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*
Cancer Research Campaign Laboratories, Beatson Institute for Cancer Research, Glasgow, United Kingdom;
Human Genome Sciences, Inc., Rockville, MD 20850;
Academic Transfusion Medicine Unit (ATMU), Department of Medicine, University of Glasgow, Glasgow Royal Infirmary, Glasgow, United Kingdom;
§
Serono Pharmaceutical Research Institute, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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Whereas many molecules are able to stimulate leukocyte chemotaxis, it
has become clear that chemokines play a central role in regulating
hemopoietic cell movement both during the establishment of inflammation
and immune responses, and also during immune surveillance and the
development of the blood system (2, 3, 4). Recently, these
proteins have also been implicated in the biology of other cell types,
such as hemopoietic stem cells, microglia, neurons, and endothelial
cells (5, 6, 7, 8). Chemokines are divided into four
subfamilies on the basis of the position of the first
two cysteine residues of the mature protein. Thus, in the CC or ß
chemokines, these two residues are juxtaposed, whereas the CXC (or 
)
and the CX3C chemokines have one and three amino
acids, respectively, between these two cysteines. The C subfamily has
only a single cysteine at this position.
The biological effects of these proteins are mediated by interactions with a family of cell surface heptahelical G protein-coupled receptors present on the target cells (9, 10). These receptors are often highly promiscuous, interacting with many chemokine ligands, usually from within the same chemokine subfamily. This has led to the receptors being named CCR, CXCR, CX3CR, or XCR,5 depending upon which ligand subfamily is recognized (9, 10, 11, 12). Interest in these proteins has intensified in the last couple of years with the demonstration that many chemokine receptors, in particular CXCR4 and CCR5, and to a lesser extent CCR3, act as coreceptors for the entry of HIV into its target cells and that the ligands for these receptors interfere with virus entry (13, 14).
Parasite- and allergen-induced inflammation is characterized by infiltration of eosinophils, T lymphocytes of the Th2 type, and occasional basophils into tissue (15, 16). The CC, or ß, chemokine receptor CCR3, is specifically expressed on these cell types and plays a central role in their infiltration (17, 18, 19, 20, 21). In response to local production of the CCR3 ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, or RANTES, these cells can adhere to, and migrate through, blood vessel endothelium (22, 23, 24, 25, 26, 27). Subsequent granule release by eosinophils and basophils brings about changes in tissue structure and integrity, often causing irreversible damage (15). Therapies that block cellular recruitment may be of benefit in allergic diseases, such as asthma and contact dermatitis, and specific targeting of the CCR3 receptor may have considerable advantage over drugs that indiscriminately inhibit leukocyte chemotaxis, such as steroids. In fact, deletion of eotaxin by homologous recombination, the use of neutralizing Abs to this ligand, or injection of a chemokine receptor antagonist Met-RANTES has been demonstrated to ameliorate allergic inflammation in a variety of animal models (28, 29, 30, 31, 32).
We report here the generation of a potent CCR3 antagonist, called Met-chemokine ß 7 (Ckß7), a modified form of the ß-chemokine referred to herein as macrophage inflammatory protein (MIP) 4 and alternatively called pulmonary and activation-regulated chemokine (PARC), dendritic cell-derived C-C chemokine (DCCK) 1, or alternative macrophage activation-associated C-C-chemokine (AMAC) 1 (33, 34, 35, 36). Met-Ckß7 is significantly more potent as a CCR3 antagonist than Met-RANTES or aminooxypentane (AOP)-RANTES (32 , 37, 38, 39) and, unlike these proteins, shows no detectable partial agonist activity.67 Furthermore, Met-Ckß7 appears to be highly specific for CCR3. This novel antagonist is able to completely inhibit eosinophil chemotaxis at concentrations as low as 1 nM. Surprisingly, the unmodified MIP4 protein (which has been reported to act as a naive T cell chemoattractant acting through a currently unidentified receptor) also exhibits CCR3 antagonistic activity in our assays, although it is considerably less potent than Met-Ckß7. Therefore, the modifications in Met-Ckß7, and specifically the introduction of a methionine in place of an alanine at the extreme amino terminus, seem to enhance a property present in the unmodified protein. Interestingly, MIP4 is able to inhibit CCR3-mediated eosinophil chemotaxis at concentrations that may be physiologically relevant. The possible significance of this observation, with respect to the biological function of MIP4, is discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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The coding sequence of Ckß7 was amplified from an adult human lung cDNA library using primers to remove the signal peptide and replace the N-terminal alanine seen in the mature protein with a methionine. The nucleotides encoding the C terminus were altered to encode either LKLMPEA or LKLNA, and both cDNAs were cloned into the pQE7 expression vector. The resulting plasmids were transformed into Escherichia coli M15 Rep4 host cells, grown at 37°C in L-Broth containing ampicillin and kanamycin and protein induced by incubation with 0.2 mM isopropyl ß-D-thiogalactoside (IPTG) for 3 h. Cells were harvested, resuspended in ET buffer (75 mM EDTA, 50 mM Tris (pH7.5)), and lysed by passing twice through a microfluidizer (Microfluidics, Newton, MA) at 6000–8000 psi. NaCl was added to 0.5 M, and the sample was centrifuged at 7000 x g for 15 min. The pellet was washed in ET plus 0.5 M NaCl and centrifuged at 7000 x g again for 15 min. These partially purified inclusion bodies were resuspended in 1.5 M guanidine hydrochloride and 50 mM Tris (pH 7.4) and were incubated overnight at 4°C and then centrifuged at 30,000 x g. The supernatant was mixed vigorously for 30 min at 4°C in 20 vol of 150 mM NaCl, 2 mM EDTA, and 50 mM sodium acetate (pH 4.5) and was left for 60 h at 4°C. This solution was clarified using a 0.16-µm sterile filter (Filtron; Pall, Port Washington, NY) and chromatographed over a strong cation exchange column (Poros HS-50; Perspective Biosystems, Framingham, MA) prewashed with 6 column vol of 250 mM NaCl and 40 mM sodium acetate (pH 5.5). Bound protein was eluted using 3 to 5 column vol of a stepwise gradient of 0.5 M, 1 M, and 1.5 M NaCl in 40 mM sodium acetate (pH 5.5). Positive fractions were pooled, diluted 3-fold with 40 mM sodium acetate (pH 5.5), and applied to a set of strong anion (Poros HQ-50) and weak cation (Poros CM-20) exchange columns in tandem mode prewashed with 150 mM NaCl and 40 mM sodium acetate (pH 5.5). The CM-20 column was eluted with a 10–20 column vol linear gradient of 0.15–1.25 M NaCl; fractions were analyzed through SDS-PAGE and positive fractions were combined. The proteins were greater than 95% pure by SDS-PAGE and reverse phase HPLC analysis. Peptide sequencing revealed the expected amino-termini of MQVGTNKEL.
Met-RANTES and AOP-RANTES were produced as previously described (37, 38). All other chemokines, including MIP4, were purchased from Peprotech (London, U.K.) or R&D Systems (Abingdon, U.K.).
Cell culture and preparation
HOS cells stably expressing human CCR3 were the generous gift of Dr. Nathaniel Landau (Salk Institute, La Jolla, CA) and were maintained in DMEM plus 10% FCS, antibiotics, and 1 µg/ml puromycin (Sigma, Poole, Dorset, U.K.). HEK293 cells stably expressing human CCR1, -2, -3, and -5 were generated by transfection with Transfectam (Promega, Southampton, U.K.), according to the manufacturer’s protocols, and selection in 0.8 mg/ml G418. Chinese hamster ovary (CHO) cells expressing human D6 are described elsewhere (40).
Eosinophils were purified from single donor leukopaks (American Red Cross, Baltimore, MD) as previously described (41), or by purifying granulocytes from peripheral blood and selecting those cells that were CD16 negative using MACS technology (Miltenyi Biotec, Bergisch, Germany). Eosinophils were greater than 90% pure as assessed on stained cytospun preparations.
Mononuclear cells were purified from samples of peripheral blood (Western Infirmary, Glasgow, U.K.). Blood containing at least 100 U/ml of preservative-free heparin was diluted 1:4 with PBS containing 0.6% acid citrate dextrose (Sigma). This was layered over Ficoll (1.077 density) in a ratio of 3:1 and centrifuged at 400 x g for 30 min at 22°C. Mononuclear cells were removed from the interface and washed three times in PBS/0.6% acid citrate dextrose.
To purify and activate T cells, heparinized blood was collected from healthy donors, separated over ficoll-hypaque, and washed four times in PBS. These cells were then adhered to plastic for 2 h at 37°C in RPMI/10% FCS, and the nonadherent cells were then stimulated for 5 days in 4 µg/ml Con A (Sigma). Cells were then repurified over ficoll-hypaque as before and stimulated at 106 cells/ml in 20 U/ml IL-2 (Peprotech) for 7–14 days. CD3+ and CD45RA+ cells were purified by positive selection from PBMCs (isolated as described above) using anti-CD3 and anti-RA Abs, respectively, and MACS technology. Purity was >95% as determined by FACS analysis.
Ca2+ flux assays
HOS-hCCR3 cells were harvested by trypsinization, washed in SR
buffer (136 mM NaCl, 4.8 mM KCl, 5 mM glucose, 1 mM
CaCl2, 0.025% BSA, and 25 mM HEPES (pH 7.6)),
and then incubated in SR with 10 µM fura-2-AM (Sigma) for 1 h at
37°C. Cells were then washed in SR and resuspended in SR to 
2
x 106 cells/ml, and 2 ml was incubated at 37°C
in a continuously stirred cuvette in a Perkin-Elmer LS50 Spectrometer.
After 2 min, fluorescence emission was recorded every 100 ms (340 nm
(
ex); 500 nm (em))
for 20 to 40 s, agonist or antagonist was added to a defined
concentration, and fluorescence was recorded every 100 ms for a further
80 to 120 s. For agonist dose-response experiments, all ligands
were compared with a full dose response performed with human eotaxin to
avoid day-to-day experimental variation. PBMCs and lymphocytes were
also loaded and analyzed using this procedure.
Eosinophils were loaded for 30 min at room temperature with 2 µM
fura-2-AM in a modified SR buffer (125 mM NaCl, 5 mM KCl, 0.5 mM
glucose, 1 mM MgCl2, 1 mM
CaCl2, 0.025% BSA, and 20 mM HEPES (pH 7.4)),
washed, and resuspended at 106 cells/ml.
Intracellular Ca2+ changes from a 2-ml sample
were measured in an F2000 spectrometer (Hitachi Instruments, San Jose,
CA) by monitoring fluorescence emission at 37°C over time (340 nm and
380 nm (ex); 510 nm
(em)).
In all experiments, the distance from the baseline emission to the highest point of the flux was calculated and converted to a percentage of the maximal flux induced in each experiment.
Eosinophil chemotaxis assays
Purified eosinophils were washed with HBSS/BSA (HBSS with 0.1% BSA) and resuspended in this medium at 5 x 106 cells/ml with 1 µM calcein-AM (Molecular Probes, Eugene, OR). After 30 min at 37°C, cells were washed in HBSS/BSA and resuspended to 5 x 106 cells/ml; 20 µl of this suspension was dispensed into each upper chamber of a 96-well chemotaxis plate filter (Neuro Probe, Cabin John, MD). Different concentrations of agonist were added to the bottom chamber, and antagonist was added to either the bottom chamber, the top and bottom chamber, or neither chamber. Cells were allowed to migrate for 3 h through the polycarbonate filter (8-µM pores; polyvinylpyrolidone-free) between the two chambers, and the number of migrated cells in the bottom chamber was quantitated using a fluorescence plate reader (Cytofluor PerSeptive Biosystems). The ratio between the number of cells migrated in the presence of agonist and the number of cells migrated in buffer alone is defined as the chemotaxis index.
Radioiodination of Met-Ckß7
Five micrograms of Met-Ckß7 was incubated in 50 µl of PBS
containing 100 µg of IODO-GEN (Pierce, Rockford, IL) and 1 mCi
Na125I (DuPont- NEN, Hounslow, U.K.) for 15 min
on ice. The reaction was then run down a D-Salt Excellulose
Desalting Column (40–100 micron), and 0.5-ml fractions were taken with
PBS. Two-microliter aliquots of the fractions were counted in a Beckman
5500B counter (Beckman, High Wycombe, U.K.), and positive fractions
were combined.
Eosinophil 125I-chemokine binding assay
Purified eosinophils (2 x 105) were placed in each well of a 96-well plate in binding buffer (1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, 0.05% sodium azide, and 50 mM HEPES (pH 7.5)). Iodinated chemokine (final concentration of 0.1 nM [125I]eotaxin or [125I]MCP4, spec. act. 2200 Ci/mmol (NEN, Boston, MA)) was added in the absence or presence of unlabeled chemokines to a final volume of 100 µl. The binding reaction was incubated for 60 min at room temperature, and the cells were then transferred to filter plates (Silent Screen with Ioprodyne membrane (Nalge Nunc, Rochester, NY)) pretreated with 0.1% polyethylenimine and washed three times with binding buffer containing 0.5 M NaCl. The plates were dried and counted after addition of 50 µl of liquid scintillant in each well. Each point was done in triplicate and is presented as the mean of these results with SE. For experiments with [125I]Met-Ckß7, 2 x 105 eosinophils were incubated in DMEM plus 10% FCS, 0.4% sodium azide, and 20 mM HEPES (pH 7.6) containing 45 nM [125I]Met-Ckß7 and with or without 500 nM unlabeled chemokine as competitor for 2 h at room temperature. Cells were then washed twice with ice cold PBS and counted in a Beckman 5500B gamma counter. Each point was done in triplicate and is presented as the mean of these results with SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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A cDNA clone, termed Ckß7, was amplified from an adult human lung cDNA library. Predictive algorithms suggest that the putative signal peptide will be cleaved from this protein to leave an amino terminus beginning AQVGT-, although QVGT- may also be produced. Production in insect cells generated a protein with AQVGTNKEL- at the N terminus, although variable amounts of truncated variants starting TNKEL- or NKEL- were also found in our studies. To produce this protein in vitro with a homogeneous N terminus, we generated a cDNA construct in which the region coding the signal sequence was removed and the nucleotides encoding the alanine residue found at the putative N terminus of the mature protein were replaced with an ATG encoding a methionine residue. This cDNA was transformed into bacteria, and the protein was purified (see Materials and Methods). Sequencing revealed the expected N termini of MQVGT-. During the production and analysis of this protein, Ckß7 was described by others and named PARC, MIP4, DCCK1, or AMAC1 (33, 34, 35, 36). These sequences were identical to Ckß7, with the exception that the predicted C terminus of Ckß7 ends LKLMPEA rather than LKLNA, a difference that may be due to allelic variation within this gene and is currently under investigation. Corroborating our observations, in two of these publications, production in COS or insect cells also demonstrated that the N terminus of the mature protein begins AQVGT- (33, 34). To assess the impact of the C-terminal sequence variation, we made a protein in bacteria that as before was engineered to contain a methionine in place of the first N-terminal alanine, but had a C terminus of LKLNA rather than LKLMPEA. Importantly, in all assays tested, these two proteins exhibited identical activity showing that the C termini differences have no affect on the properties described (data not shown). The data shown use the protein with the C terminus of LKLMPEA (since this protein has been more extensively used), and it is referred to hereafter as Met-Ckß7, with the "Ckß7" name retained to indicate that it contains the C-terminal sequence derived from our cDNA clone.
Activity of Met-Ckß7 on chemokine receptors
This protein was tested for its ability to elicit a
Ca2+ flux through chemokine receptors CCR1, -2,
-3, and -5 expressed in heterologous cells. At concentrations up to 1
µM, no signaling was detectable through these receptors whereas known
ligands signaled efficiently (data not shown). Also, 700-nM Met-Ckß7
was unable to displace any [125I]MIP1 from
the promiscuous D6 chemokine receptor (40) in binding
assays on CHO cells expressing this receptor (not shown). However, with
HOS cells stably transfected with human CCR3 (HOS-CCR3 cells),
pretreatment with 500 nM Met-Ckß7 prevented subsequent
Ca2+ fluxes induced with known CCR3 agonists
(Fig. 1
, A–D).
This activity was not seen on CCR1, -2, or -5, even when CCR3 ligands
MCP4 (that also uses CCR2) or RANTES (that also signals through CCR1
and -5) were used as agonists (not shown). To further assess the
specificity of this antagonist, we isolated mononuclear cells and
CD3+ T lymphocytes from human peripheral blood
and also prepared Con A/IL-2-activated T cells (see Materials and
Methods), and tested whether Met-Ckß7 could inhibit
Ca2+ fluxes induced with a range of chemokines.
None of the human chemokines that gave a Ca2+
flux in these various cell types (melanoma growth-stimulating activity
(MGSA), IL-8, stromal cell-derived factor (SDF) 1, RANTES, MIP1,
MIP1ß, MCP1, fractalkine, MIP3, MIP3ß, secondary lymphoid-tissue
chemokine (SLC), and IFN--inducible 10-kDa protein (IP-10)) were
antagonized by 500 nM Met-Ckß7 (not shown). Further, no agonist
activity was detected with Met-Ckß7 in these assays. One example of
these experiments using PBMCs, and human MIP1 as the agonist, is
shown in Fig. 1E. It is of note that unmodified MIP4 gave no
detectable Ca2+ flux in any of the cell types
tested (not shown).
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Next, we wanted to define the potency of this CCR3 antagonist.
Several ß-chemokines are known to act as CCR3 agonists, and
Met-Ckß7 may exert differential effects on these ligands. Thus, we
first compared the potency of these known CCR3 agonists by performing
dose-response experiments examining Ca2+ flux
into HOS-CCR3 cells at different concentrations of ligand. As shown in
Fig. 2, eotaxin, eotaxin-2, and MCP4
induced strong signals through CCR3 in the low nanomolar range and
above, with slight Ca2+ fluxes still detectable
at 1 nM. RANTES was much less potent, and MCP3 gave a barely detectable
signal, even at 100 nM. The number of CCR3 receptors on the HOS-CCR3
cells is low (not shown) and may account for the relative
ineffectiveness of RANTES and MCP3 in these assays. We then performed
dose-response experiments using a range of concentrations of Met-Ckß7
to examine its effect on a subsequent Ca2+ flux
induced with a concentration of CCR3 agonist known to give a strong
signal. As shown in Fig. 3A,
half maximal inhibition of a Ca2+ flux induced
with 25 nM eotaxin or MCP4, or 50 nM eotaxin-2, was seen at 25 nM
Met-Ckß7. With 100 nM RANTES, which only produces a weak signal into
HOS-CCR3 cells, 50% inhibition was observed at a slightly lower
concentration of Met-Ckß7 (10 nM) (not shown).
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35% (Fig. 4). In contrast, 100 nM
Met-Ckß7 reduced a 25-nM eotaxin-induced flux by 85% (Fig. 4D). It is of note that Met-RANTES and AOP-RANTES are able
to induce a moderate Ca2+ flux through CCR1 and
CCR5 and that AOP-RANTES is in fact fully active on CCR5 in these
assays5 (39). It has been shown that
AOP-RANTES (100 nM), and to a lesser degree Met-RANTES, have weak
Ca2+ mobilizing activity on CHO-CCR3 and
L1.2-CCR3 transfectants, although this activity was never superior to
50% of that induced by RANTES.6 Neither Met- nor
AOP-RANTES (at 100 nM) mobilize calcium in the HOS-CCR3 transfectants
(Fig. 4), perhaps due to the low receptor level on these cells, but
AOP-RANTES at higher concentrations (250 nM) does induce very weak, but
detectable, Ca2+ fluxes into these cells (data
not shown). On the contrary, Met-Ckß7 shows no CCR3 signaling
potential in Ca2+ flux assays with CCR3
transfectants or eosinophils, even at 1 µM.
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We extended our studies to examine whether Met-Ckß7 could
antagonize the function of CCR3 agonists on eosinophils. The
concentration of CCR3 agonist required to induce detectable
Ca2+ fluxes into these cells was considerably
lower than that required with the CCR3-transfected HOS cells used
above, most likely due to higher receptor levels on eosinophils.
Met-Ckß7 completely blocked Ca2+ fluxes into
eosinophils induced by 1 nM eotaxin or MCP4, at 50 nM or 10 nM of
Met-Ckß7, respectively (Fig. 3
B). When 10 nM RANTES or
MCP3 were used as agonists, whereas low concentrations of Met-Ckß7 (1
nM) caused a profound reduction in the magnitude of the
Ca2+ flux, complete antagonism was not achieved
even with 100 nM Met-Ckß7. This is explained by the presence of CCR1
on these cells acting as a receptor for RANTES and MCP3, further
demonstrating that Met-Ckß7 does not abrogate signaling through this
receptor.
Analysis using radio-iodinated ligands suggest a direct interaction between Met-Ckß7 and CCR3
Next, we examined the ability of unlabeled Met-Ckß7 to displace
radio-iodinated MCP4 or eotaxin from CCR3, in comparison with unlabeled
eotaxin and MCP4. The low level expression of CCR3 on the transfected
HOS-CCR3 cells necessitated the use of eosinophils in these assays.
Fig. 5A shows that Met-Ckß7
more efficiently displaced [125I]eotaxin from
these cells (IC50 6 nM) than unlabeled
eotaxin (IC50 10 nM) or MCP4
(IC50 60 nM), whereas with the displacement
of [125I]MCP4, eotaxin and Met-Ckß7 behaved
similarly (IC50 5 nM) and were more effective
than unlabeled MCP4 (IC50 25 nM) (Fig. 5B). Thus, Met-Ckß7 is able to successfully compete with
known CCR3 agonists for binding to CCR3. This is most likely due to a
direct interaction between the antagonist and the receptor. We have
been able to demonstrate this directly by radiolabeling Met-Ckß7 and
showing binding to eosinophils that could be competed by unlabeled
Met-Ckß7 and eotaxin, but not by unlabeled MIP-1 (Fig. 5C). Interestingly, unlabeled MIP4 was also able to displace
some of the labeled Met-Ckß7 from the surface of eosinophils,
suggesting that it too interacts weakly with this receptor (Fig. 5C; and see below). Subsequent experiments with radiolabeled
eotaxin also demonstrated heterologous displacement by MIP4, although
this ligand was considerably less effective than Met-Ckß7 (data not
shown).
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To further test the potency of the antagonist on eosinophil
function, we performed chemotaxis assays with purified human
eosinophils. Results from three donors are shown in Figs. 6 and 7.
Eotaxin and MCP4 induced chemotaxis of eosinophils most effectively
when 1 nM or 10 nM of the chemokine was present in the lower well,
although the maximal chemotaxis index achieved varied considerably
between donors. These concentrations are considerably lower than those
required to induce maximal Ca2+ fluxes through
CCR3 into HOS-CCR3 cells or eosinophils (Fig. 3A and not
shown), indicating that low receptor occupancy is optimal for
chemotaxis induction. Met-Ckß7 was unable to stimulate chemotaxis
over the concentrations tested but was able to efficiently block
eotaxin- and MCP4-mediated chemotaxis. The potency of the antagonist in
these assays varied slightly between donors: thus, whereas 1 nM was
sufficient to inhibit the eotaxin- and MCP4-induced chemotaxis of
eosinophils from donor A, this was not sufficient to prevent
eotaxin-induced chemotaxis of donor B’s eosinophils unless it was
added to the top and the bottom wells of the assay chamber. Curiously,
with donor C, addition of Met-Ckß7 to the bottom compartment of the
well only, reduced eosinophil chemotaxis in response to eotaxin and
MCP4, but addition of the antagonist to both upper and lower chambers
reduced chemotaxis to baseline levels. The reason for this
donor-to-donor variability is currently uncertain (see
Discussion). These data demonstrate, however, that
Met-Ckß7 is a potent inhibitor of eosinophil chemotaxis induced with
CCR3 ligands, with low nanomolar concentrations being sufficient to
completely abrogate cell migration.
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Data shown above using radio-iodinated ligands suggested that
unmodified MIP4 also demonstrates some ability to displace ligands from
human CCR3. Therefore, we next tested whether the commercially
available MIP4 protein, with an N-terminal alanine residue and LKLNA at
the C terminus, was able to affect CCR3 signaling. No CCR3-mediated
Ca2+ flux was detectable with up to 250 nM MIP4,
but, surprisingly, this protein exhibited CCR3 antagonistic activity in
Ca2+ flux assays with fura-2 loaded HOS-CCR3
cells (Fig. 8) or eosinophils (Fig. 9). However, it is not as potent as
Met-Ckß7 (5- to 10-fold less active), suggesting that the amino
acid sequence differences in Met-Ckß7 conspire to amplify a property
present in the natural protein. Thus, whereas 10 nM Met-Ckß7
completely abrogates Ca2+ fluxes induced into
eosinophils by 1 nM MCP4, only 50% inhibition is seen with MIP4 at
this concentration (Fig. 9B). However, at higher
concentrations (100 nM), MIP4 is able to completely abrogate signaling
induced with 1 nM MCP4. Similar results were obtained with abrogation
of 1 nM eotaxin signaling (Fig. 9A), with complete
inhibition observed with 50 nM Met-Ckß7 but 10% of the signal
remaining in the presence of 100 nM MIP4.
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To extend these observations, we sought to determine the potency
of MIP4 in inhibiting eosinophil chemotaxis using MCP4 or eotaxin as
agonists, the two most potent CCR3 ligands in our assays. As above,
maximal chemotaxis with these agonists was observed between 1 and 10 nM
for both donors tested (Fig. 10). When
10 nM MIP4 was added to the top and bottom chambers of the chemotaxis
assay plate, then eosinophil chemotaxis induced by 1 nM MCP4 or eotaxin
is reduced to near baseline levels, and chemotaxis induced by 10 nM of
the agonists was significantly reduced. MIP4 was unable to induce
chemotaxis when present in the lower well at concentrations ranging
from 0.1 to 100 nM (data not shown). These results corroborate the
observations obtained in the Ca2+ flux signaling
assays and show that MIP4 is able to inhibit CCR3 function, but that it
is less potent than the Met-Ckß7. The physiological relevance of this
observation, with respect to the biological function of MIP4, is
discussed below.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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Studies with purified PBMCs or lymphocytes, or with cells stably expressing exogenous chemokine receptors, show that Met-Ckß7 is highly specific for CCR3. CCRs 1, 2, and 5–7, CXCRs 1–4, CX3CR1, and D6 do not show any demonstrable interaction with this protein. However, we cannot exclude the possibility that Met-Ckß7 antagonizes other receptors. In particular, we have been unable to test the activity of this protein on the currently uncharacterized MIP4 receptor. This has been hampered by our inability to demonstrate the chemotaxis of T cells by MIP4 that has been reported elsewhere (33, 34). Using CD3+- or CD45RA+-sorted peripheral blood T cells from several donors, or Con A/IL-2-activated T cells, MIP4 (produced either in baculovirus-infected insect cells or commercially in bacteria) or Met-Ckß7 were unable to stimulate detectable chemotaxis or Ca2+ signaling at concentrations ranging from 10 µg/ml to 0.1 ng/ml (data not shown). Control chemokines, such as MIP3ß, produced robust Ca2+ signals and were efficient chemoattractants of these cells. We are uncertain as to the reason for this discrepancy between our observations and the results of others (33, 34), and we are currently investigating the possibility that the MIP4 source affects T cell chemotactic activity. Until this issue has been resolved, we are unable to test whether Met-Ckß7 interacts with the putative MIP4 receptor.
Experiments with radio-iodinated eotaxin, MCP4, and Met-Ckß7 suggest
the simple model that Met-Ckß7 exerts its antagonistic activity by
binding directly to CCR3 and sterically preventing activation by CCR3
agonists. Our data show that Met-Ckß7 is able to inhibit eotaxin- and
MCP4-induced eosinophil chemotaxis. Curiously, however, in these assays
there is variation in the extent of inhibition by Met-Ckß7 (Figs. 6 and 7). Thus, whereas eosinophil chemotaxis from donor A is inhibited
effectively with 1 nM Met-Ckß7 beneath the filter of the assay plate,
with donor C this concentration of antagonist must be added to the top
and bottom of the filter to be fully inhibitory. Indeed, even 10 nM
Met-Ckß7, when present beneath the filter only, reduces but does not
completely prevent chemotaxis. These results show that Met-Ckß7 is
markedly less potent at inhibiting chemotaxis of eosinophils from donor
C, than those from donor A, with donor B falling in between. This is
difficult to explain simply using a model of antagonism by Met-Ckß7
involving steric interference between CCR3 and agonist. Thus, other
consequences of Met-Ckß7/CCR3 interaction may be required to inhibit
eosinophil chemotaxis, such as a currently unidentified intracellular
signal, or perhaps internalization of the CCR3 protein, phenomena that
may be variable between individuals. Indeed, evidence with other
similar N-terminally modified chemokines strongly suggests that
receptor internalization is a key component in their enhanced
inhibition of receptor function (39, 42). Additionally,
different ligands for CCR3 exhibit differential effects on this process
(43). Experiments to examine these possibilities with
Met-Ckß7 are underway.
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Mechanistically, the strong homology between MIP4 and the CCR3 ligand RANTES (seen over most of the protein except the N terminus) may be responsible for a weak inhibition of CCR3 activity seen with the MIP4 protein, with the differences in the N terminus determining whether the receptor can couple to Ca2+ fluxing and induce chemotaxis. This two site model for chemokine/chemokine receptor interaction has been proposed for a number of ß-chemokines, and many studies have demonstrated that small alterations in the amino terminus dramatically affect ligand binding affinity and, in some examples, can introduce antagonist activity into proteins that previously acted as agonists (37, 38, 44, 45, 46, 47, 48). Replacing the N-terminal alanine of MIP4 with a methionine residue appears to have enhanced the interaction with CCR3, without introducing agonist activity. Comparison of Met-Ckß7 to Met-RANTES highlights an alternative explanation for the interaction of CCR3 with MIP4. Met-RANTES, a variant of RANTES that has been extended at the N-terminal by one amino acid (a methionine), is an antagonist for RANTES receptors (37, 39). If Met-Ckß7 acts in a similar fashion, then one may hypothesize that a -1 variant of MIP4, lacking the first amino acid and starting QVGT, would act as a CCR3 agonist. Interestingly, predictive algorithms of signal peptide cleavage sites of the MIP4 protein suggest that the -1 form of this chemokine is as equally likely to be produced during protein secretion as the "full-length" protein with the amino-terminal alanine. Whereas production in COS7 and insect cells consistently generates a protein starting AQVGT (see Results and Refs. 33 and 34), experiments are underway to determine whether the -1 variant of MIP4 can be produced and whether it acts as a CCR3 agonist.
The CCR3 antagonistic activity of unmodified MIP4 is particularly intriguing with respect to the in vivo function of this protein. In chemotaxis assays, concentrations of MIP4 as low as 10 nM were able to significantly reduce eosinophil chemotaxis induced by the most potent known CCR3 agonists, namely eotaxin and MCP4. This level of MIP4 protein may be achieved in vivo, especially when it is considered that chemokine immobilization on extracellular matrix components can enhance local concentrations. CCR3 antagonism may therefore reflect a property of MIP4 that is of importance in the biology of this chemokine, allowing it to use both agonism and antagonism to control leukocyte cell movement.
It has been reported that MIP4 is produced by dendritic cells and
macrophages in the secondary lymphoid tissue, where it has been
hypothesized to play a role in the selective attraction of
naïve T cells toward APCs (33). Our data suggest
that this protein may also selectively and actively exclude
CCR3-positive cells, such as basophils, eosinophils, and Th2
lymphocytes, from this particular microenvironment. Intriguingly, the
CCR3 ligands eotaxin and MCP4 have been demonstrated to act as
antagonists for CXCR3, a receptor specifically expressed on Th1
lymphocytes and activated by the ß-chemokine secondary lymphoid
tissue chemokine (SLC) (in mice only), and the -chemokines
IFN--inducible 10-kDa protein (IP-10) and Mig (19, 21, 49, 50, 51). Whereas CXCR3 interactions with eotaxin and MCP4 were
shown to be fairly weak, akin to that seen for MIP4 on CCR3, again it
may be of physiological relevance and participate in the inhibition of
Th1 T cells into sites characterized by Th2 cell influx
(49). Thus, the use of receptor antagonism may be an
emerging theme in the regulation of leukocyte movement during
inflammation and immunity. It would be of interest to examine whether
other examples of this phenomenon exist with chemokine receptors that
are specifically involved in the attraction leukocyte subsets, such as
CCR4 (known ligands are thymus and activation-regulated chemokine
(TARC) and macrophage-derived chemokine (MDC)) and CCR8 (known ligands
are I-309, TARC, and MIP-1ß) that are found preferentially on Th2
cells (19, 52, 53). Also, it would be worthwhile examining
whether CCR3 ligands, such as eotaxin and MCP4, are able to exhibit
reciprocal antagonism of the currently uncharacterized MIP4
receptor.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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Acknowledgments |
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Footnotes |
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2 R.J.B.N. and T.W.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Robert J. B. Nibbs, Cancer Research Campaign Laboratories, Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, U.K. G61 1BD. E-mail address:
4 Address correspondence and reprint requests to Dr. Theodora W. Salcedo, Human Genome Sciences, 9410 Key West Avenue, Rockville, MD 20850. E-mail address:
5 Abbreviations used in this paper: CX3CR, CX3C chemokine receptor; MCP, monocyte chemotactic protein; CXCR, CXC or chemokine receptor; Ckß7, chemokine ß 7; PARC, pulmonary and activation-regulated chemokine; MIP, macrophage inflammatory protein; DCCK, dendritic cell-derived C-C chemokine; AMAC, alternative macrophage activation-associated C-C chemokine; AOP, aminooxypentane; CHO, chinese hamster ovary.
6 J. Elsner, M. Mack, H. Bruhl, Y. Dulkies, H. Petering, D. Kimmig, P. D. Ponath, D. Schlondorff, A. Kapp, and A. E. I. Proudfoot. Differential activation of CC chemokine receptors by AOP-RANTES. Submitted for publication.
7 A. E. I. Proudfoot, R. Buser, F. Borlat, S. Alouani, D. Soler, J.-M. Schroder, C. Power, and T. N. C. Wells. Amino terminally modified RANTES analogues demonstrate differential effects on RANTES receptors. Submitted for publication.
Received for publication June 22, 1999. Accepted for publication November 10, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in Proof.References |
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), MCP4
(X), or eotaxin (
). After washing with binding buffer containing
0.5 M NaCl, the percentage of radio-iodinated ligand remaining bound
was calculated compared with assays in which no unlabeled chemokine was
added. Each point is the mean of three identical incubations and SE is
included. C, Purified eosinophils (2 x
105) were incubated for 2 h at room temperature in
azide-containing binding buffer with 45 nM
[125I]Met-Ckß7 plus 500 nM of the various unlabeled
chemokines indicated at the bottom of the graph. Cells were washed
twice with PBS and remaining [125I]Met-Ckß7 bound was
counted. Results are the mean of three identical incubations
represented as a percentage of the binding seen in the absence of
competitor, and SE is included.
