(Received for publication, November 1, 1995; and in revised form, January 11, 1996)
From the
The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma.
Eosinophil accumulation in the blood and affected tissues is a
classic characteristic of the inflammatory response to allergens and
helminths(1) . Eosinophils are considered to be important
effector cells for killing helminths, but are also considered to be
responsible for tissue damage in hypersensitivity diseases. Because of
the pathologic potential of eosinophils, identification and
pharmacologic control of the specific factors regulating their
accumulation in vivo is an important goal. In this regard, the
CC chemokines macrophage inflammatory protein-1 (MIP-1
), (
)RANTES, and monocyte chemoattractant protein (MCP)-3 have
potent eosinophil chemotactic activity in
vitro(2, 3, 4) , although so far only
RANTES has been shown to recruit significant numbers of eosinophils
when injected into animals(5) . MIP-1
, RANTES, and MCP-3
are ``broad spectrum'' chemokines, targeting neutrophils,
monocytes, lymphocytes, and basophils, in addition to eosinophils, with
different degrees of selectivity and
potency(6, 7, 8, 9) .
Jose et al.(10, 11) recently purified a novel guinea pig CC chemokine and named it eotaxin for its ability to selectively recruit eosinophils but not neutrophils when instilled in the airways and skin of guinea pigs. The guinea pig eotaxin cDNA and a related mouse cDNA have been cloned(11, 12) . Eotaxin mRNA is increased in the lung in the guinea pig model of asthma and the related mouse mRNA is increased in interleukin-4-induced tumor suppression in the mouse(11, 12) . The effect on eosinophil recruitment in vivo appears to be direct since eotaxin also chemoattracts purified guinea pig eosinophils in vitro(10) . By analogy with other chemokines, eotaxin probably acts on eosinophils by binding to one or more subtypes of 7-transmembrane-domain G protein-coupled receptors (13) .
The ability of guinea pig eotaxin to induce calcium flux and
chemotactic responses in human eosinophils (10) suggested that
a human eotaxin-like signaling system exists. Here, we report the
isolation of a cDNA and production of recombinant protein for a novel
human CC chemokine named human eotaxin that is most closely related in
sequence to human MCP-1 and guinea pig eotaxin. We show that human
eotaxin is a selective agonist for human eosinophils and for CC
chemokine receptor 3 (CC CKR3), a 7-transmembrane-domain G
protein-coupled receptor selectively expressed in
eosinophils(14, 15) . No other chemokines tested were
agonists for CC CKR3, and eotaxin was not an agonist for the related
receptors CC CKR1, CC CKR2B, and CC
CKR5()(16, 17, 18) .
Figure 1: Nucleotide and deduced aa sequences of the human eotaxin cDNA. The probable signal sequence is underlined. The stop codon is indicated by an asterisk. A potential mRNA destabilization signal is double underlined. The polyadenylation signal is underlined in bold. The nucleotide and deduced aa sequences are available from DDBJ/GenBank/EMBL under the accession number D49372.
Figure 2: Sequence alignment of human eotaxin with closely related chemokines(10, 11, 12, 25, 26, 27) . Boxed aa, identities; dashes, gaps; gp, guinea pig; mu, mouse; arrowhead, signal peptide cleavage site of recombinant human eotaxin. Numbers on top of the aligned sequence enumerate the human eotaxin sequence and are left justified. The % identity of each whole and mature sequence relative to human eotaxin is at the bottom right.
Figure 3:
Human eotaxin gene, RNA, and recombinant
protein. A, gene. Southern blots containing 20 µg of human
genomic DNA per lane digested with EcoRI (lanes 1 and 3) or HindIII (lanes 2 and 4) were
hybridized at low stringency (final wash: 0.2 SSC at 55 °C)
with the guinea pig eotaxin full-length ORF probe (left) or at
high stringency with a 217-bp probe from exon 3 of the human eotaxin
gene (right). The location of size standards in kilobase pairs
(kbp) is indicated at the left of each panel. Asterisks and arrowheads indicate restriction fragments of the
human eotaxin gene. B, RNA distribution. Lanes contain 2
µg of poly(A)
RNA from the following tissues: 1, heart; 2, brain; 3, placenta, 4,
lung; 5, liver; 6, skeletal muscle; 7,
kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon; and 16,
peripheral blood leukocytes. The RNA was analyzed by Northern blot
hybridization using a clone 141 ORF cDNA probe. The filter was washed
in 0.2
SSC at 55 °C and autoradiographed at -80
°C overnight using an intensifying screen. C, recombinant
protein. A silver-stained gel containing 3 µg of recombinant
protein produced in insect cells analyzed by SDS-PAGE is
shown.
Figure 4: Chromosomal mapping of the human eotaxin gene. A total of 20 rodent-human somatic cell hybrids were analyzed for the presence of the human eotaxin gene by using specific PCR. Karyotyping symbols are as follows: +, chromosome present in the cell line; M, chromosome present but at low frequency; D, chromosome present but with multiple deletions; blank, chromosome absent. D% is the percent discordance between the presence of a chromosome and the PCR detection of the human eotaxin gene.
Figure 5:
Eosinophil selectivity of recombinant
human eotaxin. [Ca]
was monitored by ratio fluorescence in the fura-2-loaded
leukocyte subtypes indicated to the left of each row of
tracings. Arrows mark the time of addition of chemokines at
the indicated concentration. The tracings shown are from one experiment
representative of at least three
experiments.
In contrast, the related orphan receptor CC CKR3 is expressed
selectively in eosinophils, making it an excellent candidate for an
eotaxin receptor(14, 15) . When calcium flux was
measured, three independent HEK 293 cell lines stably transfected with
CC CKR3 responded to eotaxin, but not to any other chemokines tested at
100 nM (Fig. 6A). Conversely, MIP-1,
RANTES, MIP-1
, MCP-1, and MCP-3 were agonists for their
appropriate receptors, CC CKR1, CC CKR2B, and CC CKR5, whereas eotaxin
was not (Fig. 6A and data not shown).
Figure 6:
CC CKR3 is a human eotaxin receptor. A, specificity. Each tracing represents
[Ca]
levels of
fura-2-loaded HEK 293 cell lines expressing the CC CKR indicated to its
left, measured as relative fluorescence over time. Arrows mark
the time of addition of chemokines at 100 nM coded as follows: a, MIP-1
; b, RANTES; c, MIP-1
, d, MCP-1; e, MCP-3; f, human eotaxin. The
data are from one experiment representative of at least three
experiments with 4 separate clones for CC CKR3. CC CKR3 transfectants
were tested individually with the chemokines shown also with negative
results (not shown). B, potency. Each data point represents
the peak of the calcium flux response to eotaxin in CC CKR3-transfected
HEK 293 cells. The data are from a single experiment representative of
three separate experiments with two independent cell lines expressing
CC CKR3. C, mechanism. CC CKR3 transfectants were incubated in
pertussis toxin at the concentration indicated at the left of
each tracing, and then stimulated with 25 nM eotaxin (left
arrow) and 2 µM ATP (right
arrow).
Sequential
stimulation experiments indicated that the eotaxin response underwent
homologous desensitization, whereas the response was unaffected by
prestimulation with other chemokines (Fig. 6A).
Conversely, eotaxin did not affect the response of CC CKR1, CC CKR2B,
or CC CKR5 to their respective agonists. The EC for
induction of the calcium flux response by eotaxin in CC CKR3
transfected cells was 5 nM, consistent with the sensitivity of
the native receptor to eotaxin (Fig. 6B). When stable
CC CKR3 transfectants were pretreated with pertussis toxin, 250 ng/ml,
the calcium flux response to eotaxin but not to ATP was abolished,
suggesting that a G
type G protein is involved in eotaxin
signal transduction (Fig. 6C).
We infer by analogy
with the agonists for other G protein-coupled receptors that eotaxin
induces calcium flux responses in transfected HEK 293 cells by binding
specifically to CC CKR3 on the plasma membrane. However, so far we have
been unable to show specific I-human eotaxin binding.
When we tested 10
cells and concentrations of the
radioligand ranging from 0.1 to 5 nM at 4 or 37 °C, total
binding was the same for untransfected and CC CKR3-transfected HEK 293
cells. Using identical radiolabeling and binding conditions, and
radioligands similar in specific activity to the
I-eotaxin used here, we have been able to show specific
binding of
I-MIP-1
, -MIP-1
, -MCP-1, -RANTES,
and -MCP-3 to their respective receptors CC CKR1, CC CKR2B, CC CKR5, a
mouse MIP-1
receptor, and a human cytomegalovirus CC chemokine
receptor (Refs. 23, 34, and 35, and data not shown).
We
conclude that the levels of expression of CC CKR3 and/or its affinity
for eotaxin are low, preventing detection of the binding site.
Jose et al.(10) reported that I-human RANTES
bound to guinea pig eosinophils, and that guinea pig eotaxin could
partially compete for the binding site, suggesting a common receptor
for eotaxin and RANTES. However, we have been unable to show specific
I-RANTES binding to CC CKR3-transfected cells, and RANTES
at concentrations as high as 100 nM is not an agonist for CC
CKR3 capable of inducing a calcium flux response in transfected HEK 293
cells.
The present work establishes the cDNA and deduced aa sequences, chromosome location, RNA distribution, functional expression, and receptor selectivity for human eotaxin. Previous results in guinea pig and mouse and our data in human together indicate that an eotaxin signaling system is broadly conserved in mammalian species. The only biological function currently established for eotaxin is regulation of eosinophil trafficking during allergic airway and skin inflammation in the guinea pig(10) . While our data also indicate high eosinophil selectivity for human eotaxin, more work will be needed to determine whether additional eotaxin functions exist and whether those established in the guinea pig have been conserved during mammalian evolution.
Several features of human eotaxin and its receptor CC CKR3 are unusual when compared to other chemokines and chemokine receptors. First, most chemokine receptors have multiple chemokine agonists, whereas CC CKR3 appears to be monospecific for eotaxin (Table 2). Conversely, most chemokines recognize more than one CC CKR, whereas eotaxin appears to be monospecific for CC CKR3. Second, all CC chemokines except eotaxin can activate monocytes. Third, all CC CKRs except for CC CKR3 are expressed in monocytes, whereas only CC CKR1 and CC CKR3 are expressed in eosinophils. Taken together, these features suggest that CC CKR3 is a specific factor responsible for eotaxin's high cellular selectivity for eosinophils.
Fourth, chemokines are highly inducible genes and are typically not expressed at high constitutive levels, whereas human eotaxin RNA is constitutively present at quite high levels in small intestine and colon. Eosinophils are present in large numbers in the normal gastrointestinal tract and play a major role in host defense against helminthic parasites(36) ; perhaps eotaxin regulates this function. Eotaxin may also contribute to pathogenesis of certain inflammatory bowel diseases associated with increased numbers of eosinophils(37) . It is intriguing that eotaxin RNA is also present at relatively high levels in heart. Eotaxin may have unknown functions in normal heart and may play important roles in eosinophilic endomyocardial disease, a grave complication of chronic eosinophilia(37) .
Fifth, the sequence of human eotaxin is much closer to the MCPs than would be expected given their functional divergence, and much further from guinea pig eotaxin than would be expected for orthologous genes. Even the signal peptides of human eotaxin and the MCPs are similar. It is likely that human eotaxin is the orthologue of guinea pig eotaxin. However, it could be a member of the MCP branch that has evolved to be selective for eosinophils, converging functionally with the eotaxins. Human eotaxin, like most other CC chemokines, is highly basic, containing a net charge of +11. CC CKR3, like most other chemokine receptors, has an acidic N-terminal extracellular segment, but unlike most other chemokine receptors, also has an acidic second extracellular loop. It will be interesting to study whether these charge distributions are binding determinants.
Based on the data reported here, it is reasonable to
hypothesize that eotaxin and CC CKR3 regulate eosinophil accumulation
at body sites undergoing allergic reactions, metazoan infestation, or
other types of inflammation where eosinophils are present in large
numbers. Additional studies will be needed to show this, and to
determine the importance of these molecules relative to other known
eosinophil chemoattractants, including the broader spectrum CC
chemokines MIP-1, RANTES, and MCP-3, and the other known
eosinophil chemokine receptor CC CKR1.