(Received for publication, July 24, 1995; and in revised form, August 24, 1995)
From the
The Duffy blood group antigen-erythrocyte chemokine receptor has been shown to bind to chemokines of both the C-X-C and C-C classes and to the malarial parasites Plasmodium vivax and Plasmodium knowlesi. We performed experiments to evaluate the binding properties of this receptor for the newly appreciated ``C'' and ``non-ELR C-X-C'' classes of chemokines. Binding to mouse erythrocytes was also evaluated for the first time. Whereas ELR C-X-C and C-C chemokines bound to human erythrocytes with high affinity, differences in the ability of non-ELR chemokines to act as competitive inhibitors were noted. While non-ELR chemokines were unable to displace C-X-C chemokines on human cells, they exhibited a low affinity interaction with the C-C chemokine binding site. The newly discovered C chemokine, lymphotactin, was unable to displace either C-X-C or C-C chemokines. On mouse erythrocytes, non-ELR chemokines exhibited a low affinity for both the C-X-C and C-C chemokines binding sites; again lymphotactin failed to bind. Binding competition studies using an anti-Duffy monoclonal antibody and chemokines suggested a common binding domain. These data show that the chemokine superfamily has at least four functional subdivisions, each interacting differently with the Duffy antigen-erythrocyte chemokine receptor. In addition the chemokine binding function is conserved between mouse and man. Unlike other proteins in the superfamily C and non-ELR C-X-C chemokines do not efficiently bind red blood cells, thus their role may not require clearance from circulation.
The chemokine superfamily of pro-inflammatory cytokines has
traditionally been divided into the two structural branches: C-C and
C-X-C(1, 2, 3) . This classification
has recently been expanded to include the C branch represented by the T
cell chemoattractant lymphotactin
(Ltn)()(4, 5) . Structurally, these three
classes are distinguished based on whether the first two cysteines in
the motif are adjacent (C-C), separated by an intervening residue
(C-X-C), or whether the first of the two cysteines is missing
(C). The C-X-C branch may be further subdivided into those
that contain the amino acid motif ``ELR'' preceding the
initial cysteine residue and those that do not (``non-ELR''
C-X-C). Biologically, the C-C and C-X-C chemokines tend to act
primarily, but not exclusively, on monocytes and neutrophils,
respectively(2) , whereas the C chemokine Ltn currently appears
to be a lymphocyte-specific chemoattractant(4, 5) .
It has been reported previously that erythrocytes possess a
``promiscuous'' receptor, which, unlike other known chemokine
receptors, binds chemokines of both the C-C and C-X-C
classes(6, 7) . C-C and C-X-C chemokines bind
this receptor, present at about 5000-15,000 sites per cell, with
an affinity of K
5
nM(6, 7) . While the function of this
erythrocyte receptor has not been directly demonstrated, chemokine
clearance from the circulation has been postulated. It was later shown
that this erythrocyte chemokine receptor is serologically indistinct
from the Duffy antigen which is used by Plasmodium vivax and knowlesi to bind to and invade
erythrocytes(8, 9, 10) . The recent finding
that the Duffy antigen-erythrocyte chemokine receptor (DFA-ECKR) is
also expressed by endothelial cells lining postcapillary venules and
splenic sinusoids (11) suggests additional unelucidated roles
for this protein.
Here we dissect the binding functions of the human DFA-ECKR by testing its ability to bind to the newly discovered C chemokine, lymphotactin; by assessing the ability of non-ELR C-X-C chemokines to act as competitive inhibitors of binding, and by examining receptor-ligand interactions using an anti-Duffy mAb. In addition we ascertain whether the binding functionality is conserved between mouse and men. We provide evidence for a functional division of the chemokine superfamily into at least four classes based on the differential binding interactions of the C-C, ELR C-X-C, non-ELR C-X-C, and C chemokines tested.
The results from representative
experiments using human erythrocytes showed the displacement of I-RANTES (Fig. 1A) or
I-MGSA/gro (Fig. 1B) by an
excess of unlabeled chemokines. The displacement curves for RANTES,
MGSA/gro, and MCP-1 on human erythrocytes were very similar to
those reported previously(7) , with RANTES showing a slightly
weaker binding affinity on the human red blood cells than MGSA/gro or MCP-1. Labeled RANTES could be removed from its human
erythrocyte binding sites by unlabeled I-309 and the non-ELR
C-X-C chemokines PF4 and IP-10, which showed inefficient but
consistent displacement of RANTES binding at high concentrations (Fig. 1A). By contrast, unlabeled PF4, IP-10, or I-309
did not compete for binding with
I-MGSA/gro on
human erythrocytes, even though efficient displacement was observed
with MCP-1 and RANTES (Fig. 1B). The C chemokine
lymphotactin was unable to displace either radiolabeled RANTES or
MGSA/gro from these cells (Fig. 1, A and B). Similar results were obtained in multiple experiments
using the red blood cells of different normal donors. Thus, the binding
displacement data do not seem to be a function of DFA-ECKR
heterogeneity
Figure 1:
Cross-competition of multiple
chemokines for the same binding site on human red blood cells.
Competition binding curve showing a representative displacement of I-RANTES (A) or
I-MGSA/gro (B) by the unlabeled chemokines RANTES,
MGSA/gro, MCP-1, IP-10, PF4, I-309, human Ltn (HuLtn), and mouse Ltn (MoLtn). (The experiment is
representative of n = 5).
Direct assessment of the in vivo function of DFA-ECKR awaits the characterization of this marker in non-human species. We therefore tested directly whether mouse red blood cells could bind chemokines and how this binding compared with the human system. Data from a series of homologous and heterologous binding displacement on mouse erythrocytes are presented in Fig. 2. As with human erythrocytes (Fig. 1), both C-C and C-X-C chemokines seemed to compete for binding to a single site on the surface of mouse red blood cells (Fig. 2). However, the principle difference from the human system is that the non-ELR C-X-C chemokines, as well as I-309, displace both radiolabeled RANTES (Fig. 2A) and MGSA (Fig. 2B) from the surface of the mouse erythrocytes. Although it is clear that this competition is still relatively inefficient, there is a distinct difference from the binding pattern on the human cells. We do not yet know if these binding differences are due to use of human chemokines on murine cells, as murine chemokines are not yet widely available. As with the human erythrocytes, both human and mouse Ltn failed to compete for binding (Fig. 2).
Figure 2:
Cross-competition of multiple chemokines
for binding to mouse red blood cells. Competition binding curve showing
a representative displacement of I-RANTES (A) or
I-MGSA/gro (B) by the unlabeled
chemokines RANTES, MGSA/gro, MCP-1, IP-10, PF4, I-309, human
Ltn (HuLtn), and mouse Ltn (MoLtn). (n = 3).
Scatchard analysis was performed for
the ligand displacement data using the LIGAND program(13) , and
where possible the K for each of the represented
curves in Fig. 1and Fig. 2were generated and summarized
in Table 1. The table reveals several displacement patterns.
First, the C chemokine was unable to displace
I-RANTES or
I-MGSA/gro on both human and mouse. Second, The K
and number of binding sites per cell (data not
shown) derived from any combination of labeled RANTES and MGSA/gro displaced by unlabeled RANTES, MGSA/gro or MCP-1 were
similar to each other (2-10 nM) (7) . Third,
I-RANTES displaced by unlabeled I-309 (K
= 104 nM) and PF4 (K
= 200 nM) revealed low binding affinity with
those two chemokines for the C-C binding site on human erythrocytes,
but strikingly no displacement of the C-X-C chemokine
occurred. Finally, in the mouse system, the binding affinities of I-309
and non-ELR C-X-C chemokines were slightly higher for
I-RANTES as represented by their K
values, and in contrast to human, mouse
I-MGSA/gro was displaced by I-309 (K
= 22 nM) and IP-10 (K
= 50 nM). Thus, the
heterologous displacement studies indicated that non-ELR C-X-C
chemokines did not bind the red blood cell as efficiently as the C-C
and ELR C-X-C chemokines, and the C chemokine did not bind at
all.
Figure 3:
Flow cytometric analysis of human
erythrocytes for levels of DFA-ECKR expression. Purified human
erythrocytes were preincubated with or without various concentrations
of MCP-1 and MIP-1 prior to staining with control antibodies or
the anti-Fy3 mAb followed by FITC-conjugated goat-anti-mouse IgG
F(ab)
fragment. (n =
2).
In a converse experiment, human erythrocytes
were incubated with the indicated concentrations of the anti-Fy3 or
control mAbs and then 0.5 nM radiolabeled RANTES or
MGSA/gro were added to determine their binding potential. Fig. 4shows that the preincubation of erythrocytes with
increasing concentrations of mAb Fy3 dramatically inhibited the binding
of I-RANTES and
I-MGSA/gro,
whereas a control mAb specific for CD44 did not. These data together
indicate that the anti-Fy3 mAb was a competitive antagonist for
chemokine binding.
Figure 4:
Percent
binding of radiolabeled chemokines to human erythrocytes after
incubation with the anti-Fy3 mAb. Purified human erythrocytes were
pre-incubated with various concentrations of the anti-Fy3 or an
anti-CD44 mAb prior to the addition of 0.5 nMI-RANTES or
I-MGSA/gro. The
percent inhibition of radiolabeled chemokine binding versus mAb concentration is shown. (n =
3).
In summary, we have characterized the DFA-ECKR on red blood cells, revealing new distinctions in its binding properties. The data show for the first time that while C-C and C-X-C proteins do indeed compete for binding to a shared site with equal affinities, the precise nature of the binding interaction is subtly different in that the C-C proteins are displaced by non-ELR C-X-C chemokines, whereas the C-X-C are not. We also show for the first time that the mAb anti-Fy3 is a competitive inhibitor of chemokine binding and that the newly discovered C chemokine does not bind to the DFA-ECKR. Last, we show that mouse erythrocytes also possess a promiscuous chemokine receptor, allowing for the initiation of further studies of erythrocyte chemokine binding in vivo. The data thus provide the basis for a functional division of the chemokine superfamily into four distinct classes, where binding interactions with the DFA-ECKR are ELR C-X-C > C-C > non-ELR C-X-C; no binding with the C class. This understanding should assist in the refinement of experimental models to more accurately test the physiological roles of both the chemokine ligands and the promiscuous receptor.