By
From the * Theodor Kocher Institute, University of Bern, CH-3000 Bern, Switzerland; Division of
Rheumatology, University Hospital, CH-3010 Bern, Switzerland; § Institute of Immunology and
Allergology, University Hospital, CH-3010 Bern, Switzerland; and
Hoffmann-La Roche Ltd.,
Basel, Switzerland
A novel human CC chemokine consisting of 78 amino acids and having a molecular mass of 8,778.3 daltons (VVIPSPCCMF FVSKRIPENR VVSYQLSSRS TCLKAGVIFT TKKGQQ SCGD PKQEWVQRYM KNLDAKQKKA SPRARAVA) was isolated together with three minor COOH-terminally truncated variants with 73, 75, and 76 residues. The new chemokine was termed eotaxin-2 because it is functionally very similar to eotaxin. In terms of structure, however, eotaxin and eotaxin-2 are rather distant, they share only 39% identical amino acids and differ almost completely in the NH2-terminal region. Eotaxin-2 induced chemotaxis of eosinophils as well as basophils, with a typically bimodal concentration dependence, and the release of histamine and leukotriene C4 from basophils that had been primed with IL-3. In all assays, eotaxin-2 had the same efficacy as eotaxin, but was somewhat less potent. The migration and the release responses were abrogated in the presence of a monoclonal antibody that selectively blocks the eotaxin receptor, CCR3, indicating that eotaxin-2, like eotaxin, acts exclusively via CCR3. Receptor usage was also studied in desensitization experiments by measuring [Ca2+]i changes in eosinophils. Complete cross-desensitization was observed between eotaxin-2, eotaxin and MCP-4 confirming activation via CCR3. No Ca2+ mobilization was obtained in neutrophils, monocytes and lymphocytes, in agreement with the lack of chemotactic responsiveness. Intradermal injection of eotaxin-2 in a rhesus monkey (100 or 1,000 pmol per site) induced a marked local infiltration of eosinophils, which was most pronounced in the vicinity of postcapillary venules and was comparable to the effect of eotaxin.
Leukocyte recruitment in inflammation and immunity is
regulated by a large number of CXC and CC chemokines (1). In the past few years several new proteins of this
class have been discovered and considerable information
has been gained about the functions of the known ones. It
has been shown in particular that the monocyte chemotactic proteins (MCP-1, MCP-2, MCP-3, and MCP-4) do not
only act on monocytes, as their name suggests, but also on
lymphocytes (2, 3) and basophils (4, 5), and that MCP-2,
MCP-3 and MCP-4 are potent attractants for eosinophils
(6).
Eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic diseases (9). The mechanisms by which these cells are recruited and activated is widely studied because of the
pathological consequences resulting from the release of
their phlogogenic and cytotoxic products. The challenge is
to understand the selectivity of the chemotactic process and
to develop efficient inhibitors. Several factors have been
proposed as eosinophil attractants such as the anaphylatoxin
C5a (10), platelet-activating factor (11), and more recently
RANTES (12) and IL-16 (13). None of these stimuli is selective, however, and the discovery of eotaxin, which was originally shown to attract only eosinophils and to be specific for a single chemokine receptor, CCR3 (14), was
greeted as a promising advance (17).
Within a large-scale sequencing and expression program
for the discovery of new chemokines, we have recently
identified MCP-4, a CC chemokine with powerful effects
on eosinophils (8). In this paper, we describe a chemokine
that is also active on eosinophils and is functionally very
similar to eotaxin. It attracts and activate human eosinophils and basophils via the eotaxin receptor, CCR3, and
has no activity on other leukocytes. Because of these properties, we have named the novel chemokine eotaxin-2.
Cloning, Expression, Purification and Analysis.
The EST representing CK Chemokines.
Eotaxin, MCP-3, MCP-4, RANTES, and
MIP-1 CCR3-blocking Antibody.
The anti-CCR3 monoclonal antibody 7B11 which selectively blocks the eotaxin receptor (20) was
kindly provided by Dr. Charles Mackay (LeukoSite Inc., Cambridge, MA).
Cells.
Monocytes (21), lymphocytes (3), and neutrophils (22)
were isolated from donor blood buffy coats. The lymphocytes
were cultured in the presence of IL-2 as previously described (3). Eosinophils (12) and basophils (23, 24) were purified from the
venous blood of healthy volunteers. The eosinophil preparations were >95% pure and the basophil preparations consisted of 70- 80% basophils and 20-30% lymphocytes.
[Ca2+]i Changes.
Changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were assayed in monocytes, eosinophils, lymphocytes, and neutrophils loaded with Fura-2 (25). Receptor desensitization was tested by monitoring [Ca2+]i changes in response to
sequential stimulation with chemokines (21).
In Vitro Chemotaxis.
Chemotaxis was assessed in 48-well
chambers (Neuro Probe, Cabin John, MD) using polyvinylpyrrolidone-free polycarbonate membranes (Nucleopore, Neuro
Probe, Cabin John, MD) with 5-µm pores for eosinophils, basophils, neutrophils and monocytes, and 3-µm pores for lymphocytes (3, 6). Cell suspensions and chemokine dilutions were made
in RPMI 1640 supplemented with 20 mM Hepes, pH 7.4, and
1% pasteurized plasma protein solution (Central Laboratory of the
Swiss Red Cross). Migration was allowed to proceed for 60 min
at 37°C in 5% CO2. The membrane was then removed, washed on the upper side with PBS, fixed, and stained. All assays were done in triplicate, and the migrated cells were counted in five randomly selected fields at 1,000-fold magnification. Spontaneous migration was determined in the absence of chemoattractant.
Histamine and Leukotriene C4 (LTC4) Release.
Basophils (0.1-
0.3 × 106 cells/ml) in 20 mM Hepes, pH 7.4, containing 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.5 mM glucose and 0.025% BSA were warmed to 37°C, primed with 10 ng/ml IL-3 for 5 min, and then challenged with a chemokine.
The reaction was stopped on ice after 20 min and histamine and
LTC4 were measured in the supernatants (26). Histamine release
was expressed as percent of the total cellular content (determined
after cell lysis). LTC4 generation was expressed in ng per 106 basophils.
Enzyme Release.
N-acetyl- In Vivo Activity.
A male rhesus monkey of 7.5 kg was anesthesized by injection of 10 mg/kg Ketamine (Ketalar, Parke Davis)
i.m. and 15 mg/kg Na Thiopental (Pentotal, Abbott) i.v. The
chemokines (100 pmol eotaxin, 100 and 1,000 pmol eotaxin-2 in
100 µl pyrogen-free isotonic saline) were then administered intradermally on the back, and full skin thickness punch biopsies of
8-mm diameter were taken from the injection sites after 4 h. The
biopsies were fixed in formalin, embedded in paraffin, and 5-µm
sections were prepared. The sections were stained with Giemsa
solution or hematoxylin and eosin, and the eosinophil infiltrates
were evaluated by two independent observers. In each section,
eosinophils were counted at a magnification of 630× in five randomly selected fields including a postcapillary venule, using a grid
of 0.19 × 0.19 mm, and the number of eosinophils per mm2 was
calculated. Photographs were taken at a magnification of 1,000×
with a Nikon Microphot microscope (Nikon AG, Switzerland).
The purified material contained a
protein of ~8.5 kD, as judged on SDS-PAGE. The material was treated with GluC under conditions ensuring cleavage COOH-terminally of glutamic acid, and three fragments were obtained. The first two were homogeneous
and extended from residue 1 to 18 and 19 to 54, respectively, while the third showed some COOH-terminal heterogeneity. Sequencing and mass spectrometry revealed
tree COOH-terminally truncated forms consisting of residues 55-73, 55-75, and 55-76 in addition to the main
form extending from residue 55 to 78 (Fig. 1). The mass of
the 78-residue form of eotaxin-2 is 8,778.3 and the amino
acid identities to reference chemokines were 43% for
MCP-4, 42% for MIP-1
[Ca2+]i changes,
were monitored in blood leukocytes after stimulation with
1 to 1,000 nM eotaxin-2. A marked, concentration-dependent effect was observed on eosinophils whereas neutrophils,
monocytes and T lymphocytes did not respond. Desensitization experiments were then performed with eosinophils
to gain information on receptor selectivity. As shown in
Fig. 2, eosinophil stimulation with eotaxin-2 abrogated the
response to eotaxin, attenuated the responses to MCP-3
and RANTES, but did not appreciably affect the response to MIP-1
As shown in Fig. 3, eotaxin-2 was
a highly effective attractant for eosinophils and basophils.
The concentration dependence was typically bimodal, and
the overall effect was similar to that of eotaxin. Maximum
migration was reached at 100 nM eotaxin-2 and 10 nM eotaxin which thus appears to be more potent. When the assay was performed in the presence of the anti-CCR3 antibody, eosinophil and basophil chemotaxis toward either
eotaxin or eotaxin-2 was completely prevented, indicating
that they both acted exclusively via CCR3. Eotaxin-2, like
eotaxin, did not induce migration of neutrophils, monocytes and T lymphocytes, which is in agreement with the
lack of Ca2+ mobilization observed in these cells.
In view of the marked
chemotactic activity toward basophils, the effect of eotaxin
and eotaxin-2 as inducers of release was determined (Fig.
4). Both chemokines induced a similar release of histamine
and peptido leukotrienes in IL-3 pretreated basophils from
several unselected donors. The curves relating effect to concentration show that eotaxin was somewhat more potent than eotaxin-2 in particular as inducer of LTC4 release.
Similar release responses were obtained with RANTES and
MIP-1
The ability of eotaxin-2 to act as
chemoattractant in vivo was examined in a rhesus monkey
in comparison with eotaxin. As shown in Fig. 5 A, 4 h after
injection of 100 pmol per site both chemokines induced a
similar, marked eosinophil infiltration whereas the vehicle
alone had no effect. The number of infiltrating eosinophils was ~50% higher at sites where 1,000 pmol eotaxin-2 was
applied. The effect is remarkable because the monkey used
in this experiment had a low blood eosinophil count (0.7%
of total leukocytes). As shown by representative micrographs, several infiltrating eosinophils can be recognized by
the characteristic nucleus and the Giemsa staining of the
granules. They are found in the vicinity of postcapillary venules (Fig. 5, B and C) and in association with the venular wall (Fig. 5 C).
This paper describes a chemokine which we have
termed eotaxin-2 because its function is perfectly analogous
to that of eotaxin. Both chemokines activate and attract
eosinophils and basophils, but no other leukocytes, and appear to act exclusively via CCR3. It must be mentioned,
however, that a virtually identical chemokine, MPIF-2,
was reported to inhibit colony formation by myeloid progenitor cells (18). The functional similarity between eotaxin and eotaxin-2 is astonishing because these chemokines share only 39% identical amino acids. In addition, they
differ almost completely within the short NH2-terminal sequence preceding the first cysteine, which has been identified as the receptor triggering region of several chemokines
(27). It is well established that all CXC chemokines that
activate neutrophils via the IL-8 receptors, CXCR1 and
CXCR2, share a conserved Glu-Leu-Arg motif preceding
the first cysteine that tolerates only minimal substitutions
(28, 29), and it has been shown more recently that CC
chemokines drastically lose activity toward monocytes (1)
and basophils (27, 30) upon deletion of as few as one or two
NH2-terminal amino acids. Yet the present findings show
selective activation of the same receptor, CCR3, by two
chemokines that have only one common residue (Pro-4 of
eotaxin-2 corresponding to Pro-6 of eotaxin) in the presumed receptor triggering domain. The eotaxin sequence is
actually much more similar (~60% identity) to that of
MCP-3, which binds to CCR3, but also to CCR1 and
CCR2 and even to MCP-1, a selective ligand for CCR2,
which is inactive on eosinophils.
Eotaxin is an unusually selective chemokine (17). It was
discovered as attractant for eosinophils in the bronchoalveolar lavage fluid obtained in an experimental model for
lung allergy in guinea-pigs (17, 31) and subsequently
shown to occur in mice (32) and humans (15) as well. The
present discovery of a chemokine that shares with eotaxin
receptor specificity, in vitro activities on eosinophils and
basophils, and the ability to elicit eosinophil infiltration in
vivo reveals the existence of a mechanism for the concomitant and selective recruitment of the two types of granulocytes that are characteristic of allergic inflammation.
-6 cDNA was identified in the database of Human
Genome Sciences Inc. (Rockville, MD) on the basis of the CC
motif and the homolgy to known CC chemokines (18). The
cDNA was isolated from a library derived from activated human monocytes, and the mature protein was expressed in Sf9 insect cells (CRL 1711; American Type Culture Collection, Rockville, MD). Purification was performed by cation exchange, heparin affinity, and size exclusion chromatograpy (poros 50 HS, poros 20 HE1; Perseptive Biosystem; and Sephacryl S200 HR; Pharmacia)
in the presence of protease inhibitors (20 mg/ml Pefabloc SC;
Boehringer Mannheim, 1 mg/ml leupeptin, 1 mg/ml E64, and
1 mM EDTA). The purified protein was analyzed by laser desorption mass spectrometry (matrix-assisted laser desorption ionization-time of flight) and by Edman degradation after partial
proteolysis with the endoprotease GluC (Boehringer Mannheim).
were used as standards. MCP-4 was cloned and expressed as described previously (8), and the other chemokines
were chemically synthesized by Dr. I. Clark-Lewis (Biomedical
Research Centre, University of British Columbia, Vancouver,
Canada) (19).
-D-glucosaminidase release was
assayed in monocytes (21) and elastase release in neutrophils (22)
exactly as described previously.
Eotaxin-2 Structure.
, 39% for MCP-3 and eotaxin,
and 32% for RANTES. Since the four COOH-terminal
variants could not be separated all tests were performed
with the mixture. The sequence shown in Fig. 1 corresponds to the cDNA-deduced sequence of the chemokine
MPIF-2 that was shown to inhibit the proliferation of myeloid progenitors (18), except for two substitutions, Ala35
for Gly and Ser47 for Phe.
Fig. 1.
Amino acid sequences of eotaxin-2 aligned with eotaxin, MCP-3, MCP-4, MIP-1, and RANTES. Identical amino acids are indicated by
a hyphen. The sites of COOH-terminal truncation leading to the shorter variants are indicated by arrowheads.
[View Larger Version of this Image (13K GIF file)]
. In agreement with these results the [Ca2+]i rise
induced by eotaxin-2 was abrogated by prior stimulation with eotaxin, decreased by stimulation with RANTES or
MCP-3, but was not affected by MIP-1
. Cross-desensititzation was also observed between eotaxin-2 and MCP-4
(data not shown). These results suggest that eotaxin-2 is a
selective agonist for eosinophils. The complete cross-desensitization between eotaxin-2 and eotaxin indicates that both
chemokines act mainly, if not exclusively, via CCR3. This
conclusion is in agreement with the partial desensitization of the responses to RANTES and MCP-3, which are
known to interact with at least two additional receptors,
CCR1 and CCR2. The lack desensitization of the response to MIP-1
, on the other hand rules out an interaction of eotaxin-2 with CCR1.
Fig. 2.
Cross-desensitization of human blood eosinophils. Fura2-loaded cells were stimulated sequentially at 90-s intervals with 50 nM
eotaxin-2 and another CC chemokine at the same concentration, and
[Ca2+]i dependent fluorescence changes were recorded. The tracings are
representative for three separate experiments performed under identical
conditions with cells from different donors.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
Chemotactic responses of human blood eosinophils and basophils to eotaxin
and eotaxin-2, in the presence
(open symbols) or absence (closed
symbols) of 10 µg/ml anti-CCR3
added to the cells 10 min before
loading into the chemotaxis
chamber. Numbers of migrating
cells per five high power fields
are given. One out of three similar experiments performed with
cells from different donors is
shown.
[View Larger Versions of these Images (20 + 18K GIF file)]
(data not shown). In agreement with the data of
the chemotaxis assays, mediator release induced by eotaxin
and eotaxin-2 was markedly inhibited by pretreatment of
the cells with the anti-CCR3 antibody.
Fig. 4.
(A and B) Histamine and LTC4 release by basophils pretreated with IL-3 in response to increasing concentrations of eotaxin and
eotaxin-2. Each symbol represents mean values from four experiments performed with cells from different donors. (C and D) Histamine and
LTC4 release by IL-3 pretreated basophils after stimulation with 100 nM
eotaxin or eotaxin-2 in the presence (open columns) or absence (filled columns) of anti-CCR3. Mean ± SEM of three experiments performed with
basophils from different donors.
[View Larger Version of this Image (23K GIF file)]
Fig. 5.
(A) In vivo infiltration of eosinophils. A rhesus monkey received intradermal injections of eotaxin-2 (100 or 1,000 pmol per site), eotaxin
(100 pmol per site) or pyrogen-free isotonic saline. After 4 h, punch biopsies were taken and processed for histology. Eosinophils were counted on sections stained with hematoxylin and eosin (five fields per section) in areas including postcapillary venules. (B) Micrograph of a skin site 4 h after injection
of 100 pmol eotaxin-2, stained with Giemsa solution. Clearly identifiable eosinophils are seen near the inset; one is seen in contact with the venule wall.
(C) Micrograph of a skin site 4 h after injection of 1,000 pmol eotaxin-2, stained with hemaotxylin and eosin. Two eosinophils are within the venule wall
(see also inset) and others are in the lumen and in the tissue.
[View Larger Versions of these Images (23 + 159 + 141K GIF file)]
Address correspondence to Marco Baggiolini, Theodor Kocher Institute, PO Box, CH-3000 Bern 9, Switzerland.
Received for publication 7 March 1997 and in revised form 18 April 1997.
Donor blood buffy coats were provided by the Swiss Central Laboratory Blood Transfusion Service, SRK. This work was supported by Grant 31-39744.93 of the Swiss National Science Foundation (to M. Baggiolini).We thank Dr. Ian Clark-Lewis (Biomedical Research Center, University of British Columbia, Vancouver,
Canada) for the supply of reference chemokines, Dr. Gianni Garotta (Human Genome Sciences Inc., Rockville, MD) for supplying a purified preparation of CK-6. Dr. Charles Mackay (LeukoSite Inc., Cambridge,
MA) for the anti-CCR3 monoclonal antibody 7B11, Dr. Antoinette Wetterwald (Institute of Pathophysiology, University of Bern, Switzerland) for advice on histology, and Dr. Beatrice Dewald (Theodor Kocher
Institute, University of Bern, Switzerland) for critical reading of the manuscript. We acknowledge the expert
technical assistance of Andrea Blaser and Silvia Rihs. We are grateful to Dr. Pedro Cuevas and Dr. WolfGeorg Forssmann (Ramon y Cajal Hospital, Madrid, Spain) for the application in vivo. The experiment was
approved by the Animal Ethical Committee of the Hospital and carried according to the European Union
guidelines for reducing pain and discomfort to experimental animals.
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