(Received for publication, November 28, 1995)
From the
Antagonists of multiple chemokines could be more effective than
inhibitors of specific chemokines for controlling cell migration and
inflammation. To attempt to identify such antagonists we characterized
a number of truncated analogs of regulated on activation normal T cell
expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3,
and MCP-1. On the basis of their ability to compete for binding of
their parent chemokines, three analogs were selected for
cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and
MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with
dissociation constants that were within 4-6-fold of their native
counterparts, but they did not promote detectable chemotaxis of THP-1
cells or enzyme release from purified human monocytes. The RANTES
(9-68) analog competed for the binding and inhibited the
activities of all three chemokines. In contrast, native RANTES was
specific for RANTES binding sites. However, truncation of either MCP-1
or MCP-3 did not change their respective binding specificity. MCP-3 and
MCP-3 (10-76) competed for binding of all three labeled
chemokines. MCP-1 (9-76) competed strongly for binding of labeled
MCP-1, but only weakly for the other two labeled ligands and inhibited
the activities induced by MCP-1 and MCP-3 but not RANTES. Although
RANTES (9-68) and MCP-3 (10-76) inhibited all three
chemokines, the RANTES analog was significantly more potent for
RANTES-induced activity. The results indicate that
NH-terminal residues partly determine the receptor
specificity of RANTES, and deletions within this region permit binding
to multiple chemokine receptors. The findings suggest the feasibility
of design of high affinity multi-specific CC chemokine antagonists.
Inflammation pathology involves the migration of various types
of blood cells into affected tissue sites(1) . The chemokines
are a family of protein mediators with potent chemoattractant activity
for granulocytes, monocytes and
lymphocytes(2, 3, 4) . The human chemokines
number about 20 and fall into two classes according to the relative
positions of the first two cysteines. For the CXC chemokines,
the first two cysteines are separated by one residue; and for the CC
chemokines, the first two cysteines are adjacent. Chemokines share
significant sequence similarity (24-76% identity), including
conservation of the four cysteines that are involved in two disulfide
bridges (reviewed in (2) and (5) ). Furthermore, the
structural fold of the polypeptide of the CC chemokines, MIP-1 (6) and RANTES(7) , and the CXC chemokine,
interleukin-8(8) , are very similar, suggesting that chemokines
share similarity at the level of their three-dimensional
structures(5) . Functionally the CC class acts on several types
of leukocytes including monocytes, basophils, eosinophils, and
lymphocytes but do not stimulate neutrophils. In contrast, neutrophil
activity is a distinguishing feature of members of the CXC
class(2) .
Multiple receptors mediate the complex and
overlapping functional activities of CC chemokines. Three receptors
have been identified and their polypeptide sequences deduced from cDNA
clones. These are: chemokine receptor (CKR)-1, ()which binds
MIP-1
, RANTES, and
MCP-3(9, 10, 11, 12, 13) ;
CKR-2, which binds MCP-1 and MCP-3 (14, 15) ; and
CKR-3, which has been shown to bind RANTES, MIP-1
, and
MIP-1
(12) . In addition a fourth receptor, termed
K5-5, which binds MCP-1, MIP-1
, and RANTES, has been
identified(16) . For all these receptors, either mRNA or
receptor protein is expressed by peripheral blood monocytes. However
despite this sequence information, most of our understanding of the
function and ligand specificity of the receptors has come from
experiments with cells isolated from blood or cell lines that are
representative of a particular
lineage(17, 18, 19, 20, 21, 22, 23) .
These experiments suggest that additional functional receptors are also
involved and that cross-reactive binding of several CC chemokines is a
frequently observed feature of the receptors.
The aim of the present
study is to generate and characterize receptor antagonists for CC
chemokines. With defined antagonists we can test whether cellular
infiltration and inflammatory disease progression is retarded in
vivo and evaluate the possibility that antagonists could have
therapeutic value. Previously we characterized a series of analogs of
MCP-1 and found that some truncated forms were potent antagonists for
MCP-1 in vitro(23) . Other studies indicated that
MCP-1 forms with deletions in the NH-terminal region also
had some antagonistic properties (24) . Our results (23) also demonstrated that different regions within the
NH
-terminal domain are important for binding and activity.
Based on this study, we hypothesized that modification of MCP-1 or
other chemokines that bind to several different receptors may lead to
antagonists that block multiple CC chemokine receptors. In this study
we have compared truncated analogs of three potent CC chemokines,
namely RANTES, MCP-3, and MCP-1, which have differing cellular and
receptor specificity, but all stimulate monocytes and the THP-1
monocytic cell
line(17, 19, 23, 25, 26) .
The results indicate that MCP-3 and RANTES analogs could form the basis
for antagonists with broad anti-inflammatory activity.
Figure 1:
NH-terminal sequences of
the chemokines and truncated analogs described in this study. Complete
amino acid sequences of RANTES(31) , MCP-3(32) , and
MCP-1(33, 34) have been previously
described.
The yield of purified folded protein was in the 10-100 mg range, depending on the amount of peptide-resin synthesized (0.05-0.3 mmol corresponding to 0.5-2.0 g of unpurified peptide) and the yield obtained after reverse-phase HPLC purification. The measured molecular masses were consistent with the polypeptides having the expected composition. The average masses ± S.D., with the calculated mass minus the measured mass in parentheses, were: RANTES, 7847 ± 0.2 (-0.1); RANTES (10-68), 6909.6 ± 0.6 (-1.5); RANTES (9-68), 7006.3 ± 1.0 (-1.9); RANTES (8-68), 7108.7 ± 1.3 (-0.6); RANTES (7-68), 7210.3 ± 1.5 (-0.1); RANTES (6-68), 7324.3 ± 1.1 (-1.2); MCP-3, 8934.2 ± 1.5 (-1.3); MCP-3 (2-76), 8824.7 ± 0.9 (0.3); MCP-3 (8-76), 8242.8 ± 1.1 (0.1); MCP-3 (9-76), 8154.7 ± 0.8 (-0.9); MCP-3 (10-76), 8054.0 ± 1.2 (-0.5); MCP-1, 8662.6 ± 1.1 (-0.5); MCP-1 (9-76), 7971.2 ± 0.8 (-0.1).
Figure 2:
Chemoattractant activity of chemokine
analogs. Shown is the migration of THP-1 cells toward the indicated
concentrations of RANTES (); RANTES (9-68) (
);
MCP-3 (
), and MCP-3 (10-76) (
). Control migration is
that obtained toward medium alone. The values shown represent the mean
± S.D. of triplicate determinations from one of two separate
experiments.
Figure 3:
Inhibition of chemokine-induced migration
by truncated analogs. The indicated concentrations of RANTES
(9-68) (A), MCP-3 (10-76) (B), and MCP-1
(9-76) (C) were added to 10 nM of either MCP-1
([), MCP-3 (
), or RANTES (
) in the lower wells
of the chemotaxis chamber. To compare the inhibitor activities, the
data were normalized such that the chemotactic index of THP-1 cells in
the presence of agonist alone was 100%. The values shown are the mean
± S.D. of triplicate determinations from one of three separate
experiments.
Figure 4:
Inhibition of monocyte exocytosis by the
truncated analogs. Cells were stimulated with a constant concentration
of either MCP-1 (10 nM) (), MCP-3 (10 nM)
(
), or RANTES (30 nM) (
) in the absence or the
presence of increasing concentrations of RANTES (9-68) (A), MCP-3 (10-76) (B), and MCP-1 (9-76) (C). The release of N-acetyl-
-D-glucosaminidase into the medium was
then determined. The data are expressed as percentages of control
(agonist alone) and represent the mean ± S.E. of values from
three to five experiments performed with monocytes from different
donors.
Receptor sharing and
cross-binding is known to occur amongst of members of the CC family of
chemokines. Because the truncated antagonists have lost a part of their
structure that is important for receptor activation, it is reasonable
to suggest that their receptor selectivity may be altered. To test the
specificity of the three truncated antagonists, their ability to
inhibit the activities induced by RANTES, MCP-3, and MCP-1 was
examined. The RANTES (9-68) analog completely inhibited both
MCP-3- and MCP-1-induced chemotaxis (Fig. 3A) and N-acetyl--D-glucosaminidase release (Fig. 4A). The respective IC
values were
200 nM and 820 nM for chemotaxis and 170 nM and 220 nM for release activity (Table 1). Thus
RANTES (9-68) inhibited all the chemokines, but it had the
highest potency for RANTES.
MCP-3 (10-76) was found to inhibit
MCP-3-induced monocyte N-acetyl--D-glucosaminidase release with an
IC
of 37 nM and also chemoattractant activity
IC
470 nM (Fig. 3B). Thus it was
less potent than RANTES (9-68) for RANTES-stimulated release.
However, MCP-3 (10-76) also inhibited RANTES- and MCP-1-induced
activities with similar effectiveness to its inhibition of MCP-3
elicited function. Furthermore, MCP-3 (10-76) blocked enzyme
release from monocytes (Fig. 4B). The IC
values for both RANTES- or MCP-3-induced release were around
10-fold lower than for chemotaxis of either THP-1 cells (Table 1)
or monocytes (not shown). Another MCP-3 variant, MCP-3 (9-76),
was 2-3-fold more potent than MCP-3 (10-76) in all the
inhibition assays (not shown).
As shown in Fig. 3C and Table 1, MCP-1 (9-76) inhibited MCP-1-induced
chemoattractant activity (IC = 72 nM) and
was also a potent inhibitor of MCP-3 activity (IC
=
51 nM). MCP-1 (9-76) did not inhibit RANTES promoted
activity. MCP-1 (9-76) inhibited both MCP-1 and MCP-3 induced N-acetyl-
-D-glucosaminidase release, but it did
not block RANTES-elicited release, at least over the concentration
range tested (Fig. 4C). Thus, the ability of MCP-1
(9-76) to inhibit migration and release was similar.
The RANTES (9-68) analog competed for
binding of labeled RANTES as shown in Fig. 5A. From
this data the dissociation constant (K) was
calculated to be 19 nM. Furthermore, RANTES (9-68) also
displaced both labeled MCP-3 (K
= 57
nM) and MCP-1 (K
= 120
nM) with moderate efficiency (Fig. 5, A-C, and Table 1). Although, as expected, native
RANTES competed strongly for RANTES binding (Fig. 5A),
its competition for labeled MCP-3 (Fig. 5B) and MCP-1 (Fig. 5C) was very weak and insufficient to derive a K
value. Thus, truncation of RANTES resulted in a
markedly increased affinity for both MCP-1 or MCP-3 binding sites.
Figure 5:
Receptor binding of chemokines and their
analogs. The indicated concentrations of MCP-1 (), MCP-3 (
),
RANTES (
), MCP-1 (9-76) (
), MCP-3 (10-76)
(
), or RANTES (9-68) (
) were added to either 4
nM
I-RANTES (A, D, and G),
I-MCP-3 (B, E, and H), or
I-MCP-1 (C, F, and I). The results are expressed as percentages of the maximal
specific binding that was determined as described under
``Materials and Methods.'' The data shown are representative
of two or three experiments.
MCP-3 (10-76) competed for MCP-3 binding with a K of 57 nM (Fig. 5E and Table 1) and also competed for binding of both RANTES (K
= 50 nM) and MCP-1 (K
= 69 nM) (Fig. 5, D and F, and Table 1). The competition of the MCP-3
(10-76) analog for labeled MCP-3 was only 2-fold weaker than that
of full-length MCP-3. For RANTES receptors, MCP-3 and MCP-3
(10-76) had about the same affinity. On the other hand, for MCP-1
receptors, MCP-3 (K
= 13 nM) had
about a 5-fold higher affinity than MCP-3 (10-76) (K
= 69 nM). The results indicate
that MCP-3 (10-76) had similar affinity for the binding sites of
all three chemokines. Similar results were obtained with MCP-3
(9-76) (not shown).
Consistent with previous
observations(23) , MCP-1 (9-76) competed for binding of
labeled MCP-1 with a K of 9 nM (Fig. 5I and Table 1). It also competed for
binding of labeled MCP-3, although in this case the K
(128 nM) was considerably higher (Fig. 5H and Table 1). MCP-1 (9-76) exhibited only weak binding (K
= 340 nM) to RANTES receptors (Fig. 5G and Table 1). Thus, MCP-1 (9-76)
had high affinity for MCP-1 binding sites but only low affinity for
MCP-3 or RANTES binding sites. When MCP-1 and MCP-1 (9-76) were
compared, they were similar in affinity for both MCP-3 and RANTES
binding sites, but for MCP-1 binding sites, MCP-1 (9-76) was
around 4-fold lower (Fig. 5, G-I, and Table 1).
The cross-reactivity of RANTES (9-68)
antagonist was unexpected and prompted us to evaluate other truncated
RANTES analogs for their specificity. A series of five RANTES analogs
was tested for inhibition of binding of labeled RANTES and MCP-1 (Fig. 6). There was a general correlation in the ability of the
RANTES analogs to displace the two labeled chemokines, although for the
(7-68), (8-68), and (9-68) analogs the K was around 10-fold higher for MCP-1 than for
RANTES (Fig. 6). The order of the efficiency of displacement
(low to high K
) was: RANTES (9-68) >
(8-68) > (7-68) = (10-68) >
(6-68). Only RANTES (8-68) and (9-68) completely
inhibited the binding of labeled MCP-1, with the 9-68 analog
having a 5-fold higher binding affinity. Similar results were obtained
when competition with labeled MCP-3 was tested. Thus, the results
indicate that amongst the truncated RANTES analogs tested, RANTES
(9-68) had the highest affinity for MCP-1, MCP-3, and RANTES
binding sites.
Figure 6:
Binding specificity of truncated RANTES
analogs. The indicated concentrations of RANTES (), RANTES
(6-68) (
), (7-68) (
), (8-68) (
),
(9-68) (
), and (10-68) (
) were added to THP-1
cells in the presence of either 4 nM
I-RANTES (A) or
I-MCP-1 (B). The results are
representative of three independent
experiments.
Figure 7:
The truncated analogs inhibit the
chemokine-induced [Ca]
rise.
The concentrations (nM) of the analogs are indicated on the left. The agonists, RANTES, MCP-3, and MCP-1, were used at 10
nM. Additions were made at the times shown by arrowheads. The results are representative of three
independent experiments performed with monocytes prepared from
different donors.
Figure 8:
Cross-desensitization of the
chemokine-induced [Ca]
rise by the truncated analogs. The truncated analogs were
used at 1,000 nM, and the agonists were used at 10
nM. One of three similar experiments is
shown.
Antagonists for three CC chemokines, RANTES, MCP-3, and MCP-1, were identified and further characterized to determine their specificity as inhibitors of chemokine functions and correlate this with their selectivity for receptor binding sites. These antagonists, RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76) were selected because of their high affinity for binding sites on monocytic cells.
The affinity of full-length RANTES for MCP-3 and MCP-1 binding sites was marginal at the highest concentration tested. However, the binding affinity of RANTES (9-68) to sites for both was considerably higher. MCP-3 differed in that it bound to cellular sites for all three ligands with comparable affinity. Furthermore, the MCP-3 antagonist also bound to these sites, but it did not have high affinity for any of the chemokine binding sites, including MCP-3. MCP-1 had high affinity for MCP-1 sites but competed only very weakly for RANTES or MCP-3 binding sites. However, truncation of MCP-1, unlike RANTES, did not reveal a significant change in affinity for MCP-3 and RANTES sites.
The ability to inhibit function correlated with the binding data. The truncated analogs of RANTES or MCP-3 blocked the activities of all three chemokines. The inhibitory potency of RANTES (9-68) for MCP-3 and MCP-1 activity was lower than for native RANTES. The MCP-3 antagonist had the same broad specificity as the parent agonist, but overall it did not have high potency. Subsequent experiments indicated that MCP-3 (9-76) was 2-3-fold more potent than MCP-3 (10-76) in its inhibition of function. Thus, as was the case for MCP-1(23) , the MCP-3 (9-76) analog was the optimal truncated antagonist (not shown). When the inhibitory activities of RANTES (9-68) and MCP-3 (10-76) are compared, the RANTES analog was more potent for RANTES, the MCP-3 analog was more potent for MCP-1, and both inhibited MCP-3. On average, for all three chemokines, the RANTES (9-68) was the most effective antagonist.
The results for binding and inhibition of function were consistent with the data for desensitization of calcium induction. The truncated analogs attenuated or blocked the calcium response to all three ligands with the RANTES (9-68) being the most effective. This suggests a probable link between the equilibrium binding, the inhibition of intracellular signaling events, and the inhibition of the functional responses.
RANTES (9-68) was a particularly potent inhibitor of monocyte release activity with only 4 nM required to inhibit 30 nM RANTES by 50%. In contrast, 19 nM (9-68) was required to displace 5 nM of labeled RANTES from THP-1 cells in the receptor binding studies. The reasons for this difference are not known. One possible explanation is that the binding studies were done with THP-1 cells, whereas the release activity was measured on human peripheral blood monocytes, and therefore different receptors may be involved. However, against this is the observation that the inhibition of RANTES-induced migration of both THP-1 cells and monocytes was similar (not shown). THP-1 cells did not show detectable exocytosis in response to chemokines so the release activities could not be compared. In addition, functionally important but low abundance receptors may not be detected in the binding studies.
These findings clearly indicate that the RANTES analog is binding to
receptor(s) that are not accessible to native RANTES. Truncation of
RANTES has resulted in decreased binding selectivity, which could be
due to loss of residues that normally prevent it from binding to other
receptors, for example, by a steric hindrance mechanism. For the series
of RANTES analogs with between 5 and 9 residues deleted, the affinity
for RANTES, MCP-1, and MCP-3 binding sites correlated, suggesting that
the determinants of receptor specificity are located within residues
1-6. However, for MCP-1 there was no difference in the
selectivity of the full-length or the truncated analog, suggesting that
the determinants of its receptor specificity are located elsewhere in
the protein. The selectivity and affinity of the antagonists can
probably be modified by alterations to residues outside the
NH-terminal motif. Nevertheless the results clearly
demonstrate that the binding selectivity of RANTES can be modified to
generate multiple chemokine antagonists.
The observation that RANTES
(9-68) has 50-fold higher affinity than RANTES (10-68)
demonstrates that residue 9 contributes to the receptor binding of the
RANTES antagonist. However, residues within the (10-68) domain
also contribute to binding, because the (10-68) analog did bind,
but the RANTES (1-9) peptide did not. Analogs that had residues
6-8 of RANTES intact had decreased binding. One possibility is
that flexibility of residues 6-8 region results in loss of
binding. For all three chemokines 1 or 2 residues
NH-terminal to the first cysteine were required for optimal
binding and antagonist activity. In a previous investigation of MCP-1
analogs, we concluded that residues 1-6 were critical for
receptor activation and function and residues 7-10 were important
for receptor binding. The current findings for RANTES and MCP-3 are
consistent with the model for receptor interactions proposed for MCP-1 (23) .
Our findings can be interpreted in terms of interactions with known receptor proteins. The RANTES response could be due to interactions with three known RANTES receptors, CKR-1, CKR-3, or K5-5. The truncated RANTES analog is likely to be also interacting with the CKR-2 protein, which has been shown to bind MCP-1 (14) and MCP-3 (15) but not RANTES(14) . MCP-3 also binds to CKR-1(12, 13) , which is well known to be a receptor for RANTES (9-11). Thus the observations with both MCP-3 and the MCP-3 antagonist could be explained by their interaction with these two receptors. MCP-1 and MCP-1 (9-76) responses are likely to be due to interactions with the CKR-2 receptor. The weak binding to RANTES binding sites may be due to low level cross-reactivity with CKR-2 or binding to K5-5. It is possible that not all the receptors on monocytes are represented on THP-1 cells, and therefore some receptor interactions were not included in our binding studies. Conversely, these antagonists may also interact with receptors that are only found on other cell types.
An argument could be made that a general chemokine antagonist would be the most effective anti-inflammatory molecule. Inflammation involves the infiltration of multiple cell types that is likely to occur through the interaction of different chemokines with distinct functional receptors. Blocking the infiltration of multiple effector cells could be highly effective in breaking the inflammatory cycle. Further experiments are required to test this idea. It remains to be determined which other chemokines are inhibited and to which receptor proteins they bind. Our aim was to correlate binding with inhibition of chemokine-induced function, therefore we used chemokine-responsive cells for the binding studies rather than cell lines engineered to over-express single receptors. However, to determine which receptors the antagonists bind, experiments with individual receptors are required. It will be important to know the effects of the antagonists on other cells such as basophils, eosinophils, and T lymphocytes. The chemokine antagonists described here are potent enough to demonstrate their effects in disease models and thus establish a precedent for the usefulness of chemokine receptor antagonists as anti-inflammatory agents.