A Non-peptide Functional Antagonist of the CCR1 Chemokine
Receptor Is Effective in Rat Heart Transplant Rejection*
Richard
Horuk
§,
Carol
Clayberger¶
,
Alan M.
Krensky**,
Zhaohui
Wang¶,
Hermann-Josef
Gröne**,
Christian
Weber
,
Kim S. C.
Weber
,
Peter J.
Nelson§§,
Karen
May¶¶,
Mary
Rosser¶¶,
Laura
Dunning¶¶,
Meina
Liang¶¶,
Brad
Buckman¶¶,
Ameen
Ghannam¶¶,
Howard P.
Ng¶¶,
Imadul
Islam¶¶,
John G.
Bauman¶¶,
Guo-Ping
Wei¶¶,
Sean
Monahan¶¶,
Wei
Xu¶¶,
R. Michael
Snider¶¶,
Michael M.
Morrissey¶¶,
Joseph
Hesselgesser
, and
H. Daniel
Perez
From the Departments of
Immunology and
¶¶ Discovery Research, Berlex Biosciences, Richmond,
California 94806, the Departments of ¶ Cardiothoracic Surgery and
Pediatrics, Stanford University, Stanford, California 94305, the
** Department of Experimental Pathology, German Cancer Research Center,
Heidelberg D69111, and the Institutes for

Prophylaxe der Kreislaufkrankheiten and
§§ Medical Poliklinik, University of Munich,
Munich D-80336, Germany
Received for publication, August 16, 2000, and in revised form, October 27, 2000
 |
ABSTRACT |
Chemokines like RANTES appear to
play a role in organ transplant rejection. Because RANTES is a potent
agonist for the chemokine receptor CCR1, we examined whether the CCR1
receptor antagonist BX471 is efficacious in a rat heterotopic
heart transplant rejection model. Treatment of animals with BX471 and a
subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself
ineffective in prolonging transplant rejection, is much more
efficacious in prolonging transplantation rejection than animals
treated with either cyclosporin or BX471 alone. We have examined the
mechanism of action of the CCR1 antagonist in in vitro flow
assays over microvascular endothelium and have discovered that the
antagonist blocks the firm adhesion of monocytes triggered by RANTES on
inflamed endothelium. Together, these data demonstrate a significant
role for CCR1 in allograft rejection.
 |
INTRODUCTION |
The classic signs of acute cellular rejection during organ
transplantation include the infiltration of mononuclear cells into the
interstitium (1). This cellular infiltrate consists mainly of T
lymphocytes, monocytes, and macrophages that are recruited from the
circulation into the transplanted tissue by chemotactic molecules known
as chemokines. Chemokines belong to a large family of small (8-10 kDa)
inducible chemotactic cytokines, which are characterized by a
distinctive pattern of four conserved cysteine residues (2). Currently
over 40 chemokines have been identified and classified into two major
groups, CXC and CC, dependent on the number and spacing of
the first two conserved cysteine residues. The CXC class
members include interleukin
(IL-8),1 melanoma growth
stimulatory activity, and neutrophil-activating peptide-2, whereas the
CC class includes RANTES, monocyte chemotactic protein-1, and MIP-1
(macrophage inflammatory protein-1).
A number of studies have provided evidence for a role for RANTES in
organ transplant rejection, particularly of the kidney. In a model of
reperfusion injury in the rat, RANTES levels were increased over normal
levels and remained high for more than a week, correlating with the
peak of infiltrating macrophages (3). RANTES protein was detected in
infiltrating mononuclear cells, tubular epithelium, and vascular
endothelium of renal allograft biopsy specimens from patients with
cyclosporin nephrotoxicity but not in normal kidney (1). A recent study
suggests that RANTES may play a role in graft atherosclerosis (4).
Increased levels of RANTES (both mRNA and protein) were detected in
mononuclear cells, myofibroblasts, and endothelial cells of arteries
undergoing accelerated atherosclerosis compared with normal coronary
arteries. In another recent renal transplant study, the chemokine
receptor antagonist Met-RANTES when given with low doses of cyclosporin significantly reduced renal injury including interstitial inflammation mainly by reducing the number of infiltrating monocytes (5). Mechanistically this appeared to be achieved by blocking the firm adhesion of these cells to the inflamed endothelium. In summary, these
studies strongly suggest that RANTES, through activation of specific
chemokine receptors on mononuclear cells, may play an important role in
allograft rejection.
Based on these studies, there is strong evidence in support of the
concept that the chemokine RANTES plays an important role in organ
transplant rejection. Because RANTES is a ligand for the chemokine
receptors CCR1 and CCR5, these receptors, located on circulating
mononuclear cells, may be useful therapeutic targets in transplantation
biology. These studies therefore provided the rationale for us to
establish a protocol to inhibit RANTES-mediated biological activities
by developing highly potent and specific nonpeptide CCR1 receptor
antagonists. We have previously identified a number of specific highly
potent CCR1 antagonists (6, 7) and describe here the ability of one of
these compounds, BX471, in combination with low and high doses of
cyclosporin, to significantly prolong the survival time of transplanted
heart in a rat model.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Unlabeled chemokines were human unless otherwise
indicated (Peprotech, Rocky Hill, NJ). 125I-labeled human
chemokines were obtained from PerkinElmer Life Sciences.
Cell Lines--
The human embryonic kidney (HEK) 293 cell line
was obtained from the American Type Culture Collection and was
maintained in RPMI or Dulbecco's modified Eagle's medium containing
10% heat-inactivated fetal bovine serum, 2 mM
L-glutamine, penicillin, and streptomycin in a 5%
CO2 atmosphere at 37 °C. For binding assays, the cells were harvested and washed once with phosphate buffered saline. Cell
viability was assessed by trypan blue exclusion, and cell number was
determined by counting the cells in a hemocytometer.
CCR1 Expression Vector--
Human and rat CCR1 cDNA was
obtained as described (8) and inserted into a mammalian expression
vector containing the SV40 replication origin, the human
cytomegaloviral enhancer with the puromycin-N-acetyltransferase gene (puromycin resistance)
and hygromycin B gene (hygromycin resistance) similar to that described previously (9).
CCR1-expressing Cells--
HEK 293 cells stably expressing human
or rat CCR1 were grown to confluent monolayers in T225-cm2
flasks as described previously (6). Cells were tested for their ability
to bind 125I-labeled MIP-1
and RANTES and biological
responses by changes in intracellular Ca2+ or by microphysiometry.
Chemokine Binding Studies--
The binding assays were performed
either in transfected cells or in peripheral blood mononuclear
cells by centrifugation methods as described previously (6).
Nonspecific binding was determined in the presence of either 100 nM or 1 µM unlabeled ligand. The binding data
were curve fitted with the computer program IGOR (Wavemetrics) to
determine the affinity and number of sites.
Preparation of Stock Solution of BX471--
A 25 mg/ml sterile
saline solution of BX471 in 40% cyclodextrin (Aldrich) was prepared by
dissolving the compound into 40% cyclodextrin in saline. The mixture
was shaken followed by the addition of 230 µL of concentrated HCl.
The mixture was stirred to dissolve the solute. After dissolution was
complete (1 h), the pH of the solution was pH 3.3, and 1 M
KOH was added to raise the pH to 4.5. The solution was filtered through
a 0.45-µm filter and stored at 4 °C.
Determination of Pharmacokinetic Parameters in Rats--
Male
Lewis rats (n = 6) were subcutaneously dosed with BX471
(50 mg/kg, three times per day) in a vehicle of 40%
cyclodextrin/saline for 7 days. Blood samples were collected by cardiac
puncture in EDTA-containing tubes at various times and analyzed for
drug levels as described previously (7).
Heterotopic Heart Transplant Rejection (Lewis or ACI
Rats)--
Adult male, specific pathogen-free ACI (RT1a) and Lewis
(RT1) rats (Charles River, Boston, MA) weighing 200-250 g were used in
these studies. Vascularized cardiac allografts were heterotopically transplanted into the abdomen of recipient rats using a modification (10) of the technique of Ono and Lindsay (11). Abdominal allografts were palpated on a daily basis to assess graft function, and rejection was deemed complete when palpable ventricular contractions ceased.
In Vitro Model System of Monocyte Recruitment on Microvascular
Endothelium under Physiological Flow Conditions--
The interaction
of monocytes with endothelium was studied in laminar flow assays
performed essentially as described (5, 12). Briefly, dermal
microvascular endothelial cells grown to confluence in Petri dishes
were stimulated with IL-1
(10 ng/ml) for 12 h followed by
pre-incubation with RANTES (10 nM) for 30 min at 37 °C
just prior to assay. The plates were assembled as the lower wall in a
parallel wall flow chamber and mounted on the stage of an Olympus IMT-2
inverted microscope with × 20 and × 40 phase-contrast
objectives. Isolated human blood monocytes were isolated by Nycodenz
hyperosmolaric gradient centrifugation as described (12) and
resuspended at 5 × 105 cells/ml in assay buffer
(HBSS) containing 10 mM HEPES, pH 7.4 and 0.5% human
serum albumin. Shortly before the assay, 1 mM
Mg2+ and 1 mM Ca2+ was added. The
cell suspensions were kept in a heating block at 37 °C during the
assay and perfused into the flow chamber at a rate of 1.5 dyn/cm2 for 5 min. For inhibition experiments, monocytes
were preincubated with BX471 at different concentrations (0.1-10
µM) or a Me2SO control for 10 min at
37 °C. The number of firmly adherent cells after 5 min was
quantitated in multiple fields (at least five per experiment) by
analysis of images recorded with a long integration JVC 3CCD video
camera and a JVC SR L 900 E video recorder and were expressed as
cells/mm2. The type of adhesion analyzed was restricted to
primary, i.e. direct interactions of monocytes with endothelium.
Histology, Immunohistochemistry, and Morphometry--
The hearts
were removed under deep anesthesia, quickly blotted free of blood,
weighed, and then processed as needed for histology and
immunohistochemistry. The organs were cut into 1-mm slices and either
immersion-fixed in 4% formaldehyde in phosphate-buffered saline (PBS),
pH 7.35, (PBS: 99 mM NaH2PO4, 108 mM NaH2PO4 and 248 mM
NaCl) for 24 h or fixed in methacarn for 8 h and embedded in
paraffin. Light microscopy was performed on 3-µm sections stained by
periodic acid-Schiff or Goldner-Elastica.
The ED1 monoclonal antibody (Serotec/Camon) was used on methacarn-fixed
paraffin-embedded tissue (3 µm) to stain for rat monocytes/macrophage cells. An alkaline phosphatase anti-alkaline phosphatase detection system was used for visualization (Dako). Controls that omitted the
first or second antibody for each section tested were negative.
Histopathologic Rejection Grading System (Score:
1-3)--
Rejection in the allogeneic rat heart was graded according
to Billingham (13). Mild acute rejection (score: 1) was characterized by a sparse interstitial mononuclear infiltrate often accentuated in
perivascular spaces. Moderate acute rejection (score: 2) was a
moderately dense perimyocytic mononuclear infiltrate with some myocyte
necrosis. Severe acute rejection (score: 3) featured a dense monocytic
infiltrate with focal hemorrhage and replacement of myocytes and with
occasional endothelialitis of intramural arteries. The rejection score
was calculated for every tissue block, and an average score was
calculated from the different blocks for every transplanted heart as
rejection processes tended to be focal.
Radioimmunoassay for Cyclosporin--
Cyclo-Trac SP-whole blood
radioimmunoassay for cyclosporin kits were purchased from DiaSorin
(Stillwater, MN). This method employs a specific monoclonal antibody
that measures only cyclosporin. A methanol extraction step was
performed for the standards, controls, and samples prior to assay. The
methanol extracts were then combined with 125I-labeled
cyclosporine tracer. A mixture of mouse monoclonal specific to
cyclosporin A and the second antibody (donkey anti-mouse) in a single
reagent was added. Following a 1-h incubation, the tubes were
centrifuged, decanted, and then counted. The amount of radioactivity remaining in the pellet was inversely proportional to the concentration of cyclosporin found in the sample. Each sample was assayed in duplicate, and the variation of the duplicates were < 10%,
otherwise the assay for that sample was repeated.
 |
RESULTS AND DISCUSSION |
Because RANTES appears to play an important role in organ
transplant rejection, its receptor, CCR1, is a prime therapeutic target. Empirical screening of our available compound libraries to
discover potential CCR1 antagonists yielded a number of compounds that
were potent human CCR1 antagonists (6). One of these molecules BX471
((R)-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt) had a Ki of 1 nM
for the human receptor (7).
The poor affinity of our CCR1 antagonists for mouse CCR1 (data not
shown) precluded mouse models of disease. Thus, we tested for receptor
binding of BX471 to rat CCR1 receptors by carrying out displacement
binding assays with 125I-MIP-1
as the ligand. Scatchard
analysis of displacement binding studies with BX471 on rat CCR1
revealed that the affinity of the antagonist was 121 ± 60 nM (Fig. 1), ~100 times
less effective for rat CCR1 than for human CCR1. In addition BX471 was
able to competitively displace radiolabeled RANTES from rat CCR1 with a
similar Ki (data not shown). These data demonstrated that although BX471 was not as potent an inhibitor of rat CCR1 as it
was for human CCR1, it could nevertheless compete effectively for
binding to the rat receptor at higher concentrations. Additional studies with rat peripheral blood mononuclear cells revealed that BX471
was able to displace radiolabeled MIP-1
but not MIP-1
, demonstrating no cross-reactivity for rat CCR5 (data not shown).

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Fig. 1.
The CCR1 antagonist, BX471, displaces
radiolabeled MIP-1 from rat CCR1. HEK
cells transfected with rat CCR1 were incubated with
125I-MIP-1 in the presence of increasing concentrations
of BX471. The binding reactions were terminated by centrifugation of
cells as described previously (6). Binding shown represents specific
binding. Nonspecific binding was 10% of total
125I-MIP-1 added. The results shown are from a typical
experiment (n = 3). Inset shows the
Scatchard plot of the displacement data
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We showed that BX471 is a functional antagonist of rat CCR1 by
measuring its ability to inhibit the MIP-1
-induced transient rise in
intracellular Ca2+ concentration in cells expressing rat
CCR1. We measured the change in intracellular Ca2+
concentration in response to various concentrations of MIP-1
by
fluorimetry using the indicator Fura-2. In these experiments, increasing concentrations of MIP-1
produced a transient rise in
intracellular Ca2+ that was inhibited by preincubating the
cells briefly with a 10-fold higher concentration of BX471 (data not
shown). Thus, BX471 is a potent functional antagonist of the rat CCR1
receptor, capable of inhibiting calcium transients that are mediators
of intracellular cell activation.
Based on the studies summarized above, pharmacokinetic studies with
BX471 in rats were carried out. Male Lewis rats were subcutaneously dosed with BX471 (50 mg/kg t.i.d.) in a vehicle of 40%
cyclodextrin/saline, and blood samples were withdrawn at various times
and analyzed for drug levels as described in the legend to Fig.
2. As shown in Fig. 2, peak plasma levels
following the subcutaneous administration of BX471 varied between 12 and 27 µM. Absorption was relatively rapid, with
significant plasma levels observed at 15 min post-drug. After 8 h
(trough time point), plasma drug levels were ~1-2 µM. The plasma half-life ranged between 2-3 h. Though there did not appear
to be any pattern of either enhanced clearance or accumulation of the
drug on repeated subcutaneous t.i.d.-dosing regimen (50 mg/kg), a
considerable amount of variability was observed in the rate and extent
of drug absorption on all of the days measured. These studies showed
that subcutaneous dosing of BX471 at 50 mg/kg t.i.d. seemed to give
adequate drug levels (steady-state BX471 levels observed between 200 and 500 min of each 8-h dosing regimen were 1 to 2 µM)
over a 24-h period and allowed us to test the compound in a rat
heterotopic heart transplant model.

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Fig. 2.
Plasma concentrations of BX471 following
chronic subcutaneous dosing in rats. Male Lewis rats
(n = 6) received BX471 in 40% cyclodextrin s.c. t.i.d.
for 7 days. Blood plasma levels of BX471 were measured as described
previously (7). Data are the mean ± S.E.
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RANTES is a ligand for CCR1 and appears to play a role in directing
mononuclear cells into transplanted tissue leading to inflammation and
rejection (1, 3-5). Thus, we rationalized that a CCR1 antagonist
should have some utility in prolonging the survival of organ
transplants. We tested this hypothesis directly by asking whether the
CCR1 antagonist BX471 was efficacious in a rat heterotopic heart
transplant rejection model. The mean allograft survival of animals
given BX471 alone was 8.8 ± 1.2 days compared with 6.8 ± 0.8 days for control animals with ACI grafts in LEW recipients (Fig.
3). Data from the survival times of the
animals in both studies were statistically evaluated by log rank
analysis. The mean survival times of the animals treated with BX471
were statistically significant at the 0.05 level with respect to the survival times of the control groups and log rank analysis of survival
test gave a p = 0.0048 for BX471 versus
control. Although the increase in survival time for the BX471-treated
animals was modest it was statistically significant and consistent with
what we had observed in other studies (data not shown).

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Fig. 3.
Survival plots of heart transplant
rejection. Cardiac allografts were heterotopically
transplanted into the abdomen of recipient rats (n = 10 per group), and the animals were given: (a) 40%
cyclodextrin s.c. t.i.d., (b) 50 mg/kg BX471 in 40%
cyclodextrin s.c. t.i.d., (c) cyclosporin (CsA)
in olive oil by gavage 10 mg/kg once a day for 4 days, (d)
cyclosporin in olive oil by gavage 2.5 mg/kg for the duration of the
study, (e) as in (c) above plus 50 mg/kg BX471 in
40% cyclodextrin s.c. t.i.d., (f) as in (d)
above plus 50 mg/kg BX471 in 40% cyclodextrin s.c. t.i.d. The
transplanted hearts were evaluated daily for signs of rejection over
the course of the study.
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Cyclosporin is a potent immunosuppressive agent that has been used
extensively to prevent rejection in organ transplantation (14). It
appears to suppress the immune system by inhibiting T cell activation
and proliferation, primarily by blocking the activity of the enzyme
calcineurin and by stimulating the production of the immunosuppressant
TGF-
(15, 16). However, despite its efficacy as an immunosuppressive
agent its use is associated with a progressive loss of renal function,
neurotoxicity, and an increased risk of malignancy (17-19). Thus, a
reduction in the amount of cyclosporin required to achieve sufficient
immunosuppression would be of considerable benefit in transplantion
medicine. For this reason we set out to test whether drug combination
studies in which animals were treated with BX471 and a subtherapeutic dose of cyclosporin, 2.5 mg/kg, which is by itself ineffective in
prolonging transplant rejection, would be more efficacious in
prolonging transplantation than animals treated with either cyclosporin
or BX471 alone.
The mean allograft survival of animals given 2.5 mg/kg of cyclosporin
was 7.3 ± 0.5 days compared with 17.5 ± 5.9 days for animals on the same protocol that were additionally treated with BX471
(Fig. 3). The mean allograft survival of animals given 10 mg/kg of
cyclosporin was 12.9 ± 0.7 days compared with 18.4 ± 5.4 days for animals on the same protocol that were additionally treated
with BX471 (Fig. 3). The mean survival times of the animals treated
with either 2.5 or 10 mg/kg cyclosporin plus the CCR1 antagonist BX471
were statistically significant from the mean survival times of the
animals treated with either 2.5 mg/kg or 10 mg/kg cyclosporin alone
with values of p = 0.0009 and p = 0.0148, respectively. Light microscopy and immunohistology of the rat hearts for infiltrating monocytes confirmed these survival data. Three
days after transplantation the rejection score (Table
I), and the extent of monocytic graft
infiltration were significantly reduced by the combined therapy of
BX471 and cyclosporin A (Figs. 4 and 5).
Based on the data from these studies,
BX471 given in combination with cyclosporin resulted in a clear
increase in efficacy in heart transplantation compared with cyclosporin
alone.
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Table I
Rejection score for transplanted rat hearts
Rejection grade was evaluated using the Billingham grading system (13).
Cyclosporin was administered at 2.5 mg/kg. BX471 was administered at 50 mg/kg. N = number of animals, n = number of tissue blocks examined per group. Mean ± S.E. is shown.
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Fig. 4.
Light microscopy of allogeneic heart
transplants 3 days after transplantation without (A,
B) and with (C, D)
BX471. A, in nonimmunosuppressed transplants a dense
mononuclear infiltrate was observed. Many cardiomyocytes were
vacuolated or necrotic. Interstitial edema was pronounced.
B, in the cyclosporin-treated rats the inflammatory cell
infiltrate was reduced, though still clearly evident, specifically
around venules with focal destruction of cardiomyocytes. C,
BX471-treated rats showed a focal mononuclear cell infiltrates that
were pronounced with similar morphology as to be observed in
nonimmunosuppressed transplants. D, BX471- and
cyclosporin-treated animals showed well preserved cardiac morphology
with sparse mononuclear cell infiltrates. (A-D, HE-stains;
objective × 40).
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Fig. 5.
Immunohistologic stain for ED1-positive
monocytes/macrophages in allogeneic heart transplants 3 days after
transplantation without (A, B) and
with (C, D) BX471. A,
in nonimmunosuppressed transplants, many cells of the dense mononuclear
cell infiltrate consisted of monocytes/macrophages, which were closely
juxtaposed to the cardiomyocytes. B, in the
cyclosporin-treated rats, the inflammatory cell infiltrate was focal
and was composed primarily of ED1-positive cells. C, in the
BX471-treated animals the mononuclear cell infiltrate varied
significantly, aside from the dense inflammatory cell infiltrates as
shown in Fig. 6, areas with moderately dense monocytic infiltrate
around venules were seen. D, combined treatment of BX471 and
cyclosporin resulted in a dramatic reduction in monocyte/macrophage
infiltration into the allogenic rat hearts. (A-D,
APAAP reaction in Methacarn-fixed tissue; objective × 20)
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Although we have shown that treatment with the CCR1 antagonist BX471 in
combination with cyclosporin synergistically increases transplantation
survival in a rat heart model compared with treatment with either drug
alone, it remains a formal possibility that these data are because of
drug/drug interactions that stabilize the blood plasma cyclosporin
levels rather than to the true synergism of the drug combination. To
determine whether BX471 had any effect on cyclosporin plasma levels, we
designed a pharmacokinetic study to assess this. In this study we
examined the effect of BX471 on the blood concentration of cyclosporin
in the rat after a single oral dose (2.5 mg/kg) of cyclosporin followed
by t.i.d. doses of BX471 at 50 mg/kg administered subcutaneously. We
used a radioimmunoassay to measure whole blood cyclosporin levels in
animals treated with cyclosporin in the presence and absence of BX471.
Visual inspection of the time-concentration curves suggested a slight
prolongation of elimination half-life of rats treated with BX471 (Fig.
6). However, statistical analysis of the
paired groups indicated that there was no significant difference
between the two parameters calculated (p-value for AUC was
0.224 and for the t1/2 was 0.317) and thus we can
rule out drug/drug interactions as the basis for the extended
survivability of the transplanted rat hearts.

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Fig. 6.
Effect of BX471 on cyclosporin levels in rat
whole blood. Cyclosporin levels were measured in two groups of six
Lewis cannulated rats. The first group received a single oral dose of
2.5 mg/kg cyclosporin diluted in olive oil. The second group received a
single oral dose of the same cyclosporin solution followed by
subcutaneous injection of BX471 t.i.d. at 50 mg/kg. Whole blood was
collected using EDTA as anticoagulant at 0, 1, 2, 4, 6, 8, 12, and
24 h post-dosing. The cyclosporin concentration in these samples
were analyzed by the Cyclo-Trac radioimmunoassay in rat whole blood as
described under "Experimental Procedures."
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To gain insight into the potential mechanisms of action by which the
CCR1 antagonist BX471 prolonged the rejection of transplanted rat
hearts, we asked whether it could inhibit the firm arrest and
diapedesis of monocytes on microvascular endothelium. It has been
previously shown that perfused monocytic cells show increased attachment to IL-1
-activated DMVEC following preincubation with exogenous RANTES for 30 min (5). Treatment of isolated blood monocytes
with increasing concentrations of BX471 (0.1-10 µM) showed a dose-dependent inhibition of RANTES-mediated and
shear-resistant adhesion on IL-1
-activated microvascular endothelium
in shear flow (Fig. 7A).
Consistently, the percentage of monocytes that were found to undergo or
maintain rolling interactions was dose dependently increased by
pretreatment with BX471, thus serving as an inverse measure for arrest
(Fig. 7B). BX471 also inhibited the RANTES-mediated adhesion
of T lymphocytes to activated endothelium (data not shown). These data
thus strongly suggest that the CCR1 antagonist is a potent antagonist
that can specifically inhibit mononuclear cell adhesion to activated
endothelium. It is unlikely that the CCR1 antagonist is simply
inactivating the cells because e.g. activated T lymphocytes
and monocytes treated with the CCR1 antagonist are still able to
respond to MIP-1
in calcium flux experiments (data not shown)
presumably by binding and activation of CCR5.

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Fig. 7.
Inhibition of in vitro
adhesion of monocytes to activated endothelium by BX471.
DMVEC activated with IL-1 and preincubated with RANTES were inserted
in a flow apparatus, and monocytes were perfused at a constant shear of
1.5 dyn/cm2 as described under "Experimental
Procedures" and in a previous communication (5). For inhibition
assays, monocytes were preincubated with BX471 at indicated
concentrations or a Me2SO control for 10 min at 37 °C.
A, after 5 min of accumulation, the number of adherent cells
was analyzed. Data represent the mean ± S.D. of three individual
experiments. B, as an inverse measure of adhesion, the
number of monocytes rolling at low shear was assessed in the last 30-s
interval of the 5-min period and expressed as the percentage of total
interactions within the field. After 5 min of accumulation, the number
of adherent cells was quantitated and expressed as
cells/mm2. Data represent the mean ± S.D. of three
experiments.
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The dose-responsive inhibition of monocyte arrest on activated
endothelium by BX471 observed here is in line with our previous findings that the CCR1 antagonist BX471 is able to dose responsively inhibit the RANTES- or MIP-1
-induced up-regulation of the
-2 integrin, CD11b (7). A number of studies have revealed that RANTES
treatment of monocytes results in an increased expression of CD11b and
an up-regulation of integrin avidity for their endothelial immunoglobulin family ligands (12, 20, 21). This appears to enhance the
ability of monocytes to bind to endothelial cells, which can be
partially blocked with antibodies to integrins (20). These studies
suggest that modulation of integrin expression and avidity by
chemokines may facilitate the tissue trafficking of monocytes during inflammation.
Two recent studies have demonstrated the important role of CCR1 in
organ transplantation. In the first, the efficacy of Met-RANTES, a
RANTES receptor antagonist that is thought to work in part through a
blockade of CCR1 (5) was examined in a renal transplantation study.
Following the transplantation of Fisher RT1lvl rat kidneys
into Lewis RT1 rats, the animals treated with Met-RANTES showed a
significant reduction in vascular and tubular damage relative to
untreated animals. In a more severe rejection model, where Brown-Norway
RT1 rat kidneys were transplanted into Lewis RT1 rats, Met-RANTES was
found to augment treatment with low dose cyclosporin A to significantly
reduce all aspects of renal injury. In a second study, the importance
of CCR1 was examined in heart transplantation models in mice carrying a
targeted deletion of CCR1(22). In four separate models of allograft
survival, a significant prolongation was seen in CCR1(
/
)
recipients. In one model, levels of cyclosporin that had marginal
effects in CCR1(+/+) mice resulted in permanent allograft acceptance in
CCR1(
/
) recipients.
In this study we have shown that inhibition of CCR1 by the
receptor-specific antagonist BX471 led to a significant prolongation of
cardiac allograft survival (Fig. 3). Furthermore, when the CCR1
antagonist was given in combination with a subnephrotoxic dose of
cyclosporin, it markedly enhanced the survival of the transplanted
organs (Fig. 3). It appears that the mechanism of action of the CCR1
receptor antagonist stems at least in part from its ability to inhibit
the adhesion of activated immune cells to inflamed endothelium (Fig.
7). This may be because of blocking the up-regulation of integrin
expression and adhesiveness that leads to imminent monocyte arrest. The
ability of BX471 to enhance the effects of low dose cyclosporin
treatment has clinical relevance given the dose-dependent
nephrotoxicity associated with cyclosporin therapy. A reduction in the
amount of cyclosporin required to achieve sufficient immunosuppression
could be of considerable benefit not only in transplantation but also
in the treatment of other chronic diseases such as psoriasis, allergy,
arthritis, and other autoimmune diseases.
A significant clinical advantage of BX471 may lie in its effects
directly following transplantation. Early injury to the endothelium, even when it is not lethal, can have serious consequences for survival
of the graft. Even moderate damage can lead to the loss of the
endothelial function required for adequate perfusion of the graft and
result in the enhanced production of proinflammatory cytokines. By
limiting immune effector cell emigration and the ensuing endothelial
damage during transplantation BX471 may lower the inclination toward
the development of more chronic transplant dysfunction and reduce the
requirement for immune suppressants such as cyclosporin. These results
strongly suggest that therapies directed toward the blockade of
chemokine receptors will have a positive effect on solid allograft survival.
 |
ACKNOWLEDGEMENTS |
We thank Babu Subramanyan and Elena Ho
for the cyclosporin determinations and Ron Vergona for the BX471 determinations.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Berlex Biosciences,
Dept. of Immunology, 15049 San Pablo Ave., Richmond, CA 94806. E-mail:
Horuk@pacbell.net.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M007457200
 |
ABBREVIATIONS |
The abbreviations used are:
IL-8, interleukin-8;
RANTES, regulated on activation normal T cell expressed;
HEK, human
embryonic kidney cells;
HBSS, Hanks' buffered saline solution;
t.i.d., three times a day;
MIP, macrophage inflammatory protein;
s.c., subcutaneously.
 |
REFERENCES |
1.
|
Pattison, J.,
Nelson, P. J.,
Huie, P.,
Von, L. I.,
Farshid, G.,
Sibley, R. K.,
and Krensky, A. M.
(1994)
Lancet
343,
209-211[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Schall, T.
(1994)
in
The Cytokine Handbook
(Thompson, A., ed)
, pp. 419-460, Academic Press, San Diego
|
3.
|
Takada, M.,
Nadeau, K. C.,
Shaw, G. D.,
Marquette, K. A.,
and Tilney, N. L.
(1997)
J. Clin. Invest.
99,
2682-2690[Abstract/Free Full Text]
|
4.
|
Pattison, J. M.,
Nelson, P. J.,
Huie, P.,
Sibley, R. K.,
and Krensky, A. M.
(1996)
J. Heart Lung Transplant
15,
1194-1199[Medline]
[Order article via Infotrieve]
|
5.
|
Gröne, H.-J.,
Weber, C.,
Weber, K. S. C.,
Gröne, E. F.,
Rabelinl, T.,
Klier, C. M.,
Wells, T. N. C.,
Proudfoot, A. E.,
Schlöndorff, D.,
and Nelson, P. J.
(1999)
FASEB J.
13,
1371-1383[Abstract/Free Full Text]
|
6.
|
Hesselgesser, J.,
Ng, H. P.,
Liang, M.,
Zheng, W.,
May, K.,
Bauman, J. G.,
Monahan, S.,
Islam, I.,
Wei, G. P.,
Ghannam, A.,
Taub, D. D.,
Rosser, M.,
Snider, R. M.,
Morrissey, M. M.,
Perez, H. D.,
and Horuk, R.
(1998)
J. Biol. Chem.
273,
15687-15692[Abstract/Free Full Text]
|
7.
|
Liang, M.,
Mallari, C.,
Rosser, M.,
Ng, H. P.,
May, K.,
Monahan, S.,
Bauman, J. G.,
Islam, I.,
Ghannam, A.,
Buckman, B.,
Shaw, K.,
Wei, G. P.,
Xu, W.,
Zhao, Z.,
Ho, E.,
Shen, J.,
Oanh, H.,
Subramanyam, B.,
Vergona, R.,
Taub, D.,
Dunning, L.,
Harvey, S.,
Snider, R. M.,
Hesselgesser, J.,
Morrissey, M. M.,
Perez, H. D.,
and Horuk, R.
(2000)
J. Biol. Chem.
275,
19000-19008[Abstract/Free Full Text]
|
8.
|
Neote, K.,
DiGregorio, D.,
Mak, J. Y.,
Horuk, R.,
and Schall, T. J.
(1993)
Cell
72,
415-425[Medline]
[Order article via Infotrieve]
|
9.
|
Perez, H. D.,
Vilander, L.,
Andrews, W. H.,
and Holmes, R.
(1994)
J. Biol. Chem.
269,
22485-22487[Abstract/Free Full Text]
|
10.
|
Nisco, S.,
Vriens, P.,
Hoyt, G.,
Lyu, S. C.,
Farfan, F.,
Pouletty, P.,
Krensky, A. M.,
and Clayberger, C.
(1994)
J. Immunol.
152,
3786-3792[Abstract/Free Full Text]
|
11.
|
Ono, K.,
and Lindsey, E. S.
(1969)
J. Thorac. Cardiovasc. Surg.
57,
225-229[Medline]
[Order article via Infotrieve]
|
12.
|
Weber, K. S.,
von Hundelshausen, P.,
Clark-Lewis, I.,
Weber, P. C.,
and Weber, C.
(1999)
Eur. J. Immunol.
29,
700-712[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Billingham, M. E.
(1990)
Cardiac Transplantation
, Butterworths, Boston
|
14.
|
Kahan, B. D.
(1989)
N. Engl. J. Med.
321,
1725-1738[Medline]
[Order article via Infotrieve]
|
15.
|
Schreiber, S. L.,
and Crabtree, G. R.
(1992)
Immunol. Today
13,
136-142[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Li, B.,
Sehajpal, P. K.,
Khanna, A.,
Vlassara, H.,
Cerami, A.,
Stenzel, K. H.,
and Suthanthiran, M.
(1991)
J. Exp. Med.
174,
1259-1262[Abstract]
|
17.
|
Bennett, W. M.,
DeMattos, A.,
Meyer, M. M.,
Andoh, T.,
and Barry, J. M.
(1996)
Transplant Proc.
28,
2100-2103[Medline]
[Order article via Infotrieve]
|
18.
|
Hojo, M.,
Morimoto, T.,
Maluccio, M.,
Asano, T.,
Morimoto, K.,
Lagman, M.,
Shimbo, T.,
and Suthanthiran, M.
(1999)
Nature
397,
530-534[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Cecka, M.
(1998)
Surg. Clin. North. Am.
78,
133-148[Medline]
[Order article via Infotrieve]
|
20.
|
Vaddi, K.,
and Newton, R. C.
(1994)
J. Immunol.
153,
4721-4732[Abstract/Free Full Text]
|
21.
|
Conklyn, M. J.,
Neote, K.,
and Showell, H. J.
(1996)
Cytokine
8,
762-766[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Gao, W.,
Topham, P. S.,
King, J. A.,
Smiley, S. T.,
Csizmadia, V.,
Lu, B.,
Gerard, C. J.,
and Hancock, W. W.
(2000)
J. Clin. Invest.
105,
35-44[Abstract/Free Full Text]
|
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