In Vivo Inhibition of CC and CX3C Chemokine-induced
Leukocyte Infiltration and Attenuation of Glomerulonephritis
in Wistar-Kyoto (WKY) Rats by vMIP-II
By
Shizhong
Chen,*
Kevin B.
Bacon,
Li
Li,*
Gabriela E.
Garcia,*
Yiyang
Xia,*
David
Lo,*
Darren A.
Thompson,§
Michael A.
Siani,§
Tadashi
Yamamoto,
Jeffrey K.
Harrison,¶
and
Lili
Feng*
From the * Department of Immunology, The Scripps Research Institute, La Jolla, California 92037;
Neurocrine Biosciences, San Diego, California 92121; § Gryphon Sciences, South San Francisco,
California 94908; the
Department of Pathology, Instutute of Nephrology, Niigata University School
of Medicine, Niigata 951, Japan; and the ¶ Department of Pharmacology and Therapeutics, University
of Florida, Gainesville, Florida 32610
 |
Abstract |
Chemokines play a central role in immune and inflammatory responses. It has been observed
recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such
viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II
has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate
the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by
an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte
chemoattractant protein 1-, macrophage inflammatory protein 1
-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated
leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the
glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by
some viruses may serve as useful templates for the development of antiinflammatory compounds.
Key words:
vMIP-II;
CX3CR1;
chemokine;
glomerulonephritis;
inflammation
 |
Introduction |
The recruitment and activation of leukocytes at sites of
pathogenesis or injury is the hallmark of inflammation.
Although physiological inflammation is required for host
defense and wound repairs, reactions that are disproportionate
to the magnitude of the immune challenges are at the core
of most inflammatory and autoimmune diseases. It is increasingly evident that chemokines play a crucial role in these physiological and pathological processes (1), and have been regarded
as rational targets for the development of antiinflammatory
reagents (2). However, the approach of antichemokine therapy has been hampered by the pleiotropy and redundancy of
the chemokine system. Not only are multiple chemokines with overlapping activities frequently induced in inflammatory diseases, but often many different chemokine receptors are
expressed by the activated leukocytes. Consequently, it may
be difficult to control inflammation with an agent designed to
neutralize the activity of a single chemokine. Molecules that
have the capacity to bind and antagonize multiple types of
chemokine receptors may provide a rational approach to
overcome difficulties associated with this potential redundancy. Recently, a chemokine analogue encoded by human herpesvirus 8 has been identified and termed vMIP-II
(3). In vitro, vMIP-II competes with native chemokines in
the binding of a number of human CC and CXC chemokine receptors and blocks the actions of these chemokines
on human monocytes (Mo; reference 4). To investigate the in
vivo antiinflammatory activity of this broad-spectrum chemokine antagonist, we used vMIP-II in a well-established kidney inflammatory disease model, anti-glomerular basement
membrane antibody-induced experimental glomerulonephritis (anti-GBM GN) in Wistar-Kyoto (WKY) rats (6). We
found that, at nanomolar concentrations, vMIP-II effectively attenuated leukocyte infiltration to the kidney and significantly reduced the ensuing renal injury in the treated rats.
 |
Materials and Methods |
Synthesis of vMIP-II and Fractalkine.
Synthetic chemokines were
generated by native chemical ligation of peptides synthesized by
solid-phase methods on a peptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City, CA; reference 7). The resulting chemokines were purified by reverse-phase HPLC and characterized by electrospray mass spectrometry. The purified synthetic
chemokines were reconstituted to 0.1 mg/ml in PBS before use.
Induction, Treatment, and Analysis of Anti-GBM GN in WKY
Rats.
At day 0, male WKY rats (Charles River Laboratories,
Wilmington, MA), 10-12 weeks of age and 200-220 g of body
weight, were given one intravenous injection of anti-GBM antibody (7) at a dose of 25 µl/100 g body weight. These rats were then
given two daily intravenous injections of PBS or vMIP-II (12.5 µg/
injection/rat, 25 µg total per day) for a period of 6 d, starting from
day 0. On days 3 and 5, 24-h rat urine excretion was collected. Different groups of rats were killed on days 4 and 6 to collect blood and kidney tissues. Proteinuria was assayed by the sulfosalicylic method
(8). Urine and blood creatinine was determined using a creatinine
diagnostic kit (Sigma Chemical Co., St. Louis, MO).
RNA Analysis.
Glomeruli were prepared from rat kidneys as
previously described (8). Total RNA was isolated from glomeruli
using a one-step method (9). 5 µg of total RNA from each sample was used for RNase protection assay, following a previously
described protocol (10). Riboprobes for chemokines MIP-1
,
MIP-1
, MCP-1, RANTES, and the housekeeping gene L32 are
described elsewhere (Xia, Y., S. Chen, Y. Wang, G. Ku, C.B.
Wilson, D. Lo, and L. Feng, manuscript in preparation).
Western Blot Analysis and ELISA.
The protein levels of MIP-1
and RANTES in rat glomeruli were analyzed by Western blot
analysis as described previously (8), with some modification. Isolated glomeruli from each rat were solubilized in 20 mM PBS
containing 0.5% Triton X-100, 10 mM EGTA, 1 mM PMSF,
and 10 µM leupeptin. After centrifugation, the supernatants were
collected and enriched by binding to heparin Sepharose CL-6B beads (Pharmacia Biotech, Piscataway, NJ). The protein contents of the eluted lysates were determined by a BCA protein assay kit from Pierce (Rockford, IL). 100 µg of protein from each sample was electrophoresed in a NuPAGE gel (Novex, San Diego, CA)
and transferred to a nitrocellulose membrane. The protein blot
was first probed with anti-MIP-1
or anti-RANTES antibody
and then with horseradish peroxidase-conjugated second antibody. Antibody binding was detected by the addition of chemiluminescent substrate (SuperSignal Kit; Pierce) and exposure to autoradiograph film (Wolf X-ray Corp., West Hempstead, NY).
The protein level of MCP-1 in the glomerular lysate was quantitated by ELISA and expressed as nanograms of MCP-1/milligrams of protein. Anti-rat MCP-1 mAb B4 and C4 (PharMingen, San Diego, CA) were used as paired antibodies for ELISA,
and recombinant rat MCP-1 (PeproTech, Rocky Hill, NJ) was
used to generate the standard curve.
Preparation and Chemotaxis Analysis of Inflammatory Leukocytes
from Nephritic Glomeruli.
Inflammatory leukocytes were isolated
from nephritic glomeruli following the method of Cook et al.
(11). The chemotaxis assay was performed as previously described
(12). In brief, glomerular inflammatory leukocytes were resuspended at 2 × 106/ml in DMEM plus 10% heat-inactivated FCS.
25 µl of increasing concentrations of chemoattractant was placed
in the lower wells of a 48-well chemotaxis chamber (NeuroProbe, Cabin John, MD) and separated from 50 µl of cell suspension in the top wells by an 8- or 5-µm pore-size polyvinylpyrolidone-free polycarbonate filter. After incubation at 37°C for 2 h,
sedimented cells on the top surface of the filter were wiped off
and migrated cells on the undersurface were fixed in methanol
and stained using Diff-QuikTM. Results are expressed as mean ± SEM cell number per five high-power fields (×400) and are representative of n = 3 experiments performed in duplicate.
Competitive Binding Assays.
A filtration protocol was used for
equilibrium binding of 125I-labeled fractalkine. 5 × 105 cells were
incubated with 0.2 nM 125I-labeled fractalkine in the presence of
unlabeled fractalkine or vMIP-II in the following buffer for 2 h at
22°C: 25 mM Hepes, 80 mM NaCl, 1 mM CaCl2, 5 mM MgCl2,
and 0.5% BSA, adjusted to pH 7.4. The reactions were aspirated
onto polyethyleneimine-treated GF/C filters (Packard, Meriden,
CT) using a 96-well cell harvester (Packard). The filters were
washed twice in 25 mM Hepes (pH 7.4), 500 mM NaCl, 1 mM
CaCl2, 5 mM MgCl2, and counted on a Packard Top Counter. The
resulting data was analyzed using GraphPad PrismTM software
(GraphPad Software, Sorrento Valley, CA).
Histopathology.
Kidney tissue samples were fixed in 10% neutralized buffered formalin (NBF) or methanol-Carnoy (Methacarn) fixative solution, and were embedded in paraffin. For light
microscopy examination, 5-µm paraffin sections of NBF-fixed
tissues were stained with periodic acid-Schiff reagent. The number of crescentic glomeruli per 100 glomeruli of each rat was calculated and expressed as a percentage. For staining of CD8+ and
ED1+ infiltrates, 5-µm paraffin sections of methacarn-fixed tissues were dewaxed and microwave-heated in 10 mM of sodium
citrate (pH 6.0) at 800 watts for 10 min. The slides were reacted
with mAb MRC-OX8 against rat CD8 (PharMingen) or mAb
ED-1 against rat macrophages (M
s; Chemicon, Temecula, CA),
and goat anti-mouse second antibody. Antibody binding was detected by an alkaline phosphatase antialkaline phosphate kit and
developed with a New Fuchsin substrate (DAKO Corp., Carpinteria, CA). Positively stained cells per 100 glomeruli of each rat
were counted and expressed per glomerular cross-section.
 |
Results and Discussion |
Anti-GBM GN in WKY rats is characterized by an accumulation of CD8+ cells and ED1+ Mo/M
s in the glomeruli (6), and CC chemokines are likely to play an important
role in this form of renal inflammation. We first examined the
expression of CC chemokines MCP-1, MIP-1
, MIP-1
,
and RANTES in normal and nephritic glomeruli of WKY
rats. Normal glomeruli had very little mRNA and protein
expression of these chemokines (Fig. 1). Intravenous injection of anti-GBM antibody induced a profound mRNA expression of MCP-1, MIP-1
, and RANTES in the glomeruli (Fig. 1 a). The induction was prominent 3 d after the
antibody injection, persisted through day 7, and started to subside by day 9. Compared with MCP-1, MIP-1
, and
RANTES, the induction of MIP-1
mRNA expression was
not as significant. Western blot analysis of MIP-1
and
RANTES protein (Fig. 1 b) and ELISA analysis of MCP-1
protein (Fig. 1 c) confirmed that the protein levels of these
CC chemokines correlated with their mRNA levels in the
glomeruli. In addition to CC chemokines, we found that the CX3C chemokine, fractalkine, was also induced in the
glomeruli of anti-GBM GN of WKY rats.1

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Fig. 1.
Analysis of CC chemokine expression in the glomeruli of anti-GBM GN in WKY rats. GN was induced in WKY rats by intravenous injection of anti-GBM antibody on day 0. On days 0, 3, 5, 7, and 9, the rats were
killed and glomerular samples were prepared for analysis. (a) RNase protection analysis of MCP-1, MIP-1 , MIP-1 , and RANTES mRNA expression. Each lane represents a single rat sampled. Probes contain polylinker regions and are longer than the protected bands. Rat ribosomal L32 gene was
used as a housekeeping gene. (b) Western blot analysis of MIP-1 and
RANTES protein expression. Each lane represents samples pooled from three
rats. (c) ELISA analysis of MCP-1 protein level. Each data point represents
samples pooled from three rats and is expressed as mean ± SD.
|
|
The expression of multiple chemokines coincides temporally with the influx of CD8+ cells and ED1+ Mo/M
s
into the glomeruli of anti-GBM GN of WKY rats. In vitro, MCP-1, MIP-1
, RANTES, and fractalkine all induced
strong migratory responses in cells prepared from the glomeruli of WKY rats 3 d after the anti-GBM antibody injection (Fig. 2 a). The antagonistic activity of vMIP-II against
these chemokines was investigated. vMIP-II efficiently inhibited chemotactic activities of MCP-1, MIP-1
, and
RANTES on activated leukocytes isolated from nephritic glomeruli (Fig. 2 a). In addition to the CC chemokines, the
chemotactic activity of fractalkine was also inhibited by
vMIP-II (Fig. 2 a). Fractalkine represents a new class of
chemokine (13, 14), and its receptor, CX3CR1, is the first
receptor identified for this class of chemokines (15, 15a). To
confirm that the vMIP-II inhibition of fractalkine activity
was through CX3CR1 binding, whole cell competitive
binding assays were carried out. vMIP-II displaced 125I-labeled
fractalkine binding from HEK293 cells transfected with rat
CX3CR1 cDNA (data not shown) and from nephritic glomeruli-derived cells (Fig. 2 b). Our findings indicate that
vMIP-II is a CX3CR1 antagonist, and extend the spectrum
of chemokine antagonism of vMIP-II to include that of the
CX3C chemokine.

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Fig. 2.
vMIP-II inhibition of chemotactic activity of CC and CX3C
chemokines and displacement of 125I-labeled fractalkine in a competitive
binding assay. (a) Chemotaxis of inflammatory leukocytes from the glomeruli of WKY rats with anti-GBM GN. The chemotactic responses, expressed as numbers of migratory cells per five high-power fields (Numbers/
5 × hpf) of the glomerular infiltrates to different concentrations of MCP-1,
MIP-1 , RANTES, and fractalkine with the presence of varying amounts
of vMIP-II ( , 0 nM; , 3 nM; , 10 nM; , 30 nM) were shown. (b)
Competitive binding assay of 125I-labeled fractalkine. 125I-labeled fractalkine
binding to the glomerular cells from WKY rat with anti-GBM GN was
performed with the presence of varying amounts of fractalkine or vMIP-II.
|
|
The in vivo activity of vMIP-II was investigated next.
WKY rats with anti-GBM GN were treated with vMIP-II
or with PBS as a control. In the control group, anti-GBM
GN led to a prominent glomerular and periglomerular accumulation of CD8+ cells and ED1+ Mo/M
s (Fig. 3, a and
c). This infiltration was significantly attenuated by vMIP-II
treatment (Fig. 3, b and d). Consequently, the severe glomerular hypercellularity and crescentic formation characteristic of anti-GBM GN (Fig. 3 e) were markedly reduced in
the vMIP-II treatment group (Fig. 3 f ). Quantitative study indicated that the glomerular accumulation of CD8+ and
ED1+ infiltrates and frequency of crescentic glomeruli in the
vMIP-II treatment group were <50% of those in the control
rats (P <0.001, student's t test; Fig. 4). As a result of the attenuation of inflammatory lesions in the kidney, normal renal function was largely maintained in anti-GBM GN WKY
rats treated with vMIP-II. 24-h urinary protein of the vMIP-II-treated group was mild, being less than one-third that of
the control group (P <0.001; Fig. 5 a), and the serum creatinine levels in the experimental group were also significantly lower than the control group (P <0.001; Fig. 5 b).

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Fig. 3.
Photomicrographs (original magnification, ×400) of the glomeruli from WKY rats with anti-GBM GN that were treated with PBS or with
vMIP-II. (a-d). Kidney sections of PBS- (a and c) or vMIP-II-treated rats (b and d) immunohistochemistry stained for CD8+ cells (a and b) or ED1+ Mo/
M s (c and d). Sections were sampled on day 4 after anti-GBM antibody injection. (e-f ) Periodic acid-Schiff staining of kidney section of PBS- (e) or
vMIP-II-treated rats (f ). Sections were sampled on day 6 after anti-GBM antibody injection.
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Fig. 4.
Quantitation of CD8+
(a), ED1+ (b) cell infiltration, and
crescent formation (c) in the glomeruli from WKY rats with anti-GBM GN that were treated with
PBS (control) or vMIP-II. Kidney
sections, stained as shown in Fig.
3, were examined, and 100 glomeruli per section were counted.
Each data point represents sections sampled from three rats and is expressed as mean ± SD. *P <0.001, student's t test.
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Fig. 5.
(a) Proteinuria (milligrams of urine protein per 24 h) and (b)
serum creatinine levels (arbitrary units) in WKY rats with anti-GBM GN
that were treated with PBS (control) or vMIP-II. Results were sampled from
six rats per group and expressed as mean ± SD. *P <0.001, student's t test.
|
|
In this study, we demonstrated by assessing a number of
disease parameters that vMIP-II has antiinflammatory activity in anti-GBM GN in WKY rats. vMIP-II treatment attenuated leukocyte infiltration in the kidney, suppressed
the onset of inflammation, and protected the kidney from
inflammatory injury. The protection was not due to simple
interference in the binding of rabbit anti-GBM antibody to
rat kidneys. Immunofluorescent staining revealed rabbit
IgG binding along the capillary walls of glomeruli in a linear pattern, with no discernible difference in the intensity
between the control and experimental groups (data not
shown). The attenuation of leukocyte infiltration cannot be
attributed to a depletion of CD8+ cells or M
s by vMIP-II
treatment. Flow cytometry profiles of blood CD8+ cells and
ED1+ Mo were indistinguishable between the vMIP-II- and
PBS-treated rats (data not shown). Consistent with its in
vitro activity, the antiinflammatory activity of vMIP-II is
probably a direct result of its interference with the chemotactic recruitment of leukocytes into the kidney. Kledal et
al. found that vMIP-II binds to human chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR4, and antagonizes the action of MIP-1
, MIP-1
, and RANTES
on freshly prepared human Mo, and they suggested that
vMIP-II may help to prevent leukocyte recruitment in response to viral infection (4). Extending these findings, we
showed that vMIP-II inhibited the chemotactic activity of
rat chemokines MCP-1, MIP-1
, RANTES, and fractalkine on activated leukocytes isolated from nephritic glomeruli of WKY rats with anti-GBM GN. In particular, ours
is the first report of the antagonistic activity of vMIP-II
against fractalkine receptor. MCP-1, MIP-1
, RANTES,
and fractalkine were dramatically induced in the nephritic
glomeruli of WKY rats with anti-GBM GN (Fig.1).1 As a
broad-spectrum chemokine antagonist, vMIP-II could interfere with the activities of these chemokines in vivo, and
thus prevent lymphocyte and M
recruitment into the diseased kidney. In addition to leukocyte recruitment, MCP-1
has recently been found to mediate direct effects upon resident renal cells and to play a critical role in crescent formation and deposition of type I collagen in a murine crescentic nephritis model (16). It is possible that vMIP-II can
interfere with the MCP-1 effect on resident renal cells and
help to improve the renal function in inflammatory GN. Bacon et al. reported that RANTES could directly activate
T cells and induce proliferation (17), an effect that seems to
be mediated through a receptor different from the G protein-coupled chemokine receptors. It remains to be determined whether vMIP-II can inhibit the T cell activation
function of RANTES as well.
Extensive efforts have been expended in the search and
development of antichemokine therapeutic agents (18),
and this in turn has contributed to the understanding of
chemokine functions. In this respect, antichemokine and
antichemokine receptor antibodies have constituted a major part of the validation of the critical role of chemokines
in inflammatory diseases (21). On the other hand, for therapeutic interventions, antichemokine antibodies or reagents
specific for a single ligand may not be effective. The bulk
of data suggest that more than one chemokine is responsible for the recruitment of any individual cell type in inflammatory diseases, and the key chemokine may vary from
disease to disease. Moreover, the key chemokine may vary
with the progression of an inflammatory disease. Thus, the
antagonism of one particular chemokine is prone to complications. Fujinaka et al. (22) have recently shown that
MCP-1 plays an important role in the anti-GBM GN in WKY rats, and that the injection of anti-MCP-1 mAb significantly suppresses Mo/M
infiltration and reduces proteinuria during the early phase of GN. However, the same
treatment is ineffective during the later stages of GN, and
the authors suggested that other chemotactic factors may be
important during later stages of the disease. Agents with
broad-spectrum antagonism to chemokines are therefore
more desirable for inflammatory disease interventions. Viruses have coevolved with the host defense system and
must constantly develop countermeasures to interfere with
the physiological function of the immune system (23, 24).
The rapidity of the viral genetic cycle coupled with the
life-and-death selection imposed by the hostile host has optimized the molecular mimicry mechanism. Although detrimental to the physiological functions of the host immune system, viral antagonists thus evolved may be powerful reagents for treating pathological conditions and valuable
prototypes for rational design of chemokine antagonists.
The application of vMIP-II may be just one example of
our "mimicry of viral mimicry."
 |
Footnotes |
Address correspondence to Lili Feng, Department of Immunology, IMM5, The Scripps Research Institute,
10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: 619-784-8262; Fax: 619-784-8558; E-mail:
llfimm{at}scripps.edu
Received for publication 13 March 1998 and in revised form 17 April 1998.
1
Feng, L., S. Chen, G. Garcia, Y. Xia, M.A. Siani, P. Botti, J.K. Harrison,
C.B. Wilson, and K.B. Wilson, manuscript in preparation.
We thank Carolyn Douglas for expert technical assistance.
This work was supported by the National Institutes of Health grant DK-49832-01A2 to L. Feng. This is
publication No. 11484-IMM from The Scripps Research Institute, La Jolla, CA.
 |
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