(Received for publication, September 7, 1994; and in revised form, October 31, 1994)
From the
Interleukin-8 (IL-8) is a member of the CXC branch of the
chemokine superfamily and activates neutrophils but not monocytes. The
related CC chemokine branch, which includes monocyte chemoattractant
protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes
but not neutrophils. Examination of the sequences of the CXC chemokines
reveals that the highly conserved leucine, corresponding to Leu in IL-8, is always replaced by tyrosine in CC chemokines. There
is also a high degree of conservation among the CXC chemokines of the
adjacent Val
residue, which points out from the same side
of the
-sheet as Leu
. In RANTES, Val
is
also replaced by a tyrosine. In order to investigate the role of these
residues in controlling cell specificity, we have made the single
mutants Leu
Tyr, Val
Tyr and
the double mutant Leu
Tyr,Val
Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All
three mutants have lower potency and efficacy in chemotaxis and calcium
mobilization assays using neutrophils. The mutants also show lowered
affinity to both IL-8 receptors A and B expressed recombinantly in
HL-60 cells and to neutrophils in [
I]IL-8
competition assays. Additionally, the Leu
Tyr
mutation introduces a novel monocyte chemoattractant activity into
IL-8. We therefore studied the displacement of
[
I]MIP-1
by IL-8 Leu
Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1
ligand
with an affinity only 12-fold less than MIP-1
itself. This
suggests that mutations in this region of IL-8 are involved in receptor
binding and activation and in the control of specificity between CC and
CXC chemokines.
Chemokines (or chemotactic cytokines) are small molecular weight
proteins of 8-10 kDa involved in cell recruitment and activation
during inflammation. Interleukin-8 (IL-8) ()is a CXC
chemokine that is involved in the recruitment of neutrophils but not
monocytes in acute inflammation(1, 2, 3) .
This contrasts with members of the CC chemokines, such as MCP-1 and
RANTES, which are chemoattractants for monocytes and are involved in
chronic inflammation (4, 5, 6) . The
receptors for these groups of chemokines are different. IL-8 binds to
two distinct seven-transmembrane-spanning receptors on neutrophils,
IL-8R-A (7) and IL-8R-B(8) . IL-8R-A is specific and
only binds IL-8 with nanomolar potency, whereas the IL-8R-B binds most
of the CXC chemokines. RANTES, MCP-1, and macrophage inflammatory
protein-1
(MIP-1
) bind to a CC chemokine
receptor(9, 10) . Apart from the spacing of the
cysteine residues at the amino terminus, the molecular basis of the
specificity of interaction between CXC and CC chemokines and their
receptors is not known. A region in the amino terminus of IL-8 (29, 30) consisting of the amino acid sequence
Glu-Leu-Arg has been shown to be important in binding and activation of
the IL-8 receptors. We are therefore searching for other determinants
of molecular specificity.
The three-dimensional structure of IL-8
has been solved by NMR and x-ray
crystallography(11, 12) . The quaternary structure is
a dimer made up of two antiparallel -helices lying on top of a
six-stranded
-sheet(13) . In the central
-sheet of
IL-8 2 residues, Leu
and Val
, point upward.
These residues are conserved as small hydrophobic amino acids in CXC
chemokines, and the Leu
equivalent is always a tyrosine in
CC chemokines (Fig. 1). The equivalent residues in RANTES are
both tyrosines, Tyr
and Tyr
, and these
residues have been suggested to be important in the selectivity of
MCP-1(14) .
Figure 1:
Multiple sequence alignment between
members of the human CXC and CC class of the chemokine superfamily. The
CXC group contains interleukin-8 (IL-8)(15, 33) ,
-interferon induced protein (
-IP-10)(34) , platelet
factor 4 (PF-4)(35) , melanoma growth stimulatory activity
(MGSA/GRO
)(36) , macrophage inflammatory protein-2
(MIP-2
and
)(37) , neutrophil-activating peptide 2
(NAP-2)(38) , and ENA-78(39) . The CC group contains
monocyte chemoattractant protein (MCP-1, 2, and
3)(40, 41) , macrophage inflammatory protein-1
(MIP-1
and
)(42) , RANTES(16) , and I-309 (18) .
We have mutated residues 25 and 27 of IL-8 to
tyrosines (Fig. 2), and we show that the mutant proteins are
less potent in neutrophil chemotaxis, calcium mobilization, and IL-8
receptor binding assays. The mutation Leu
Tyr in
IL-8 introduces a monocyte chemotaxis activity. In addition, the mutant
is able to displace radiolabeled MIP-1
from the shared
MIP-1
/RANTES receptor. These data confirm the importance of this
region of the protein in determining cell type selectivity.
Figure 2:
Sequence alignment of human IL-8 and
RANTES. Sequences are for the monocyte derived form (72-amino acid
form) of IL-8 (15) and the platelet derived form of
RANTES(16) . These show 21.5% identity and 41.5% sequence
similarity. The positions of Leu and Val
in
IL-8 and the corresponding tyrosines 28 and 30 in RANTES are shown in boldface type.
Examination of the final RANTES models showed that two
tyrosines, 28 and 30, point out from the -sheet (Fig. 3).
An alignment of CXC and CC chemokines showed that the equivalent
residues in IL-8 (Leu
and Val
), are always
small hydrophobic residues, whereas in CC chemokines they are changed
mainly to tyrosines or charged amino acids (Fig. 1). Residues 25
and 27 of IL-8 were changed to tyrosines in this study.
Figure 3:
Homology models of RANTES based on the NMR
data of IL-8 (C) and MIP-1 (D) showing the
positions of Tyr
and Tyr
. The monomers of
IL-8 (A) and MIP-1
(B) are also shown. All of
the structures are oriented in the same direction, showing their
similarity. The Leu
and Val
residues for IL-8
and the Tyr
and Glu
residues for MIP-1
along with the two tyrosines in RANTES point out from the
-sheet.
The oligonucleotides were kinased and allowed to self-anneal by slowly reducing the temperature from 95 °C to 40 °C. They were ligated to form the NdeI-SacI fragment, which was inserted into the trp-IL-8 plasmid, and the vector was introduced into E. coli B cells by transformation. The sequences of the mutant constructions were verified by the dideoxy chain termination method. Cells were grown overnight at 37 °C in Terrific Broth (Promega) to allow auto-induction of IL-8. The three IL-8 mutants were also highly expressed in this system.
Figure 4:
Purification of recombinant human IL-8 and
the three IL-8 mutants from E. coli lysate. All four proteins
have been purified to homogeneity as described under ``Materials
and Methods.'' A 4-20% acrylamide gel was run under reducing
conditions and stained with Coomassie Brilliant Blue R-250 after
electrophoresis. Samples are IL-8 (lane 1), Leu
Tyr (lane 2), Val
Tyr (lane 3), Leu
Tyr,Leu
Tyr (lane 4), and molecular weight standards (lane
5).
For the chemotaxis assay, a
48-well micro-Boyden chamber (NeuroProbe, Cabin John, MD) was used.
Serial dilutions of the chemoattractants (RANTES, IL-8, and mutant IL-8
proteins) were made in medium (RPMI 1640 with 2 mML-glutamine, 25 mM Hepes, and 10% heat
inactivated fetal calf serum). 25 µl of chemoattractant was added
to the lower chamber of the assay wells and covered with a
polyvinylpyrrolidone-free polycarbonate membrane with pore size of 3
µm for neutrophils and 5 µm for monocytes(20) . A
50-µl solution containing 10 cells/ml was then added to
the top wells. The assay plates were incubated at 37 °C for 20 min
for neutrophils and 30 min for monocytes. The upper surface of the
membranes was then washed with PBS buffer, and the cells on the
underside of the membrane were fixed in methanol. The membranes were
stained with a mixture of Field's A and B stains (Bender and
Hobein) and air-dried. The cells on the under surface of the membranes
were then counted using a Zeiss Axiophot microscope and the VIDAS image
analyzer software (KONTRON Electronics, Zurich, Switzerland).
The CC chemokine receptor-1 (CC-CKR-1) was isolated by PCR using
primers based on the published sequence (9) and subcloned into
pcDNAI vector (Invitrogen). CsCl gradient-purified plasmid
DNA (30 µg) of CC-CKR-1-pcDNAI constructs was transfected into Cos7
cells using the same method as for HL-60 cells. Cells were selected in
Dulbecco's medium containing 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin,
and resistant cells were analyzed by measuring the binding of
[
I]MIP-1
.
Figure 5:
Chemotactic activity of IL-8 and mutants
on human neutrophils. The chemotaxis index (stimulated
migration/control random migration) was determined at varying
concentrations of chemoattractants. The data are shown for IL-8
(), Val
Tyr (
), Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). Each point represents three measurements.
Similar results were obtained with three different donors. RANTES was
inactive in this assay.
We then studied the effect of these mutants
on the mobilization of intracellular Ca in
neutrophils. IL-8 shows a dose-response curve with a midpoint at 10
nM and a maximal calcium mobilization of 250 nM (Fig. 6). The Val
Tyr mutant shows
similar behavior to IL-8. The Leu
Tyr and the
double mutants, however, were less efficacious and show lowered
potency, with a midpoint at around 300 nM.
Figure 6:
Mobilization of intracellular calcium by
IL-8 and the mutant proteins using human neutrophils. The assay was
carried out for IL-8 (), Val
Tyr (
),
Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). Similar results were obtained
in two separate experiments with different
donors.
Receptor binding
was assayed by displacement of [I]IL-8 from its
receptors on neutrophils. Complete displacement of the radioligand by
cold IL-8 is observed, and the protein has a dissociation constant of
1.1 ± 0.2 nM. (Fig. 7). In the Val
Tyr mutant, the binding is lowered to 3.2 ± 0.3
nM, and the Leu
Tyr mutant is 100-fold
less potent, with a dissociation constant of 104 ± 25
nM. The ability of the double mutant Leu
Tyr,Val
Tyr to displace IL-8 from its receptor was
lowered still further, showing a dissociation constant of 225 ±
60 nM. These values can be converted into free energy values
for the perturbation of the receptor ligand complex, using the
equation,
G =
-RTln(K
(mut)/K
(wt)) (24) , where mut and wt correspond to the mutant and wild type
protein respectively. The ligand binding is lowered by 0.6
kcal
mol
for the Val
Tyr
mutation, 2.5 kcal
mol
for the Leu
Tyr mutation, and 2.9 kcal
mol
for
the double mutation. Neutrophils contain both IL-8 receptors A and
B(7, 8) . We have therefore measured the displacement
of [
I]IL-8 from HL-60 cells transfected with
either the A or the B receptor. IL-8 shows equal affinity for both
receptors, 1.4 ± 0.1 nM for the IL-8R-A and 1.9
± 0.3 nM for the IL-8R-B ( Fig. 8and Fig. 9). The Leu
Tyr mutant shows a decrease
in affinity for both receptors, giving values of 170 ± 10 nM for the IL-8R-A and 41 ± 2 nM for the IL-8R-B.
When the second tyrosine is introduced, there is a further decrease of
affinity for the IL-8R-B, with a binding constant of 100 ± 10
nM. On the A receptor, there is a small improvement in
affinity, giving an IC
of 130 ± 12 nM.
Figure 7:
Equilibrium binding cold displacement of
[I]IL-8 by IL-8 and mutant proteins binding to
IL-8 receptors on human neutrophils. Binding was performed at 4 °C
using varying concentrations of chemokine. The data are shown for IL-8
(
), Val
Tyr (
), Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). The points represent the means of triplicate
measurements. The maximal response represents 6000 cpm. Similar data
were obtained in three different experiments with three different
donors.
Figure 8:
Equilibrium binding cold displacement of
[I]IL-8 by IL-8 and mutant proteins binding to
recombinant HL-60-IL-8RA cells. Binding was performed at 4 °C using
varying concentrations of chemokine. The data are shown for IL-8
(
), Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). The points
represent the means of triplicate measurements. The maximal response
represents 15,000 cpm. Similar data were obtained in three different
experiments.
Figure 9:
Equilibrium binding cold displacement of
[I]IL-8 by IL-8 and mutant proteins binding to
recombinant HL-60-IL-8RB cells. Binding was performed at 4 °C using
varying concentrations of chemokine. The data are shown for IL-8
(
), Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). The points
represent the means of triplicate measurements. The maximal response
represents 4000 cpm. Similar data was obtained in three different
experiments.
In a chemotaxis assay using human monocytes, RANTES gives a
bell-shaped curve with a maximum at 1 nM and a maximal
efficacy of 2.5 (Fig. 10), whereas IL-8 is inactive. The
Leu
Tyr mutant shows chemotactic activity, which is
similar to RANTES both in potency and efficacy. The Leu
Tyr,Val
Tyr mutant shows 10-fold lower
potency than RANTES (maxima at 10 nM). The Val
Tyr mutant was completely inactive in the monocyte
chemotaxis assay, as was IL-8. We next studied the displacement of
MIP-1
from the MIP-1
/RANTES receptor or CC-CKR-1 by the
mutant chemokines (Fig. 11). MIP-1
was chosen as the ligand
because of the reported difficulties in getting reproducible
displacement of RANTES from the shared receptor(9) . MIP-1
can displace the radioactive ligand from the receptor, showing an
IC
of 0.97 ± 0.03 nM. IL-8 is incapable of
competing the ligand, up to concentrations of 1000 nM. The
mutant Leu
Tyr is able to displace
[
I]MIP-1
with an IC
of 12
± 0.5 nM, which is only a factor of 12 less than
MIP-1
itself.
Figure 10:
Chemotactic activity of RANTES and mutant
IL-8 proteins on freshly isolated human monocytes. The data are shown
for RANTES (), Val
Tyr (
), Leu
Tyr (
), and Leu
Tyr,Val
Tyr (
). Each point represents three measurements,
and this experiment is representative of two others. Wild type IL-8 was
inactive in this assay.
Figure 11:
Equilibrium binding cold displacement by
MIP-1 and IL-8 L25Y of [
I]MIP-1
to
Cos7 cells transfected with the CC-CKR-1 receptor. Data is shown for
IL-8 (
), Leu
Tyr (
), and MIP-1
(
). The points represent the means of duplicate measurements
with a superimposed three-parameter fit. The maximal response
corresponds to 15,000 cpm. Similar data were obtained in each of three
separate experiments.
The chemokine family of proteins was originally identified based on their abilities to attract a variety of lymphocytes and on a conserved spacing of cysteines throughout the protein. The family can also be subdivided on the basis of function, the CXC chemokines being primarily neutrophil attractants and the CC chemokines being monocyte and macrophage attractants. One of the central questions to be addressed is whether there are any particular amino acid residues, apart from the cysteines themselves, that control the molecular specificity of CXC chemokines relative to CC chemokines.
The
three-dimensional structure of IL-8 was originally solved by NMR (11) . Model-building studies showed that the sequence of the
CC chemokine, MCP-1 could be fitted to this model without any gross
distortions of structure(13) . Recently, the three-dimensional
structure of another CC-chemokine, MIP-1 has been
solved(17) , and this confirms that the fold of the monomer
unit is very similar for all chemokines. However, at high
concentrations, there are significant differences in the way the
monomers associate for CC compared with CXC chemokines.
We therefore
constructed a three-dimensional model of RANTES based on the structure
of IL-8(11) . We focused our attention on the inner
-sheet, which in the dimeric IL-8 structure is at the subunit
interface. Alignments of the CC and CXC chemokines were carried out,
which showed that Leu
in IL-8 is always a small
hydrophobic amino acid in the CXC chemokines but is replaced by
tyrosine in CC chemokines. This suggested a role in the control of
receptor-ligand specificity between the two chemokine subfamilies.
Mutation of either this residue or Val
, which also points
in a similar direction in the models, to tyrosine residues (as found in
RANTES) results in a decrease in affinity for the IL-8 receptors on
neutrophils and a concomitant decrease in the physiological response of
neutrophils. The mutation Leu
Tyr has the more
dramatic effect, showing a 100-fold drop in receptor binding,
equivalent to 2.5 kcal
mol
and a large decrease
in potency in both the neutrophil chemotaxis and calcium mobilization
assays. The introduction of a tyrosine at position 27 perturbs the
binding of ligand by 0.6 kcal
mol
and has a
2-fold effect on chemotaxis with little effect on calcium mobilization.
The energetic effects of the two mutations in the receptor binding
assays on neutrophils are almost
additive(26, 28, 29) . When the individual
receptors are studied, it can be seen that the effect of the mutation
at residues 25 and 27 is to lower the affinity of IL-8 for both
receptors to a similar degree. There are, however, subtle differences
in the additivity of the two mutations, which presumably reflect
differences in the conformation of the receptor ligand complex at this
point. Most importantly, mutating Leu
to the completely
conserved tyrosine in CC chemokines introduces a novel monocyte
chemoattractant property into the protein, which is not seen in IL-8.
This result is confirmed for the double mutation but not for the
Val
Tyr mutant. This confirms that the effect we
see here is not simply caused by misfolding of the protein, which would
simply inactivate the protein in all assays. Furthermore, we have
confirmed that the mutant Leu
Tyr can displace
[
I]IL-8 from the MIP-1
/RANTES receptor
recombinantly expressed in Cos7 cells, confirming this as a possible
receptor involved in the chemotactic response.
Earlier studies (14) investigated the effects of mutating the equivalent
residues, Tyr and Arg
in MCP-1. Only
Arg
Val and the double mutant were characterized,
and although both of these proteins show reduced activity against
monocytes, only the double mutant was able to attract neutrophils. This
suggests that Tyr
was the important residue in specificity
control, the equivalent of Leu
in IL-8. A recent study of
MCP-1(30) , shows the mutants Tyr
Asp, and
Arg
Leu are less active than wild type, and the
effect of mutating Arg
was much larger(14) .
The region of IL-8 important in binding to the receptor and
subsequent signaling has been extensively analyzed by mutagenesis and
peptide synthesis(31, 32) . These studies showed that
out of all of the charged residues, in IL-8 only the amino-terminal
Glu-Leu
-Arg
sequence was absolutely
required. Although they confirmed the nonimportance of Glu
and Arg
in the action of IL-8, they did not
investigate the role of hydrophobic side chains in the interaction with
the receptor. Our data show that the side chains on the opposite face
of the
-sheet, namely Leu
and Val
, are
important in the interaction not only with the neutrophil IL-8
receptors but also with the monocyte CC chemokine receptors. The
identification of the receptor used by monocytes will require the use
of recombinant receptor clones, and such studies are under way.
Our
initial interpretation of this data was to suggest that the receptor
bound into the groove formed by the IL-8 dimer and that these side
chains interfered with such binding. However, three recent pieces of
evidence argue against this. First, analytical ultracentrifugation
studies and cross-linking studies show that IL-8 and MCP-1 are monomers
at physiologically relevant concentrations(33) . Second, IL-8,
which contains N-methyl-leucine 25, is always monomeric and
yet remains active. Third, the recently published structure of the CC
chemokine, MIP-1 has a very similar monomer structure to IL-8 but
has completely different dimer packing, which would place the
equivalent amino acids to Leu
and Val
away
from the dimer interface(17) . The subunit packing of RANTES
has now been shown to be similar to that of MIP-1
(34) . (
)
We have identified two positions, Leu and
Val
, in IL-8 that are involved in the interaction of the
molecule with its receptors on neutrophils. In addition, mutation at
one of these positions, Leu
introduces a novel CC-CKR-1
binding and monocyte chemotactic activity into IL-8. The molecular
details of the interaction between the IL-8 mutants with the two IL-8
receptors and the CC chemokine receptor that are responsible for this
interesting change in bioactivity are currently being studied in our
laboratory.