultured T cells to a subsequent stimulation with SLC
(Fig. 7B). SLC did not induce calcium flux in neutrophils or
monocytes although these types of cells responded vigorously to
subsequent treatment with IL-8 or MCP-1, respectively (data not
shown).
Previously, we have
characterized specific receptors for novel chemokines, TARC, LARC, and
ELC, by using chemokines fused with the secreted form of alkaline
phosphatase with the (His)6 tag (SEAP) (18-20). To prepare
labeled SLC convenient for binding experiments, we employed the same
strategy and prepared SLC-SEAP. The alkaline phosphatase allowed
quantitative determination of specific binding whereas the
(His)6 tag was useful for one-step affinity purification
with a nickel agarose column. In displacement-type experiments where
fresh PBL were incubated with a fixed concentration of SLC-SEAP and
increasing concentrations of unlabeled SLC, SLC competed with SLC-SEAP
with an IC50 of 0.12 nM (Fig.
8A). In heterologous competition experiments
where fresh PBL were incubated with a fixed concentration of SLC-SEAP
in the presence of indicated unlabeled chemokines at 200 nM, only SLC, but not any other CC chemokines so far
tested, inhibited the binding of SLC-SEAP, indicating that resting PBL
express a class of receptors highly specific for SLC (Fig.
8B).
To determine the
chromosomal localization of the SLC gene, we examined a
panel of somatic cell hybrids each containing a single human chromosome
by PCR using SLC-specific primers. The results showed that the gene was
present on chromosome 9 (Fig. 9A). A similar
PCR analysis using GeneBridge 4 radiation hybrids further placed the
SLC gene near the chromosomal markers D9S1978(WI-8765) and
AFM326vd1 that are located at 9p13 (data not shown). Consistently, the
analysis on the Stanford G3 panel localized the SLC gene
between markers NIB64 and D9S1978 (data not shown). Previously, we have mapped the gene for another novel CC chemokine ELC at the same locus
(18). Therefore, we examined mega-YAC clones constituting a contig
containing these markers for the presence of the SLC and
ELC genes by PCR. Fig. 9B schematically shows the
marker contents of each YAC clone. The SLC and
ELC genes were found to exist within 1 megabase pair. To
further narrow down the region, we examined BAC clones with ~120-kb
inserts derived from this region by PCR and identified one clone,
235d21, that contains both the SLC and ELC genes.
Thus, the two genes are closely linked, probably within 100 kb.
In the present study, we have described a novel human CC chemokine
designated as SLC from secondary
lymphoid-tissue chemokine, which was originally
identified in the EST data base (Fig. 1). Even though SLC shows
relatively low homologies to other CC chemokines (21-33% identity),
SLC retains a number of amino acid residues conserved in most CC
chemokines in addition to the four properly spaced cysteine residues
(Fig. 2). However, in comparison with other CC chemokines, SLC has a
carboxyl-terminal extension of about 30 amino acids which is
characterized by an extra pair of cysteine residues and a high content
of basic amino acids (Fig. 2). The murine CC chemokine JE is also known
to have a carboxyl terminus extended by about 50 amino acids (35).
However, the carboxyl-terminal extension of JE is characterized by a
high content of serine/threonine and has no homology with that of
SLC.
SLC is expressed constitutively at high levels in lymphoid tissues such
as lymph nodes, Appendix, and spleen (Fig. 3). The recombinant SLC was
a highly efficient chemotactic factor for two human T cell lines, Hut78
and Hut102, and a highly potent one for resting PBL and cultured normal
T cells expanded with PHA + IL-2 (Figs. 5 and 6). SLC was, however, not
chemotactic for neutrophils, monocytes, or a monocytic cell line THP-1.
SLC also induced vigorous transient calcium mobilization in cultured normal T cells expanded with PHA + IL-2 (Fig. 7). Thus, SLC appears to
act selectively on lymphocytes and especially T cells. Consistent with
its activity on lymphocytes, SLC binds to fresh PBL with a high
affinity (Fig. 8). Importantly, the class of receptors expressed on
fresh PBL is highly specific for SLC and not shared by any other CC
chemokines tested so far (Fig. 8).
In contrast to fresh PBL, cultured normal T cells expanded with PHA + IL-2 did not show a typical bell-shaped dose-response curve but showed
a broad peak in the chemotaxis assay (Fig. 6). A similar tendency was
also seen in the chemotactic responses of established T cell lines,
Hut78 and Hut102 (Fig. 5A). Furthermore, SLC was much more
potent for fresh PBL and cultured normal T cells than for Hut78 and
Hut102 (compare Figs. 5 and 6). These discrepancies may indicate that
there are at least two (and even more) classes of receptors for SLC
with distinct affinities. Thus, fresh PBL may express mainly the high
affinity class of receptors for SLC; normal T cells expanded with PHA + IL-2 may express both the high and low affinity classes of receptors
for SLC; and Hut78 and Hut102 may express mainly the low affinity class
of receptors for SLC. Identification of the SLC receptors will help us
examine such possibilities.
The SLC gene (SCYA21) has been localized at human
chromosome 9p13 and between markers D9S1978(WI-8765) and AFM326vd1
(Fig. 9). This is striking because most other CC chemokine genes are known to cluster at chromosome 17q11.2 (25). However, we have recently
identified three novel CC chemokines, TARC, LARC, and ELC, whose genes,
SCYA17, SCYA20, and SCYA19,
respectively, have been localized distinctly at chromosomes 16q13,
2q33-37, and 9p13, respectively (15-20). In fact, the SLC
and ELC genes have been localized at the same chromosomal
locus and within a region of about 100 kb (Fig. 9). Thus, they may
represent a new cluster of chemokine genes present at chromosome
9p13.
Thus far, we have identified four novel CC chemokines, TARC, LARC, ELC,
and SLC that appear to act specifically on lymphoid cells. TARC is
expressed mainly in the thymus and acts selectively on T cells of the
CD4+ lineage especially via its specific receptor CCR4 (15,
20). LARC is expressed mainly in the liver and lung and acts
selectively on lymphocytes via its specific receptor CCR6 which is
expressed on both T and B cells (16, 19). ELC is expressed strongly in
most lymphoid tissues such as thymus, lymph nodes, and spleen and
functions via its specific receptor CCR7/EBI1 which is known to be
expressed on activated T and B cells (18). SLC is thus another
lymphocyte-specific CC chemokine mainly expressed in secondary lymphoid
tissues such as lymph nodes, Appendix, and spleen and acts via a class
of receptors not shared by any other CC chemokines so far tested. Our
recent experiments have shown that SLC acts on both T cells and B
cells. Identification of the SLC receptors will help us define the
exact types of cells responding to SLC.
Even though SLC and ELC are only weakly homologous (32% identity), the
presence of their genes at the same chromosomal locus might suggest
their functional relationship. In this study, however, the limited
supply of recombinant ELC protein (18) precluded experiments such as
cross-competition and desensitization between SLC and ELC. Thus, the
functional relationship between SLC and ELC remains to be seen.
Collectively, it is tempting to speculate that TARC, LARC, ELC, and SLC
represent a new category of CC chemokines that play specific roles in
the immune system most probably by regulating migration and homing of
particular subsets of lymphocytes within particular lymphoid tissue
microenvironments, since selective migration and homing of lymphocytes
into and/or within lymphoid tissues have been shown to be highly
sensitive to pertussis toxin (36, 37) and likely to be regulated by
chemokines (3, 4).
We are grateful to Drs. Yorio Hinuma,
Masakazu Hatanaka, and Retsu Miura for constant support and
encouragement.
Shionogi Institute for Medical Science,
Laboratory of Genetic Resources,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cells
,
MIP-1
, MCP-1, MCP-2, MCP-3, RANTES, IL-8, and lymphotactin were
purchased from Peprotech (Rocky Hill, NJ).
-RACE primer, 5
-CCTTCTTGCATCTTGGGTTCAGGCTTC; 3
-RACE
primer, 5
-GAAGCCTGAACCAAGATGCAAGAAGG; AP1 primer
(CLONTECH) which is complementary to part of the
cDNA adaptor ligated at both ends of the cDNA), 2.5 units of
TAKARA LA Taq (Takara, Kyoto, Japan), 1 × buffer
supplied with the polymerase, and 0.55 µg of TaqStart antibody
(CLONTECH). The PCR conditions were 94 °C for 1 min, 5 cycles of 94 °C for 30 s and 72 °C for 2 min, 5 cycles of 94 °C for 30 s and 70 °C for 2 min, and 25 cycles
of 94 °C for 30 s and 68 °C for 2 min. The amplification products were cloned into pCR-II vector (Invitrogen) by T-A ligation and sequenced on both strands by using gene-specific primers and commercially available primers.
80 °C
with an intensifying screen for 8, 24, 48, and 96 h.
peptide, making colonies containing
recombinant bacmids white in the presence of 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside. The resulting recombinant bacmid
was transfected into Sf9 cells using Cellfectin reagent (Life
Technologies, Inc.), and the recombinant viruses were obtained. High
FiveTM cells (Invitrogen) were infected with the
recombinant viruses at multiplicity of infection of 10-20. The culture
supernatants collected 2 days after infection were mixed with 0.1 volume of 500 mM MES, pH 6.5, and applied to 1-ml
cation-exchange Hitrap-S column (Pharmacia) equilibrated with 50 mM MES, pH 6.5, and 100 mM NaCl. The column on
the FPLC system (Pharmacia) was eluted with 45 ml of a linear gradient
of 0.1-1.0 M NaCl in 50 mM MES at a rate of 1 ml/min. The fractions containing recombinant SLC were pooled and
injected into a reverse-phase high performance liquid chromatography
column (4.6 × 250 mm Cosmocil 5C4-AR-300, Cosmo Bio, Tokyo,
Japan) equilibrated with 0.05% trifluoroacetic acid. The column was
eluted with 60 ml of a linear gradient of 0-60% acetonitrile in
0.05% trifluoroacetic acid at a flow rate of 1 ml/min. Fractions
containing recombinant SLC were pooled and lyophilized. Protein
concentrations were determined by BCA kit (Pierce). Endotoxin levels,
which were determined by the Limulus amoebocyte lysate assay
(QCL-1000, BioWhittaker, Walkersville, MD), were always <4 pg/µg of
recombinant SLC. The amino-terminal sequence analysis was done on a
protein sequencer (Shimazu, Tokyo, Japan).
-SalI-SLC primer
(5
-GCCGTCGACACAGACATGGCTCAGTCACTGGCT-3
) and
3
-SLC-XbaI primer
(5
-GCCTCTAGATGGCCCTTTAGGGGTCTGTGACCG-3
), digested with
SalI and XbaI, subcloned into
SalI-XbaI sites of the
pDREF-SEAP(His)6 vector (16) to make pDREF-SLC-AP.
293/EBNA-1 cells were transfected with pDREF-SLC-AP using
LipofectAMINETM (Life Technologies, Inc.). After 96 h,
the supernatants were centrifuged, filtered (0.45 µm), and stored at
4 °C with 20 mM HEPES, pH 7.4, and 0.02% sodium azide.
Alkaline phosphatase (AP) activity was measured by using Great EscApe
Detection Kit (CLONTECH) and expressed with
relative light units/s. In binding assays, PBL (2 × 105) were incubated with SLC-SEAP (4 × 106 relative light units/s) in the absence or presence of
increasing concentrations of SLC or 200 nM of various
chemokines at 16 °C for 1 h in 200 µl of RPMI 1640 containing
1% bovine serum albumin, 20 mM HEPES, pH 7.2, and 0.02%
sodium azide. After washing five times, cells were lysed in 50 µl of
1% Triton X-100 in 10 mM Tris-HCl, pH 8.0. Lysates were
heated at 65 °C for 10 min to inactivate cellular phosphatases.
After centrifugation, SEAP activity in 25 µl of the lysate was
determined by the chemiluminescent assay as described above. All assays
were done in duplicate.
-GCCTTGCCACACTCTTTCTC and
5
-CAAGGAAGAGGTGGGGTGTA). The PCR conditions were 35 cycles of 95 °C for 30 s, 65 °C
for 30 s, and 72 °C for 1 min. PCR products were
electrophoresed on 2% agarose gel. The SLC product was 218 bp in
length.2 Four yeast artificial chromosome
(YAC) clones of a contig containing a chromosome marker AFM326vd1 were
purchased from Research Genetics (Huntsville, AL), and their DNAs were
prepared as described previously (25). Using these DNAs, PCR was
carried out with the SLC primers and ELC primers (18). Furthermore,
previously selected bacterial artificial chromosome (BAC) clones
(Research Genetics) containing the DNA fragments of human chromosome
9p133 were examined by PCR using the SLC and
ELC primers. Primer sequences for AFM326vd1 and the nearby markers were
taken from the Whitehead Institute for Biomedical Research/MIT YAC data
base.
Cloning of SLC cDNA
- and 3
-RACE
using primers designed from W84422 and human fetal cDNA
commercially available for RACE-PCR (CLONTECH) as
templates. The 5
-RACE further extended 53 nucleotides beyond the 5
end of the overlapping ESTs. Sequence comparison of the RACE fragments with the ESTs showed no misincorporation of nucleotides during the
RACE-PCR. The full-length cDNA is 852 bp with an open reading frame
of 402 bp starting with the first methionine and encoding a polypeptide
of 134 amino acid residues (Fig. 2A). The
nucleotide sequence flanking the first methionine codon conforms well
to Kozak's rule (32). The 3
-noncoding sequence contains a canonical polyadenylation signal (AATAAA) and a single copy of ATTTA that may be
an mRNA destabilization signal (33).
Fig. 1.
Alignment of the SLC cDNAs and ESTs.
Below the SLC mRNA are the 5- and 3
-RACE cDNAs with the
gene-specific primers indicated by closed boxes. The
arrows indicate the positions, lengths, and orientations of
the ESTs. The GenBankTM accession number and tissue origin
of each EST are also indicated.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Nucleotide and deduced amino acid sequences
of the full-length SLC cDNA. A, the cDNA and deduced
amino acid sequences. The arrow indicates the predicted
cleavage site of the signal sequence. A potential mRNA
destabilization element is indicated by dots. The
polyadenylation signal is underlined. B, amino
acid alignment of SLC with other human CC chemokines. The mature form of SLC and 14 other human CC chemokines in Swiss-Prot were aligned using the Clustal W program with a BLOSUM residue weigh table and
default settings of gap penalties for pairwise and multiple alignments.
Cysteine residues are marked with stars, and 100% conserved
residues are boxed. Percent identity to SLC is indicated on
the right.
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
Expression of SLC mRNA in various human
tissues. Multiple tissue blots and immune blots obtained from
CLONTECH were probed with the
32P-labeled SLC cDNA.
[View Larger Version of this Image (49K GIF file)]
Fig. 4.
Purification of recombinant SLC.
A, the elution profile of SLC by reverse-phase high
performance liquid chromatography. Pooled culture supernatants of
insect cells infected with the SLC recombinant virus were loaded onto
cation-exchange Hitrap-S and eluted with a gradient of NaCl. The
fractions containing SLC were pooled and loaded onto Cosmocil
5C4-AR-300 and eluted with a gradient of acetonitrile. SLC was eluted
as indicated by the arrow. B, gel electrophoresis
of purified SLC. Purified SLC was electrophoresed on a 15-25%
gradient SDS-polyacrylamide gel and visualized by silver staining.
Positions of the size markers are shown on the right
(kDa).
[View Larger Version of this Image (13K GIF file)]
Fig. 5.
Chemotactic response of human T cell lines to
SLC. A, dose-response experiments. Hut78 (closed
circles) and Hut102 (closed squares) were stimulated
with indicated concentrations of SLC in a 48-well chemotaxis chamber.
The assay was done in triplicate, and the number of migrated cells in
five high-power (× 400) fields (hpfs) was counted for each
well. Each point represents mean ± S.E. from three separate
experiments. B, a checkerboard-type analysis. In chemotaxis
assay using Hut78 cells, SLC was added to upper and/or lower wells at
10 nM as indicated. The assay was done in triplicate. The
data shown are representative of two separate experiments.
C, effect of pertussis toxin. Hut78 and Hut102 cells were
pretreated without or with 500 ng/ml PT at 37 °C for 90 min. The
chemotactic assay was carried out as above using 10 nM SLC. Data are representative of two separate experiments.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Chemotactic response of peripheral blood
leukocyte subsets to SLC. Indicated cells were stimulated without
(closed squares) or with SLC (closed circles) or
control chemokines (closed diamonds, closed inverted
triangles, or closed triangles) at indicated concentrations in the chemotaxis assay. All assays were done in triplicate. Each point represents mean ± S.E. of three separate experiments. Controls are IL-8 (closed diamonds) for
neutrophils, MCP-1 (closed inverted triangles) for monocytes
and THP-1, and RANTES (closed triangles) and MCP-1
(closed inverted triangles) for cultured T cells.
[View Larger Version of this Image (17K GIF file)]
Fig. 7.
Calcium mobilization in cultured T cells by
SLC. A, dose-response experiments. Cultured normal T cells
expanded with PHA + IL-2 were loaded with Fura-2/AM and stimulated with
SLC as indicated by the arrowheads. The results shown are
representative of three separate experiments. The scale (0.2) is shown
as a bar on the right. B,
desensitization experiments. Cultured T cells were loaded with
Fura-2/AM and stimulated with chemokines as indicated by the
arrowheads. The results shown are representative of three separate experiments. The scale (0.2) is shown as a bar on
the right.
[View Larger Version of this Image (11K GIF file)]
Fig. 8.
Specific binding of SLC-SEAP to freshly
isolated PBL. A, PBL (2 × 105 cells) were
incubated with SLC-SEAP (4 × 106 relative light
units/s) in the absence or presence of increasing concentrations of SLC
at 16 °C for 1 h. After washing, amounts of cell-bound SLC-SEAP
were determined enzymatically. Assays were done in duplicate. The
results shown are representative of four separate experiments. The
calculated IC50 is 0.12 nM. B, PBL
(2 × 105 cells) were incubated with SLC-SEAP (4 × 106 relative light units/s) in the absence or presence
of indicated chemokines at 200 nM at 16 °C for 1 h.
After washing, amounts of cell-bound SLC-SEAP were determined
enzymatically. Each histogram represents mean ± S.E. of four
separate experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 9.
Chromosomal localization of the
SLC and ELC genes. A, PCR analysis
of a DNA panel of somatic cell hybrids, each containing a single unique
human chromosome, for the presence of the SLC gene.
Lanes are labeled 1-22, X, and
Y to indicate the human chromosome retained in each hybrid.
DNA controls from human (H), Chinese hamster (C),
and mouse (M), negative control without DNA (N), and positions of size markers (S) are indicated.
B, the YAC and BAC clones derived from the p13 region of
human chromosome 9 are shown schematically on the right of
chromosome 9 idiogram. Closed circles indicate the presence
of chromosomal markers and chemokine genes. The distances between the
markers are not known.
[View Larger Version of this Image (40K GIF file)]
*
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must therefore be hereby marked
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To whom correspondence should be addressed. Tel.:
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1
The abbreviations and other trivial names used
are: TARC, thymus and activation-regulated chemokine; LARC, liver and
activation-regulated chemokine; ELC, EBI1-ligand chemokine; SCM-1,
single C motif 1; EST, expressed sequence tags; SLC, secondary
lymphoid-tissue chemokine; FCS, fetal calf serum; PBMC, peripheral
blood mononuclear cells; PBL, peripheral blood lymphocytes; PHA,
phytohemagglutinin; IL, interleukin; PT, pertussis toxin; MIP,
macrophage inflammatory protein; MCP, monocyte chemoattractant protein;
RANTES, regulated upon activation, normal T cell-expressed and
secreted; RACE, rapid amplification of cDNA end; PCR, polymerase
chain reaction; SEAP, secreted form of alkaline phosphatase; YAC, yeast
artificial chromosome; BAC, bacterial artificial chromosome; G-protein,
heterotrimeric guanine nucleotide-binding regulatory protein; CCR, CC
chemokine receptor; EBI1, Epstein-Barr virus-induced gene 1; kb,
kilobase(s); bp, base pair(s); MES, 2-morpholinoethanesulfonic acid;
FPLC, fast protein liquid chromatography; contig, group of overlapping clones; AP, alkaline phosphatase.
2
The radiation hybrid mapping data were analyzed
by accessing the server at
http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl and
http://www-shgc.stanford.edu/, respectively.
3
M. Ridanpää, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.