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Sir William Dunn School of Pathology, University of Oxford, Oxford; and
Imperial College School of Medicine at the National Heart and Lung Institute, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1),3 and
eotaxin; CXC () chemokines such as IL-8 and growth-related oncogene
(GRO)- (alternatively known as melanoma growth stimulating
activity); and the C chemokine lymphotactin. Chemokines exert their activity by interacting with seven-transmembrane domain receptors expressed in different cell subsets, thus determining the leukocyte subtype that predominates in different types of inflammation. Chemokines are thought to form a chemical gradient in an immobilized phase via electrostatic interactions with negatively charged proteoglycans, which may increase the specificity of chemokine action (6, 7). There is evidence that the binding site of chemokines for proteoglycans and specific chemokine receptors (CKRs) are distinct (8, 9).
The activity of chemokines is tightly regulated to prevent excessive inflammation that can cause disease, and these molecules represent potential targets for therapeutic intervention in a wide range of diseases. The production of soluble versions of cytokine receptors containing only the extracellular binding domain represents a physiologic and therapeutic strategy to block the activity of some cytokines (10, 11). However, the seven-transmembrane domain structure of CKRs makes the construction of soluble, inhibitory CKRs difficult, and thus antagonists based on mutated chemokines, blocking peptides, or Abs are alternative inhibitors of the chemokines under evaluation (1, 3).
Poxviruses, a family of complex DNA viruses (12), have evolved
unique strategies for evasion of the host immune response (13, 14, 15, 16) and
are the only virus family known to produce secreted versions of
receptors for cytokines such as TNF, IL-1ß, IFN-/ß, and IFN-
.
These poxvirus proteins bind cytokines with high affinity and block
their activity by preventing interaction with receptors on the target
cell. Inactivation of virus genes encoding these cytokine inhibitors
has profound effects on viral pathogenesis. Poxvirus cytokine receptors
were initially identified from sequence similarity to the extracellular
binding domain of cellular cytokine receptors, but binding and
functional analysis led to the identification of the IFN-/ßR
encoded by vaccinia virus (VV), which has very limited similarity to
known cellular counterparts (17, 18).
Here we report that a soluble 35-kDa protein encoded by VV strain Lister has chemokine binding activity, and similar proteins are produced by 11 orthopoxviruses representing three species: VV, cowpox, and camelpox viruses. During the preparation of this manuscript, Graham et al. (19) reported that the T1/35-kDa family of poxvirus-secreted proteins binds CC and CXC chemokines and modulates the influx of inflammatory cells into virus-infected tissues. However, the mechanism by which the T1/35-kDa proteins modulate chemokine activity was not demonstrated. Here we show that the virus chemokine binding protein (vCKBP) binds CC chemokines with high affinity and inhibits their ability to induce signal transduction and cell migration in vitro. However, vCKBP does not bind or inhibit CXC or C chemokines. We demonstrate that the vCKBP inhibitory mechanism is the blockade of the interaction of chemokines with cellular receptors, thus representing the first soluble protein identified that sequesters chemokines and blocks their activity. Furthermore, the therapeutic application of this novel soluble chemokine inhibitor is illustrated in a guinea pig skin model by the ability of vCKBP to block eotaxin-induced eosinophil infiltration in vivo, a feature of allergic inflammatory reactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The growth conditions of human U937 and
TK-143B cells, and the sources of VV strains have
been described previously (20). The Evans and Lister strains used in
this study and a Lister virus lacking the gene encoding the 35-kDa
protein (Lister
35K or V357) (21) were provided by A. H. Patel
(Institute of Virology, Glasgow, U.K.).
Reagents
Radioiodinated recombinant human IFN- (90 µCi/µg) and
RANTES, MIP-1, IL-8, and GRO- (2200 Ci/mmol) were obtained from
DuPont-New England Nuclear (Boston, MA). Recombinant human
[125I]MIP-1 (2000 Ci/mmol) used for determination
of affinity constants was obtained from Amersham (Little Chalfont,
U.K.). Recombinant human RANTES, MIP-1, monocyte chemoattractant
protein-1 (MCP-1), MCP-2, MCP-4, eotaxin, IL-8, GRO-, 78-amino acid
epithelial cell-derived neutrophil activator, IFN--inducible
protein-10, platelet factor 4, neutrophil-activating peptide-2 (NAP-2),
stromal cell-derived factor-1, and lymphotactin, mouse eotaxin, and
rat RANTES and MIP-1 were purchased from PeproTech (Rocky Hill, NJ).
Recombinant mouse MIP-1 and human eotaxin, GRO-, and I309 were
obtained from R&D Systems (Minneapolis, MN). Recombinant human IFN-
was purchased from Genzyme (Cambridge, MA). Leukotriene B4
(LTB4) was purchased from Cascade Biochem Ltd (Reading,
U.K.). The following were gifts: guinea pig eotaxin from Drs. G.
Andrews and H. J. Showell (Pfizer Central Research, Groton, CT),
human IL-5 from Dr. T. N. C. Wells (Glaxo-Wellcome Molecular,
Geneva, Switzerland), and human C5a from Dr. J. Van Oostrum (Ciba
Geigy, Summit, NJ). Heparin (6 kDa) and heparan sulfate from porcine or
bovine intestinal mucosa, respectively, were purchased from Sigma
Chemical Co. (St. Louis, MO). Rabbit antiserum to purified VV Lister
35-kDa protein, provided by A. H. Patel (Institute of Virology),
or VV Western Reserve (WR) B15R protein expressed in baculovirus have
been described previously (21, 22).
Preparation of medium for binding and biologic assays
Supernatants from orthopoxvirus-infected TK-143B cells or baculovirus-infected Sf cells were harvested 1 or 3 days postinfection, respectively, and prepared as previously described (22). Baculovirus supernatants were concentrated and dialyzed against PBS as described previously (18). VV supernatants were inactivated with 4,5',8-trimethylpsoralen and UV light for cell migration assays (23).
Binding assays
The binding medium was RPMI 1640 containing 20 mM HEPES (pH 7.4)
and 0.1% BSA. Cross-linking experiments with
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or ethylene
glycol-bis-succinamidyl succinate (EGS; 1 mg/ml) to
125I-labeled chemokines (0.4 nM) or
[125I]IFN- (2 nM) were performed in a volume of 25
µl as previously described (20, 24). Samples were analyzed by
SDS-PAGE in 16- or 20-cm-long gels with 12 or 14% acrylamide. In the
competition assays with U937 cells, supernatants were preincubated with
100 pM [125I]chemokine in 100 µl for 1 h at 4°C.
Subsequently, 2.5 x 106 U937 cells were added in 50
µl and incubated for 2 h at 4°C. Bound
[125I]chemokine was determined by phthalate oil
centrifugation as previously described (22). A scintillation proximity
assay (Amersham) (25) was used for determination of the affinity
constant. Purified 35K-Fc (200 pg) was incubated with
[125I]MIP-1, with or without unlabeled chemokines, in
100 µl for 2 h at room temperature. Protein A-fluoromicrospheres
containing scintillant were added in 50 µl and incubated for 2
h, and bound [125I]MIP-1 was determined by
scintillation proximity assay in a beta counter. Nonspecific binding,
determined in the presence of a 1000-fold excess of unlabeled MIP-1
or as binding to 200 pg of purified B8R-Fc, was subtracted and
represented 3.8 to 7.5% of the total counts per minute bound. Binding
data were analyzed by the LIGAND program (26). The number of binding
sites for MIP-1 per molecule of vCKBP was calculated from the number
of binding sites deduced from the Scatchard analysis and from the
concentration and m.w. of recombinant 35K-Fc.
Construction of recombinant baculovirus
The 35-kDa gene was obtained by PCR using VV Lister DNA and oligonucleotides 5'-ATCGGTACCAATTATGAAACAATATATCGTCC-3', which inserts a KpnI site, and 5'-GTTGGATCCTCAGACACACGCTTTGAGTTTTG-3', containing a BamHI site. The 782-bp fragment was sequenced and cloned into pAcCL29-1 (27), generating pAc35K. The sequence contained His148 instead of Arg148 (21), but both sequences are natural variants of the VV Lister 35-kDa protein and bind chemokines with the same specificity. Recombinant 35-kDa protein containing a C-terminal six-histidine tag (35K.His) was produced in the baculovirus system. The 35-kDa gene was obtained by PCR using pCOS35K-Fc DNA (see Construction of 35K-Fc fusion and expression below) and oligonucleotides 5'-TCAGAATTCATTATGAAACAATATATCGTCC-3', containing an EcoRI site, and 5'-ATCCTCGAGGACACACGCTTTGAGTTTTG-3', containing an XhoI site. The 778-bp fragment was cloned into pBAC-1 (Novagen, Madison, WI), generating pAc35K.His. The sequence was confirmed and contained the Arg148 variant of the Lister 35-kDa protein. The recombinant baculoviruses Ac35K and Ac35K.His were produced from pAc35K and pAc35K.His, respectively, as described previously (20). The 35K.His protein was purified by metal chelate affinity chromatography (Ni-NTA resin; Qiagen, Inc., Chatsworth, CA). The recombinant baculoviruses AcB15R and AcB8R have been described (20, 22).
Construction of 35K-Fc fusion and expression
The VV Lister 35-kDa gene fused to the Fc region of human IgG1 was constructed in pCOSFCLINK (a gift from Dr. P. R. Young, SmithKline Beecham Pharmaceuticals, King of Prussia, PA). The insert was obtained by PCR using VV Lister DNA and the oligonucleotides 5'-TCAGAATTCATTATGAAACAATATATCGTCC-3', which introduces an EcoRI site, and 5'-ATCGGTACCGACACACGCTTTGAGTTTTG-3', which inserts Gly and Thr via a KpnI site after amino acid 258 of the 35-kDa protein before the hinge region of human IgG1, which was provided by pCOSFCLINK, generating pCos35K-Fc. The sequence was confirmed and contained the Arg148 variant of the Lister 35-kDa protein. The 35K-Fc fusion protein was purified from supernatants of CHO cells stably transfected with pCOS35K-Fc on a protein A-Sepharose column (Pharmacia, Piscataway, NJ). The B8R-Fc fusion protein will be described elsewhere (J. A. Symons and G. L. Smith, unpublished).
Measurement of calcium mobilization in human eosinophils and neutrophils
Changes in intracellular calcium flux were determined in purified human neutrophils and eosinophils as described previously (28). Briefly, granulocytes were separated from mononuclear leukocytes, RBCs, and platelets using a combination of dextran sedimentation and density centrifugation over Percoll-plasma gradients. For neutrophil experiments, granulocytes comprised >95% neutrophils, and the remaining cells were a mixture of eosinophils and mononuclear cells. For eosinophil experiments, blood was obtained from healthy atopic volunteers, and eosinophils were purified (>98%) from the isolated granulocytes by immunomagnetic separation using anti-human CD16 microbeads (Midimacs, Miltenyl Biotec, Teddington, U.K.) (29). Purified cells were loaded with fura-2/AM (neutrophils, 1 µM; eosinophils, 2.5 µM) and, after two washes, resuspended at 106 cells/ml in Ca2+/Mg2+-free PBS containing 10 mM HEPES (pH 7.4), 10 mM glucose, and 0.25% BSA. Aliquots of cells were taken, and the external calcium concentration was adjusted to 1 mM. Changes in fluorescence were monitored at 37°C using a spectrometer (LS50, Perkin-Elmer Corp., Norwalk, CT) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm.
Chemotaxis assay
Cell migration was evaluated in 10-mm tissue culture inserts (Life Technologies, Grand Island, NY) placed in 24-well plates. A 0.5-ml aliquot of test reagents diluted in RPMI 1640 with 1 mg/ml BSA was placed in the lower compartment, and 0.5 ml of U937 cell suspension (4 x 106 cells/ml) was placed in the upper chamber, separated by a polycarbonate filter (8-µm pore size). After incubation at 37°C for 2 h, the filter was washed, fixed, and stained with 4'6,-diamidino-2-phenylindole (DAPI) (30). The number of migrating cells in five high powered fields was counted using a fluorescence microscope.
Bioassay of eosinophil accumulation in guinea pig skin
Accumulation of endogenous peripheral blood eosinophils. Sedated guinea pigs were pretreated for 1 h with human IL-5 (18 pmol/kg; i.v. injection) to induce blood eosinophilia (31). Guinea pig eotaxin, human C5a, or LTB4, with or without purified 35K.His protein, was then injected intradermally (50 µl/site) into individual sites in the shaved dorsal skin of the animals. After 2 h, the animals were killed, and the skin sites were excised (11 mm in diameter) for quantification of eosinophil accumulation by measurement of eosinophil peroxidase as previously described (31).
Accumulation of 111In-labeled eosinophils
Eosinophils were purified from the peritoneal cavity of horse serum-treated donor animals and radiolabeled with 111InCl3 as previously described (28). 111In-labeled eosinophils (5 x 106 cells/animal) were injected i.v. into recipient animals, and after 10 min, guinea pig eotaxin and human C5a, with or without purified 35K.His protein, were injected intradermally (100 µl/skin site; two sites per treatment) into individual sites in the shaved dorsal skin. After 2 h, the animals were killed, and the skin sites were excised (17 mm in diameter) for quantification of eosinophil accumulation by measurement of 111In counts using a gamma counter (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although no CKR homologues were found in the VV genome sequence
(32), we searched for the expression of soluble chemokine binding
proteins by orthopoxviruses. Binding assays with
[125I]RANTES were performed with media from cultures
infected with 13 strains of VV, rabbitpox, or buffalopox viruses
(considered VV strains), cowpox virus (Brighton Red strain),
elephantpox virus (considered a cowpox virus strain), or camelpox
virus, followed by chemical cross-linking with EDC. A
[125I]RANTES-vCKBP complex was observed with several
viruses, including VV Evans (Fig. 1
c), but not with the
best characterized VV strains WR and Copenhagen (Fig. 1a). The size of the ligand-vCKBP complex ranged from
33 to 37 kDa, suggesting a vCKBP size of 25 to 29 kDa after subtraction
of the 8-kDa monomeric RANTES. The variation in vCKBP size may reflect
different polypeptide lengths or degrees of glycosylation among
viruses. The different size predicted for vCKBP from orthopoxviruses by
Graham et al. (19) in similar experiments (41 kDa) might be due to the
use of higher resolution gels in this study. The soluble vCKBP was
expressed before (early) and after (late) viral DNA synthesis by VV
rabbitpox and United Soviet Socialists Republic (USSR) (Fig. 1b). The RANTES-vCKBP complex was 37 to 44 kDa when
the cross-linker EGS was used (Fig. 1b), suggesting
that two RANTES molecules could form a complex with vCKBP. Similar
results were observed with [125I]MIP-1, another CC
chemokine (Fig. 1c). MIP-1 can dimerize, but there
is controversy about binding of CC chemokines as monomers or dimers to
cellular CKRs (4). Cross-linking of media from all viruses shown in
Figure 1a to [125I]IL-8 (not shown) or
[125I]GRO- (Fig. 1c and data not shown) was
negative or very weak, suggesting vCKBP specificity for CC, but not
CXC, chemokines.
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Although the predicted vCKBP size was 25 to 29 kDa, its expression
profile in orthopoxviruses (Fig. 1
, a and
c) correlated with the expression of a 35-kDa major
secretory protein in VV Lister, Evans, and rabbitpox, but not in WR,
Tian-Tan, or Wyeth (21, 33). The 35-kDa gene is present in cowpox (34),
but is truncated by a frameshift mutation within the signal peptide in
VV Copenhagen, where it is called B29R or C23L (32), and in VV WR,
where it produces a 7.5-kDa protein (21, 35). The expression of vCKBP
before (early) and after (late) viral DNA synthesis (Fig. 1b) was also consistent with the transcriptional
regulation of the 35-kDa gene from the strong early/late promoter p7.5,
widely used in VV expression vectors (36).
The absence of RANTES-binding activity in supernatants from
cultures infected with a VV Lister mutant lacking the 35-kDa gene
(Lister35K) (21) indicated that the 35-kDa protein is the vCKBP
encoded by VV Lister (Fig. 2a). This was confirmed
by showing specific binding of [125I]RANTES to the 35-kDa
protein produced from recombinant baculovirus (Ac35K) and when fused to
the Fc region of human IgG1 (35K-Fc), but not to the controls AcB15R
(VV IL-1ßR) (22), AcB8R (VV IFN-R) (20), or B8R-Fc (VV IFN-R
fused to human IgG1; J. A. Symons and G. L. Smith,
unpublished; Fig. 2a). Additional evidence was the
neutralization of the RANTES binding activity of VV Lister and Ac35K
supernatants by antiserum against purified 35-kDa protein (21), but not
by antiserum against the VV IL-1ßR B15R (22) (Fig. 2b).
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R (24), but not the VV
IFN-R (B8R) (20), was demonstrated to bind a wide range of
chemokines by interaction with their proteoglycan binding domain (19, 37). Here we demonstrate that VV expresses a 35-kDa vCKBP that does not
bind IFN- and is not B8R by showing that 1) the binding of
[125I]RANTES to natural (VV Lister) or recombinant (Ac35K
and 35K-Fc) 35-kDa protein was inhibited by unlabeled RANTES, but not
IFN- (Fig. 2a); 2) [125I]IFN- was
cross-linked to the natural IFN-R expressed from all three viruses,
WR, Lister, and Lister35K, or the recombinant IFN-R (AcB8R), but
not to the 35-kDa protein (Ac35K; Fig. 2c); 3) the
molecular sizes of monomeric IFN- complexed to IFN-R were 60 and
52 kDa for the natural and recombinant IFN-R, respectively (20),
which were higher than the 35-kDa size of the RANTES-vCKBP complex and
could not be explained by the size difference between IFN- (17 kDa)
and RANTES (8 kDa; Fig. 2, a and c); 4)
the 35-kDa size of the RANTES-vCKBP complex was smaller than the 50-kDa
size predicted for the RANTES-IFN-R complex, as reported for the
myxoma IFN-R/M7T (37); and 5) the binding of
[125I]IFN- to the natural (VV Lister) or recombinant
(AcB8R) VV IFN-R was inhibited by unlabeled IFN- but not by
RANTES (Fig. 2c). It is also demonstrated that VV
IFN-R does not bind RANTES by the inability of RANTES to bind
rIFN-R (AcB8R and B8R-Fc) (Fig. 2a) and the lack
of binding of RANTES to supernatants from cells infected with
Lister35K, which still express IFN-Rs (Fig. 2, a and
c). In addition, a higher molecular size of the
soluble IFN-R, known to be encoded by all the orthopoxviruses
included in this study (20), complexed with [125I]RANTES,
[125I]GRO-, or [125I]IL-8 was not
observed (Fig. 1, a and c, and data not shown),
indicating that the IFN-R from other strains of VV, cowpox, or
camelpox viruses does not bind CC or CXC chemokines. Binding specificity and affinity of vCKBP for chemokines
The binding specificity of vCKBP was further investigated in
cross-linking experiments (Fig. 3). The
vCKBP expressed from recombinant baculovirus, VV Lister, cowpox virus,
or camelpox virus bound human [125I]RANTES, and this
binding was effectively competed in a dose-dependent manner by all
human CC chemokines tested, including eotaxin, a specific
chemoattractant of eosinophils. In contrast, human CXC chemokines or
the C chemokine lymphotactin were not bound by vCKBP. The failure of
high doses of GRO- or IL-8 to block [125I]RANTES
binding was consistent with the negative or very weak cross-linking of
vCKBP from orthopoxviruses to [125I]GRO- or
[125I]IL-8 (Fig. 1c and data not shown),
suggesting a very low, and probably physiologically insignificant,
affinity for CXC chemokines. The vCKBP also bound CC chemokines from
mouse and rat. This vCKBP binding specificity was confirmed in
experiments cross-linking [125I]MIP-1 to VV Lister
supernatants in the presence of unlabeled chemokines (not shown). These
results demonstrate that vCKBP from VV, cowpox, and camelpox viruses
bind CC, but not CXC, chemokines.
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,
and quantification of bound MIP-1 was achieved using a scintillation
proximity assay (25). The interaction of MIP-1 to 35K-Fc was
saturable and of high affinity: in two experiments
Kd values of 103 ± 4 pM (Fig. 4a) and 128 ± 9
pM (not shown) were obtained. The high affinity interaction of vCKBP
for CC chemokines (Kd = 115 pM) was
similar or 100-fold higher than the affinity of chemokines for cellular
CKRs (1, 4, 5). Scatchard analysis of the binding data predicted 4.2
binding sites for MIP-1 in the 35K-Fc protein in both experiments,
suggesting 2.1 binding sites for chemokines in the 35-kDa protein. This
was consistent with the binding of two chemokine molecules to vCKBP, as
suggested by cross-linking studies (Fig. 1c). The
interaction of [125I]MIP-1 with 16.5 pM 35K-Fc was
inhibited by a similar dose of purified recombinant 35-kDa protein,
which was produced in baculovirus and containing a C-terminal
six-histidine tag (35K.His), suggesting that both proteins bind
MIP-1 with similar affinities (Fig. 4b).
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in the presence of
increasing doses of unlabeled chemokine competitors (Fig. 4c). These data showed that vCKBP binds CC chemokines
with different affinities: MIP-1 > eotaxin > RANTES >
MCP-1 > I309. The lower affinity for I309 probably reflects the
amino acid sequence of this chemokine being the most divergent of the
CC chemokine group (4). Competition experiments with other chemokines
corroborated, in a different quantitative binding assay, the
specificity of vCKBP for CC, but not CXC or C, chemokines and extended
these studies to 19 chemokines (Fig. 4d). The vCKBP does not bind to the proteoglycan binding site of chemokines
Chemokines interact with glucosaminoglycans such as heparin or
heparan sulfate through their carboxyl terminus (6, 9). This
interaction is thought to facilitate chemokine localization to the
endothelial cells and does not interfere with chemokines binding to
their receptors. To ascertain whether vCKBP bound to the proteoglycan
binding site of chemokines, [125I]MIP-1 was
preincubated with various doses of heparin or heparan sulfate before
incubation with the 35K-Fc protein. Figure 4e shows that
heparin or heparan sulfate did not interfere with MIP-1 binding to
vCKBP at doses up to 10 µg/ml and had a minor effect at higher doses
up to 200 µg/ml, equivalent to a 6 x 105-fold molar
excess of heparin over MIP-1.
The vCKBP blocks binding of CC chemokines to cell surface receptors
Biologic activity of vCKBP for CC chemokines was shown by the
ability of supernatants from orthopoxviruses and recombinant
baculovirus Ac35K to inhibit the binding of radioiodinated RANTES and
MIP-1, but not GRO-, to cellular receptors (Fig. 5a). This again
demonstrated the specificity of vCKBP for CC chemokines. As previously
reported (38, 39), excess unlabeled RANTES failed to block the binding
of [125I]RANTES to cells (Fig. 5a). The
binding of [125I]MIP-1, but not
[125I]GRO-, to U937 cells was inhibited in a
dose-dependent manner by supernatants containing the 35-kDa protein
(Fig. 5b). The potent inhibitory activity of vCKBP
was illustrated by the complete blockade of MIP-1 binding to cells
in the presence of 0.1 to 1 µl of medium, equivalent to the amount of
vCKBP synthesized by only 200 to 2000 cells. Furthermore, 10 or 50 pM
purified recombinant 35K.His protein produced in baculovirus
blocked the binding of 100 pM [125I]MIP-1 to
U937 cells by 50 and 95%, respectively (Fig. 5c). These results showed that the soluble
vCKBP blocks the binding of CC chemokines to their cellular
receptors and were consistent with the high affinity interaction of
MIP-1 and other CC chemokines with recombinant 35K-Fc.
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The blockade of CC chemokine binding to cell receptors suggested
that vCKBP would inhibit their biologic activity. This was demonstrated
by the ability of vCKBP to inhibit the rapid and transient increase in
intracellular calcium in eosinophils by the CC chemokines MCP-4 and
eotaxin (Fig. 6a). This
inhibitory effect was selective for these agonists, since vCKBP had no
effect on the calcium response induced by C5a in eosinophils (not
shown). In contrast, vCKBP had no effect on the elevation of
intracellular calcium levels in human neutrophils in response to the
CXC chemokines IL-8, GRO-, and NAP-2 (Fig. 6b).
This demonstrated specificity of vCKBP for CC, but not CXC, chemokines
in a biologic assay.
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(Fig. 6c). In contrast, Lister35K and
AcB8R supernatants did not. Blockade of eotaxin chemotactic activity by vCKBP in vivo
The blockade of chemokine activity by vCKBP was demonstrated in
vivo in a guinea pig skin model of eotaxin-induced eosinophil
infiltration (31). Purified recombinant 35K.His bound guinea pig
eotaxin, as determined by cross-linking assay (not shown). The vCKBP
inhibited local eosinophil infiltration induced by intradermal
injection of eotaxin, but not other agents, in a guinea pig model using
eosinophil peroxidase as an index of eosinophil numbers (Fig. 7a). The inhibitory
effect of various doses of vCKBP on eosinophil accumulation in the skin
in response to eotaxin was determined in guinea pigs injected i.v. with
111In-labeled eosinophils (Fig. 7b). The
potency of this inhibitor in vivo was illustrated by the complete
blockade of eotaxin activity by a threefold molar excess of vCKBP and
50% inhibition by an equal molar concentration (Fig. 7, a
and b). This emphasizes the potential therapeutic
application of vCKBP in inflammatory diseases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to known cellular CKRs, which are hydrophobic proteins with
seven transmembrane domains, the 35-kDa protein is a cysteine-rich,
acidic (pI 4.2), soluble protein with no sequence similarity to
cellular counterparts. Many of the poxvirus-encoded cytokine receptors
mimic the extracellular binding domain of known cellular counterparts;
thus, vCKBP may represent a viral version of an unidentified cellular
molecule, as has been proposed for the VV IFN-/ßR (18).
Chemokine binding proteins closely related to the VV Lister 35-kDa protein are found in other VV strains, the leporipoxviruses Shope fibroma virus (S-T1) and myxoma virus (M-T1), racoonpox virus, cowpox virus, and camelpox virus (Ref. 19 and this report), and are predicted to be produced by strains of variola virus, the cause of smallpox (G3R) (40, 41). Interestingly, VV WR gene A41L is predicted to encode a secretory protein more distantly related to the VV Lister 35-kDa protein (33, 42) and may represent another vCKBP that binds chemokines not included in this study.
Concerning the specificity of vCKBP for different chemokines, we demonstrate unequivocally that it is a specific inhibitor of CC, but not CXC or C, chemokines. This is shown by cross-linking experiments with cold competitors, in more quantitative binding experiments, and by determination of affinity constants in studies in which 19 chemokines were analyzed. Most importantly, the CC specificity was confirmed in biologic assays showing that vCKBP does not block the interaction of CXC to cellular receptors or their capacity to transduce signals. This contradicts the conclusion of Graham et al. (19), who reported that it had a broad specificity for CC and CXC chemokines. However, consistent with our results, these researchers found that excess IL-8 could not compete the cross-linking of RANTES to vCKBP, but the effect on the biologic activity of CXC chemokines was not investigated.
The mechanism by which vCKBP inhibits chemokine activity might be its
interaction with the proteoglycan binding domain of chemokines, which
is thought to facilitate chemokine localization to endothelial cells,
as suggested by Graham et al. (19). Alternatively, vCKBP might interact
with the receptor binding domain of chemokines and thus block binding
to cellular receptors, as reported for poxvirus soluble receptors (13, 14). The data in this paper show that vCKBP blocks CC chemokine
activity by binding chemokines with high affinity, at a site different
from the proteoglycan binding domain, and preventing their interaction
with cellular receptors. Consistent with this, the affinity of vCKBP
for human MIP-1 (Kd = 115 pM) is
similar or 10- to 100-fold higher than the affinity of most chemokines
for cellular CKRs, which normally ranges from 1 to 10 nM (1, 4, 5). The
vCKBP also binds eotaxin, RANTES, and MCP-1 with high affinity
(Kd = 1–15 nM). The lower affinity of
RANTES for myxoma M-T1 protein reported by Graham et al. (19)
(Kd = 73 nM) might be due to the use of
human chemokines with the M-T1 protein from myxoma virus, whose natural
host is the rabbit. There is precedent for myxoma virus-soluble
cytokine receptors binding human cytokines with much lower affinity
than rabbit cytokines (43, 44).
Evidence for the binding of vCKBP to two chemokine molecules was found
in cross-linking experiments using EGS and in Scatchard analysis of the
binding of MIP-1 to 35K-Fc. MIP-1 and IL-8 can dimerize, but
there is controversy about binding to cellular CKRs and the biologic
activity of chemokines as monomers or dimers (4). If vCKBP does bind to
dimeric chemokines, this might enhance the inhibitory activity of
vCKBP.
Poxviruses have evolved two different mechanisms to block chemokine
activity. First, the soluble IFN-R from myxoma virus (M-T7), but not
VV strain WR, binds the heparin-binding domain of a wide range of
chemokines (19, 37), and this additional function of M-T7 is consistent
with an increased leukocyte infiltration to sites of myxoma virus
replication in rabbits infected with a deletion mutant lacking this
protein (45). The myxoma M-T7 protein may prevent the correct
localization of chemokines in vivo, which form a gradient by
interacting with proteoglycans at the surface of endothelial cells. We
show here that the soluble IFN-R from 19 orthopoxviruses (20),
including strains of VV, cowpox, and camelpox viruses, does not bind CC
or CXC chemokines. Second, we show that orthopoxviruses encode a vCKBP
that binds chemokines with high affinity at a site different from the
proteoglycan binding domain and blocks their interaction with
cellular CKRs.
The expression of a potent, soluble, CC-specific vCKBP by poxviruses suggests an important role for CC chemokines in anti-viral defense. Nonetheless, deletion of the 35-kDa protein in VV rabbitpox did not greatly affect the outcome of infection in mice and rabbits (33). Graham et al. (19) now report an increased leukocyte influx in the skin of rabbits infected with VV rabbitpox mutant lacking the 35-kDa gene, compared with wild-type VV rabbitpox. However, construction of a revertant virus in which the 35-kDa gene is reintroduced into the VV rabbitpox mutant genome is required to support this conclusion. This VV rabbitpox mutant was constructed by transfecting DNA from VV Lister into VV rabbitpox-infected cells (33), and other mutations might have been introduced in the regions flanking the 35-kDa gene, which includes a gene encoding a TNFR homologue (32). Additional studies with this or alternative models of poxvirus infection are required to fully understand the role of vCKBP in poxvirus pathogenesis.
The important role of chemokines and CKRs in the pathogenesis of virus
infections is emphasized by the expression of 1) chemokine-like
proteins by molluscum contagiosum virus (46), Kaposi’s
sarcoma-associated herpes virus (47, 48), and murine CMV (49),
representing potential alternative ways to modulate chemokine activity;
and 2) seven-transmembrane domain CKRs by large DNA viruses such as
herpesviruses and poxviruses (14). Moreover, the MIP-1 knockout
mouse is less able to clear influenza virus infection (50), and RANTES,
MIP-1, and MIP-1ß increase resistance to HIV infection (2, 3).
Poxvirus proteins that counteract the immune system have been optimized during the evolution of viruses with their hosts; are providing insights into the physiologic role of immune regulatory molecules such as cytokines (51) or chemokines; and represent potential sources of immunomodulatory proteins and new strategies of immune modulation. In this report a novel soluble chemokine inhibitor from poxviruses with therapeutic potential is described. Eosinophil accumulation in response to eotaxin is a feature of allergic inflammatory reactions, such as those occurring in allergic asthma. The potent eotaxin inhibitory effect of vCKBP in vivo emphasizes its potential therapeutic application in inflammatory diseases. The vCKBP has binding specificity for CC chemokines, but the soluble inhibitor or variants from other poxviruses may provide the structural scaffolding needed to design specific soluble inhibitors of other chemokines.
The finding of a soluble vCKBP also has implications for infectious agents such as HIV, in which CKRs play an important role in determining transmission and disease progression (2, 3). Inhibition of the interaction of the HIV envelope with the chemokine binding domain of CKRs has recently emerged as a new target for intervention. The vCKBP might be engineered to create soluble molecules that bind to the domain of gp120, which interacts with the CKR to block HIV infection at an early stage (2, 3). A similar approach might yield therapeutic agents to block attachment of the malarial parasite to the Duffy Ag on erythrocytes and initiation of the infection (52).
The poxvirus chemokine inhibitor reported here represents a new and exciting virus immune evasion mechanism that provides insights into virus pathogenesis, the function of key immune regulatory molecules, and new strategies for therapeutic intervention in immune responses and disease.
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Geoffrey L. Smith, Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford, United Kingdom OX1 3RE.
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; GRO-, growth-related oncogene ; CKR, chemokine receptor; VV, vaccinia virus; vCKBP, virus chemokine binding protein; MCP, monocyte chemoattractant protein; NAP-2, neutrophil activating peptide-2; LTB4, leukotriene B4; WR, Western Reserve; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; EGS, ethylene glycol-bis-succinamidyl succinate.
Received for publication August 1, 1997. Accepted for publication September 30, 1997.
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M. Saraiva, P. Smith, P. G. Fallon, and A. Alcami Inhibition of Type 1 Cytokine-mediated Inflammation by a Soluble CD30 Homologue Encoded by Ectromelia (Mousepox) Virus J. Exp. Med., September 16, 2002; 196(6): 829 - 839. [Abstract] [Full Text] [PDF]
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J. A. Symons, D. C. Tscharke, N. Price, and G. L. Smith A study of the vaccinia virus interferon-{gamma} receptor and its contribution to virus virulence J. Gen. Virol., August 1, 2002; 83(8): 1953 - 1964. [Abstract] [Full Text] [PDF]
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N. Bartlett, J. A. Symons, D. C. Tscharke, and G. L. Smith The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence J. Gen. Virol., August 1, 2002; 83(8): 1965 - 1976. [Abstract] [Full Text] [PDF]
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D. C. Tscharke, P. C. Reading, and G. L. Smith Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes J. Gen. Virol., August 1, 2002; 83(8): 1977 - 1986. [Abstract] [Full Text] [PDF]
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 B. T. Seet and G. McFadden Viral chemokine-binding proteins J. Leukoc. Biol., July 1, 2002; 72(1): 24 - 34. [Abstract] [Full Text] [PDF]
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J. F. Panus, C. A. Smith, C. A. Ray, T. D. Smith, D. D. Patel, and D. J. Pickup Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: A soluble, secreted CD30 homologue PNAS, June 11, 2002; 99(12): 8348 - 8353. [Abstract] [Full Text] [PDF]
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C. Gubser and G. L. Smith The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox J. Gen. Virol., April 1, 2002; 83(4): 855 - 872. [Abstract] [Full Text] [PDF]
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A. Alcami and G. L. Smith The vaccinia virus soluble interferon-{gamma} receptor is a homodimer J. Gen. Virol., March 1, 2002; 83(3): 545 - 549. [Abstract] [Full Text] [PDF]
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V. P. Smith and A. Alcami Inhibition of Interferons by Ectromelia Virus J. Virol., February 1, 2002; 76(3): 1124 - 1134. [Abstract] [Full Text] [PDF]
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 C. M. Lilly and B. L. Daugherty A Novel LPS-Inducible CCR3 Activator . Why So Many CCR3 Ligands? Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 673 - 675. [Full Text] [PDF]
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C. Penido, H. C. Castro-Faria-Neto, A. Vieira-de-Abreu, R. T. Figueiredo, A. Pelled, M. A. Martins, P. J. Jose, T. J. Williams, and P. T. Bozza LPS Induces Eosinophil Migration via CCR3 Signaling Through a Mechanism Independent of RANTES and Eotaxin Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 707 - 716. [Abstract] [Full Text] [PDF]
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C. G. Beck, C. Studer, J.-F. Zuber, B. J. Demange, U. Manning, and R. Urfer The Viral CC Chemokine-binding Protein vCCI Inhibits Monocyte Chemoattractant Protein-1 Activity by Masking Its CCR2B-binding Site J. Biol. Chem., November 9, 2001; 276(46): 43270 - 43276. [Abstract] [Full Text] [PDF]
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 S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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A. Ng, D. C. Tscharke, P. C. Reading, and G. L. Smith The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence J. Gen. Virol., September 1, 2001; 82(9): 2095 - 2105. [Abstract] [Full Text] [PDF]
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A. Bridgeman, P. G. Stevenson, J. P. Simas, and S. Efstathiou A Secreted Chemokine Binding Protein Encoded by Murine Gammaherpesvirus-68 Is Necessary for the Establishment of a Normal Latent Load J. Exp. Med., August 6, 2001; 194(3): 301 - 312. [Abstract] [Full Text] [PDF]
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H. R. Luttichau, J. Gerstoft, and T. W. Schwartz MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8 J. Leukoc. Biol., August 1, 2001; 70(2): 277 - 282. [Abstract] [Full Text] [PDF]
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F. Almazan, D. C. Tscharke, and G. L. Smith The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication J. Virol., August 1, 2001; 75(15): 7018 - 7029. [Abstract] [Full Text]
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B. T. Seet, R. Singh, C. Paavola, E. K. Lau, T. M. Handel, and G. McFadden Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor PNAS, July 19, 2001; (2001) 171069398. [Abstract] [Full Text] [PDF]
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M. Saraiva and A. Alcami CrmE, a Novel Soluble Tumor Necrosis Factor Receptor Encoded by Poxviruses J. Virol., January 1, 2001; 75(1): 226 - 233. [Abstract] [Full Text]
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F. J. Culley, A. Brown, D. M. Conroy, I. Sabroe, D. I. Pritchard, and T. J. Williams Eotaxin Is Specifically Cleaved by Hookworm Metalloproteases Preventing Its Action In Vitro and In Vivo J. Immunol., December 1, 2000; 165(11): 6447 - 6453. [Abstract] [Full Text] [PDF]
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V. P. Smith and A. Alcami Expression of Secreted Cytokine and Chemokine Inhibitors by Ectromelia Virus J. Virol., September 15, 2000; 74(18): 8460 - 8471. [Abstract] [Full Text]
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V. van Berkel, J. Barrett, H. L. Tiffany, D. H. Fremont, P. M. Murphy, G. McFadden, S. H. Speck, and H. W. Virgin IV Identification of a Gammaherpesvirus Selective Chemokine Binding Protein That Inhibits Chemokine Action J. Virol., August 1, 2000; 74(15): 6741 - 6747. [Abstract] [Full Text]
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V. P. Smith, N. A. Bryant, and A. Alcamí Ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins J. Gen. Virol., May 1, 2000; 81(5): 1223 - 1230. [Abstract] [Full Text]
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C. L. Afonso, E. R. Tulman, Z. Lu, L. Zsak, G. F. Kutish, and D. L. Rock The Genome of Fowlpox Virus J. Virol., April 15, 2000; 74(8): 3815 - 3831. [Abstract] [Full Text]
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T. L. Born, L. A. Morrison, D. J. Esteban, T. VandenBos, L. G. Thebeau, N. Chen, M. K. Spriggs, J. E. Sims, and R. M. L. Buller A Poxvirus Protein That Binds to and Inactivates IL-18, and Inhibits NK Cell Response J. Immunol., March 15, 2000; 164(6): 3246 - 3254. [Abstract] [Full Text] [PDF]
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 P. M. Murphy, M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors Pharmacol. Rev., March 1, 2000; 52(1): 145 - 176. [Abstract] [Full Text] [PDF]
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R. S. B. Milne, C. Mattick, L. Nicholson, P. Devaraj, A. Alcami, and U. A. Gompels RANTES Binding and Down-Regulation by a Novel Human Herpesvirus-6 {beta} Chemokine Receptor J. Immunol., March 1, 2000; 164(5): 2396 - 2404. [Abstract] [Full Text] [PDF]
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C. M. Parry, J. P. Simas, V. P. Smith, C. A. Stewart, A. C. Minson, S. Efstathiou, and A. Alcami A Broad Spectrum Secreted Chemokine Binding Protein Encoded by a Herpesvirus J. Exp. Med., February 7, 2000; 191(3): 573 - 578. [Abstract] [Full Text] [PDF]
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 J. G. Cyster Chemokines and Cell Migration in Secondary Lymphoid Organs Science, December 10, 1999; 286(5447): 2098 - 2102. [Abstract] [Full Text]
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G. Camarda, G. Spinetti, G. Bernardini, C. Mair, N. Davis-Poynter, M. C. Capogrossi, and M. Napolitano The Equine Herpesvirus 2 E1 Open Reading Frame Encodes a Functional Chemokine Receptor J. Virol., December 1, 1999; 73(12): 9843 - 9848. [Abstract] [Full Text]
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A. Carfi, C. A. Smith, P. J. Smolak, J. McGrew, and D. C. Wiley Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus PNAS, October 26, 1999; 96(22): 12379 - 12383. [Abstract] [Full Text] [PDF]
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P. Fleming, N. Davis-Poynter, M. Degli-Esposti, E. Densley, J. Papadimitriou, G. Shellam, and H. Farrell The Murine Cytomegalovirus Chemokine Homolog, m131/129, Is a Determinant of Viral Pathogenicity J. Virol., August 1, 1999; 73(8): 6800 - 6809. [Abstract] [Full Text]
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D. Wilcock, S. A. Duncan, P. Traktman, W.-H. Zhang, and G. L. Smith The vaccinia virus A40R gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface J. Gen. Virol., August 1, 1999; 80(8): 2137 - 2148. [Abstract] [Full Text]
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A Alcami, A Khanna, N. Paul, and G. Smith Vaccinia virus strains Lister, USSR and Evans express soluble and cell- surface tumour necrosis factor receptors J. Gen. Virol., April 1, 1999; 80(4): 949 - 959. [Abstract]
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B. T. Seet, J. Barrett, J. Robichaud, B. Shilton, R. Singh, and G. McFadden Glycosaminoglycan Binding Properties of the Myxoma Virus CC-chemokine Inhibitor, M-T1 J. Biol. Chem., August 3, 2001; 276(32): 30504 - 30513. [Abstract] [Full Text] [PDF]
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J. M. Burns, D. J. Dairaghi, M. Deitz, M. Tsang, and T. J. Schall Comprehensive Mapping of Poxvirus vCCI Chemokine-binding Protein. EXPANDED RANGE OF LIGAND INTERACTIONS AND UNUSUAL DISSOCIATION KINETICS J. Biol. Chem., January 18, 2002; 277(4): 2785 - 2789. [Abstract] [Full Text] [PDF]
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B. T. Seet, R. Singh, C. Paavola, E. K. Lau, T. M. Handel, and G. McFadden Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor PNAS, July 31, 2001; 98(16): 9008 - 9013. [Abstract] [Full Text] [PDF]
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