By
From the * Biomedical Research Centre and Department of Biochemistry and Molecular Biology, and Department of Oral Biology and Department of Microbiology, University of British Columbia,
Vancouver, British Columbia V6T 1Z3, Canada
An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.
Monocyte chemoattractant protein (MCP1)-1 is a
chemoattractant cytokine (chemokine) (1) that promotes the migration and activation of monocytes (2, 3). It
has been associated with several inflammatory diseases (4),
but a causal relationship has been difficult to prove. Monocyte infiltrates are prominent in rheumatoid arthritis (RA)
and their products, such as cytokines that amplify the inflammatory response and enzymes that destroy connective
tissue (5), are readily detected in diseased joints. MCP-1 is
produced by both synovial cells and infiltrated monocytes in RA (6). Thus, the inhibition of MCP-1 function
could control inflammation by preventing monocyte accumulation in the joints.
To test the antiinflammatory effect of the MCP-1 antagonist, MCP-1(9-76) (9, 10), we chose a mouse model for
RA. Previous studies had shown that murine monocytes
respond to both human and mouse MCP-1 (11). The
MRL-lpr mouse strain was chosen to test the antagonist because it has a genetic predisposition to arthritis with similar
characteristics to human RA including cell infiltration, pannus formation, bone and cartilage breakdown, and the
presence of serum RF (12). The disease normally develops towards the end of the animal's life span (13); however, injection with CFA initiates early onset and increases the severity of arthritis, making the MRL-lpr mouse a practical
experimental model for testing potential therapeutics (12).
The Proteins.
MCP-1, the antagonist, MCP-1(9-76), and the
control peptide, MCP-1Ala, were chemically synthesized and characterized as described (9, 10). MCP-1Ala is an analogue of MCP-1
that had all the cysteines (residue numbers 11, 12, 36, and 52) replaced by alanines.
Arthritis Induction and Treatment.
Both male and female MRL-lpr
mice were used at 13-14 wk of age and were bred at the University of British Columbia (Vancouver, Canada) from stock originally
obtained from the Jackson Labs. (Bar Harbor, ME). On Day 0 of
each experiment, all groups of mice were injected with CFA intradermally into a thoracic and an inguinal site with 0.05 ml CFA
supplemented to 10 mg/ml with heat inactivated Mycobacterium
tuberculosis H37 RA (Difco, Detroit, MI) (12). Either immediately
or after a delay, depending on the experiment, mice were injected
either intravenously, intraperitoneally, daily or not at all, with the
appropriate antagonist or control protein. The chemokine analogue
treatment was continued for 30 d. The ankle width was determined
with a micrometer. For evaluation of the incidence of arthritis,
the symptoms of impaired mobility, presence of erythema, or
swelling were scored as either + or Histopathological Analysis.
At day 30 after CFA priming, the
hind paws were fixed in buffered formalin. After decalcification
in 10% formic acid for 48 h, the tissues were processed for paraffin embedding. Serial sections of the tarso-metatarsal joints were cut
to a thickness of 5 mm and stained with hematoxylin and eosin.
Sections were examined by an individual without knowledge of the
experimental protocol. A minimum of 10 sections/joint were
assessed and scored to provide a semiquantitative measure of subsynovial inflammation (0, normal; 1, focal inflammatory infiltrates;
2, inflammatory infiltrate that dominated the cellular histology),
synovial hyperplasia (0, normal; 1, a continuous, minimum three-layer thick, synovial lining seen in one joint; 2, minimum three-layer thick, synovial lining detected in several joints), pannus
formation and cartilage erosion (0, normal; 1, pannus partially
covered cartilage surfaces without evident cartilage loss; 2, pannus
connected to evident cartilage loss), bone destruction (0, normal;
1, detectable destruction of bone by the pannus or osteoclast activity; 2, the pannus or osteoclast activity had destroyed a significant part of the bone), and finally, overall pathology was the
overall assessment derived by the summation of the values for
these criteria. Statistical analysis of the histopathology indices was
done using the Student's t test.
The human
MCP-1(9-76) antagonist competed for binding of labeled
MCP-1 to receptors on human monocytic cells with a dissociation constant (Kd) of 8.3 nM (9). In vitro it inhibited
MCP-1 (10 nM) with an ID50 of 70 nM (10). When tested
for competition binding of a number of chemokines, the
specificity of the antagonist was similar to that of native
MCP-1. It is likely that the inhibitory effects of the MCP-1
antagonist are due to its binding to the human CC chemokine
receptor (CCR)2 (14). The effects on mouse cells are probably due in part to blocking of the corresponding receptor,
murine CC chemokine receptor 2, which appears to be similar to the human CCR2 in its specificity (15). Using a
murine myelomonocytic cell line, WEHI 274, the Kds of
human MCP-1 and the MCP-1 antagonist were 39 nM
and 58 nM, respectively. For the in vivo experiments, we
injected MCP-1(9-76) so that theoretically there was a 13-fold (0.5 mg/kg) or a 54-fold (2.0 mg/kg) excess over the Kd
measured on mouse cells, estimated on the basis of an average exchangeable fluid volume of 2 ml per mouse. MCP-1 was at a 72-fold excess. The control MCP-1Ala analogue
had the same sequence as the antagonist except that all four
cysteines were replaced by alanines. This analogue lacks the
two essential disulfide bridges (Clark-Lewis, I., and J.-H.
Gong, unpublished data), did not bind to MCP-1 receptor,
and neither induced detectable chemotaxis, nor inhibited
MCP-1 chemotaxis (data not shown). Thus the control,
MCP-1Ala, was neither an agonist nor an antagonist.
To test the effect of the antagonist on the onset of
disease, mice were primed with intradermal CFA on day 0. When the MCP-1 antagonist was given intraperitoneally at
a dose of 2.0 mg/kg daily, it resulted in significant reduction of swelling of the ankle joint (Fig. 1 a). Controls that
received the same dose of a closely related but inactive analogue, MCP-1Ala, developed similar swelling to that of
untreated controls, but showed a trend towards a delayed
onset. Although not significant, the effect was likely to be
due to the daily injection protocol. In subsequent experiments, the controls were all injected with the control peptide, MCP-1Ala. In a separate experiment where the antagonist treatment was stopped at 15 d (Fig. 1 b), onset was
inhibited during the treatment interval, but swelling became apparent by 20 d and rose to untreated control levels
by 24 d. Mice that received a fourfold lower intraperitoneal dose of the antagonist did not have a significant reduction
in swelling, suggesting that for maximal effect, the concentration of antagonist must be maintained at pharmacological levels. Analysis of the sera for anti MCP-1 antibodies
showed that low titers (1:160) were present in both groups
(not shown). The production of anti-MCP-1 antibodies
was not considered a major factor in these studies.
Histological analysis of the ankle joints was performed at day 30 for
all the experiments described. Shown in Fig. 2 are photomicrographs taken from representative antagonist-treated
and control animals, from the experiment described in Fig.
1 a. The effects observed in control mice that were not
given MCP-1 antagonist included infiltration of mononuclear cells into the subsynovial tissue (Fig. 2 a), synovial hyperplasia, pannus formation (Fig. 2 b), and bone erosion
(Fig. 2 c). In contrast, mononuclear cell infiltration and
bone and cartilage pathology was absent in the MCP-1 antagonist-treated example. Only minor thickening of the
subsynovium can be seen (Fig. 2 d). Analysis of the histological results (Fig. 3) for the experiment described in Figure 1 a, indicates that the group that received 2.0 mg/kg
antagonist had significant lower subsynovial inflammation,
synovial hyperplasia, pannus formation and cartilage erosion, bone destruction, and overall histopathology. The histopathology results for the antagonist-treated group compared favorably to age-matched animals that have not been
primed with CFA and have no disease symptoms (not shown).
Interestingly, compared to the controls, the group (Fig. 1 a)
that received 0.5 mg/kg had significantly lower overall histopathology (Fig. 3), even though joint swelling was not
significantly reduced. This suggests that the antagonist is
affecting cellular infiltration and pathology even in the
absence of an apparent effect on externally measurable
symptoms.
To more completely
describe this effect and to provide some insight into possible mechanisms, we examined the effect of full-length functional MCP-1 in this model. Native MCP-1 treatment significantly enhanced the disease, as indicated by the earlier
and higher incidence of disease (Fig. 4 a) and magnitude of
swelling (Fig. 4 b), compared to the inactive control protein. Thus, MCP-1 is causing hyperresponsiveness to arthritis in the MRL-lpr mouse, and is having the opposite effect
to the antagonist when both are compared to the inactive control peptide. In contrast to the experiments described in
Figs. 3 and 6, for this experiment, histopathological analysis
was done 7 d after onset of symptoms and the time varied
from animal to animal. At this time point there was no significant difference in the histopathology between the MCP-1
and control groups, although the trend was toward more
histopathology in the MCP-1 group, which is consistent
with the observed enhancement of swelling (not shown). The MCP-1 antagonist significantly reduced inflammatory
infiltration, hyperplasia, bone destruction, and overall histopathology in this experiment.
Although the experiments described so far
demonstrate that the MCP-1 antagonist is capable of preventing the onset of arthritis, they do not show whether
the antagonist inhibits disease that is already evident. The
postonset situation is a much more stringent test of the ability of the antagonist to inhibit disease, as cell infiltration
and inflammatory events are already going on when the antagonist treatment is started (12). Another reason to address
the postonset effects is that this more closely reflects the
clinical situation where symptoms are already apparent when a patient presents with RA. To test the effects of the antagonist on existing disease, we primed the mice at day 0, but
then delayed treatment until after significant swelling was
apparent. The results for representative individuals from the
control peptide- (Fig. 5 a), and antagonist- (Fig. 5 b) treated
groups are shown. The individuals from the control group
generally had recurrent swelling after symptoms first became apparent. This is typical of the disease in this model.
Individuals from the treated group showed an immediate reduction of swelling and duration of inflammatory episodes. Some degree of relapse of the antagonist-treated animals was apparent at later time points, and the duration and
persistence of this was highly variable. Nevertheless, when
all the animals were taken into account (not shown), the
results indicated that the antagonist greatly reduced the symptoms.
MCP-1 antagonist treatment significantly reduced joint
histopathology by day 28. Thus, synovial hyperplasia, subsynovial inflammation, cartilage erosion, and overall histopathology were significantly reduced in the antagonist treated
group (Fig. 6). This indicates that the external observation
of reduction of swelling in mice treated after disease onset
is reflected in the inflammatory disease in the joints, as
measured by histopathology.
In this study, we have shown that a human MCP-1 receptor antagonist greatly reduces the symptoms and histopathology of chronic arthritis in a disease model. Infiltration of the subsynovium by monocytes is prominent in
human RA, and thus, the diminished cellular infiltration
observed in these studies is probably due to inhibition of
endogenous MCP-1 receptors (see below). The results indicate that the effect of the antagonist is reversible, because when the antagonist treatment was stopped, the swelling
symptoms return. Moreover, antagonist treatment inhibited
the disease after symptoms were already apparent, suggesting that there is turnover of cells that infiltrate the lesion
and that inhibition of further infiltration leads to reversal of
symptoms and pathology.
Native MCP-1 accelerated the onset and enhanced the
symptoms of joint inflammation. Predictions could have
been made, not only for the hyperresponsiveness that was
observed, but also for inhibition similar to that seen with
the antagonist. In vitro, MCP-1 results in migration only if
a gradient is formed and if at high concentrations, MCP-1
inhibits migration by collapsing the gradient (16). Moreover, both the agonist and the antagonist desensitize MCP-1
receptor signaling in vitro (4, 9). Nonresponsiveness has
also been reported in vivo for IL-8 (17). It is unlikely that
the enhancement we have observed in vivo with MCP-1 is
simply nonspecific, since the control protein, which is similar chemically, did not cause this effect. Rather, it is more
likely that the systemic levels of MCP-1 in these mice are
insufficient to cause nonresponsiveness of the target cells to
MCP-1. Our interpretation for the enhancement of the onset and swelling is that the injected MCP-1 accumulates in
the tissues and causes activation of monocytes and other target cells, whereas the MCP-1 that remains in the vascular
compartment is eliminated rapidly. A gradient of MCP-1 from the ablumenal side (high) to the lumenal side of the
endothelium (low) could result in migration of responsive
cells from the blood into the tissues. Monocyte activation
could lead to some of the pathological effects observed
with MCP-1.
The results suggest that the in vivo action of the antagonist is dependent on its receptor binding and its inability to
cause activation. However, it cannot be directly proven
that the receptor blocking competition that occurs in vitro,
is also the mechanism in vivo. A case could be made for a
more complex mechanism. Physiologically, the CC chemokine receptor system is redundant in that multiple CCRs,
which each bind several ligands, are coexpressed (18). Thus, other chemokines and their receptors can induce
similar activities in vitro. Since only CCR2 receptor is inhibited by the MCP-1 antagonist, then the question arises:
why are we not seeing migration in response to other
chemokines, such as RANTES, that bind to CCR1, CCR3,
CCR4, and CCR5, but not CCR2 (18)? RANTES is also
produced in inflamed joints along with the MCP-1 (19).
The answer is not known. However we have found that a RANTES antagonist, RANTES (9-68), also inhibits the
onset of arthritis in the model described here and, to a similar extent, as the MCP-1 antagonist. The fact that two antagonists that bind different receptors have similar effects
suggests that the independence of the receptor actions that
is observed in vitro is not directly translated to the in vivo
situation. It is possible that the long-term presence of antagonist not only prevents cells from responding to chemokine, but also prevents migration by another mechanism. The antagonists do not stimulate detectable receptor signaling, but likely promote receptor internalization, and there
may be separate negative regulatory effects caused by receptor occupancy. Some of the possible mechanisms for
nonresponsiveness of chemokines have been reviewed (4).
Some that could apply to the MCP-1 antagonist are heterologous desensitization of receptors, failure to stimulate adhesion molecules (20), changes to the cytoskeleton (21), or
general interference of the formation of endogenous chemokine gradients due to saturation of glycosaminoglycan interaction sites (22). Another possibility is that chemokine
production is turned off by the presence of excess antagonist. This could occur if MCP-1-responsive cells have not
migrated, and therefore cannot amplify chemokine and cytokine production. Further work will be necessary to determine the in vivo mechanisms of chemokine antagonist
action. Nevertheless, our results indicate that the in vitro
patterns of receptor specificity and chemokine function do
not always correspond to in vivo effects.
Infiltrated monocytes are thought to be important in the
pathology of RA (5). Although MCP-1 primarily acts on
monocytes, it is also known to stimulate basophils (23) and
T lymphocytes (24), indicating that these cells could also be
stimulated by MCP-1 and/or be inhibited by the MCP-1
antagonist. Whatever target cells are important in this arthritis model, the results suggest that blocking MCP-1 receptors breaks a critical link in the chain of inflammatory
events. Furthermore the antagonist prevents arthritis onset
and also alleviates existing disease, suggesting the potential for MCP-1 antagonists or other cytokine inhibitors in the
therapy of the human disease. Antibody therapy represents
an alternative approach to inhibition of ligand function
(25). Beneficial effects in RA of antibodies that block tumor necrosis factor- Receptor antagonists that inhibit ligand binding and
function is a conventional pharmacological approach that
relies on competition between the antagonist and the natural agonist(s) for specific receptor binding sites. Antagonists
and agonists for seven transmembrane receptors, a class that
includes all the chemokine receptors, form the basis for
many widely used pharmaceuticals (27). Targeted receptors
include, for example, those for histamine, epinephrine, and
serotonin. Most of these antagonists are nonpeptide in nature and are orally active, a major advantage for therapeutic use. Nevertheless, nonpeptide antagonists of peptide ligands, including neurokinins, cholecystokinin, and angiotensin, have
also been developed (27). Despite the fact that chemokines
are larger than these peptides, our results have indicated
that the major binding site on MCP-1 and other chemokines is relatively small (9, 28), suggesting that nonpeptide
antagonists for chemokine receptors may be a future possibility.
. Statistical analysis of the incidence was carried out with the one-tailed Fisher Exact test. For
quantifying swelling, ankle widths were measured with a micrometer. The statistical comparison of paired sets of ankle width
measurements was carried out using the Student's t test.
Receptor Interactions of the MCP-1 Antagonist.
Fig. 1.
The MCP-1 antagonist prevents the onset of the symptoms
of arthritis. The ankle widths for both hind legs of each animal were measured with a micrometer on the indicated days, and the results are presented as the mean change from the day 0 measurement, ± SEM. *Values
are significantly different from the control MCP-1Ala analogue (P
<0.05). (a) MRL-lpr mice received CFA on day 0 and were injected intraperitoneally daily for 30 d with either MCP-1 antagonist, 2.0 mg/kg,
n = 23, ; MCP-1 antagonist, 0.5 mg/kg, n = 9,
; control MCP-1Ala
analogue, 2.0 mg/kg, n = 10,
; or no further treatment, n = 20,
. (b)
Mice were either injected intravenously daily for 15 d with MCP-1 antagonist, 0.5 mg/kg, n = 6,
; or received no further treatment, n = 8,
. Results shown are from one of two similar experiments.
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
The effects of MCP-1 antagonist on the joint histopathology. Representative photomicrographs of histology from the experiment in Fig. 1
a are shown. For the control group: (a) marked subsynovial infiltration by mononuclear cells resembling rheumatoid nodule formation (×20 objective);
(b) pannus formation and synovial hyperplasia (×40) objective; (c) bone erosion (arrow; ×20 objective). For the antagonist (2.0 mg/kg) -treated group: (d)
indicates some stromal thickening of the subsynovium, but absence of infiltrating cells or joint destruction (×20 objective). as, articular surface; b, bone;
bm, bone marrow; bv, blood vessel; jc, joint cavity; mc, mononuclear cell infiltrate; p, pannus; sh, synovial hyperplasia; si, subsynovial inflammation; ss,
subsynovium.
[View Larger Version of this Image (148K GIF file)]
Fig. 3.
Summary of the effects of MCP-1 antagonist on joint histopathology. Shown are the mean ± the SEM of the scores, from the experiment in Fig. 1 a, determined as described in Materials and Methods. *Groups that were significantly different (P <0.05) from the control MCP-1Ala analogue for each histopathological parameter.
[View Larger Version of this Image (31K GIF file)]
Fig. 4.
The effects of the functional MCP-1 agonist on disease incidence and joint swelling. The mice were given CFA treatment at day 0 and divided into three groups. The groups received daily injection of 2 mg/kg of either native MCP-1 (1-76), n = 24, ; MCP-1 antagonist,
n = 19,
; or the inactive control peptide, n = 23,
. (a) The incidence
as percentage of mice that showed visible erythema or swelling on the days
indicated. (b) The change in the ankle widths (mean ± SEM). *Significantly different from controls (P <0.05).
[View Larger Version of this Image (23K GIF file)]
Fig. 6.
The effect of postonset treatment with MCP-1 antagonist on
joint histopathology. Histopathological assessments of the experiment outlined in Fig. 5 were made 28 d after CFA priming. All the mice that developed significant swelling are included. The data is presented as for
Fig. 3. Shown are the mean ± SEM of the assessment values for the indicated parameters. *The antagonist and the controls are significantly different, P <0.05.
[View Larger Version of this Image (20K GIF file)]
Fig. 5.
The effect of treatment with MCP-1 antagonist after disease
onset. Animals were injected with CFA once at day 0 and examined daily
for the visual appearance of erythema and swelling around the joints as
measured with a micrometer. 23 of 40 mice developed swelling at different times, and they were injected daily with 2.0 mg/kg of either the
MCP-1 antagonist or the MCP-1Ala control, on the day indicated by the
arrows, until day 28. (a) The swelling measurements of three representative examples from the 13 controls (,
,
). (b) Three representative
examples from the 10 antagonist-treated mice (
,
, X).
[View Larger Version of this Image (29K GIF file)]
activity have been described (26).
However, a potential disadvantage of the antibody approach in typical pathological situations is that targeting just
one ligand may not be effective. On the other hand, with
the antagonist approach, all the ligands for the receptor are
blocked. For example, the MCP-1 antagonist described
here blocks not only MCP-1 activity (9), but also MCP-2
(Gong, J.-H. and I. Clark-Lewis, unpublished observations) and MCP-3 (10).
Address correspondence to Dr. Ian Clark-Lewis, Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Phone: 604-822-7805; FAX: 604-822-7815; E-mail: ian{at}brc.ubc.ca
Received for publication 28 October 1996 and in revised form 2 April 1997.
1Abbreviations used in this paper: CCR, human CC chemokine receptor; MCP, monocyte chemoattractant protein; RA, rheumatoid arthritis.We wish to thank Luen Vo, Jennifer Anderson, Philip Owen, and Peter Borowski for their technical assistance with the chemical synthesis.
This work was supported by grants from the Arthritis Society of Canada and the British Columbia Health Research Fund. I. Clark-Lewis is the recipient of a Scientist Award from the Medical Research Council of Canada.
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