By
From the * Infectious Disease Unit, Allergy and Immunology Unit, Massachusetts General Hospital,
and Harvard Medical School, Boston, Massachusetts 02114; and the § Gladstone Institute of
Cardiovascular Disease; and Department of Medicine, University of California, San Francisco,
California 94141
The chemokines are a large family of cytokines that control the recruitment of leukocytes in immune and inflammatory responses. We describe the isolation of a novel murine CC chemokine that, based on its biological and structural features, we have named monocyte chemoattractant protein (MCP)-5. MCP-5 mapped to the CC chemokine cluster on mouse chromosome 11 and was most closely related to human MCP-1 in structure (66% amino acid identity). Purified recombinant MCP-5 protein was a potent chemoattractant for peripheral blood monocytes, was only weakly active on eosinophils at high doses, and was inactive on neutrophils. MCP-5 induced a calcium flux in peripheral blood mononuclear cells, but not in purified murine eosinophils or neutrophils. Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes. MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5. Constitutive expression of MCP-5 mRNA was detected predominantly in lymph nodes, and its expression was markedly induced in macrophages activated in vitro and in vivo. Moreover, MCP-5 expression was upregulated in the lungs of mice following aerosolized antigen challenge of sensitized mice, and during the host response to infection with Nippostrongylus brasiliensis. These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.
The monocyte chemoattractant proteins (MCP)1 and
eotaxin constitute an important subfamily of CC or
Chemokines induce leukocyte migration and activation
by binding to specific G protein-coupled seven transmembrane spanning cell surface receptors (17). There have been
five human CC chemokine receptor (CCR) genes cloned,
now being referred to as CCR1 through CCR5. Each of
these has an orthologue in the mouse. Human CCR2a and
CCR2b are splice variants of the same gene. The chemokine and leukocyte selectivity of CCRs overlap extensively;
a given leukocyte often expresses multiple chemokine receptors, and more than one chemokine typically binds to
the same receptor.
While chemokines often have overlapping activities in
vitro, differences in the timing and location of chemokine
production in vivo imply that the redundancy found in
vitro may not be biologically relevant. This is supported by
chemokine inactivation experiments conducted in animal
models of infection and inflammation, such as the targeted
deletion of the macrophage inflammatory protein (MIP)-
1 To fully appreciate the role of chemokines in regulating
inflammation, the entire spectrum of chemokines needs to
be delineated and their functional role analyzed in the context of in vivo immune responses. In this report, we describe the cloning and functional characterization of a new
member of the MCP subfamily of Isolation of the Murine MCP-5 Gene.
The human MCP-4 cDNA
(2) was 32P labeled and used as a probe to screen a 129SV mouse
genomic library (Stratagene Inc., La Jolla, CA). Approximately
106 phages were plated, transferred to GeneScreen Plus (DupontNew England Nuclear, Wilmington, Delaware), hybridized for
18 h at 50°C in a low stringency hybridization buffer (0.6 M
NaCl, 80 mM Tris HCl, 4 mM EDTA, 0.1% [wt/vol] sodium
pyrophosphate, 0.1% [wt/vol] SDS, 10× Denhardt's, 100 µg/ml
denatured herring sperm DNA), and washed at 60°C in 1× SSC/
0.1% SDS for 40 min. To exclude JE, the putative murine orthologue of human MCP-1, the filters were rehybridized as described above with a full-length murine JE cDNA fragment, and
washed at 65°C in 0.1× SSC/0.1% SDS for 40 min. 15 plaques
were identified that hybridized more strongly with the human
MCP-4 probe than with the murine JE probe. These plaques
were analyzed by PCR (30 cycles, 45°C annealing) using a set of
degenerate oligomers made from highly conserved regions of
murine and human MCP and eotaxin proteins (5 5 Chromosomal Localization.
PCR primers in intron 2 and exon
3 of the MCP-5 gene (5 RNA Analysis.
RNA was isolated from the organs of a
BALB/c mouse by lysing the tissue in guanidinium isothiocyanate and pelleting the RNA through a 5.7 M CsCl2 cushion. The
poly(A)+ fraction was isolated from total RNA by oligo dT cellulose chromatography (Pharmacia, Piscataway, NJ). RNA STAT-60
(Tel-Test "B", Inc., Friendswood, TX) was used to isolate RNA
from mouse leukocytes and cell lines. 10 µg of total RNA was
fractionated on a 1.2% agarose gel containing 0.7% formaldehyde,
transferred to GeneScreen, and hybridized with 32P-dCTP Klenow-labeled random primed cDNA probes encoding MCP-5, JE, and the ribosomal protein (rp) L32 as a control of RNA loading. The membranes were hybridized under conditions of high
stringency (50% formamide, 10% dextran sulfate, 5× SSC, 1×
Denhardt's solution, 1% SDS, 100 µg/ml denatured herring sperm
DNA, and 20 mM Tris at 42°C) and washed at 55°C in 0.2×
SSC/0.1% SDS for 40 min. SVEC cells, an SV-40 virus immortalized murine endothelial cell line, and RAW 264.7 (American
Type Culture Collection, Rockville, MD) were cultured for 6 and
18 h without additions, or with the addition of 200 U/ml murine
IFN Mouse Models of Pulmonary Inflammation.
The aerosolized OVA
model was performed as described (22). Briefly, BALB/cJ mice
between 5 and 10 wks of age were immunized with 10 µg of
OVA (Sigma Chemical Co., St. Louis, MO) and 1 mg aluminum
hydroxide intraperitoneally on days 0, 7, and 14. Sham-immunized mice received aluminum hydroxide alone. Mice underwent
aerosol challenge with OVA (50 mg/ml in sterile saline) 7-10 d
after the final immunization. Mice were killed at 3, 6, 24, and 48 h after challenge, and lungs were harvested for RNA extraction.
A minimum of three mice were included in each group at each
time point. The Nippostronglyus brasiliensis (Nb) model was performed as described (23). Briefly, 12-wk-old female BALB/cJ
mice were injected with 750 third stage Nb larvae and the lungs
harvested at 7, 10, and 14 days for RNA extraction.
Purification of Monocytes, Macrophages, Eosinophils, and Neutrophils.
PBMC were obtained from 4 normal donors by density
gradient centrifugation using 1.077 Histopaque (Sigma Chemical
Co.). Murine eosinophils were isolated from the spleens of IL-5
transgenic mice by negative selection through a MACS magnet
(Miltenyi Biotech, Auburn, CA) (24). The resulting eosinophil
purity was >90% as determined by microscopic examination of
Diff Quick (Baxter Scientific, McGaw Park, IL)-stained cytospin
preparations; contaminating cells were mononuclear. Neutrophils
and macrophages were isolated from the peritoneal cavities of
mice, and further purified by centrifugation in self-forming Percoll gradients as described (25). Neutrophil preparations were
typically >90% pure with <10% mononuclear cell contamination.
Chemotaxis.
Eosinophils, neutrophils, and mononuclear cells
were suspended in HBSS with 0.05% BSA at 2.5, 1, and 5 × 106
cells/ml, respectively, and placed in the top of a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD). A polycarbonate filter with 5-µm pores (eosinophils and mononuclear
cells) and a polyvinylpyrrolidine-free filter with 3-µm pores (neutrophils) separated the cells from buffer alone or buffer containing
purified recombinant murine eotaxin, human MCP-1, and human IL-8 (PeproTech Inc., Rocky Hill, NJ), or murine MIP-1 Calcium Flux in Leukocytes.
Purified cells (107/ml in HBSS
with 0.05% BSA) were loaded with 5.0 µM of the acetoxymethyl
ester of fura-2 (fura-2 AM) (Molecular Probes Inc., Eugene, OR)
for 60 min at 37°C in the dark. Loaded cells were washed twice
and resuspended in a buffer containing 145 mM NaCl, 4 mM
KCl, 1 mM NaHPO4, 0.8 mM MgCl2, 1.8 mM CaCl2, 25 mM
Hepes, and 22 mM glucose. 2 ml of cells (5 × 106 cells/ml) were
placed in a continuously stirring cuvette at 37°C in a dual-wavelength excitation source fluorimeter (Photon Technology Inc.,
South Brunswick, NJ). Changes of cytosolic-free calcium were
determined after addition of the chemokines by monitoring the
excitation fluorescence intensity emitted at 510 nm in response to
sequential excitation at 340 and 380 nm. The data are presented as the relative ratio of fluorescence at 340/380 nm.
Calcium Flux Responses in Chemokine Receptor Transfected
Cells.
Human embryonic kidney (HEK)-293 cells stably expressing human CCR2b, CCR1, and CCR3 and murine CCR2
and CCR5 with the "FLAG" epitope at the extreme NH2 terminus, were prepared as described (26). For calcium fluorimetry,
cells were grown to log phase, loaded with the calcium-specific
dye indo-1 AM (Molecular Probes Inc.), and assayed by spectrofluorimetry for changes in the concentration of intracellular calcium in response to addition of chemokines (27).
To isolate novel mouse CC chemokine genes, a murine
genomic library was sequentially screened with a human
MCP-4 cDNA probe under conditions of low stringency
and a mouse JE cDNA probe under conditions of high
stringency. 15 plaques were identified that hybridized more
strongly with the human MCP-4 probe than with the murine JE probe. These plaques were purified and further analyzed by PCR using a set of degenerate primers. A PCR
product was generated from five of these clones, and all
contained the same novel sequence that had a high degree
of homology with the MCP subfamily. Southern blot analysis of mouse genomic DNA established a restriction map (Fig. 1 A) and revealed that MCP-5 was a single copy gene
(data not shown). One of the five overlapping MCP-5 genomic clones was partially sequenced to determine the intron/exon structure of the MCP-5 gene (Fig. 1 A), and to
confirm the sequence of the PCR products. Like other CC
chemokines, the MCP-5 gene contained three exons and
two introns.
The chromosomal localization of MCP-5 was determined
by single-strand conformation polymorphism (SSCP) analysis using a set of PCR primers in intron 2 and exon 3 that
detected a polymorphism when using DNA from C57BL/
6J and Mus spretus (Fig. 1 B). A panel of genomic DNA from
94 interspecific backcrossed animals was used to map the
MCP-5 gene based on this SSCP polymorphism. The
MCP-5 gene cosegregated with Scya7 (fic) and Scya11 (eotaxin) in this cross, placing it between D11Mit markers 7 and
36 on chromosome 11 (data not shown). A comparison to the
consensus map from the mouse genome database revealed
this to be the region of chromosome 11 containing the CC
chemokine gene cluster designated Scya1-11. The MCP-5
gene has been assigned the designation Scya12.
To determine the complete
structure of the MCP-5 cDNA, 5 Sequence analysis revealed that murine MCP-5 was a
novel chemokine most homologous to human MCP-1. In
fact, MCP-5 is structurally more similar to MCP-1 than JE,
the putative murine homologue of MCP-1. This holds true
even when JE's unique 49-amino acid serine/threoninerich, highly glycosylated COOH-terminal extension is excluded from the comparison. The mature MCP-5 protein
is 66% identical to the mature human MCP-1 protein,
while JE is 55% identical to human MCP-1. MCP-5 is
unique among the MCP proteins in that its NH2-terminal amino acid is predicted to be glycine, a feature that it shares with human eotaxin (15).
The chemotactic
activity of purified recombinant MCP-5 was evaluated on
human peripheral blood monocytes, mouse eosinophils, and mouse neutrophils (Fig. 2). MCP-5 was a potent
chemoattractant for human peripheral blood monocytes, as
was mouse JE and human MCP-1 (Fig. 2 A). MCP-5 had
minimal activity on murine eosinophils and only at doses
Chemokines induce cell migration and activation by binding to specific G protein-coupled seven transmembrane
cell surface receptors on leukocytes. Signaling through
these receptors results in a transient rise in intracellular calcium. To determine if MCP-5 induces a calcium flux in
responding leukocytes, and thus further examine the leukocyte specificity of MCP-5, purified leukocyte subsets
were loaded with the calcium sensitive dye fura-2, and their response to MCP-5 was monitored by fluorimetry.
Concordant with the chemotaxis data presented above,
MCP-5 induced a dose dependent calcium flux in mononuclear cells (Fig. 3 A), but not eosinophils (even at 50 µg/ml
or 5 µM) (Fig. 3 B), or neutrophils (Fig. 3 C). As controls,
the purified eosinophils responded appropriately to eotaxin
and MIP-1
Rapid successive exposure to
the same ligand is known to desensitize the signaling capacity of G protein-linked receptors. This was true for MCP-5
which, at a concentration of 10 nM, completely desensitized PBMC to a subsequent MCP-5 (10 nM) challenge
(data not shown). Exposure of cells to different ligands that
use the same receptor signaling pathway can also result in
desensitization. MCP-5 (10 nM) was able to completely
desensitize the cells to a subsequent stimulation with JE (10 nM) or human MCP-1 (10 nM) (Fig. 3 A); however, these
cells were still responsive to the unrelated ligand FMLP.
This desensitization effect was dose dependent; when the
initial dose of MCP-5 was lowered to 0.05 nM, the mononuclear cells were no longer desensitized to subsequent
stimulation with either 10 nM JE or MCP-1 (data not
shown). Likewise, mononuclear cells initially stimulated
with 10 nM JE or MCP-1 were desensitized, although usually not completely, to a subsequent stimulation with 10 nM
MCP-5 (Fig. 3 A). As would be expected from the lack of activity of MCP-5 on eosinophils and neutrophils, initial
treatment of these cells with MCP-5 (100 nM) had no effect
on the ability of eosinophils to respond to eotaxin (10 nM)
or MIP-1 The signaling and desensitization studies performed on mononuclear cells suggested
that MCP-5 activated the same receptor as MCP-1 and JE.
Furthermore, the inability of MCP-5 to induce a calcium
flux in murine eosinophils suggested that MCP-5 does not
signal through the two receptors known to be expressed on eosinophils, CCR3, and CCR1 (21). To directly test these
hypotheses, we examined the ability of MCP-5 to activate
a number of cloned human CC chemokine receptors stably
expressed in HEK-293 cells. MCP-5 (100 nM) reproducibly induced a robust intracellular calcium flux in cells expressing CCR2 (Fig. 4), but not CCR1, CCR3, or CCR5
(data not shown). In control experiments, we confirmed
functional expression of the cloned receptors in these
MCP-5 unresponsive targets by demonstrating that the
CCR1 and CCR5 cell lines responded to MIP-1
To investigate the expression of murine MCP-5 in normal
murine tissues, a Northern analysis of mRNA obtained from
mouse organs was performed (Fig. 6). This analysis revealed an ~550-bp transcript that corresponds to the mature mouse MCP-5 mRNA. The highest levels of constitutive MCP-5 expression were observed in normal lymph nodes. MCP-5 mRNA levels were also seen in breast and
salivary glands containing lymph nodes, heart, lung, thymus, brain, small intestines, kidney, and colon. However,
no expression was detected in spleen, skeletal muscle, bone
marrow, or liver. The expression of MCP-5 and JE were
compared in these mouse tissues since MCP-5 had a similar
biological profile to JE and signaled through the same receptor. Highest constitutive expression of JE was seen in
the salivary gland with low levels detected in other organs (e.g., lymph nodes) (Fig. 6). The ~550- and 800-bp JE
transcripts correspond to known 3
The expression of MCP-5 was examined in purified leukocyte subsets isolated from normal mice (Fig. 7 A). MCP-5
expression was not seen in eosinophils, bone marrow-derived
mast cells, or resident peritoneal macrophages. Stimulation
of these mast cells with either IgE or Con A did not induce
MCP-5 expression. Con A upregulated the expression of
JE (Fig. 7 A), and both treatments induced the expression of
murine CCR1 (21). Activated macrophages elicited into the
peritoneal cavity by thioglycollate expressed significant levels of MCP-5 mRNA. Neutrophils elicited into the peritoneum by treatment with sodium casein also expressed
MCP-5 mRNA; however, expression from contaminating
elicited macrophages cannot be excluded.
Since it appeared that activated macrophages were a primary source of MCP-5 mRNA, we evaluated the ability of
various stimuli to induce MCP-5 mRNA in the RAW
264.7 macrophage cell line. IFN The expression of MCP-5 was also examined in cytokine stimulated murine endothelial cells (SVEC) to further
explore the regulation and cellular sources of this chemokine. MCP-5 was not induced in SVEC endothelial cells
by IFN To examine the regulation of MCP-5 during in vivo immune responses, we examined its expression
in the lungs of mice following infection with Nb and in a
murine model of OVA-induced pulmonary inflammation
(Fig. 8). Following infection with Nb, MCP-5 lung mRNA
levels were markedly increased by day 7, peaked by day 10, and returned to baseline by day 14. The kinetics of MCP-5
mRNA accumulation preceded the peak recruitment of
pulmonary macrophages, neutrophils, and eosinophils that
characterize the granulomatous immune response to this
nematode pathogen (23). MCP-5 mRNA was also detected in OVA-immunized mice 3 h after aerosol challenge (Fig. 8), and remained elevated at 48 h (data not shown).
MCP-5 mRNA remained at prechallenge levels in challenged sham-immunized mice (Fig. 8). Again, the kinetics
of MCP-5 mRNA accumulation preceded the peak accumulation of pulmonary macrophages, lymphocytes, and
eosinophils that characterize the immune response in this model (22). Like MCP-5, JE was induced in both murine
models of pulmonary inflammation; however, in the OVA
model, levels of JE mRNA returned to baseline by 48 h, while
MCP-5 levels remained elevated (data not shown).
As the complexity of the chemokine system continues to
increase, it is apparent that to fully appreciate the role of
chemokines in development, homeostasis, and host response
to infection and inflammation, the entire spectrum of chemokines needs to be known, and their functions deciphered in animal models. In this report, we describe the
molecular isolation and functional characterization of a
novel mouse Although JE is widely regarded as the murine orthologue
of human MCP-1, MCP-5 is more homologous to human
MCP-1 than is JE. It is noteworthy that both murine
MCP-5 and human MCP-1 were more potent and more
efficacious agonists of human CCR2b than JE. In contrast,
JE was a better agonist of murine CCR2 than murine
MCP-5. This suggests that there may be as yet an unidentified receptor for MCP-5 in the mouse and perhaps additional MCP-like ligands (e.g., MCP-5) and receptors in
humans.
Like other The mature NH2-terminal amino acid is thought to be
important for the biological activity and leukocyte selectivity of the MCP and eotaxin proteins. For example, addition
of a single amino acid before MCP-1's NH2-terminal
glutamine, or the deletion of this glutamine residue, reduces
MCP-1's biological activity on monocytes 100-1,000-fold
(32). Furthermore, deletion of this NH2-terminal glutamine residue converts MCP-1 from an activator of basophils to an eosinophil chemoattractant (33). However, if
the NH2-terminal glutamine of MCP-1 is replaced by other
small amino acids, like asparagine and alanine, it still retains
activity on monocytes (32). We have also found that an
NH2-terminal extension of eotaxin or MCP-4 completely
inactivated these molecules (2, 16). Our findings that
MCP-5 shares a receptor with MCP-1, -3, and -4, namely
CCR2, even though it does not share the NH2-terminal glutamine, is consistent with the hypothesis that the NH2terminal residue of this family of molecules is not the sole
determinant of leukocyte specificity.
Understanding the regulation of chemokine expression
in vitro and in vivo is essential in order to begin to appreciate the mechanisms whereby this family of molecules controls the recruitment of immune cells in the host response.
In this regard, the expression of MCP-5 was examined in
normal tissues, cytokine activated cells in vitro, and inflammatory responses in vivo. The pattern of MCP-5 expression differs considerably from other related chemokines. For example, MCP-5 was expressed at the highest constitutive levels in the lymph nodes of normal mice, while JE
was expressed at highest levels in the salivary gland. MCP-4
and eotaxin are constitutively expressed at the highest levels
in organs with large epithelial surfaces, such as the small
and large intestines and the lungs (2, 15). MCP-5 was induced in thioglycollate elicited macrophages and in a macrophage cell line induced by IFN The expression of MCP-5 was also examined in two
murine models of inflammation. A kinetic pattern of expression was observed consistent with the role of MCP-5
in recruiting the early mononuclear phase of the inflammatory response. In the Nb model, MCP-5 mRNA expression peaked at day 10 and was back down to baseline by
day 14. Nb larvae pass through and molt in the lung during
the first few days of infection. The time of peak histologic
change in the lungs is at day 14, and is characterized by focal aggregates of monocytes, neutrophils, and eosinophils
(23). In the aerosolized ovalbumin model, MCP-5 mRNA
expression was detected 3 h after antigen challenge, a time
when the inflammatory infiltrate is just beginning to be
seen (22). We have determined that several chemokines
such as eotaxin, JE, MIP-1 These findings add to the growing understanding of the
chemokine system by describing the isolation of MCP-5, a
potent monocyte chemoattractant, and CCR2 agonist that
is expressed in activated macrophages and models of pulmonary inflammation. As such, the functional role of MCP-5
will need to be considered when trying to understand the
in vivo immune response to allergens and pathogens.
-chemokines that share structural and functional features.
Four human MCP proteins (-1, -2, -3, and -4) have been
identified that share ~65% amino acid identity (1, 2). Of
the four human MCP proteins identified to date, only two
have been identified in the mouse: JE (3, 4), the putative
orthologue of human MCP-1, and MARC/FIC (5, 6), the
putative orthologue of human MCP-3. Human MCP-1, -2, -3, and -4 are all active on monocytes (2, 7, 8), T cells (8-
10), and basophils (2, 11, 12). In addition, human MCP-2,
-3, and -4 chemoattract eosinophils (2, 8, 12, 13), and human MCP-3 is chemotactic for dendritic cells (14). Eotaxin, although highly related in sequence to the MCP
proteins, is inactive on monocytes, basophils, and lymphocytes and is unique in that it specifically attracts eosinophils
(15, 16). MCP-1, MCP-4, and eotaxin are similarly regulated in a variety of cells. For example, in epithelial and endothelial cells, MCP-1, MCP-4, and eotaxin are induced
by TNF
, IL-1, and IFN
(1, 2, 15). IFN
induces the secretion of MCP-2 from mononuclear cells and fibroblasts,
and MARC/FIC is secreted from activated mast cells (1,
5). Other CC chemokines (e.g., RANTES and MIP-1
/
) are more distantly related in sequence to those of the
MCPs and eotaxin, although they chemoattract the same
spectrum of leukocyte subsets, with variable selectivity.
gene (18). Despite the fact that all MIP-1
activities described in vitro are shared by other
-chemokines, including receptor usage, MIP-1
-deficient mice do not mount a
normal response to viral infections.
-chemokines, murine
MCP-5. The data described below provide evidence that
this novel chemokine is a potent monocyte chemotactic
factor that signals through CCR2. Further, we demonstrate
that MCP-5 is a product of activated macrophages, and its
expression is increased in murine models of pulmonary inflammation.
oligomer in
exon 1: CTTCTGKGYCTGCTGYTCA, and 3
oligomer in
exon 2: ACAGCYTYYYDGGGACA). 5 of the 15 plaques amplified an 800-bp PCR product, were subcloned into pCRII vector (Invitrogen, San Diego, CA), and sequenced using Sequenase
(United States Biochemical, Cleveland, OH).
and 3
Rapid Amplification of cDNA Ends of Murine MCP-5
cDNA.
The 5
and 3
ends of the cDNA for murine MCP-5
were isolated using a 3
oligomer in exon 2 (CTGGCTGCTTGTGATTCTCCTGT), a 5
oligomer at the end of exon 1 (CAGTCCTCAGGTATTGGCTGG), and the Marathon cDNA
Amplification Kit (Clontech, Palo Alto, CA). The rapid amplification of cDNA ends (RACE) products were cloned and sequenced, and oligomers were made to amplify the full length cDNA from IFN
-treated RAW 264.7 cell poly A+ RNA (5
oligomer: AGCTTTCATTTCGAAGTCTTTG, and 3
oligomer: TAGATTCGGTTTAATTGGCCC). The PCR products
were cloned and sequenced.
sense oligomer: TTACAGGTCAGGTCCCCTACT, and 3
anti-sense oligomer: CTCCTTATCCAGTATGGTCCTG) were used to amplify genomic DNA
from 94 interspecific backcross animals (C57BL/6JEi × SPRET/
Ei)F1 × SPRET/Ei (Jackson Laboratory, Bar Harbor, ME) (19).
The 32P-radiolabeled PCR products were analyzed on a nondenaturing 5% acrylamide gel as described (20).
(Genentech, Inc., San Francisco, CA), 5 ng/ml murine IL-1
(Genzyme Corp., Cambridge, MA), or 10 ng/ml murine IL-4
(Genzyme Corp.). Mouse bone marrow-derived mast cells were
activated with IgE anti-TNP and TNP-BSA, or 2.5 mg/ml of Con A for 4 h as described (21).
,
murine MIP-1
, murine JE, and murine KC (R&D Sys. Inc.,
Minneapolis, MN). Murine MCP-5 was expressed and purified
from Escherichia coli by PeproTech as the predicted mature 82-
amino acid protein beginning with the NH2-terminal glycine. NH2-terminal sequence analysis of the purified recombinant
MCP-5 preparation confirmed its homogeneity and the NH2terminal glycine. Cells were incubated at 37°C for 30 (neutrophils), 60 (eosinophils), or 90 min (mononuclear cells), and the
cells that migrated across the filter and adhered to the bottom side
of the filter were stained with Diff-Quick.
MCP-5 Genomic Structure and Chromosomal Localization.
Fig. 1.
Genomic organization, chromosomal mapping, nucleotide
sequence, and predicted amino acid sequence of the murine MCP-5 gene. (A) Partial restriction map and genomic organization of the mouse MCP-5
gene. The mature mRNA is shown schematically below with the positions of the start codon (ATG) and stop codon (TGA). The scales are
shown below each drawing. (B) SSCP polymorphism. PCR primers in
intron 2 and exon 3, indicated by arrows in A, were used to amplify genomic DNA from C57BL/6J, Mus spretus, or the C57BL/6J × M. spretus
heterozygote, and analyzed using SSCP. The complete raw data for this
cross with references and notes are available on the World Wide Web at
http://www.jax.org/resources/documents/cmdata/BSS11data.html and the
Mouse Genome Database accession number is MGD-CREX-697. (C)
MCP-5 cDNA sequence. The filled triangles indicate the intron/exon borders. The single underline indicate the ATTTA sequences that have
been reported to decrease mRNA stability. The double underlined sequence indicates the predicted polyadenylation signal. The sequence has
been deposited in GenBank/EMBL/DDBJ under the accession U66670. The arrow indicates the predicted site for a signal peptidase cleavage.
[View Larger Versions of these Images (57 + 11 + 50K GIF file)]
and 3
RACE was performed using RNA isolated from IFN
-treated RAW 264.7 cells. Once the ends of the cDNA were determined,
a full-length cDNA was isolated using reverse transcriptase
(RT)-PCR and was sequenced (Fig. 1 C). The cDNA was
514 bp long with an open reading frame that encoded 104 amino acids. The 5
region of the cDNA encoded a 22-
amino acid hydrophobic leader sequence with a predicted cleavage site at a position similar to the other MCPs and
eotaxin, resulting in a mature protein of 9.3 kD with a pI
of 9.4. The 3
untranslated region contained a single polyadenylation signal of a rare type also found in human eotaxin (ATTAAA) (15) and four mRNA destabilization signals (ATTTA) (Fig. 1 C) that have been reported to
decrease the mRNA stability of other cytokine mRNAs (29).
1,000 ng/ml. These cells were very responsive to the
positive controls eotaxin and MIP-1
(peak chemotaxis at 50 ng/ml or 5 nM), and only minimally responsive to the
negative controls JE and MIP-1
(Fig. 2 B). Murine neutrophils exhibited no response to MCP-5, but had a strong
response to the controls mouse KC and human IL-8 (Fig.
2 C). These results demonstrate that purified MCP-5 was a
potent, dose-dependent, chemotactic agent for peripheral
blood monocytes.
Fig. 2.
Chemotactic response of leukocytes to recombinant murine MCP-5. Human peripheral blood mononuclear cells (A), mouse eosinophils (B), and mouse neutrophils (C) were exposed to increasing concentrations of the indicated chemokines in a modified Boyden chamber, and the number
of cells that migrated through the membrane was determined. Data are the number of cells/×400 field. The results shown are representative experiments
(n = 9 PBMC, n = 7 eosinophils, and n = 3 neutrophils) and presented as the mean ± standard error of eight fields counted of replicate wells.
[View Larger Version of this Image (22K GIF file)]
, but did not respond to MIP-1
(Fig. 3 B) or
JE (data not shown), and the purified neutrophils responded appropriately to KC and MIP-1
(Fig. 3 C). The
half-maximal effective concentration of the MCP-5 induced mononuclear cell calcium transient was ~10 ng/ml
(1 nM) (data not shown). These results demonstrate that
MCP-5 induces a calcium flux in mononuclear cells, but
not in eosinophils or neutrophils.
Fig. 3.
Calcium flux responses of leukocytes to MCP-5. Fura-2-loaded
cells were exposed sequentially to the indicated chemokines with their
concentrations in parentheses. Calcium flux is reported as ratio fluorescence of fura-2 in human peripheral blood mononuclear cells (A), murine
eosinophils (B), and murine neutrophils (C). The results shown are representative experiments (n 4 for all cell types).
[View Larger Version of this Image (38K GIF file)]
(100 nM), or neutrophils to respond to KC (10 nM). These results suggest that MCP-5 shares a receptor
with JE and MCP-1 that is distinct from the receptors utilized by MIP-1
, eotaxin, and KC.
, and the
CCR3 line responded to eotaxin (data not shown). Doseresponse experiments demonstrated that MCP-5 was an
excellent ligand for human CCR2b (EC50
5 nM), as was
MCP-1, and both were more potent ligands than JE (Fig. 4
A-C). Since the MCP-5 we have described is a murine
protein, it was of interest to directly compare it to JE for
activation of the murine MCP-1 receptor (CCR2). As
shown in Fig. 4 B, JE induced a more robust intracellular
calcium flux than MCP-5 in HEK-293 cells stably expressing murine CCR2. No response was seen to MCP-5 in
cells transfected with murine CCR5, although these cells
did respond to the positive control MIP-1
(data not
shown). These data raise the possibility that the natural murine receptor for MCP-5 is yet to be cloned. Desensitization studies using cells transfected with human CCR2b revealed that MCP-5 blocked subsequent responses to MCP-5,
MCP-1, and JE (Fig. 5). Similar results were obtained
when the initial agonist was MCP-1 or JE (Fig. 5). However, MCP-1 did not completely desensitize the CCR2b
transfectants to a subsequent MCP-5 stimulation (Fig. 5).
These data are consistent with the desensitization responses observed on human PBMC (Fig. 3), and with the hypothesis that MCP-5 is a full agonist for the human MCP-1 receptor (CCR2b).
Fig. 4.
Identification of CCR2 as a functional MCP-5 receptor.
HEK-293 cells stably expressing human CCR2b (A-C) or murine
CCR2 (D and E) were loaded with indo-1 AM, and intracellular calcium concentrations were monitored by ratio fluorescence in response
to the indicated concentrations of MCP-5 (A and D), JE (B and E), and
MCP-1 (C). Shown is one of three similar experiments.
[View Larger Version of this Image (32K GIF file)]
Fig. 5.
MCP-5 induces desensitization of CCR2b. HEK-293 cells
stably expressing human CCR2b were exposed to MCP-5 (25 nM) (A),
and subsequently challenged with MCP-5 (100 nM), MCP-1 (100 nM),
or JE (100 nM). Intracellular calcium concentrations were monitored by
ratio fluorescence in response to the indicated ligand. Identical experiments were performed in which the CCR2b expressing cells were initially exposed to MCP-1 (25 nM) (B), or JE (25 nM) (C). Shown is one
of three experiments.
[View Larger Version of this Image (23K GIF file)]
splice variants (4).
Fig. 6.
Expression of MCP-5 in normal mouse organs. Northern
analysis of 10 µg total RNA isolated from various tissues of a normal BALB/c mouse. Salivary gland and breast tissue contained lymph nodes.
The blot was hybridized sequentially with MCP-5, JE, and rpL32 cDNA
probes and exposed for 29 d, 21 d, and 18 h, respectively. The positions
of 18s and 28s RNA are indicated on the left.
[View Larger Version of this Image (52K GIF file)]
Fig. 7.
MCP-5 mRNA expression in murine leukocytes and endothelial cells. (A) Northern analysis of total RNA isolated from eosinophils purified from the spleens of CD5-IL5 transgenic mice (Eos), bone marrow-derived mast cells either untreated (Ctl) or stimulated with IgE antiTNP and TNP-BSA (IgE), or Con A, resident peritoneal macrophages
(RM) (93% mononuclear, 7% granulocytes), elicited peritoneal macrophages (EM
) (76% mononuclear, 24% neutrophils), and elicited peritoneal neutrophils (95% neutrophils, 5% mononuclear). Blots were hybridized with MCP-5, JE, and rpL32 cDNA probes and exposed for 14 d,
3 d, and 18 h, respectively. (B) Northern analysis of total RNA isolated
from SVEC cells treated for 6 and 18 h with IFN
(200 U/ml), IL-4 (10 ng/ml), and IL-1
(5 ng/ml) and 10 µg of total RNA isolated from
RAW 264.7 macrophage cell line, either untreated or treated for 18 h with
IFN
(200 U/ml) or the indicated concentrations of LPS. Blots were hybridized sequentially with MCP-5, JE, and rpL32 cDNA probes and exposed for 7 d, 3 d, and 18 h, respectively.
[View Larger Versions of these Images (55 + 34K GIF file)]
and LPS induced MCP-5
expression (Fig. 7 B), while IL-4 and TNF
had no apparent effect (data not shown). JE was similarly expressed in
activated macrophages and induced in RAW cells by LPS
and IFN
(Fig. 7 B).
, IL-4, or IL-1
, but JE was induced by IFN
and
IL-4 (Fig. 7 B), and eotaxin was induced by IFN
(24).
Fig. 8.
MCP-5 mRNA expression in murine models of pulmonary
inflammation. Northern analysis of total RNA isolated from the lungs of nonimmunized nonchallenged mice (NN), sham-immunized mice challenged with aerosolized OVA (SC), OVA-immunized mice challenged
with aerosolized OVA (IC), or from the lungs of mice at 7, 10, and 14 d
after infection with Nb. All OVA challenged tissue was harvested at 3 h
(n = 3). Each lane represents an individual mouse. Blots were hybridized
sequentially with MCP-5, JE, and rpL32 cDNA probes and exposed for
14 d, 3 d, and 18 h, respectively.
[View Larger Version of this Image (47K GIF file)]
-chemokine, most closely related to the MCP and eotaxin subfamily of
-chemokines, which we have
named MCP-5. We have demonstrated that MCP-5 is expressed constitutively in lymph nodes of normal mice, and its
expression is increased in activated macrophages and in the
lungs of mice following allergen challenge and Nb infection. Purified recombinant MCP-5 protein was potently
chemotactic for monocytes and induced a calcium flux in
these cells. In addition, we identified CCR2 (the MCP-1
receptor), which is expressed preferentially on monocytes
and activated lymphocytes (30, 31) as an MCP-5 receptor.
-chemokines, MCP-5 was inactive on neutrophils. However, we were also interested in testing the
activity of MCP-5 on another granulocyte, the eosinophil.
Aside from MCP-1, which lacks activity on eosinophils,
the other MCP proteins characterized to date, MCP-2, -3, and -4, are active on eosinophils. We were surprised that
MCP-5 had essentially no activity on eosinophils since it
shares an NH2-terminal glycine with human eotaxin. The small amount of chemotaxis seen at high doses of MCP-5
(1 and 10 µg/ml) was not associated with a detectable calcium flux in these cells. Furthermore, MCP-5 was not a
functional ligand for human CCR1 and CCR3, the only two
CC chemokine receptors that have been demonstrated to
be expressed on eosinophils. Therefore, the weak chemotaxis of eosinophils induced by high doses of MCP-5 may
be secondary to a low affinity interaction with a different receptor that does not induce a calcium flux or induces one
below the limit of detection.
and LPS. However, MCP-5 was not expressed in activated mast cells, though
these cells expressed MARC/FIC and JE. Furthermore,
while we did not detect MCP-5 expression in activated endothelial cells, these cells express MCP-1, JE, MCP-4, and
eotaxin after stimulation with inflammatory cytokines like
IL-1, IL-4, TNF, and IFN
(2, 15, 24). These results suggest that MCP-5 has a unique pattern of expression that reflects its unique role in regulating the immune and inflammatory responses.
, and MARC/FIC are also upregulated following antigen challenge, whereas the level of RANTES is unchanged (22). While MCP-5 is not directly
chemotactic for eosinophils, its association with pulmonary
eosinophilia in these two models may be due to its role in
recruiting the initial waves of mononuclear cells which
control the subsequent recruitment of eosinophils.
Address correspondence to Andrew D. Luster, Infectious Disease Unit, Massachusetts General Hospital-East, Building 149, 13th Street, Charlestown, MA 02129.
Received for publication 16 September 1996
This work was supported by National Institutes of Health grants to A.D. Luster (CA69212), I.F. Charo (HL52773), and J. MacLean (AI01245), a Cancer Research Institute/Benjamin Jacobson Family Investigator Award to A.D. Luster and a Fogarty International Fellowship to E.A. Garcia-Zepeda.We would like to thank L. Rowe from the Jackson Laboratory for her help with the chromosome mapping, M. Rothenberg for the generous gift of mast cell RNA, hybridomas, and for his helpful discussions, R. Coffman for the generous gift of lungs infected with Nb, C. Sanderson for the IL-5 transgenic mice, C. Franci for calcium fluorimetry on transfected cell lines, B. Rollins for the JE cDNA clone, and K. O'Connell for the SVEC cells.
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