A 1.6-kilobase pair cDNA was isolated from a
human T-cell-derived expression library that encodes a novel eosinophil
chemoattractant (designated ecalectin) expressed during allergic and
parasitic responses. Based on its deduced amino acid sequence,
ecalectin is a 36-kDa protein consisting of 323 amino acids. Although
ecalectin lacks a hydrophobic signal peptide, it is secreted from
mammalian cells. Ecalectin is not related to any known cytokine or
chemokine but rather is a variant of human galectin-9, a member of the
large family of animal lectins that have affinity for
-galactosides. Recombinant ecalectin, expressed in COS cells and insect cells, exhibited potent eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo in a
dose-dependent manner but not neutrophils, lymphocytes, or
monocytes. The finding that the ecalectin transcript is present in
abundance in various lymphatic tissues and that its expression
increases substantially in antigen-activated peripheral blood
mononuclear cells suggests that ecalectin is an important
T-cell-derived regulator of eosinophil recruitment in tissues during
inflammatory reactions. We believe that this is the first report of the
expression of an immunoregulatory galectin expressed by a T-cell line
that is selective for eosinophils.
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INTRODUCTION |
Eosinophil accumulation is a common feature of many inflammatory
diseases such as bronchial asthma, allergic rhinitis, helminth infection, and atopic dermatitis (1). Although eosinophils are
proinflammatory cells that normally reside in the circulation, they are
induced to extravasate into tissues by a diverse array of
chemoattractants and viability-enhancing factors. The eotaxins (2-8)
are the most specific of the known peptide eosinophil chemoattractants (ECA),1 but
granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2,
IL-3, IL-5, IL-8, IL-16, RANTES, macrophage inflammatory protein
1
, monocyte chemotactic peptide (MCP)-2, MCP-3, MCP-4, complement
component 3a (C3a), and C5a also can regulate the chemotaxis, viability, growth, differentiation, and/or activation of certain populations of eosinophils (9-18, 20, 22-25). Platelet-activating factor (26) and 5-lipoxygenase metabolites of arachidonic acid (27-29)
are lipid mediators with potent chemotactic activity for eosinophils.
Because these regulatory factors often work in concert, an eosinophil
generally has to be cytokine-primed to respond to an ECA.
Increased numbers of eosinophils in tissues that contain
antigen-activated T-cells have been observed by several groups
(30-34). However, it has been assumed that T-cells do not directly
recruit eosinophils into tissues because they do not produce
substantial amounts of an eotaxin. Most ECA are positively charged
proteins that are small in size. For example, human eotaxin-1 consists of only 74 amino acids, yet 16 of them are either Lys or Arg (5, 6). We
previously noted that concanavalin A-activated or antigen-activated CD4+ T-cells from patients infected with Schistosoma
mansoni (35) produce an apparently novel 30-40-kDa ECA with a
relatively neutral isoelectric point. The virus-transformed human
T-cell line STO-2 also appears to spontaneously produce this ECA (36).
Using blocking antibodies, we concluded that the T-cell-derived ECA was
not IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor. We now describe the purification, molecular cloning, and functional expression of ecalectin, a novel ECA produced by T-cells. Ecalectin is
a variant of a protein, designated human galectin-9 (37), of unknown
function recently found in the tumor of a patient with Hodgkin's
lymphoma.
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EXPERIMENTAL PROCEDURES |
Partial Purification of Ecalectin and Generation of
Ecalectin-specific Antibodies--
The STO-2 cell line was previously
derived by transformation of normal human T-cells with human T-cell
lymphoma-leukemia virus (35). Fluorescence-activated cell sorting
analysis revealed that the cell line is of T-cell origin. It expresses
CD2, CD3, CD4, CD5, and CD8 but lacks granulocyte/macrophage and B-cell markers such as CD16, CD19, and Leu7. Supernatants from STO-2 cells
(2 × 10
5/ml) that had been cultured for ~72 h in
SF-02 serum-free medium (Sanko-Junnyaku Co., Tokyo, Japan) supplemented
with 0.1% human serum albumin, 100 units/ml IL-2 (Tokushima Research
Institute, Tokushima, Japan), 50 µM 2-mercaptoethanol, 100 µg/ml
streptomycin, 100 units/ml penicillin, and 5 µg/ml fungizone were
adjusted to pH 5.0 with 50 mM sodium acetate buffer. The
resulting conditioned medium (5 liters) was applied to replicate
2.6 × 40-cm columns of CM-Sepharose (Amersham Pharmacia Biotech)
that had been equilibrated with 50 mM sodium acetate, pH
5.0. Each ion exchange column was washed extensively with equilibration
buffer to remove the major albumin contaminate in the conditioned
medium. Bound ecalectin was eluted by exposing the column to 20 mM sodium phosphate and 0.1 M NaCl, pH 7.0. After dialysis of the pooled eluate (~300 ml) against 1% glycine,
the material was subjected to preparative isoelectric focusing. Those
40-ml fractions having proteins with a pI of 7-8 were collected on a
Rotofor system (Bio-Rad). The ecalectin-enriched material was then
concentrated ~10-fold in an ultrafiltration apparatus with a YM-5
membrane (Amicon Corp., Lexington, MA). After the material was dialyzed
against phosphate-buffered saline (PBS), it was fractionated further on
a 1 × 30-cm column of Superose-12 (Amersham Pharmacia Biotech)
equilibrated with the same buffer. Based on a calibration of the gel
filtration column, most of the ECA activity was 30-40 kDa. The
ecalectin-enriched fractions were precipitated with 60% saturated
ammonium sulfate, dissolved in 20 mM sodium phosphate (pH
7.0) containing 30% ammonium sulfate, and applied to a 4.6 mm × 7.5-cm reverse phase column of TSKgel phenyl-5PW (Tosoh Co., Tokyo,
Japan) equilibrated in the same buffer. The column was subjected to a
linear gradient, decreasing the ammonium sulfate concentration in the
buffer from 30 to 0%. SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis of a portion of the final biologically active
fraction failed to reveal a Coomassie Blue-stainable protein of a
defined molecular weight. Nevertheless, New Zealand White rabbits were
immunized with the resulting partially purified ecalectin in Freund's
complete adjuvant, and after booster injections in Freund's incomplete adjuvant, sera were collected.
Once the ecalectin cDNA described below was isolated and its
amino acid sequence was deduced, a more specific antibody was generated
in rabbits using an anti-peptide approach. A 13-mer peptide
(AFSSSQAPYLSPA) corresponding to residues 2-14 of human ecalectin was
synthesized by Peptide Inc. (Osaka, Japan). A Cys residue was then
added to the C terminus of the ecalectin peptide to facilitate the
coupling of the resulting 14-mer peptide through its thiol group to
m-maleimidobenzoic acid N-hydroxysuccinimide ester-activated keyhole limpet hemocyanin (Sigma). A rabbit was immunized with the coupled peptide, sera were collected, and the relevant antibodies were purified using an affinity column consisting of the same peptide coupled to 2-fluoro-1-methylpyridinium toluene 4-sulfonate cellufine (Seikagaku Corp., Tokyo, Japan).
Construction and Screening of a cDNA Library from a T-cell
Line--
A STO-2 cell cDNA expression library was constructed
with the ZAP ExpressTM cDNA Gigapack cloning kit
(Stratagene, La Jolla, CA) according to the manufacturer's
instructions. In brief, ~5 µg of poly(A)+ mRNA was
isolated from 1 × 108 STO-2 cells with the FastTrack
RNA isolation kit from Invitrogen (San Diego, CA). The first DNA strand
was synthesized with Moloney murine leukemia virus reverse
transcriptase and a 50-mer oligo(dT) primer
(5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3') containing
an internal XhoI site. Synthesis was carried out in the
presence of 5-methyl-CTP to methylate internal XhoI sites in
the cDNAs, and then EcoRI adaptors were ligated onto the
5'-ends of the resulting cDNAs. After digestion with
XhoI (Stratagene), the cDNAs were size-fractionated on a
1-ml Sephacryl S-500 HR (Life Technologies, Inc.) gel filtration column
that had been equilibrated in 0.1 mM EDTA, 25 mM NaCl, and 10 mM Tris-HCl, pH 7.5. cDNAs
of >500 base pairs were pooled, directionally cloned into the
bacteriophage expression vector, and then packaged into phage particles
with Gigapack III Gold extract. Escherichia coli (strain
XL-1 Blue MRF') was transfected with the resulting phage to obtain a
cDNA library with a titer of ~1 × 1011
plaque-forming units/ml. The STO-2 cell cDNA library was then probed with the picoBlueTM immunoscreening kit from
Stratagene and the newly generated antibodies (~10 ng/ml) directed
against human ecalectin.
Generation and Purification of Recombinant Ecalectin--
The
ExAssist/XLOLR system (Stratagene) was used for the cloning and
expression of ecalectin in COS-1 cells because it allows for efficient
excision of pBK-CMV phagemid from the ZAP express vector. With the use
of plasmid miniprep isolation kits from Qiagen (Santa Clara, CA),
plasmid DNA was purified from the clones selected by the
immunoscreening procedure. To determine which immunoreactive clones
actually encoded a functional ECA, in each instance ~5 µg of
purified cDNA were transfected into COS cells via the DEAE-dextran method (38) or the SuperFect method employing a polycationic reagent
from Qiagen. Transfectants were cultured in RPMI 1640 medium
supplemented with 10% (FCS) and 50 µM 2-mercaptoethanol at 37 °C
for 3 days, and then the resulting supernatants were examined as
described above for ECA activity. For a negative control, replicate COS
cells were transfected in a similar fashion with a pCMV
-gal
cDNA (Stratagene). The ecalectin cDNAs, isolated from the three
immunoreactive clones that exhibited ECA activity in the in
vitro assay described below, were sequenced in both directions on
an Applied Biosystems automated DNA sequencer as described by Sanger
and co-workers (39) using vector-specific primers and newly created
oligonucleotides corresponding to internal nucleotide sequences in the
ecalectin cDNA.
Recombinant human ecalectin was also expressed in
Spondoptera frugiperda 9 (Sf9) insect
cells using the approach we previously used to generate a recombinant
tryptase zymogen (40). Briefly, the ecalectin cDNA was liberated
from pBK-CMV by digesting the mammalian cell expression plasmid with
EcoRI and NotI. The resulting cDNA was then
inserted into the corresponding restriction sites of pVL1393
(PharMingen, San Diego, CA) downstream of the promoter of the
polyhedrin gene. Plasmid DNA, purified by CsCl density gradient
centrifugation, was mixed with linearized BaculoGoldTM DNA
(PharMingen) and calcium phosphate, and the resulting DNA solution was
added to Sf9 insect cells. The recombinant baculovirus that
contained the ecalectin cDNA was identified by a plaque assay, amplified, and used to infect fresh Sf9 cells. Sf9 cells
were cultured at 27 °C in TNM-FH medium (Life Technologies)
supplemented with 10% heat-inactivated FCS. After 3 days of culture,
the infected cells (~7 × 106) were collected by
centrifugation and suspended in 1 ml of 20 mM Tris-HCl, pH
7.5, containing 150 mM NaCl (TBS) and varied protease inhibitors (100 µg/ml phenylmethylsulfonyl fluoride (Sigma), 2 µg/ml leupeptin (Sigma), 1 µg/ml pepstatin A (Sigma), 2 µg/ml aprotinin (Sigma), and 1 mM EDTA). The cell suspension was
sonicated on ice and centrifuged to remove the debris, and the
resulting supernatant was applied to a 1-ml lactose column (Seikagaku
Corp.). After the affinity column was washed extensively with TBS to
remove contaminating insect cell-derived proteins, recombinant
ecalectin was eluted by exposing the column to 200 mM
lactose in TBS. Aliquots of the eluate fractions were evaluated for
their protein content. Buffer containing SDS and 2-mercaptoethanol was
added to each, and the sample was boiled for 5 min and then subjected
to SDS-PAGE. Gels were silver-stained to evaluate the number of
proteins in each column fraction. Protein blots, prepared from
replicate gels, were stained with the above anti-peptide antibody and
the ECLTM development system (Amersham Pharmacia Biotech)
to demonstrate the presence of the recombinant ecalectin.
In Vitro and in Vivo Chemotaxis--
Eosinophil chemotaxis of
native, COS cell-derived recombinant, and insect cell-derived
recombinant ecalectin was evaluated in vitro as described
(35, 36). In brief, CD16
eosinophils were isolated by
subjecting peripheral blood human mononuclear cells from healthy
volunteers to a discontinuous density gradient of Percoll (Amersham
Pharmacia Biotech), followed by immunomagnetic treatment of the
obtained cells with anti-CD16 immunoglobulin (DAKO A.S., Glostrup,
Denmark). The purity and viability of the obtained eosinophils were
>97 and 95%, respectively. Chemotactic activity was evaluated with a
48-well chamber (Neuro Probe Inc., Cabin John, MD) containing a
polyvinylpyrolidone-free membrane with 5-µm standard pore sizes.
Human eosinophils (0.5-1 × 106/ml) and varied
concentrations of a test chemoattractant were placed in the top and
bottom chambers, respectively. Each assay was performed in triplicate.
After a 1-2-h incubation at 37 °C in a humidified atmosphere of 5%
CO2, the membrane separating the two chambers was removed
and placed in Diff-Quik® stain (Baxter Healthcare Corp.,
McGaw Park, IL), and the stained eosinophils that had migrated through
the membrane were counted under the microscope. Human C5a (Sigma), IL-5
(Genzyme, Cambridge, MA), RANTES (Genzyme), and eotaxin-1 (Seikagaku
Corp.) were used as positive controls.
The ability of ecalectin to attract peripheral blood human neutrophils,
monocytes, and lymphocytes in vitro was evaluated in a
similar fashion. Neutrophils were obtained by centrifuging human
peripheral blood cells at 400 × g for 20 min on a
discontinuous density gradient of Percoll. After the contaminating
erythrocytes were lysed with ammonium chloride, the purity of the
resulting neutrophils was ~99%. Monocytes and lymphocytes were
separated from one another by briefly culturing the resulting
monocyte/lymphocyte-enriched fraction in 75-cm2 plastic
culture flasks. The nonadherent lymphocytes were subjected to a second
plastic adherence step. As assessed immunohistochemically with
fluorescence-activated cell sorting and anti-CD14 immunoglobulin (DAKO
A.S.), the monocyte contamination in the resulting lymphocyte preparation was 1% or less. The flasks containing the adherent monocytes were washed several times with PBS. Ice-cold RPMI 1640 containing 5% FCS and 0.5% EDTA were added, and the flasks were shaken on ice for 30 min. The recovered cells were almost pure monocytes. Chemotaxis was carried out essentially as described above
for eosinophils except that the membranes used in some of the assays
had 3- and 8-µm standard pore sizes. Human C5a and IL-8 (Genzyme)
served as positive controls in the neutrophil chemotaxis assay. Human
lymphotactin (Genzyme) and MCP-1 (Genzyme) served as positive controls
in the lymphocyte and monocyte chemotaxis assays, respectively.
For evaluation of its ECA activity in vivo, purified insect
cell-derived recombinant ecalectin was dialyzed extensively against PBS
to remove the lactose. PBS containing 1% human serum albumin (1.0 ml) with or without purified ecalectin (~10
7
M) was then injected into the peritoneal cavities of
replicate C57BL/6 mice. 14-24 h later, each mouse was sacrificed, 6 ml
of PBS containing 2% FCS was injected into the peritoneal cavity, and
the obtained cells in the peritoneal exudate were centrifuged onto
slides. The resulting slides were stained with Diff-Quick, and the
number of eosinophils that had extravasated into the peritoneal cavity
of the mouse was determined.
DNA and RNA Blot Analyses--
To determine if there are
multiple ecalectin-like genes in the human genome, 6-µg samples of
human blood-derived genomic DNA (Noven, Madison, WI) were incubated
overnight at 37 °C with 30-60 units of BamHI,
BglII, EcoRI, HindIII,
KpnI, NotI, PstI, SalI, XbaI, or XhoI (New England Biolabs). After
electrophoretic separation of the resulting digests in a 0.9% agarose
gel (41), a DNA blot was prepared and probed under conditions of high
stringency with [
-32P]dCTP-labeled probes that
correspond to residues 1-460 or residues 1073-1579 (sequence
corresponding to the 3'-untranslated region) of the human ecalectin
cDNA.
Premade blots containing either total RNA (OriGene Technologies,
Rockville, MD) or poly(A)+ RNA
(CLONTECH, Palo Alto, CA) from various human
tissues were analyzed with the radiolabeled ecalectin probes. RNA blots
were washed twice for 15 min each at room temperature in 2× SSC
containing 0.1% SDS and then twice for 30 min each at 60 °C in
0.1× SSC containing 0.1% SDS. After autoradiography, the images were
analyzed with a BAS-2000. 32P imaging apparatus
(Fuji Film Co., Tokyo, Japan).
To determine if the steady-state level of the ecalectin transcript in a
T-cell can be antigen-regulated, heparinized peripheral blood was
obtained from two volunteers sensitive to Dermatophagoides farinae, and the peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. The obtained cells
(5 × 106/ml) were suspended in RPMI 1640 medium
supplemented with 5% FCS and cultured for 48 h in the absence or
presence of 5 µg/ml of D. farinae. Under these in
vitro conditions, the T-cells in the peripheral blood mononuclear
cell preparation should be the cells that are preferentially activated.
Total RNA was isolated from control and D. farinae-stimulated cells with RNeasy Midi kits from Qiagen. For
blot analysis of each sample, ~5 µg of denatured total RNA were
electrophoresed in a 1.4% agarose/formaldehyde gel, and the separated
RNA was blotted onto HybondTM nylon membranes (Amersham Pharmacia
Biotech) (42). After UV-cross-linking of the RNA, the membranes were
incubated at 68 °C for 30 min in QuikHyb (Stratagene) containing 40 µg/ml of denatured salmon sperm DNA (Stratagene) alone and then for
2 h in the same hybridization buffer containing a radiolabeled
ecalectin probe. To ensure that approximately equal amounts of RNA were
loaded in the individual lanes of the blot, the same blot generally was
reprobed with the 548-base pair fragment (CLONTECH)
that consists of residues 256-804 of a human
glyceraldehyde-3-phosphate dehydrogenase cDNA (43). Supernatants
from antigen-activated T-cells from normal donors were also evaluated
by SDS-PAGE/immunoblot analysis with the anti-peptide antibody
described above for determining whether or not immunoreactive ecalectin
was exocytosed from these nontransformed cells.
 |
RESULTS |
Isolation and Characterization of Human Ecalectin Transcript and
Protein--
32 immunoreactive clones were obtained when the
expression cDNA library prepared from the human T-cell line was
screened with anti-ecalectin immunoglobulin directed against the
semipurified factor. When the cDNAs from these clones were
individually transfected into COS cells and the supernatants of the
transfectants were analyzed 2 days later, three of the clones exhibited
potent ECA activity in the in vitro chemotaxis assay.
Subsequent nucleotide sequence analysis revealed that these clones
encoded the same protein. The consensus nucleotide and deduced amino
acid sequences of the STO-2 cell-derived ecalectin cDNAs are
depicted in Fig. 1. If the
translation-initiation and translation-termination sites begin at
residues 60 and 1028, respectively, the open reading frame of the
transcript encodes a 36-kDa protein consisting of 323 amino acids. The
ecalectin transcript has a typical polyadenylylation signal sequence at
residues 1576-1581. 59 and >560 nucleotides are present in its 5'-
and 3'-untranslated regions, respectively. Repetitive sequences are
often found in the 3'-untranslated regions of transcripts that have
short half-lives (44). Although no repetitive "AUUUA" motif resides
in the 3'-untranslated region as it does in eotaxin-1 and most other
chemokine and cytokine transcripts, four copies of a "CCCUCC" motif
reside between residues 1341 and 1484 in the 3'-noncoding portion of
the ecalectin transcript. Native ecalectin is secreted from the STO-2
cell line. Although the deduced amino acid sequence of the
STO-2-derived transcript indicates that ecalectin contains large
numbers of Phe, Leu, and Val residues, this ECA lacks a hydrophobic
signal peptide at its amino terminus. Ecalectin is also predicted to
have a pI of ~8.1. Ecalectin has four Cys residues and three
potential Asn-linked glycosylation sites at positions 34, 79, and
137.

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Fig. 1.
Nucleotide sequence of an STO-2 cell-derived
ecalectin cDNA and its predicted amino acid sequence. The
nucleotide sequence of the ecalectin cDNA is shown in the
upper line, and the predicted amino acid sequence
of its coding region is shown in the bottom line.
The stop codon is marked by an asterisk. The repetitive
"CCCUCC" motifs in the transcript, corresponding to the CCCTCC
motifs in the cDNA, are boxed. The putative
polyadenylylation signal sequence in the 3'-untranslated region of the
transcript is underlined. Potential N-linked
glycosylation sites are double underlined. The
nucleotide sequences of the other two isolated ecalectin cDNAs were
the same except that one lacked the first 6 nucleotides and the other
contained a shorter poly(A)+ tail.
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Expression of the Ecalectin Transcript in Cells and
Tissues--
Blot analysis of genomic DNA digested separately with 10 different restriction enzymes indicated multiple genes in the human genome encode ecalectin-like transcripts. Nevertheless, the 5' (Fig.
2, lanes 3,
5, 6, 8, and 10) and 3'
(data not shown) ecalectin probes often hybridized strongly to one
major DNA fragment in the digested genomic DNA. When varied human
tissues were analyzed with these probes, the ~1.8-kb ecalectin
transcript or its homolog was found in relative abundance in lymphoid
tissues (Fig. 3). Because of the
prominence of the ecalectin transcript in T-cell-enriched tissues, we
investigated whether or not its level increases in peripheral blood
mononuclear cells after antigen stimulation. As shown in Fig.
4 for one of the two allergic patients,
these cells contained a small amount of ecalectin mRNA. However,
the amount of ecalectin transcript increased ~50- and ~90-fold in the two experiments after the cells from the patients were exposed to
D. farinae for 48 h. Although the steady-state level of
a transcript does not always correlate with the amount of protein that
the cell produces, these RNA blot findings suggest that the T-cells in
the preparation dramatically increased their expression of ecalectin
when they were exposed to the relevant antigen. SDS-PAGE/immunoblot analysis with the peptide antibody directed against the N terminus of
ecalectin revealed that immunoreactive ecalectin was present in the
conditioned medium of activated, nontransformed human T-cells (Fig.
4).

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Fig. 2.
Genomic blot analysis. A DNA blot
containing human genomic DNA that had been digested with
BamHI (lane 1), BglII
(lane 2), EcoRI (lane
3), HindIII (lane 4),
KpnI (lane 5), NotI
(lane 6), PstI (lane
7), SalI (lane 8),
XbaI (lane 9), or XhoI
(lane 10) was probed under conditions of high
stringency with a radiolabeled fragment that corresponds to residues
1-406 of the human ecalectin cDNA. Size markers are indicated on
the left.
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Fig. 3.
Expression of ecalectin transcript in human
tissues and cells. In panels A and
B, two RNA blots containing 20 µg of total RNA from brain
(lane 1), heart (lane 2),
kidney (lane 3), spleen (lane
4), liver (lane 5), peripheral blood
lymphocytes (lane 6), lung (lane
7), small intestine (lane 8), muscle
(lane 9), stomach (lane
10), testis (lane 11), and placenta
(lane 12) were sequentially analyzed under
conditions of high stringency with human ecalectin (A) and
glyceraldehyde-3-phosphate dehydrogenase (B) probes. In
panels C and D, a third RNA blot
containing 2 µg of poly(A)+ RNA from spleen
(lane 1), lymph node (lane
2), thymus (lane 3), peripheral blood
leukocytes (lane 4), bone marrow (lane
5), and fetal liver (lane 6) was
sequentially analyzed under conditions of high stringency with the
human ecalectin (C) and glyceraldehyde-3-phosphate
dehydrogenase (D) probes. The arrows on the
right of panels A and C
indicate the prominent ecalectin transcript. Molecular weight markers
are indicated on the left of each blot. Although the probe
used in the experiments depicted in panels A and
C corresponded to residues 1-406 of the ecalectin cDNA,
similar findings were obtained with the 500-base pair probe that
corresponds to the 3'-untranslated region of the ecalectin cDNA
(data not shown).
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Fig. 4.
Induction of ecalectin expression in
antigen-activated peripheral blood human mononuclear cells. A blot
containing 5 µg of total RNA from peripheral blood human mononuclear
cells isolated from a patient allergic to D. farinae before
(lane 1) and after (lane 2)
exposure to D. farinae was sequentially analyzed for the
presence of ecalectin mRNA (A) and
glyceraldehyde-3-phosphate dehydrogenase mRNA (B). The
migration position of 28 S rRNA is indicated. The arrow on
the right points to the ecalectin transcript. C,
SDS-PAGE/immunoblot analysis with anti-ecalectin Ig was carried out on
conditioned medium from resting (lane 1) and
activated (lane 2) peripheral blood mononuclear
cells isolated from another patient. The arrows point to
immunoreactive ecalectin.
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Bioactivity of Recombinant Ecalectin in Vitro and in
Vivo--
Recombinant ecalectin was spontaneously secreted from COS
cells but not insect cells (Fig. 5).
Nevertheless, because insect cells express measurable amounts of
ecalectin by silver staining analysis of an SDS-PAGE gel (Fig.
5A), we attempted to affinity-purify the immunoreactive
(Fig. 5B) recombinant protein from the lysates of these
cells. The observation that the ecalectin cDNA appears to encode a
protein with carbohydrate-binding domains suggested that the
recombinant protein probably would bind to a lactose column. Thus,
soluble lysates of insect cells were subject to lactose affinity
chromatography. After the column was exposed to 200 mM
lactose, a single ~36-kDa silver-stained protein (Fig. 5C)
was eluted, which was recognized by anti-ecalectin antibody (Fig.
5D). The culture supernatant of COS cells transfected with an ecalectin cDNA (data not shown) and purified, recombinant, insect cell-derived ecalectin (Fig. 6)
exhibited dose-dependent ECA activity. In our in
vitro system, the optimal chemotactic activity of IL-5,
RANTES, eotaxin-1, and C5a for freshly isolated peripheral blood
eosinophils was achieved at 10
11 M,
10
8 M, 10
8 M, and
10
8 M, respectively. Insect cell-derived
ecalectin was active at 10
10 M, and its
activity at 10
8 M was approximately one-half
of that of eotaxin-1. Although IL-5 was active at a lower molar
concentration, in comparative assays carried out on replicate
eosinophils from the same patients, recombinant ecalectin induced
~4-fold more eosinophils to migrate through the membrane than the
optimal dose of recombinant IL-5. COS cell-derived (data not shown) and
insect cell-derived (Fig. 7) recombinant ecalectin did not induce chemotaxis of peripheral blood neutrophils, lymphocytes, or monocytes. 4-24 h after C57BL/6 mice were given insect
cell-derived ecalectin, 15.1 ± 2.7% (n = 3; mean
±S.D.) of the cells in the peritoneal cavity exudates consisted of
eosinophils. In contrast, the number of eosinophils was below detection
(<1 eosinophil/500 cells) in the exudates of control mice given buffer lacking ecalectin. Thus, ecalectin has potent ECA activity in vitro and in vivo.

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Fig. 5.
Expression of human ecalectin in insect cells
and affinity purification of the resulting recombinant protein.
Lysates of insect cells before (lane 1) and after
(lane 2) they had been induced to express human
ecalectin were subjected to SDS-PAGE gel analysis. The gel depicted in
A was silver-stained to detect all of the proteins in these
cells. The protein blot in B was prepared from the replicate
gel; it was stained with the peptide antibody directed against the N
terminus of human ecalectin. In C and D, the
soluble lysate of ecalectin-expressing insect cells was applied to a
lactose affinity column. After the column was washed with equilibration
buffer, it was exposed to buffer containing 200 mM lactose.
Samples of the resulting six fractions were subjected to SDS-PAGE gel
analysis. The gel depicted in C was stained with silver, and
the corresponding protein blot in D was stained with
anti-ecalectin antibody. Molecular weight makers are shown on the
left of each. The arrows point to recombinant
ecalectin.
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Fig. 6.
ECA activity of recombinant ecalectin.
In the in vitro assay used in these experiments, a porous
membrane separates the top chamber that contains human eosinophils from
the bottom chamber that contains medium with or without an ECA. After
the incubation, the membrane that separates the two compartments in
each well was removed and stained with Diff-Quik. Depicted in
A is the stained side of the membrane that faces the bottom
chamber of cells exposed to conditioned medium from a mock transfectant
(left panel), conditioned medium from an ecalectin
transfectant of COS cells (center panel), or culture medium
supplemented with an optimal amount of eotaxin-1 (right
panel). The colored cells are the eosinophils that had migrated
through the black and white holes in the membrane. Depicted in
B are the quantitative chemotaxis data from multiple
experiments using insect cell-derived ecalectin that had been purified
by lactose affinity chromatography. In these experiments, the ECA
activity of ecalectin ( ) was similar to that obtained with an
optimal dose of RANTES ( ) and IL-5 ( ) and was approximately
one-half of that obtained with an optimal dose of eotaxin-1 ( ). The
number of eosinophils that had migrated through the membrane is
indicated on the y axis.
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Fig. 7.
Analysis of the chemotactic activity of
recombinant human ecalectin for neutrophils, lymphocytes, and
monocytes. The ability of purified, insect cell-derived ecalectin
(~10 8 M) to attract peripheral blood
neutrophils (A), lymphocytes (B), and monocytes
(C) in the in vitro assay was determined. PBS was
used as a negative control in each assay. IL-8, lymphotactin, and MCP-1
were used as positive controls for the chemotaxis of neutrophils,
lymphocytes, and monocytes, respectively. The numbers of neutrophils,
lymphocytes, and monocytes that had migrated through the membranes are
indicated on the y axis. The depicted results are
the mean ± S.D. of an experiment in which each assay
was carried out in triplicate.
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DISCUSSION |
We previously reported that antigen-activated T-cells produce an
ECA with unique biochemical properties (35, 36). We now describe the
isolation, cloning, and expression of this ECA, which is discovered to
be a member of the galectin family of lectins. Polyclonal antibodies
prepared against the partially purified factor from T-cell conditioned
medium were used to screen a cDNA expression library prepared from
a T-cell line to obtain candidate cDNAs that encoded the novel ECA
(Fig. 1). To confirm that the isolated cDNAs encoded a protein
possessing ECA, the cDNAs were expressed in COS cells, and the
resulting supernatants from the transfectants were evaluated for their
chemotactic activity. Because native ecalectin was isolated from the
conditioned medium of a T-cell line and because recombinant ecalectin
was isolated from the conditioned medium of a COS cell transfectant,
ecalectin can be secreted by two very different types of mammalian
cells. Nevertheless, because we were unable to induce insect cells to
exocytose recombinant ecalectin, we cannot rule out the possibility
that some ecalectin is retained by activated T-cells in
vivo. Recombinant ecalectin induced the specific chemotaxis of
eosinophils (Figs. 6 and 7). On a weight or molar basis, ecalectin
exhibited an ECA that is approximately one-half as potent as that of
eotaxin-1. Native ecalectin was isolated from a human T-cell line,
whereas recombinant ecalectin was isolated from both COS cells and
insect cells. Only one silver-stained protein was detected after insect
cell-derived ecalectin was purified by lactose affinity chromatography
(Fig. 5). Although this finding suggests that ecalectin does not
require an insect cell-derived cofactor to exert its chemotactic
activity, we still cannot rule out the possibility that an
unrecognizable low molecular weight peptide, oligosaccharide, or
glycolipid stays tightly bound to the galectin during the affinity
chromatography step or after it is dissolved in medium.
Genomic blot analysis revealed that there are a number of closely
related ecalectin-like genes in the human genome (Fig. 2). As assessed
by RNA blot analysis, ecalectin (Figs. 3 and 4) is expressed by
T-cell-rich lymphoid tissues or cells. However, a relatively high level
of the ecalectin transcript was also found in the stomach RNA sample.
Patients with chronic gastritis often have large numbers of activated
T-cells and eosinophils in their stomach (45). Because the stomach RNA
was obtained from a commercial source, it was not possible for us to
ascertain whether some of the transcripts in the sample originated from
activated T-cells. Nevertheless, because the ecalectin was found in the
stomach, as well as in the lung and small intestine (Fig.
3A), it is likely that T-cells are not the only cell type
that expresses ecalectin.
A homology search of the GenBankTM protein and nucleotide
data bases revealed that STO-2 cell-derived ecalectin is not related to
any known cytokine or chemokine. Rather, it is related to the galectin
family of carbohydrate-binding proteins (46). Based on a comparison of
its deduced amino acid sequence (Fig. 8),
ecalectin most closely resembles the proteins designated human (37) and mouse (54) galectin-9. Mouse galectin-9 cDNA was isolated from an
embryonic kidney cDNA library, and expression studies revealed that
its transcript is particularly abundant in liver. Although the mouse
galectin-9 transcript is present in the thymus, in situ hybridization and immunohistochemical analyses revealed that this galectin is expressed primarily by thymus epithelial cells rather than
by T-cells (54). Micromolar concentrations of recombinant mouse
galectin-9 induced cultured T-cells to undergo apoptosis (55). Because
the ecalectin-expressing STO-2 cell line is a T-cell line that is not
undergoing apoptosis and because the ecalectin transcript is not
abundant in human liver (Fig. 3A), either ecalectin is not
the human homolog of mouse galectin-9 or this galectin evolved in the
two species to carry out different functions.

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Fig. 8.
Comparison of the deduced amino acid sequence
of human ecalectin with those of related galectins.
Dashes and dots in the amino acid sequences
indicate identical residues and insertions, respectively. The numbers
indicate the residues in human ecalectin. The solid and
dashed thick lines under
the sequences indicate the putative residues that correspond
to first and second carbohydrate-binding regions, respectively, in
ecalectin. The conserved amino acids reported to be essential for
lectin activity are shaded. Human ecalectin and human
galectin-9 differ in their amino acid sequences at residues 5, 88, 135, 238, and 281. The depicted amino acid sequences of human galectin-1,
human galectin-2, human galectin-3, rat galectin-4, rat galectin-5,
human galectin-7, rat galectin-8, mouse galectin-9, and human
galectin-9 have been reported by others (37, 47-54). The first 100 amino acids of human galectin-3 are not shown in this
alignment figure.
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The relationship of ecalectin and the Hodgkin's lymphoma protein
designated human galectin-9 (37) also remains to be determined. A
BLASTN (56) computer search of the GenBankTM/EMBL
nonredundant nucleic acid sequence data base, focusing on the six
regions that differ in the ecalectin and human galectin-9 transcripts,
revealed clones that correspond to one or the other transcript. Thus,
the differences in the nucleotide sequences between the ecalectin and
galectin-9 transcripts are not cloning artifacts. Relevant to our
study, one of the ecalectin-like expressed sequence tags in the data
base originated from the human Jurkat T-cell line (accession number
EST63150). The inability of Türeci and co-workers (37) to find an
allelic variant of galectin-9 in a preliminary reverse
transcription-polymerase chain reaction analysis of the peripheral
blood of normal patients coupled with the finding that four of the five
amino acid differences in galectin-9 and ecalectin are substantial
raises the possibility that ecalectin represents a new galectin.
However, because ecalectin and galectin-9 differ only in a few amino
acids, because the 5'-untranslated regions of their transcripts are
identical, because the 3'-untranslated regions of their transcripts
differ by only one nucleotide at residue 1547, and because galectin-9
is also expressed in a peripheral blood cell, the most likely
interpretation of the data from the two studies is that ecalectin and
galectin-9 are two allelic variants of the same human gene.
Galectins differ from Ca2+-dependent C-lectins.
Galectins have conserved sequences that enable them to interact with
-galactoside residues. As noted in Fig. 7, those residues thought to
be the most important for carbohydrate binding in galectin-2 (57) are present in ecalectin. Galectin-1 and galectin-2 are dimers, whereas the
other galectins are monomers. Galectin-1, -2, -3, -5, and -7 have a
single carbohydrate-binding domain, whereas galectin-4, -8, and -9 have
two homologous carbohydrate-binding domains. In this regard, ecalectin
resembles the latter family of galectins. Recombinant ecalectin readily
binds to a lactose column (Fig. 5, C and D), as
do other galectins. How ecalectin selectively induces eosinophil
chemotaxis remains to be determined at the molecular level. However,
the finding that ecalectin has two putative domains that both bind
carbohydrate suggests that this galectin might function as a bridging
protein interacting with two or more galactoside-containing molecules
on the surface of the eosinophil. Because ecalectin has four Cys
residues (Fig. 1), it could have two disulfide bonds. However,
structural analysis of crystallized galectin-2 (57) suggests that these
residues do not form disulfide bonds.
Because all galectins lack a hydrophobic signal peptide, they are often
found in the cytosol. It has been proposed that galectins have
intracellular functions. However, galectins can reside on the surface
of a cell and often are spontaneously secreted by a mechanism that has
not been deduced. For example, galectin-3 (49) is the Mac-2 antigen
found abundantly expressed on the surface of inflammatory macrophages
(58). When peripheral blood mononuclear cells are exposed to antigen,
ecalectin mRNA increases dramatically (Fig. 5), and the amount of
ECA recovered in the conditioned medium increases accordingly (35).
Exocytosis of ecalectin could occur via a pathway that does not involve
post-translational modification of the protein. However, ecalectin has
two sites in its amino terminus at residues 27 and 57 that might be
susceptible to IL-1
-converting enzyme (59). Thus, some ecalectin may
be released into the conditioned medium by the same proteolytic
mechanism that T-cells use to generate soluble IL-1
.
Many galectins are developmentally regulated (46). An ecalectin-like
expressed sequence tag has been identified in Jurkat T-cells. However,
our finding that the steady-state level of ecalectin mRNA is low in
human peripheral blood mononuclear cells (Fig. 4) until these cells are
activated with antigen may explain why there has been no published in
depth report of ecalectin expression in a T-cell line. Although members
of the large galectin family are expressed in numerous cell types, some
galectins exhibit immunoregulatory activities, and some galectins are
expressed by certain hematopoietic cells. Galectin-1, expressed in
abundance in muscle, can induce T-cells to undergo apoptosis (60).
Galectin-3 has been localized in macrophages, basophils (61),
eosinophils (62), and mast cells (61). Immunoelectron microscopic
studies have indicated that much of the galectin-3 found in a mast cell
is granule-associated. Because it readily binds to the oligosaccharides
attached to both IgE and its receptor, galectin-3 can induce most, if
not all, Fc
RI-bearing mast cells and basophils to
degranulate (63). Galectin-3 prevents T-cells from undergoing apoptosis
(64), induces neutrophils to increase their production of superoxide (65) and to adhere to laminin (66), and potentiates the production of
IL-1 by human monocytes (67). Because both galectin-1 and galectin-3
bind to laminin, these two galectins could influence cell
proliferation, cell-cell interactions, and cell-matrix interactions in vivo. Erythrocytes express galectin-5, which exhibits
weak agglutinin activity (51).
While our study was in progress, galectin-9 was identified during
an analysis of the antigens that are selectively expressed by a patient
with Hodgkin's lymphoma (37). Although the cellular origin of the
galectin-9 transcript was not identified in the patient's tumor,
galectin-9 mRNA or a related transcript also was found to be
expressed in an undefined, peripheral blood leukocyte. Hodgkin's
disease, which accounts for nearly 1% of all cancers in United States,
is a neoplastic disorder primarily involving lymphoid tissue (68).
Neoplastic Reed-Sternberg cells are found in affected tissue and are
surrounded by a variable inflammatory infiltrate of B-cells,
histiocytes, plasma cells, stromal cells, neutrophils, T-cells, and
eosinophils. Although different types of Hodgkin's lymphoma have been
identified, eosinophils regularly appear in those patients with the
"nodular sclerosis" form of the disease. Interestingly, the
galectin-9 transcript was isolated from a cDNA library prepared
with mRNA isolated from a patient with the "nodular sclerosis"
form of Hodgkin's lymphoma (37). While it has been proposed that the
increased number of eosinophils in this subtype of Hodgkin's lymphoma
is primarily a consequence of increased production of IL-5 and/or
granulocyte-macrophage colony-stimulating factor by the Reed-Sternberg
cells (19, 21, 68, 69), galectin-9 and/or its variant ecalectin could
work in concert with the two viability-enhancing cytokines to cause the
prominent eosinophilia. If galectin-9 and ecalectin are allelic variants of the same gene, galectin-9 probably originates, in part,
from the antigen-activated T-cells present in Hodgkin's lymphoma.
Whatever its mechanism of action at the molecular level, ecalectin
appears to be a newly recognized factor by which antigen-activated T-cells regulate eosinophil extravasation into tissues. Because the
eotaxins are expressed primarily by epithelial and endothelial cells
rather than by T-cells (3), ecalectin could be the dominant ECA
expressed during certain T-cell-mediated inflammatory reactions in
humans.
We thank Drs. Takashi Yokota, Hisao
Masai, Tastuo Kinashi, and Maiko Fukuoka for valuable suggestions and
Junko Ohmoto for technical assistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB005894 (ecalectin transcript and protein).