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*
Tokyo Research Laboratories, Kyowa Hakko Kogyo, Tokyo, Japan; and
Toxicological Research Laboratories, Kyowa Hakko Kogyo, Yamaguchi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, to eosinophils and acted on cell lines
transfected with CCR-3, suggesting that eotaxin-3 recognized CCR-3.
IL-13 as well as IL-4 up-regulated eotaxin-3 mRNA in HUVEC, whereas
neither TNF-, IL-1
, IFN-
, nor TNF- plus IFN- did. The
expression profile of eotaxin-3 is different from those of eotaxin,
RANTES, and monocyte chemoattractant protein-4, which are potent
eosinophil-selective chemoattractants and are induced by either TNF-
or TNF- plus IFN-. These results suggest that eotaxin-3 may
contribute to the eosinophil accumulation in atopic
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In general, leukocyte extravasation from the bloodstream into tissues is a regulated process that involves a series of coordinated interactions between leukocytes and vascular endothelial cells (VEC)3. This process is divided into the following four steps (8, 9): in step 1, the initial interaction between leukocytes and VEC, namely rolling, comprises transient and reversible adhesion mediated mainly by cell adhesion molecules, selectins, and carbohydrate ligands; in step 2, the leukocytes rolling on VEC come into contact with chemoattractants such as chemokines and platelet-activating factor present on the surface of VEC, which stimulate leukocytes to induce integrin adhesiveness; in step 3, the activated leukocytes adhere firmly to VEC through the interaction of integrins with their endothelial counterreceptors, such as ICAM-1 and VCAM-1; and in step 4, the leukocytes adhered to VEC transmigrate through vascular walls and move to the center of inflamed tissues.
The selective recruitment of specific classes of leukocyes to inflamed tissues has been explained by inducible expression of specific molecules on VEC in steps 1, 2, and 3. As to IL-4-induced accumulation of eosinophils, IL-4-induced expression of P-selectin (10) and L-selectin ligands (11, 12) in step 1 and that of VCAM-1 (13, 14) in step 3 have been detected.
Eosinophil-active chemokines identified to date comprise eotaxin
(15, 16), eotaxin-2 (17), RANTES (18, 19), monocyte chemoattractant protein (MCP)-2 (20, 21), MCP-3 (20, 21, 22), MCP-4 (23, 24, 25),
and macrophage inflammatory protein (MIP)-1 (19), which
belong to the CC subfamily. Among them, eotaxin has been most
characterized and is thought to be a potent eosinophil-selective
chemoattractant because it acts on a single receptor, CCR3, highly
expressed on eosinophils (26, 27). However, eotaxin
expression was not up-regulated in IL-4-stimulated HUVEC
(28). Eotaxin has not been found in the culture medium of
IL-4-stimulated HUVEC but has been in that of IL-4-stimulated dermal
fibroblasts (29). Sanz et al. (30) reported
that eotaxin-dependent accumulation of eosinophils was observed in rats
in response to IL-4, but no evidence has been presented for
IL-4-induced expression of eotaxin on VEC. Furthermore, eosinophil
recruitment to the sites of inflammation was not completely blocked in
eotaxin-deficient mice (31). Thus, chemokines that are
induced on VEC by IL-4 and involved in the leukocyte-activation step
(step 2) in the selective recruitment of eosinophils to inflammatory
sites have not been definitely identified so far.
In this study, we have explored novel molecules expressed selectively on HUVEC stimulated by IL-4 using a differential display technique and have identified a novel CC chemokine that attracts human eosinophils. We have named this chemokine eotaxin-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVEC were purchased from Kurabo (Osaka, Japan). Recombinant
human TNF- and IL-4 were obtained from Genzyme (Cambridge, MA), and
IL-1, IL-13, and IFN- were obtained from R&D Systems
(Minneapolis, MN). Recombinant human eotaxin, MIP-1, MCP-3, RANTES,
and IL-8 were purchased from PeproTech (London, U.K.).
125I-eotaxin and
125I-MIP-1 were obtained from Amersham
Pharmacia Biotech (Uppsala, Sweden).
Fluorescent differential display
HUVEC were propagated in F-12K medium supplemented with 100
µg/ml heparin, 30 µg/ml endothelial cell growth supplement (Becton
Dickinson, Franklin Lakes, NJ), and 10% FCS in tissue culture plastic
flasks precoated with 2 mg/ml gelatin. The confluent HUVEC monolayers
of passage 6 were treated for 17 h with either 10 ng/ml TNF- or
100 U/ml IL-4. HUVEC cultivated without the addition of these cytokines
were used as controls. Total cellular RNA was extracted from the cells
by the method of Chomczynski and Sacchi (32) and was
treated with DNase I. Then, 2.5 µg of RNA was mixed with 50 pmol of
FITC-labeled 3'-anchored oligo(dT) primer
(5'-GT15A-3') and then subjected to first-strand
synthesis with Superscript II reverse transcriptase (Life Technologies,
Rockville, MD). The PCR reactions for differential display were
performed as described by Ito et al. (33) using 80
different arbitrary primers (Operon Technologies, Alameda, CA). The PCR
products were electrophoresed on a high-resolution denaturing gel and
then analyzed with a FluorImager (Molecular Dynamics, Sunnyvale, CA).
The differentially regulated bands were excised from the gel,
reamplified, cloned into plasmid pT7Blue(R) (Novagen, Madison, WI), and
then sequenced using a dye primer cycle sequencing kit and a 377 DNA
sequencer (Perkin-Elmer, Norwalk, CT).
Isolation of full-length cDNA of eotaxin-3
A cDNA library was constructed from
poly(A)+ RNA prepared from HUVEC treated with
IL-4 for 17 h. First-strand synthesis was performed using a 3'
oligo(dT) primer that contained a NotI site. After synthesis
of the second strand, the DNAs were blunt-ended with T4 DNA polymerase,
ligated with EcoRI adaptors, and then digested with
NotI. The cDNAs of 0.4–2.0 kb fragments were inserted into
vector ZAP Express (Stratagene, La Jolla, CA). Then they were
packaged in phage particles using packaging extract (Stratagene). After
the library had been amplified with Escherichia coli
XL1-Blue MRF' as the host strain, 
1 x
106 phages were plated with the host strain and
plaque hybridization was performed using a
32P-labeled DNA fragment covering the 3'-terminal
portion of eotaxin-3 cDNA (Fig. 1
A), as described
(34). Positive plaques were isolated and converted to
plasmid DNAs by in vivo excision using E. coli XLOLR as the
host strain. The plasmid carrying the full-length eotaxin-3 cDNA was
named pHVC002.
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For chromosomal mapping of the human eotaxin-3 gene, the DNAs of
25 human-hamster somatic cell hybrids (BIOSMAP Somatic Cell Hybrid
PCRable DNAs; BIOS Laboratories, New Haven, CT) and those of 93
radiation hybrids (GeneBridge 4 Mapping Panel; Research Genetics,
Huntsville, AL) were analyzed by PCR using the primers indicated in
Fig. 1
A. The reaction products were electrophoresed on a 2%
agarose gel. Radiation hybrid mapping data were analyzed by accessing
the server at http://www-genome.wi.mit.edu (Whitehead Institute/MIT
Center).
Expression of recombinant eotaxin-3 by baculovirus-infected insect cells
The BamHI-PstI 370-bp fragment encoding eotaxin-3 was excised from pHVC002 and then cloned into the baculovirus transfer vector, pVL1393 (PharMingen, San Diego, CA) to obtain pVL-HVC. The plasmid was cotransfected with BaculoGold viral DNA (PharMingen) into Sf9 cells to construct a recombinant baculovirus, and then the recombinant virus was purified by plaque purification as described (35). Confluent monolayers of Sf21 cells grown in serum-free medium, Ex-Cell 400 (JRH Biosciences, Lenexa, KS) were infected with the recombinant virus (multiplicity of infection = 10) and then incubated at 27°C.
Purification of the recombinant eotaxin-3 protein
The culture medium of the recombinant baculovirus-infected Sf21 cells was applied to a heparin-Sepharose (Amersham Pharmacia Biotech) column preequilibrated with 50 mM sodium phosphate, pH 6.5. The column was washed with 10-bed volumes of the same buffer, and then bound proteins were eluted with a linear gradient of 0–1 M NaCl in 50 mM sodium phosphate, pH 6.5. Each fraction was analyzed by SDS-PAGE followed by silver staining, and the fractions containing the recombinant eotaxin-3 protein were pooled and diluted with 1.6 volumes of 50 mM sodium phosphate, pH 6.5. Then, SP Sepharose (Amersham Pharmacia Biotech) preequilibrated with 50 mM sodium phosphate, pH 6.5, containing 0.4 M NaCl was added, followed by gentle mixing at 4°C for 12 h. The resin was packed into a column and washed with 50 mM sodium phosphate, pH 6.5, containing 0.4 M NaCl, and then the bound proteins were eluted with 1 M NaCl. The eluted proteins were applied to a PD-10 column (Amersham Pharmacia Biotech) preequilibrated with 50 mM sodium phosphate, pH 7.3, containing 0.4 M NaCl. The recombinant eotaxin-3 protein thus purified was concentrated to an appropriate concentration using a Centricon-3 (Millipore, Bedford, MA). Protein concentrations were determined by the method of Bradford (36) using BSA as a standard. N-terminal sequence analysis was performed with a protein sequencer (PPSQ-10; Shimadzu, Tokyo, Japan).
Preparation of human leukocytes
Human granulocytes and PBMC were separated from the venous blood of healthy donors by Percoll (1.085 g/ml) gradient centrifugation at room temperature as described (37). To isolate granulocytes, RBC were removed by hypotonic lysis after centrifugation. To enrich eosinophils, CD16-positive cells, i.e., neutrophils, were removed, and to enrich neutrophils, very late Ag-4-positive cells, i.e., eosinophils, were removed from the granulocyte fraction, respectively, using an immunomagnetic bead technique, as described (37). The contents of the leukocytes determined by analysis of Diff-Quik (International Reagents, Kobe, Japan)-stained cytocentrifugation preparations were as follows: the PBMC fraction contained 70% lymphocytes and 30% monocytes, the neutrophil-rich preparation 95% neutrophils, and the eosinophil-rich preparation 95% eosinophils.
Construction of cell lines stably expressing CCR3
A plasmid pcDNA-CCR3 carrying human CCR3 gene was generously provided by Dr. Kouji Matsushima (University of Tokyo, Tokyo, Japan). A CMV promoter on the plasmid was replaced with a CAG promoter on plasmid pAdexCAwt (38) as follows. The SalI-ClaI 1.8-kb fragment was excised from pAdexCAwt and cloned into pBluescriptII KS(+). The resultant plasmid was digested with SalI, treated with T4 DNA polymerase, and digested with HindIII. The 1.8-kb fragment encoding CAG promoter was excised and ligated with the NruI-HindIII 5.8-kb fragment of pcDNA-CCR3 to construct pcDNA (CAG)-CCR3. K562 cells were transfected with pcDNA(CAG)-CCR3 by electroporation and grown in the presence of 0.8 mg/ml G418 sulfate to obtain G418-resistant clones. From those clones, a CCR3-transfectant was selected, which showed specific binding to 125I-eotaxin.
Ca2+ mobilization assay
Peripheral blood cells were loaded with 1 nmol fura-2/AM (Wako Pure Chemicals, Osaka, Japan) per 1 x 106 cells for 30 min at 37°C in 1 ml of loading buffer (1 mM CaCl2, 1 mM MgCl2, 125 mM NaCl, 5 mM KCl, 0.5 mM glucose, 20 mM HEPES, pH 7.5) containing 0.025% BSA. The cells were washed with the loading buffer containing 0.5% BSA, resuspended in the same buffer at 1 x 106 cells/ml, and then placed in a continuously stirred cuvette at 37°C. As for the CCR3-transfected K562 cells, HBSS was used for loading fura-2/AM, washing, and resuspension. The cells were excited at 340 and 380 nm in a fluorescence spectrophotometer (CAF-110; Jasco Corp., Tokyo, Japan) and then the relative ratio of the fluorescence (340 nm/380 nm) emitted at 500 nm was recorded.
Competition binding assaying of 125I-chemokine
Cells were mixed with either 0.1 nM
125I-eotaxin or
125I-MIP-1 for 1 h at 37°C in binding
buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM
MgCl2, 0.5% BSA, 0.02% sodium azide) containing
various concentrations of the respective unlabeled chemokine. After
incubation, the cells were collected by centrifugation at 8,000 x
g for 3 min, suspended in 150 µl of washing buffer
(binding buffer plus 0.5 M NaCl), and then centrifuged at 16,000
x g for 3 min. The resulting pellets were transferred in
300 µl of the same buffer to polystyrene tubes for counting.
In vitro chemotaxis assay
Chemotaxis for eosinophils was assessed in 96-well microplate chambers (Neuro Probe, Cabin John, MD) using polycarbonate filters with 5-µm pores (Neuro Probe). Cell suspensions and chemokine dilutions were made with RPMI 1640 medium supplemented with 1% (v/v) FCS. Aliquots (350 µl) of chemokines were placed in the wells of the lower compartment, and 200-µl aliquots of the eosinophil suspension (1 x 105 cells/ml) were placed in the upper wells of the chamber. In some experiments, various concentrations of chemokines were mixed with the cells in the upper compartment (checkerboard analysis). The chamber was incubated for 60 min at 37°C in humidified air containing 5% CO2. After incubation, nonmigrating cells were scraped from the upper surface of the filter and then the lower compartment to which the filter was attached was centrifuged at 200 x g for 10 min. The migrated eosinophils were counted essentially as described (39). After removing the medium carefully, the cells were lysed with 50 µl of 0.3% (w/v) cetyltrimethylammonium bromide, followed by the addition of 100 µl of eosinophil peroxidase substrate (2.2 mM o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO), 8.8 mM hydrogen peroxide in 50 mM sodium citrate, pH 5.0) to each well. After 5 min at room temperature, the color reaction was terminated by the addition of 50 µl of 4 N H2SO4. The OD490 was monitored and converted to the number of migrating cells using purified eosinophils as a standard. Assays for PBMC and neutrophils were performed on chemo TX-96 chambers (Neuro Probe) using 5-µm pore polyvinylpyrrolidone-free polycarbonate filters. Cell suspensions and chemokine dilutions were prepared as described above. Aliquots (28 µl) of chemokines were placed in the wells of lower compartment, whereas 60 µl aliquots of the PBMC suspension (3 x 107 cells/ml) or neutrophil suspension (3 x 106 cells/ml) were put on the upper wells of the chamber. The chamber was incubated for 90 min for PBMC or for 60 min for neutrophils at 37°C, and migrated cells were collected as described above. After removing the medium carefully, 20 µl of RPMI 1640 containing 10% FCS and 10 µl of XTT (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) solution (Boehringer Mannheim, Mannheim, Germany) were added to each well and incubated at 37°C for 3 h. The value of the OD490 minus OD655 was determined and converted to the number of migrating cells using purified PBMC or neutrophils as a standard. These cell-counting procedures were found to be highly reproducible and comparable to a direct cell-counting method by microscopic observation.
In vivo assessment of leukocyte recruitment
Thirty micromolar eotaxin-3, purified as described above, and 10 µM eotaxin in PBS were diluted to 8.5 µM and 1 µM, respectively, with pyrogen-free isotonic saline. These chemokine preparations (100 µl each) and 100 µl isotonic saline as a control were then each administered intradermally at two sites on the back of a male cynomolgus monkey that had been anesthetized by injection of 10 mg/kg ketamine. Then, full skin thickness punch biopsies of 8-mm diameter were taken from the injection sites after 4 h. The biopsies were fixed in 10% formalin and then processed by standard histologic techniques. After embedding in paraffin blocks, the pieces were cut into 3-mm sections. The sections were stained with Giemsa solution plus hematoxylin and eosin, and the leukocyte infiltrates were evaluated by two independent observers. In each section, eosinophils and neutrophils that had migrated out of the vessels were counted at 400-fold magnification in five randomly selected fields including postcapillary venules using a grid of 0.22 x 0.22 mm, and the number of eosinophils and neutrophils per mm2 were calculated.
Northern blot analysis
Total RNA from HUVEC with or without cytokine treatment was
electrophoresed on a formaldehyde-1.0% agarose gel and then
transferred to a Biodyne A membrane (Pall BioSupport, East Hills, NY).
The digoxigenin-labeled cDNA of eotaxin-3 was synthesized by
amplification of a region corresponding to the sequence from 190 to 383
in Fig. 1A, using a PCR digoxigenin labeling mix
(Hoffmann-LaRoche, Basel, Switzerland), and hybridization was performed
according to the method described in the manufacturer’s manual.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three mRNA samples were prepared from IL-4-stimulated,
TNF--stimulated, and unstimulated HUVEC, respectively, and then were
subjected to differential display analysis. HUVEC cultivated without
the addition of these cytokines were used as a control. On the analysis
of 80 different PCR reactions, two bands were identified that were
abundant only for the sample from IL-4-stimulated HUVEC. These
materials were reamplified, cloned, sequenced, and then subjected to
database analysis (data not shown). One was a part of dUTP
pyrophosphatase cDNA. This may be a technical artifact because
up-regulation of this mRNA was not observed in IL-4-stimulated HUVEC by
RT-PCR (data not shown). The other had a novel sequence exhibiting
significant homology to the carboxyl-terminal halves of some known CC
chemokines. To isolate the full-length cDNA of the latter, we
constructed a IL-4-treated HUVEC cDNA library and obtained several
independent positive clones. The nucleotide sequence of the most
5'-extended one among them is shown in Fig. 1A. The
full-length cDNA contains an open reading frame consisting of 94 aa
with a predicted molecular mass of 10,647. The predicted polypeptide
sequence has a highly hydrophobic amino-terminal region (Fig. 1A), characteristic of a signal peptide with a putative
cleavage site between Ala23 and
Thr24. The predicted mature protein of 71 aa
shows significant homology to human CC chemokines (Fig. 1B).
Four cysteine residues conserved in the CC chemokine subfamily are
present at the proper positions. We designated this novel CC chemokine
as eotaxin-3 because it was functionally similar to eotaxin and
eotaxin-2 (Refs. 15, 16, 17 and see below). The expression
level of eotaxin-3 mRNA was dependent on the concentration of IL-4
(Fig. 2A). Similar expression
level was observed when HUVEC at passage 3, 4, 6, 12, and 15 were
stimulated with IL-4 (data not shown). Furthermore, the expression of
eotaxin-3 mRNA was found to continue at least for 24 h by
stimulation with 100 U/ml IL-4 (Fig. 2B). No known CC
chemokine from other species showing extensive homology with eotaxin-3
was found, suggesting an ortholog of eotaxin-3 has not been identified
in other species.
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The chromosomal localization of the human eotaxin-3 gene was
investigated by PCR using human-hamster somatic cell hybrids. The
expected 152-bp fragment, corresponding to the carboxyl-terminal and
3'-untranslated regions of eotaxin-3 (Fig. 1A), was
amplified with two kinds of somatic cell DNA fragments containing human
chromosome 7 (data not shown). To map the eotaxin-3 gene more
precisely, radiation hybrid mapping was conducted. The result was
0100000110 0000000011 0101010010 0011010000 0110111001 1010000100
0000110010 1100001000 1101001010 000 (Whitehead Institute/MIT Center
for Genomic Reseach order), where 0 and 1 represent negative and
positive results in the PCR assay, respectively. These results indicate
that the gene is located at 7q11.2 between chromosomal markers D7S489
and D7S669.
Preparation of the recombinant eotaxin-3 protein
The recombinant eotaxin-3 protein was purified from the culture
medium of baculovirus-infected insect cells. On SDS-PAGE, 10-kDa
major material was detected, showing about 95% purity (Fig. 3). Because this material was not
detected in the culture medium of the insect cells infected with a
control virus which had no recombinant eotaxin-3 cDNA (data not shown),
this should be a product of the recombinant eotaxin-3 gene. N-terminal
amino acid sequence analysis of the purified eotaxin-3 protein showed
that the mature form of human eotaxin-3 started at
Thr24 (data not shown).
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We monitored changes in the free cytosolic
Ca2+ level
([Ca2+]i) in leukocytes
after stimulation with eotaxin-3 (Fig. 4). Like eotaxin, a rapid and drastic
increase in [Ca2+]i was
observed when eotaxin-3 acted on eosinophils. The dose of eotaxin-3
conferring a maximal response on eosinophils was 10 nM, which was
10-fold higher than that of eotaxin (Fig. 4A). The
increase in [Ca2+]i was
not observed toward PBMC and neutrophils stimulated with an excess
concentration of eotaxin-3, unlike MCP-3 or IL-8 (Fig. 4B).
Desensitization experiments were then performed with eosinophils to
determine the receptor selectivity (Fig. 4C). Preexposure of
eosinophils to eotaxin-3 completely blocked the responses to eotaxin
and MIP-1. Furthermore, activation of eosinophils by eotaxin-3 was
completely prevented by prior stimulation with eotaxin, but was hardly
affected by preexposure to MIP-1. These relationships between
eotaxin-3 and MIP-1 were almost the same as those between eotaxin
and MIP-1.
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). Eotaxin-3 was a potent inhibitor of
125I-eotaxin binding to eosinophils, with an
ID50 value of 10 nM, which was 10-fold
higher than that of eotaxin. On the other hand,
125I-MIP-1 binding was hardly affected by the
addition of eotaxin-3 up to 300 nM. Eotaxin and MIP-1 have been
shown to recognize CCR3 and CCR1, respectively (26, 27).
Taken together, these results suggest that eotaxin-3 preferentially
recognizes a receptor for eotaxin, CCR3, on eosinophils. The receptor
usage of eotaxin-3 was examined using K562 cells stably expressing CCR3
(Fig. 6, A and B).
The cells also exhibited calcium flux responses when stimulated with
eotaxin-3 as well as eotaxin. Furthermore, eotaxin-3 competitively
inhibited 125I-eotaxin binding to the K562 cells
as eotaxin did. Eotaxin-3 had no effect on K562 cells not expressing
CCR3 (data not shown). These results also suggest that eotaxin-3
recognizes CCR3. We cannot yet explain why
[Ca2+]i in eosinophils
after stimulation with MIP-1 was completely prevented by prior
stimulation with either eotaxin or eotaxin-3, although they were not
potent inhibitors of 125I-MIP-1 binding (Fig. 4C, Fig. 5B).
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Eotaxin-3 induced strong chemotaxis on eosinophils with a
typically bimodal manner, but not for PBMC and neutrophils (Fig. 7). We observed that MCP-3 and RANTES
preferentially acted on monocytes and lymphocytes, respectively, by
Diff-Quik staining of the migrated cells (data not shown). Checkerboard
analysis revealed that the migration of eosinophils toward eotaxin-3
was "chemotactic" rather than "chemokinetic" (data not
shown).
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10-fold lower than that of eotaxin as described
above, each monkey was injected intradermally with 850 pmol of
eotaxin-3, 100 pmol eotaxin, and isotonic saline alone. Histological
assessment and quantitative image analysis of skin biopsies were
performed at 4 h after injection as described (17).
In the three monkeys, infiltrating eosinophils that exhibited the
characteristic nucleus and Giemsa staining of granules were observed in
association with the venular walls at the sites of injection of either
eotaxin or eotaxin-3, whereas isotonic saline alone had no effect (Fig. 8, Table I). Nonspecific dermal recruitment of
neutrophils was apparent in all specimens, including those injected
with isotonic saline alone (Table I). Furthermore, recruitment of
monocytes and lymphocytes at the sites injected with eotaxin and
eotaxin-3 were not observed.
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Because eotaxin-3 mRNA was expressed in IL-4-stimulated HUVEC, the
changes in its mRNA level after treatment with various cytokines were
studied. Eotaxin-3 mRNA was up-regulated by another Th2 cytokine,
IL-13, having biological activity similar to that of IL-4
(40), but not by either TNF-, IL-1, a Th1 cytokine,
IFN-, or TNF- plus IFN- (Fig. 9). TNF- and IL-1 proteins used
could induce VCAM-1 mRNA, respectively, and TNF- plus IFN- could
induce RANTES mRNA in HUVEC, indicating that these cytokines were
effective (data not shown).
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These results strongly suggest that eotaxin-3 shows Th2-associated expression in VEC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, on eosinophils.
Because CCR3 has been shown to be expressed on basophils
(41) and Th2 cells (42), eotaxin-3 should
also act on these cells. Most CC chemokine genes are clustered on chromosome 17q11.2, and the genes of thymus and activation-regulated chemokine, liver and activation-regulated chemokine, EBII-ligand chemokine, and secondary lymphoid-tissue chemokine were mapped to 16q13, 2q33–37, 9p13, and 9p13, respectively (43, 44). The eotaxin-3 gene has been localized to 7q11.2, where the eotaxin-2 gene is mapped (45). This suggests that eotaxin-3 may be evolutionarily related to eotaxin-2.
Eosinophils are involved in allergic diseases, such as asthma, rhinitis, and atopic dermatitis (46). Elucidation of the mechanisms underlying the accumulation of eosinophils in inflamed tissues is of critical importance for understanding the onset and progress of these eosinophilic diseases. Eosinophils predominantly reside in tissues (47). However, eosinophils infiltrating from the bloodstream are likely involved in these diseases because IL-4, a central mediator of allergic inflammation, acts on VEC and induces cell adhesion molecules, such as P-selectin, L-selectin ligand, and VCAM-1, which are responsible for eosinophil infiltration (10, 11, 12, 13, 14). Chemokines that are involved in the IL-4-dependent recruitment of eosinophils have not been definitely identified yet. Eotaxin, MCP-4, and RANTES, which have been well characterized as potent eosinophil-selective chemoattractants, were not induced on stimulation of HUVEC with IL-4 (24, 28, 48). Because eotaxin-3 mRNA was significantly induced in HUVEC stimulated with IL-4, eotaxin-3 protein may be involved in the IL-4-dependent activation of eosinophils on VEC. We could not detect the expression of eotaxin-3 protein both in IL-4-stimulated HUVEC and their culture medium (data not shown). The eotaxin-3 protein might be unstable or inefficiently expressed in the cultivated cells. Recently, eosinophil accumulation through the action of eotaxin was observed in rat skin stimulated with IL-4 (30). Eotaxin was found in the culture medium of IL-4-stimulated human dermal fibroblasts but not in that of IL-4-stimulated HUVEC (29). Therefore, in atopic diseases, it is possible to speculate that eosinophils are activated by eotaxin-3 expressed on VEC, and then extravasate from the bloodstream to an inflamed tissue, and then migrate to the center of the tissue through the action of eotaxin.
Eotaxin-3 mRNA was not induced in HUVEC stimulated with IFN-. On the
contrary, eotaxin, MCP-4, and RANTES mRNAs were induced in HUVEC
stimulated with either IFN- or TNF- plus IFN- (24, 28, 48), suggesting that these three chemokines play roles at the
site where Th1 cells predominantly exist. An inhalant allergen patch
test on atopic dermatitis patients revealed that the expression of IL-4
mRNA was increased, but that of IFN- mRNA was not, within 24 h.
However, the level of IFN- mRNA increased more than that of IL-4
mRNA within 48 h (49). In this case, eotaxin-3 might
contribute to the early phase response, whereas eotaxin, MCP-4, or
RANTES might mainly act in the late phase to maintain the inflammatory
response.
BCL-6, a repressor of IL-4-induced transcription, was shown to bind to a site recognized by an IL-4-responsive transcription factor, Stat6 (50). Recently, BCL-6-deficient mice were generated in which IL-4 expression is dramatically enhanced. Furthermore, the mice were found to develop myocarditis and pulmonary vasculitis, which were accompanied by cellular infiltrates in the heart and lungs composed of mononuclear cells and polymorphonuclear cells, mostly eosinophils (50). Taking the results obtained in this study into consideration, over-expressed IL-4 may induce eotaxin-3 at the inflamed sites in the heart and lungs of the mice, which may contribute to the accumulation of eosinophils in these inflamed tissues.
In this study, we have shown that a novel chemokine designated as eotaxin-3 mRNA is induced on VEC by IL-4, and the recombinant protein preferentially activates eosinophils. These results suggest that it may play an important role in inflammatory allergic diseases such as asthma and atopic dermatitis. It will be important to obtain definitive evidence for the involvement of eotaxin-3 in the onset and progress of these allergic diseases either by determining the eotaxin-3 levels in allergic patients or by analyzing experimental animal allergic models.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tatsunari Nishi, Tokyo Research Laboratories, Kyowa Hakko Kogyo, 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan. E-mail address:
3 Abbreviations used in this paper: VEC, vascular endothelial cells; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; [Ca2+]i, free cytosolic Ca2+ concentration.
Received for publication November 17, 1998. Accepted for publication May 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1- integrins, and 2-integrins. J. Cell Biol. 125:1417. to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1. J. Immunol. 145:2886.[Abstract]
induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489.4 and 2 integrin-dependent eosinophil accumulation in rat skin in vivo: delayed generation of eotaxin in response to IL-4. J. Immunol. 160:3569.This article has been cited by other articles:
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 M. B. Ruiz-Arguello, V. P. Smith, G. S. V. Campanella, F. Baleux, F. Arenzana-Seisdedos, A. D. Luster, and A. Alcami An Ectromelia Virus Protein That Interacts with Chemokines through Their Glycosaminoglycan Binding Domain J. Virol., January 15, 2008; 82(2): 917 - 926. [Abstract] [Full Text] [PDF]
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 P. C. Fulkerson, C. A. Fischetti, and M. E. Rothenberg Eosinophils and CCR3 Regulate Interleukin-13 Transgene-Induced Pulmonary Remodeling Am. J. Pathol., December 1, 2006; 169(6): 2117 - 2126. [Abstract] [Full Text] [PDF]
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 A. M. Das, K. G. Vaddi, K. A. Solomon, C. Krauthauser, X. Jiang, K. W. McIntyre, X. X. Yang, E. Wadman, P. Welch, M. Covington, et al. Selective Inhibition of Eosinophil Influx into the Lung by Small Molecule CC Chemokine Receptor 3 Antagonists in Mouse Models of Allergic Inflammation J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 411 - 417. [Abstract] [Full Text] [PDF]
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T. Morokata, K. Suzuki, Y. Masunaga, K. Taguchi, K. Morihira, I. Sato, M. Fujii, S. Takizawa, Y. Torii, N. Yamamoto, et al. A Novel, Selective, and Orally Available Antagonist for CC Chemokine Receptor 3 J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 244 - 250. [Abstract] [Full Text] [PDF]
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D. K. Fritz, C. Kerr, L. Tong, D. Smyth, and C. D. Richards Oncostatin-M Up-Regulates VCAM-1 and Synergizes with IL-4 in Eotaxin Expression: Involvement of STAT6 J. Immunol., April 1, 2006; 176(7): 4352 - 4360. [Abstract] [Full Text] [PDF]
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S. M. Pope, N. Zimmermann, K. F. Stringer, M. L. Karow, and M. E. Rothenberg The Eotaxin Chemokines and CCR3 Are Fundamental Regulators of Allergen-Induced Pulmonary Eosinophilia J. Immunol., October 15, 2005; 175(8): 5341 - 5350. [Abstract] [Full Text] [PDF]
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 S. Yamamoto, I. Kobayashi, K. Tsuji, N. Nishi, E. Muro, M. Miyazaki, M. Zaitsu, S. Inada, T. Ichimaru, and Y. Hamasaki Upregulation of Interleukin-4 Receptor by Interferon-{gamma}: Enhanced Interleukin-4-Induced Eotaxin-3 Production in Airway Epithelium Am. J. Respir. Cell Mol. Biol., October 1, 2004; 31(4): 456 - 462. [Abstract] [Full Text] [PDF]
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U. Forssmann, I. Hartung, R. Balder, B. Fuchs, S. E. Escher, N. Spodsberg, Y. Dulkys, M. Walden, A. Heitland, A. Braun, et al. n-Nonanoyl-CC Chemokine Ligand 14, a Potent CC Chemokine Ligand 14 Analogue That Prevents the Recruitment of Eosinophils in Allergic Airway Inflammation J. Immunol., September 1, 2004; 173(5): 3456 - 3466. [Abstract] [Full Text] [PDF]
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 I. Oynebraten, O. Bakke, P. Brandtzaeg, F.-E. Johansen, and G. Haraldsen Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments Blood, July 15, 2004; 104(2): 314 - 320. [Abstract] [Full Text] [PDF]
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 N. Torpey, S. E. Maher, A. L. M. Bothwell, and J. S. Pober Interferon {alpha} but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells J. Biol. Chem., June 18, 2004; 279(25): 26789 - 26796. [Abstract] [Full Text] [PDF]
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V. Petkovic, C. Moghini, S. Paoletti, M. Uguccioni, and B. Gerber Eotaxin-3/CCL26 Is a Natural Antagonist for CC Chemokine Receptors 1 and 5: A HUMAN CHEMOKINE WITH A REGULATORY ROLE J. Biol. Chem., May 28, 2004; 279(22): 23357 - 23363. [Abstract] [Full Text] [PDF]
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U. Atasoy, S. L. Curry, I. Lopez de Silanes, A.-B. Shyu, V. Casolaro, M. Gorospe, and C. Stellato Regulation of Eotaxin Gene Expression by TNF-{alpha} and IL-4 Through mRNA Stabilization: Involvement of the RNA-Binding Protein HuR J. Immunol., October 15, 2003; 171(8): 4369 - 4378. [Abstract] [Full Text] [PDF]
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P. Ogilvie, S. Paoletti, I. Clark-Lewis, and M. Uguccioni Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes Blood, August 1, 2003; 102(3): 789 - 794. [Abstract] [Full Text] [PDF]
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K. Y. Larbi, J. P. Dangerfield, F. J. Culley, D. Marshall, D. O. Haskard, P. J. Jose, T. J. Williams, and S. Nourshargh P-selectin mediates IL-13-induced eosinophil transmigration but not eotaxin generation in vivo: a comparative study with IL-4-elicited responses J. Leukoc. Biol., January 1, 2003; 73(1): 65 - 73. [Abstract] [Full Text] [PDF]
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C. Langdon, C. Kerr, L. Tong, and C. D. Richards Oncostatin M Regulates Eotaxin Expression in Fibroblasts and Eosinophilic Inflammation in C57BL/6 Mice J. Immunol., January 1, 2003; 170(1): 548 - 555. [Abstract] [Full Text] [PDF]
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 A. Shinkai, M. Komuta-Kunitomo, N. Sato-Nakamura, and H. Anazawa N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3 Protein Eng. Des. Sel., November 1, 2002; 15(11): 923 - 929. [Abstract] [Full Text] [PDF]
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C. M. Litterst and E. Pfitzner An LXXLL Motif in the Transactivation Domain of STAT6 Mediates Recruitment of NCoA-1/SRC-1 J. Biol. Chem., September 20, 2002; 277(39): 36052 - 36060. [Abstract] [Full Text] [PDF]
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H. Tachimoto, M. Kikuchi, S. A. Hudson, C. A. Bickel, R. G. Hamilton, and B. S. Bochner Eotaxin-2 Alters Eosinophil Integrin Function via Mitogen-Activated Protein Kinases Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 645 - 649. [Abstract] [Full Text] [PDF]
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S. N. Georas, L. A. Beck, and C. Stellato What Is Eotaxin Doing in the Pleura? . Insights into Innate Immunity from Pleural Mesothelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 2002; 26(4): 384 - 387. [Full Text] [PDF]
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K. Watanabe, P. J. Jose, and S. M. Rankin Eotaxin-2 Generation Is Differentially Regulated by Lipopolysaccharide and IL-4 in Monocytes and Macrophages J. Immunol., February 15, 2002; 168(4): 1911 - 1918. [Abstract] [Full Text] [PDF]
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S. L. Cuvelier and K. D. Patel Shear-dependent Eosinophil Transmigration on Interleukin 4-stimulated Endothelial Cells: A Role for Endothelium-associated Eotaxin-3 J. Exp. Med., December 10, 2001; 194(12): 1699 - 1709. [Abstract] [Full Text] [PDF]
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C. M. Lilly and B. L. Daugherty A Novel LPS-Inducible CCR3 Activator . Why So Many CCR3 Ligands? Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 673 - 675. [Full Text] [PDF]
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J. Hoeck and M. Woisetschlager Activation of Eotaxin-3/CCL26 Gene Expression in Human Dermal Fibroblasts Is Mediated by STAT6 J. Immunol., September 15, 2001; 167(6): 3216 - 3222. [Abstract] [Full Text] [PDF]
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N. W. Lukacs, K. K. Tekkanat, A. Berlin, C. M. Hogaboam, A. Miller, H. Evanoff, P. Lincoln, and H. Maassab Respiratory Syncytial Virus Predisposes Mice to Augmented Allergic Airway Responses Via IL-13-Mediated Mechanisms J. Immunol., July 15, 2001; 167(2): 1060 - 1065. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, A. Herbst, and P. F. Weller Eotaxins . Contributing to the Diversity of Eosinophil Recruitment and Activation Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 653 - 657. [Full Text] [PDF]
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N. Berkman, S. Ohnona, F. K. Chung, and R. Breuer Eotaxin-3 but Not Eotaxin Gene Expression Is Upregulated in Asthmatics 24 Hours after Allergen Challenge Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 682 - 687. [Abstract] [Full Text] [PDF]
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A. Mathew, J. A. MacLean, E. DeHaan, A. M. Tager, F. H.Y. Green, and A. D. Luster Signal Transducer and Activator of Transcription 6 Controls Chemokine Production and T Helper Cell Type 2 Cell Trafficking in Allergic Pulmonary Inflammation J. Exp. Med., May 7, 2001; 193(9): 1087 - 1096. [Abstract] [Full Text] [PDF]
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M. Stassen, C. Muller, M. Arnold, L. Hultner, S. Klein-Hessling, C. Neudorfl, T. Reineke, E. Serfling, and E. Schmitt IL-9 and IL-13 Production by Activated Mast Cells Is Strongly Enhanced in the Presence of Lipopolysaccharide: NF-{{kappa}}B Is Decisively Involved in the Expression of IL-9 J. Immunol., April 1, 2001; 166(7): 4391 - 4398. [Abstract] [Full Text] [PDF]
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 R. K. Mehlotra, L. R. Hall, M. A. Haxhiu, and E. Pearlman Reciprocal Immunomodulatory Effects of Gamma Interferon and Interleukin-4 on Filaria-Induced Airway Hyperresponsiveness Infect. Immun., March 1, 2001; 69(3): 1463 - 1468. [Abstract] [Full Text] [PDF]
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F. H. Falcone, H. Haas, and B. F. Gibbs The human basophil: a new appreciation of its role in immune responses Blood, December 15, 2000; 96(13): 4028 - 4038. [Full Text] [PDF]
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H. Tachimoto, M. M. Burdick, S. A. Hudson, M. Kikuchi, K. Konstantopoulos, and B. S. Bochner CCR3-Active Chemokines Promote Rapid Detachment of Eosinophils from VCAM-1 In Vitro J. Immunol., September 1, 2000; 165(5): 2748 - 2754. [Abstract] [Full Text] [PDF]
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 P. M. Murphy, M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors Pharmacol. Rev., March 1, 2000; 52(1): 145 - 176. [Abstract] [Full Text] [PDF]
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M. R. Mayer and M. J. Stone Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin J. Biol. Chem., April 20, 2001; 276(17): 13911 - 13916. [Abstract] [Full Text] [PDF]
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I. Sabroe, M. J. Peck, B. J. Van Keulen, A. Jorritsma, G. Simmons, P. R. Clapham, T. J. Williams, and J. E. Pease A Small Molecule Antagonist of Chemokine Receptors CCR1 and CCR3. POTENT INHIBITION OF EOSINOPHIL FUNCTION AND CCR3-MEDIATED HIV-1 ENTRY J. Biol. Chem., August 18, 2000; 275(34): 25985 - 25992. [Abstract] [Full Text] [PDF]
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), and
changes of the relative fluorescence ratio were measured. Duplicate
experiments were performed, and the values whose error ranges were
within 5% were averaged. B, Fura-2-loaded human blood
PBMC and neutrophils were stimulated with 0.8 µM eotaxin-3 or 0.1
µM of another CC chemokine, as indicated by arrows. The results are
representative of two separate experiments. C,
Cross-desensitization of human blood eosinophils. Fura-2-loaded
eosinophils were stimulated sequentially at 90-s intervals with 0.8
µM eotaxin-3 and 0.1 µM of another CC chemokine. The chemokines
were added at the times indicated by arrows. The results are
representative of two separate experiments.
) for 1
h at 37°C. The bound 125I-labeled chemokines were
measured. Three independent experiments were performed, and the results
were expressed as the mean ± SD. C represents 0 nM
chemokine.
), MCP-3 (
), or RANTES (
) was
placed in the lower wells. Migrated cells were collected and counted by
measuring the eosinophil peroxidase activity for eosinophils, and
dehydrogenase activity in mitochondria for PBMC and neutrophils. Three
independent experiments were performed, and the results were expressed
as the mean ± SD. C represents 0 nM chemokine.
