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*
Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center,
Molecular and Developmental Biology Graduate Program, Children’s Hospital Research Foundation, and
Division of Immunology, Department of Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45229
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2). However, none of these mediators selectively promote
eosinophil recruitment. In contrast, a group of human chemokines,
designated eotaxins, with eosinophil-selective chemoattractant activity
has been identified.
Eotaxin was initially discovered using a biological assay in guinea
pigs designed to identify the molecules responsible for
allergen-induced eosinophil accumulation in the lungs. Using an in vivo
chemotaxis assay in guinea pig skin, the partial amino acid sequence
for the protein responsible for eosinophil chemoattraction in the
bronchoalveolar fluid in allergen-challenged guinea pigs was determined
(3). This facilitated the genetic cloning of the genes and
cDNA for guinea pig, murine, and human eotaxin, and the identification
of eotaxin as a member of the CC chemokine family most homologous to
the macrophage chemoattractant protein (MCP) subfamily
(4, 5, 6, 7). This subfamily of eotaxin and MCP chemokines is
clustered on human chromosome 17q11, a region that also contains other
CC chemokines (such as MIP-1, I-309, RANTES, and hemofiltrate CC
chemokine (HCC)-1 and -2, Ref. 8). Interestingly, this
region has been recently linked to asthma susceptibility
(9). Using genomic analyses, rather than biological
assays, two additional chemokines have been identified in the human
genome that encode for CC chemokines with eosinophil-selective
chemoattractant activity, and have hence been designated eotaxin-2 and
eotaxin-3 (10, 11, 12, 13). Eotaxin-2 and eotaxin-3 are only
distantly related to eotaxin-1 because they are only 
30% identical
in sequence and are located in a different chromosomal position
(7q11.23). The specific activity of all three human eotaxin chemokines
is mediated by the selective expression of the eotaxin receptor, CCR3,
a seven-transmembrane-spanning G protein linked genetically polymorphic
receptor, primarily expressed on eosinophils (14, 15, 16, 17).
CCR3 is a promiscuous receptor; it interacts with multiple ligands
including MCP-2, -3, -4, RANTES, and HCC-2 (MIP-5, leukotactin);
however, the only ligands that signal exclusively through this receptor
are the eotaxin chemokines, accounting for the cellular selectivity of
eotaxin. Interestingly, other cells involved in allergic responses, Th2
cells and basophils, also express CCR3 (18, 19, 20); however,
the significance of CCR3 expression on these cells has been less
clearly demonstrated than on eosinophils.
A variety of approaches have been used to determine the biological role of the eotaxin chemokines. Eotaxin-1 is induced by allergen challenge in the human respiratory tract and eotaxin-1 and eotaxin-2 are over-expressed in allergic inflammatory tissue (21, 22, 23). However, the relative contribution of the eotaxin chemokines compared with other chemoattractants has not been extensively addressed. Substantial progress has been made by analysis of the role of chemokines in mice. In particular, Ab neutralization experiments and eotaxin-1 gene targeting in mice have revealed a nonredundant role for eotaxin-1 in allergic lung inflammation and in the regulation of eosinophil homing to the gastrointestinal tract (24, 25, 26, 27). Eotaxin-1 gene-targeted mice have a marked deficiency of tissue-dwelling eosinophils at baseline (25) and this has recently facilitated the identification of a role for eosinophils in postnatal mammary gland development (28). Although eotaxin-1 gene-targeted mice have a marked deficiency in tissue-dwelling eosinophils, they still have resident eosinophils, especially in the gastrointestinal tract when IL-5 is overexpressed in vivo (29). Understanding the full biological and pathological role of eosinophils will likely be facilitated by identification and characterization of the eotaxin-2 and eotaxin-3 homologues in mice. We now report the characterization of murine eotaxin-2 cDNA and protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nucleotide sequence of human eotaxin-2 (CC chemokine ligand (CCL)-24 according to new classification (30)) was used to screen the public expressed sequence tag (EST) database by use of BLASTN searches. The cDNA clones AI536479 and AI099007, encoding a putative murine homologue of eotaxin-2, were obtained from Genome Systems (St. Louis, MO) and subjected to sequencing. The predicted cleavage site of the mature form of eotaxin-2 was determined by use of the SignalP server at http://www.cbs.dtu.dk/services/SignalP/.
Genomic DNA analysis
The eotaxin-2 gene was amplified by PCR from genomic DNA isolated from a wild-type mouse. The primers used were located just upstream of the ATG (5'-CTG TGC CTG ACC TCC AGA AC-3') and at the end of the open reading frame (ORF) (5'-CTA AAC CTC GGT GCT ATT GC-3'). The 2.2-kb PCR product was subcloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced. Location of exons was determined by homology with the cDNA sequence. All splice donor and acceptor sites conform with the established consensus.
RNA preparation and Northern blot analysis
RNA from multiple tissues (stomach, jejunum, liver, kidney, spleen, lung, thymus, brain, and testis) was isolated from BALB/c mice using the Trizol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. RNA was electrophoresed in an agarose-formaldehyde gel, transferred to Gene Screen transfer membranes (NEN, Boston, MA) in 10x SSC, and cross-linked by UV radiation. The murine eotaxin-2 cDNA fragment was isolated from clone AI099007 with EcoRI and NotI digestion; cDNA fragments were radiolabeled with 32P using the Klenow reaction with random priming. Membranes were hybridized and washed under standard conditions and exposed to film for 3–7 days.
IL-4 overexpression
Wild-type or STAT-6 gene-targeted mice (31) expressing the murine IL-4 transgene under the control of lung-specific Clara cell 10 promoter (32) were generated by mating F1 crosses of IL-4-transgenic mice and STAT-6 gene-targeted mice with STAT-6 gene-targeted mice. The IL-4 transgene and the STAT-6 gene-targeted locus were screened by PCR analysis (31, 32) and RNA was isolated from whole lungs. In other experiments, ether-anesthetized wild-type mice were treated with 2 µg intranasal rIL-4 (PeproTech, Rocky Hill, NJ) complexed to 10 µg anti-IL-4 mAb (BVD4-1D11 (33); a gift from DNAX Research Institute, Palo Alto, CA) in 20 µl of saline on three occasions separated by 2 days. Following 24 h after the last dose, whole lung RNA was isolated.
Murine models of asthma
A mouse model of allergic lung disease was established using methods described previously (29, 34). In brief, mice were lightly anesthetized with Metofane inhalation (methoxy-fluorane; Pittman-Moore, Mundelein, IL) and 100 µg (50 µl) Asperigillus fumigatus (Bayer Pharmaceuticals, Spokane, WA) or 50 µl of normal saline alone was applied to the nasal cavity using a micropipette with the mouse held in the supine position. After instillation, mice were held upright until alert. After three treatments per week for 3 wk, mice were sacrificed between 18 and 20 h after the last intranasal challenge. Alternatively, mice were sensitized and challenged with OVA as previously described (24).
Eotaxin protein production
Recombinant murine eotaxin-2 protein was produced by PeproTech
in Escherichia coli from a recombinant strain carrying a
synthetic gene sequence encoding eotaxin-2 preceded with a synthetic
leader sequence, MKKKSLAMVTIP. The leader sequence is
underlined and was removed by cleavage with aminopeptidase, an enzyme
that that cleaves the amino acid penultimate to proline, therefore
yielding eotaxin-2 (IP). Alternatively, the leader sequence was removed
by treatment with cyanogen bromide that cleaves following a methionine,
thus yielding eotaxin-2 (VTIP). The protein was purified by sequential
column chromatography and the sequence and/or purity was verified by
mass spectroscopy, Edman sequencing, HPLC, and SDS-PAGE. Additionally,
eotaxin-2 (IDSVTIP) was purchased from R&D Systems (Minneapolis, MN). A
comparable eotaxin-2 amount in the three preparations was shown by
SDS-PAGE separation followed by Coomassie staining. Recombinant murine
eotaxin-1, KC and MIP-1 were obtained from PeproTech.
Chemotaxis assay
Chemotactic responses were determined by transmigration through
either bare filters (macrophages) or respiratory epithelial cells
(granulocytes) as previously described (35). In brief,
A549 cells (American Type Culture Collection, Manassas, VA) were grown
as monolayers in tissue-culture flasks in DMEM (Life Technologies)
supplemented with 10% FCS, penicillin, and streptomycin. Cell
monolayers were trypsinized, centrifuged, and resuspended in fresh
medium before culture on permeable filters (polycarbonate filters with
3-µm pores) in Transwell tissue-culture plates (Corning Costar,
Cambridge, MA). Cells (1.5 x 105) in
100-µl volumes were grown to confluence on the upper surface of the
filters for 2 days and treated with 10 ng/ml TNF- for 18 h.
Leukocytes (1 x 106) in HBSS plus 0.5% BSA
(low endotoxin; Sigma, St. Louis, MO) were placed in the upper chamber
and the chemokine (in HBSS plus 0.5% BSA) was placed in the lower
chamber. Eosinophils were obtained by use of splenocytes from
IL-5-transgenic mice (4); neutrophils were obtained from
the peritoneal cavity 3 and 18 h after injection of 9% (w/v)
casein (Sigma). Transmigration was allowed to proceed for 3 h for
neutrophils and 2.5 h for splenocytes. Macrophages were obtained
from the peritoneal cavity of mice 72 h after injection of 3%
(w/v) thioglycollate (Becton Dickinson, Mountain View, CA). Cells were
placed in the upper chamber of the bare filters with 5-µm pores and
allowed to migrate toward the chemokines placed in the bottom chamber
for 3 h. Finally, cells in the lower chamber were counted in a
hemacytometer, cytocentrifuged, stained by immersion for 2 min in
Harleco Wright-Giemsa stain (EM Science, Gibbstown, NJ) followed by
DiffQuick (Fisher Scientific, Pittsburgh, PA) and the differential
white cell analysis was determined microscopically.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human eotaxin-2 and eotaxin-3 cDNA sequences were used to
screen the public mouse EST databank. Interestingly, over the course of
this study, two murine ESTs that were homologous to human eotaxin-2,
but no eotaxin-3 homologues, have been identified. This suggests that
the mouse genome may in fact contain only two eotaxin genes. The mouse
eotaxin-2 homologues were derived from mammary gland cDNA libraries. An
additional EST derived from porcine small intestine was also homologous
to eotaxin-2 (70% identity over 385 bp). The two mouse EST clones
(accession numbers AI536479 and AI099007) were subjected to sequencing
and the consensus sequence of the ORF was found to have considerable
homology to human eotaxin-2 (58%); homology to other chemokines was
significantly less (e.g., 35.1 and 35.6% homology to murine eotaxin-1
and human eotaxin-3, respectively). Using the EST sequences, the
longest 5' untranslated region (UTR) was found to contain 53 bp,
whereas the 3' UTR contained 183 bp. The nucleotide sequence of the
consensus of the two clones verified by sequencing is shown in Fig. 1
A. To elucidate the genomic
organization of the eotaxin-2 gene, genomic DNA was amplified by PCR
using primers immediately upstream of the start codon and at the end of
the ORF. A 2.2-kb fragment was sequenced and compared with the cDNA
sequence. This analysis demonstrated that the DNA encoding eotaxin-2
ORF is located on three exons (Fig. 1B). The three exons
contain 73, 118, and 169 bp of the ORF, respectively (Fig. 1A). The introns are 0.2 and 1.6 kb in size. This is
comparable to the human eotaxin-2 gene, which can be located under
accession number AC005102 (the complete sequence of a bacterial
artificial chromsome containing human chromosome 7q11.23-q21.1
including eotaxin-2 and eotaxin-3 genes). The cDNA contains an ORF that
encodes for a protein of 119 amino acids (Fig. 2). The deduced polypeptide sequence
contains a highly hydrophobic amino-terminal region characteristic of a
signal peptide with a predicted cleavage site between
Ser26 and Val27. The
predicted mature protein of 93 amino acids has a molecular mass of
10,335 Da and an isoelectric point of 10.4. There are two potential
sites for N-glycosylation at positions 54 and 115 and one
for O-glycosylation at threonine in position 28. The mature
protein is a CC chemokine with four cysteine residues in the proper
arrangement and is most homologous to human eotaxin-2 (59.1%) and only
38.9 and 38.2% homologous to murine eotaxin-1 and human eotaxin-3,
respectively. An alignment of murine and human eotaxin-2 (Fig. 2)
reveals an equal number of amino acids and comparable conservation of
homology throughout the protein sequence (including the leader
sequence) and conservation of the amino-terminal valine in the mature
protein.
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Northern blot analysis of multiple organs from healthy mice
revealed the presence of readily detectable eotaxin-2 mRNA in the
jejunum and spleen and to a lesser extent in the liver and lung (Fig. 3). The following three bands were
evident: 0.8, 1.1, and 1.8 kb. Interestingly, while the 0.8-kb species
was of the highest intensity in the spleen; the 0.8- and 1.1-kb species
were of equal intensity in the jejunum. No specific hybridization was
noted in the kidney, thymus, brain, or testis. In comparison, eotaxin-1
was expressed more ubiquitously, with readily detectable expression in
the stomach, jejunum, lung, and thymus (Fig. 3).
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Eotaxin-1 was originally described as an allergen-induced gene
product in the rodent lung and has subsequently been shown to be
directly induced by allergen challenge in humans (36).
However, although human eotaxin-2 has been shown to be up-regulated in
allergic inflammatory tissue (23) and allergen-induced
late phase responses in the skin (37), the ability of
allergen to directly induce this gene product in the lung has not been
determined. Therefore, we were interested in determining whether
eotaxin-2 mRNA was induced in the lung in response to allergen
challenge using a mouse model of asthma. We subjected mice to repeated
challenges with intranasal A. fumigatus allergen; these
conditions induce eotaxin-1 expression (Fig. 4) and promote eosinophil recruitment
into the lung. This allergen challenge protocol was found to induce a
marked increase in the level of eotaxin-2 mRNA compared with saline
challenged mice (Fig. 4). To demonstrate that eotaxin-2 induction was
not specific to the A. fumigatus allergen, OVA-sensitized
mice were subjected to intranasal challenge with OVA. Eotaxin-2 mRNA
was also increased by this allergen (data not shown).
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We were next interested in determining whether murine eotaxin-2 is
induced by IL-4 because this cytokine is critically involved in the
pathogenesis of allergic lung disease (38). In addition,
IL-4 has been shown to induce murine eotaxin-1 and human eotaxin-1–3
(4, 12, 39, 40). To assess this, we examined the lungs of
IL-4 lung-transgenic mice. Interestingly, these mice had markedly
increased levels of eotaxin-2 mRNA compared with wild-type mice (Fig. 5). Similar to eotaxin-2, eotaxin-1 mRNA
was also induced by the IL-4 transgene. However, RANTES mRNA was not
modulated by IL-4 expression. To confirm the ability of IL-4
over-expression in the lungs to induce eotaxin production, wild-type
mice were treated with intranasal IL-4 directly. Administration of IL-4
was also found to induce eotaxin-1 and eotaxin-2 mRNA in the lungs and
this effect was dose-dependent (data not shown).
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). In contrast, the baseline expression of RANTES
was not dependent on STAT-6. Eotaxin-2 is an eosinophil-specific chemokine
We were next interested in determining the chemoattractant
activity of the murine eotaxin-2 protein. We first examined this by
analysis of leukocyte transmigration in response to a concentration
gradient of eotaxin-2. Because wild-type mice do not have appreciable
numbers of eosinophils, splenocytes were isolated from IL-5-transgenic
mice. Although splenocytes from these mice contain a mixed population
of cells (30–40% eosinophils), the only cells that were able to
transmigrate in response to eotaxin-2 were eosinophils (Fig. 6A and data not shown). For
example, murine eosinophils exhibited a strong chemotactic response to
murine eotaxin-2. A dose response was seen between doses of 1 and 10
ng/ml with a plateau demonstrated at doses of 10–1000 ng/ml (Fig. 6A). In contrast, eotaxin-1 exhibited a bell-shaped dose
response with a peak visible at 10 ng/ml and a decrease in chemotactic
activity at higher doses (Fig. 6B). The experiments in Fig. 6, A and B, were performed on separate occasions;
when eotaxin-1 and -2 were compared in the same experiment, the
efficacy and potency of the two chemokines was not significantly
different (paired t test, p = 0.32,
n = 3). Checkerboard analysis with varying
concentrations of eotaxin-2 in the upper and lower chambers revealed
only chemotaxis without any chemokinesis (data not shown). High doses
of chemokines have recently been proposed to induce reverse chemotaxis
(41); however, at doses up to 1 or 5 µg/ml of eotaxin-2
or eotaxin-1, respectively, no reverse chemotaxis was induced (data not
shown). To test the selectivity of eotaxin-2, chemotactic responses
were also determined for purified neutrophils (Fig. 6C) and
macrophages (Fig. 6D). Recombinant eotaxin-2 (at doses up to
1000 ng/ml) had no activity on these cell types even though they
responded to control chemokines MIP-1 and KC.
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It has been suggested that ligand-induced activation of the
eotaxin receptor is dependent upon the chemokine’s amino terminus
(8). Therefore, we were interested in determining the
functional role of the amino terminus of eotaxin-2. Interestingly,
sequence analysis of the recombinant eotaxin-2 protein indicated that
the first two amino-terminal amino acids were deleted compared with the
predicted mature protein (based on the consensus sequence for signal
peptide cleavage). Therefore, it was of interest to also examine the
activity of eotaxin-2 with a mature amino terminus. We subsequently
produced a small-scale preparation of eotaxin-2 containing an amino
terminus starting with the amino-acid sequence VTIP, and compared its
function to the original preparation of eotaxin-2 (IP). Additionally,
during the final preparation of this manuscript, recombinant murine
eotaxin-2 became commercially available (from R&D Systems); this
protein preparation contained three additional amino acids on the amino
terminus, designated eotaxin-2 (IDSVTIP). We subsequently examined the
activity of all three preparations of eotaxin-2 by testing their
ability to induce eosinophil transmigration. Eotaxin-2 (VTIP) reached a
plateau at lower concentrations (1 ng/ml) than eotaxin-2 (IP) (10
ng/ml) indicating higher potency (paired t test,
p = 0.04, n = 3; Fig. 7); however, the maximal chemotactic
response was not significantly different between both forms of
eotaxin-2 (paired t test, p = 0.78,
n = 3). Interestingly, the amino-terminally extended
eotaxin-2 (IDSVTIP), displayed no chemotactic activity at doses up
to 100 ng/ml and only weak chemotactic activity at 1000 ng/ml (Fig. 7).
At a higher dose (10,000 ng/ml) of this preparation of eotaxin-2
(IDSVTIP), chemotactic activity was comparable to 1 ng/ml eotaxin-2
(VTIP) and 10 ng/ml eotaxin-2 (IP) (data not shown). These data
indicate that the rank order of potency of the different eotaxin-2
variants is VTIP > IP > IDSVTIP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30% homologous to
each other. This structural diversity provided an important distinction
that allowed designation of the presently described
eosinophil-selective CC chemokine, which is 59% homologous to human
eotaxin-2 (but only 38% homologous to eotaxin-1 or eotaxin-3), as
murine eotaxin-2. Identical with human eotaxin-2 (10),
murine eotaxin-2 contains 119 amino acids including a predicted leader
sequence with 26 amino acids. In comparison, eotaxin-1 (murine and
human) and eotaxin-3 are smaller chemokines with 94 and 97 amino acids,
respectively, and both contain a 23 amino acid leader sequence
(3, 4, 12, 13). Of note, similar to the genes for all
three human eotaxins (13, 42), murine eotaxin-2 consists
of a three-exon gene. Analysis of the positions of the intron/exon
sites reveals identical locations between murine and human eotaxin-2,
further substantiating that these genes are homologues.
Experimental analysis of the role of chemokines in homeostatic
processes and immunity has been facilitated by studies in rodents. This
has been particularly useful for analysis of chemokine-induced
eosinophil trafficking because eotaxin-1 was originally discovered
using a guinea pig model of allergic lung inflammation and subsequently
shown to have conserved structure and function in mice and humans. For
example, eotaxin-1 in mice and man has been demonstrated to be an
allergen-induced gene product in the respiratory tract where expression
directly correlates with eosinophil tissue recruitment, and
neutralization of its activity diminishes eosinophil chemoattraction in
vitro and in vivo (26, 36). The eotaxin-1 gene-targeted
mice have an impairment in eosinophil recruitment into the respiratory
tract following allergen challenge (24) and a marked
deficiency of eosinophils throughout the gastrointestinal tract at
baseline and following oral allergen challenge (29, 43).
However, although tissue eosinophils are reduced in eotaxin-1-deficient
mice and in mice depleted of eotaxin by Ab neutralization, these mice
still have residual tissue eosinophils. In particular, mice
over-expressing IL-5 in the absence of the eotaxin-1 gene, have
substantial levels of eosinophils in the jejunum compared with
wild-type mice (29). Taken together, these results
indicate the importance of eotaxin-1-dependent and -independent
pathways in the regulation of eosinophil tissue recruitment. In the
present study, murine eotaxin-2 is demonstrated to have shared and
distinct features compared with eotaxin-1. In particular, both genes
are shown to be allergen-induced and increased by IL-4 via a
STAT-6-dependent pathway in the lung. Additionally, while both
chemokines are constitutively expressed, their tissue expression
pattern is distinct. Eotaxin-1 appears to be more ubiquitously
expressed in the gastrointestinal tract (Fig. 3) (29)
compared with eotaxin-2, which is predominantly expressed in the
jejunum. Furthermore, eotaxin-1 is expressed in the thymus (Fig. 3)
where it regulates eosinophil trafficking to the medulla
(25). Thus, eotaxin-1 and eotaxin-2 are likely to
coregulate eosinophil trafficking during homeostatic, allergen-induced,
and IL-4-associated responses; however, their tissue-specific and
temporal roles are likely to be distinct.
To demonstrate direct eosinophil chemotactic activity, we have isolated eosinophils from CD2-driven IL-5-transgenic mice and measured eosinophil chemotaxis using a transmigration assay in vitro. Despite the large numbers of eosinophils present in the hematopoietic organs of these mice, there are few tissue eosinophils except in the gastrointestinal tract (29, 44). This benign phenotype suggests that other signals (in addition to IL-5) are necessary for eosinophil tissue recruitment and activation. By using eosinophils from these mice, we have shown that eotaxin-2 is a potent chemoattractant for eosinophils. By combining the results of comparative experiments between eotaxin-1 and eotaxin-2, both chemokines are demonstrated to have comparable activity. Whereas eotaxin-1 displays a typical bell-shaped curve with a peak activity at 10 ng/ml, eotaxin-2 reaches a plateau at 10 ng/ml and does not have diminished activity at doses as high as 1000 ng/ml. It is likely that the eosinophils isolated from IL-5-transgenic mice have been primed in vivo by IL-5 exposure and that this potentiates their responsiveness to both eotaxin-1 and eotaxin-2. Consistent with this, IL-5 activates human eosinophils and primes them to respond to RANTES and eotaxin-1 in vitro (45, 46, 47). We have also examined the chemoattractant activity of eotaxin-2 on neutrophils and macrophages in vitro and observed no activity under conditions where control chemokines induced chemoattraction. Although we have not ruled out the possibility that eotaxin-2 interacts with macrophages, neutrophils, or other cells under other conditions, these results have consistently shown that eotaxin-2 is a strong eosinophil chemoattractant relative to other cell types.
The results presented also give insight into the structure-function of eotaxin-2. The predicted amino-terminal amino acids of mature murine eotaxin-2 start with VTIP. Consistent with this, amino-acid sequence analysis of human eotaxin-2 produced in Sf9 insect cells infected with a recombinant baculovirus has revealed that the human protein begins with VVIP, indicating conservation with the predicted site in the mouse protein. Interestingly, our initial preparation of murine eotaxin-2 produced in bacteria started with IP because the VT was removed during purification by the aminopeptidase cleavage. In other experiments, we were able to produce eotaxin-2 with an intact native amino terminus (starting with VTIP). Interestingly, this protein was more potent displaying a reduced ED50, compared with the amino-terminally truncated eotaxin-2. Finally, recently murine eotaxin-2 became commercially available; however, the commercial source indicated that their preparation of murine eotaxin-2 contained three additional amino-terminal amino acids (IDS-VTIP) compared with the predicted mature protein. Interestingly, this preparation of eotaxin-2 was considerably less potent (>1000-fold) than either of the other two eotaxin preparations. Thus, the amino terminus of murine eotaxin-2 is involved in interacting with its receptor. The amino terminus of numerous chemokines has been demonstrated to be critically involved in triggering receptor activation (48). For example, removal of the first two amino acids of mature human eotaxin-1 by CD26, a dipeptidyl peptidase, results in markedly reduced eosinophil chemoattractive activity (49).
In summary, we have characterized the sequence, expression pattern, and gene organization of murine eotaxin-2 (CC chemokine ligand-24). Our results have several biological messages concerning eosinophils and chemokines. First, the mouse is demonstrated to contain two functional eotaxin chemokines, indicating that analysis of eosinophil trafficking in experimental murine models needs to account for both of these activities. Second, a differential pattern of constitutive expression of eotaxin-1 and eotaxin-2 is demonstrated, suggesting usage of distinct homeostatic chemokines by eosinophils in different tissues. Third, eotaxin-2 expression is increased in response to allergen challenge and transgenic overexpression of IL-4 in the lung. This response is similar to eotaxin-1, however, distinct from RANTES, another eosinophil-active CCR3 ligand. Fourth, STAT-6 is demonstrated to be required for the induction of eotaxin-1 and eotaxin-2 expression by chronic IL-4 stimulation. This is the first study to show that chemokine expression induced by continuous signaling through the IL-4 receptor remains dependent upon STAT-6. Last, we demonstrate the functional importance of the amino terminus of eotaxin-2. It is hoped that further analysis of this murine chemokine will contribute to understanding the broad functional role of chemokines and eosinophils.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marc E. Rothenberg, Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229.
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; EST, expressed sequence tag; MCP, macrophage chemoattractant protein; ORF, open reading frame; UTR, untranslated region.
Received for publication June 26, 2000. Accepted for publication August 28, 2000.
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 F. J. Culley, A. M. J. Pennycook, J. S. Tregoning, T. Hussell, and P. J. M. Openshaw Differential Chemokine Expression following Respiratory Virus Infection Reflects Th1- or Th2-Biased Immunopathology J. Virol., May 1, 2006; 80(9): 4521 - 4527. [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. M. Pope, P. C. Fulkerson, C. Blanchard, H. S. Akei, N. M. Nikolaidis, N. Zimmermann, J. D. Molkentin, and M. E. Rothenberg Identification of a Cooperative Mechanism Involving Interleukin-13 and Eotaxin-2 in Experimental Allergic Lung Inflammation J. Biol. Chem., April 8, 2005; 280(14): 13952 - 13961. [Abstract] [Full Text] [PDF]
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P. C. Fulkerson, N. Zimmermann, L. M. Hassman, F. D. Finkelman, and M. E. Rothenberg Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-{gamma} J. Immunol., December 15, 2004; 173(12): 7565 - 7574. [Abstract] [Full Text] [PDF]
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 N. M. Heller, S. Matsukura, S. N. Georas, M. R. Boothby, P. B. Rothman, C. Stellato, and R. P. Schleimer Interferon-{gamma} Inhibits STAT6 Signal Transduction and Gene Expression in Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 573 - 582. [Abstract] [Full Text] [PDF]
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 N. E. King, M. E. Rothenberg, and N. Zimmermann Arginine in Asthma and Lung Inflammation J. Nutr., October 1, 2004; 134(10): 2830S - 2836S. [Abstract] [Full Text] [PDF]
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N. E. King, N. Zimmermann, S. M. Pope, P. C. Fulkerson, N. M. Nikolaidis, A. Mishra, D. P. Witte, and M. E. Rothenberg Expression and Regulation of a Disintegrin and Metalloproteinase (ADAM) 8 in Experimental Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 257 - 265. [Abstract] [Full Text] [PDF]
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N. Zimmermann, A. Mishra, N. E. King, P. C. Fulkerson, M. P. Doepker, N. M. Nikolaidis, L. E. Kindinger, E. A. Moulton, B. J. Aronow, and M. E. Rothenberg Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways J. Immunol., February 1, 2004; 172(3): 1815 - 1824. [Abstract] [Full Text] [PDF]
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N. M. Nikolaidis, N. Zimmermann, N. E. King, A. Mishra, S. M. Pope, F. D. Finkelman, and M. E. Rothenberg Trefoil Factor-2 Is an Allergen-Induced Gene Regulated by Th2 Cytokines and STAT6 in the Lung Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 458 - 464. [Abstract] [Full Text] [PDF]
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 L. A. Gildea, R. Gibbons, F. D. Finkelman, and G. S. Deepe Jr. Overexpression of Interleukin-4 in Lungs of Mice Impairs Elimination of Histoplasma capsulatum Infect. Immun., July 1, 2003; 71(7): 3787 - 3793. [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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L. Zhang, M. P. Soares, Y. Guan, S. Matheravidathu, R. Wnek, K. E. Johnson, A. Meisher, S. A. Iliff, J. S. Mudgett, M. S. Springer, et al. Functional Expression and Characterization of Macaque C-C Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic Anti-macaque CCR3 Monoclonal Antibodies J. Biol. Chem., September 6, 2002; 277(37): 33799 - 33810. [Abstract] [Full Text] [PDF]
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X.-Z. Shang, B.-C. Chiu, V. Stolberg, N. W. Lukacs, S. L. Kunkel, H. S. Murphy, and S. W. Chensue Eosinophil Recruitment in Type-2 Hypersensitivity Pulmonary Granulomas : Source and Contribution of Monocyte Chemotactic Protein-3 (CCL7) Am. J. Pathol., July 1, 2002; 161(1): 257 - 266. [Abstract] [Full Text] [PDF]
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D. Broide Fast Flowing Eosinophils . Signals for Stopping and Stepping Out of Blood Vessels Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 637 - 640. [Full Text] [PDF]
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M. T. Borchers, T. Ansay, R. DeSalle, B. L. Daugherty, H. Shen, M. Metzger, N. A. Lee, and J. J. Lee In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration J. Leukoc. Biol., June 1, 2002; 71(6): 1033 - 1041. [Abstract] [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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 J. R. Crosby, H. H. Shen, M. T. Borchers, J. P. Justice, T. Ansay, J. J. Lee, and N. A. Lee Ectopic expression of IL-5 identifies an additional CD4+ T cell mechanism of airway eosinophil recruitment Am J Physiol Lung Cell Mol Physiol, January 1, 2002; 282(1): L99 - L108. [Abstract] [Full Text] [PDF]
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C. Penido, H. C. Castro-Faria-Neto, A. Vieira-de-Abreu, R. T. Figueiredo, A. Pelled, M. A. Martins, P. J. Jose, T. J. Williams, and P. T. Bozza LPS Induces Eosinophil Migration via CCR3 Signaling Through a Mechanism Independent of RANTES and Eotaxin Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 707 - 716. [Abstract] [Full Text] [PDF]
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D. C. Webb, A. N. J. McKenzie, and P. S. Foster Expression of the Ym2 Lectin-binding Protein Is Dependent on Interleukin (IL)-4 and IL-13 Signal Transduction. IDENTIFICATION OF A NOVEL ALLERGY-ASSOCIATED PROTEIN J. Biol. Chem., November 2, 2001; 276(45): 41969 - 41976. [Abstract] [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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 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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