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The chemokine receptors CXCR1CXCR2 modulate antigen-induced arthritis by regulating adhesion of neutrophils to the synovial microvasculature.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 8, August 2008, pp 2329–2337
DOI 10.1002/art.23622
© 2008, American College of Rheumatology
The Chemokine Receptors CXCR1/CXCR2 Modulate
Antigen-Induced Arthritis by Regulating Adhesion of
Neutrophils to the Synovial Microvasculature
Fernanda M. Coelho,1 Vanessa Pinho,1 Flávio A. Amaral,1 Daniela Sachs,1 Vı́vian V. Costa,1
David H. Rodrigues,1 Angélica T. Vieira,1 Tarcı́lia A. Silva,1 Daniele G. Souza,1
Riccardo Bertini,2 Antônio L. Teixeira,1 and Mauro M. Teixeira1
Objective. The chemokine receptors CXCR1 and
CXCR2 play a role in mediating neutrophil recruitment
and neutrophil-dependent injury in several models of
inflammation. We undertook this study to investigate
the role of these receptors in mediating neutrophil
adhesion, subsequent migration, and neutrophildependent hypernociception in a murine model of monarticular antigen-induced arthritis (AIA).
Methods. AIA was induced by administration of
antigen into the knee joint of previously immunized
mice. Intravital microscopy studies were performed to
assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic
pressure meter. Neutrophil accumulation in the tissue
was measured by counting neutrophils in the synovial
cavity and assaying myeloperoxidase activity. Levels of
tumor necrosis factor ␣ (TNF␣) and the chemokines
CXCL1 and CXCL2 were quantified by enzyme-linked
immunosorbent assay. Histologic analysis was per-
formed to evaluate the severity of arthritis and leukocyte infiltration.
Results. Antigen challenge in immunized mice
induced production of TNF␣, CXCL1, and CXCL2 and
also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment
with reparixin or DF2162 (allosteric inhibitors of
CXCR1/CXCR2) decreased neutrophil recruitment, an
effect that was associated with marked inhibition of
neutrophil adhesion. Drug treatment also inhibited
TNF␣ production, hypernociception, and the overall
severity of the disease in the tissue.
Conclusion. Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the
adhesion of neutrophils to synovial microvessels. As a
consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies
suggest a potential therapeutic role for the modulation
of CXCR1/CXCR2 receptor signaling in the treatment
of arthritis.
Supported by the Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico (CNPq, Brazil), the Fundação do Amparo a
Pesquisas do Estado de Minas Gerais (FAPEMIG, Brazil), and the
European Union’s Sixth Framework Programme (INNOCHEM
project grant LSHB-CT-2005-518167).
1
Fernanda M. Coelho, MSc, Vanessa Pinho, PhD, Flávio A.
Amaral, MSc, Daniela Sachs, PhD, Vı́vian V. Costa, PhD, David H.
Rodrigues, MSc, Angélica T. Vieira, MSc, Tarcı́lia A. Silva, PhD,
Daniele G. Souza, PhD, Antônio L. Teixeira, MD, PhD, Mauro M.
Teixeira, MD, PhD: Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais, Brazil; 2Riccardo Bertini, PhD: Dompé
Pharma SpA Research Center, L’Aquila, Italy.
Address correspondence and reprint requests to Mauro M.
Teixeira, MD, PhD, Departamento de Bioquı́mica e Imunologia,
Instituto de Ciências Biológicas, Universidade Federal de Minas
Gerais, Avenida Antônio Carlos, 6627 Pampulha, 31270-901 Belo
Horizonte, Minas Gerais, Brazil. E-mail: mmtex@icb.ufmg.br.
Submitted for publication December 28, 2007; accepted in
revised form April 14, 2008.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints that affects 0.5–1.0% of the
adult population worldwide and is associated with significant morbidity (1,2). Cytokines are directly implicated in many of the immune processes that are associated with the pathogenesis of RA. In recent years, the
targeted blockade of these cytokines has been a major
therapeutic advance in the management of RA. Nevertheless, cytokine-based therapy must be administered via
a systemic route, current treatments are costly, and many
patients fail to respond to blockade of either tumor
necrosis factor ␣ (TNF␣) (3) or interleukin-1␤ (4).
Moreover, minor adverse events, including injection-site
2329
2330
COELHO ET AL
reactions, are common (5), and susceptibility to serious
infection is a commonly reported risk (6,7). Thus, new
therapeutic options for the treatment of arthritis are
clearly needed.
The neutrophil is the most abundant of all leukocytes in the joints of patients with active RA (8,9) and
yet is a relatively understudied cell in this disease.
Neutrophils are attracted into diseased joints by the
chemoattractants commonly detected in rheumatoid synovial fluid (8,9). More direct evidence for the involvement of neutrophils in the pathogenesis of RA has come
from studies of animal models of disease (9–11). Indeed,
recent evidence has shown a key role for neutrophils in
both the initiation and progression of the disease in the
K/BxN mouse model (9).
Among the mediators of inflammation that have
been shown to activate neutrophils and induce their
recruitment in vivo, much interest has been placed on
the role of CXC chemokines (12). Previous studies
demonstrated that blockade of the action of Glu-LeuArg motif–positive CXC chemokines or their receptors,
CXCR1 and CXCR2, appears to be a valid strategy for
the treatment of neutrophil-associated injuries in several
models of inflammation (10,12–15).
We hypothesized that CXCR1/CXCR2 would be
a major inducer of neutrophil adhesion to the synovial
microvascular endothelium and hence would mediate
the migration of these cells into the joints, in an antigeninduced arthritis (AIA) mouse model. As a corollary of
this hypothesis, blockade of CXCR1/CXCR2 would be
accompanied by inhibition of neutrophil accumulation
in the joints, leading to prevention of neutrophildependent joint injury in this mouse model. To test our
hypothesis, we investigated the effect of treatment with
the CXCR1/CXCR2 allosteric inhibitor reparixin
(12,13) and its long-acting derivative, DF2162 (10), in
mice with AIA.
MATERIALS AND METHODS
Animals. Eight-to-10–week-old male C57BL/6J (wildtype) mice were obtained from the Centro de Bioterismo of
the Universidade Federal de Minas Gerais (UFMG) in Brazil
and kept in the animal facilities of the Laboratório de Imunofarmacologia, Department of Biochemistry and Immunology
at UFMG. The mice were maintained with filtered water and
food ad libitum in a controlled environment (stable temperature and humidity). All animal care and handling procedures
were in accordance with the guidelines of the International
Association for the Study of Pain (16), and all experiments
received prior approval from the UFMG ethics committee
(certificate 166/2006).
Arthritis induction. The mice were immunized intradermally at the base of the tail with 500 ␮g of methylated
bovine serum albumin (mBSA; Sigma, St. Louis, MO) in 100 ␮l
of an emulsion of saline and an equal volume of Freund’s
complete adjuvant (CFA; Sigma) on day 0 (17). Fourteen days
later, antigen challenge was performed in the mice. Each
mouse received an injection of 10 ␮g of mBSA (10 ␮g mBSA
in 10 ␮l sterile saline) in the left knee joint. At different time
points (6 hours, 24 hours, 48 hours, and 7 days) after antigen
challenge, the mice were killed. The knee cavity was washed
with phosphate buffered saline (PBS) (2 ⫻ 5 ␮l), and the
periarticular tissue was removed from the joint for evaluation
of cytokines, chemokines, and myeloperoxidase (MPO) activity.
The total number of leukocytes in the tissue was
determined by counting the leukocytes in a Neubauer chamber
after staining tissue samples with Turk’s solution. Differential
counts were obtained from cytospin preparations (Shandon
III; Thermo Shandon, Frankfurt, Germany) by evaluating the
percentage of each leukocyte on a slide stained with MayGrünwald-Giemsa stain.
Experimental protocol. Initial experiments evaluated
the kinetics of neutrophil influx, using intravital microscopy,
and also evaluated cytokine production after intraarticular
antigen challenge in immunized mice. To evaluate the role of
CXCR1/CXCR2, mice were treated with reparixin (30 mg/kg
subcutaneously [SC]; Dompé Pharma, L’Aquila, Italy) 40
minutes before and 3 and 6 hours after challenge with antigen.
Control mice received saline (200 ␮l SC). Preliminary experiments in mice (13) have shown that a dose of 30 mg/kg
reparixin is the maximally effect dose for preventing neutrophil
influx.
The levels of cytokines and chemokines and the number of neutrophils were evaluated at 24 hours after antigen
challenge. Hypernociception related to joint inflammation was
evaluated 6 and 24 hours after antigen challenge. In order to
evaluate the role of CXCR1/CXCR2 in mediating neutrophil
rolling and adhesion to the synovial microvasculature, intravital microscopy was carried out 24 hours after challenge,
preceded 30 minutes prior to the procedure by treatment with
reparixin (30 mg/kg intravenously [IV]) or saline (100 ␮l IV, as
control).
Further experiments were carried out using the longacting reparixin analog DF2162 (10). DF2162 was resuspended
in 0.05% carboxymethylcellulose and administrated orally at a
dose of 15 mg/kg, using an administration schedule similar to
that for reparixin. All parameters in mice treated with DF2162
were evaluated 24 hours after antigen challenge.
Intravital microscopy of the knee joint. Intravital microscopy was performed in the synovial microcirculation of the
mouse knee, as described previously (18). Briefly, the left hind
limb was placed on a stage, with the knee slightly flexed and
the patellar tendon mobilized and partly ressected. The intraarticular synovial tissue of the knee joint was then visualized
for the determination of leukocyte rolling and adhesion.
A 20-fold objective was used to select 2–4 regions of
interest in each mouse. To measure the leukocyte–endothelial
cell interactions, the fluorescent marker rhodamine 6G
(Sigma) was injected IV as a single bolus of 0.15 mg/kg
immediately before the measurements. Rhodamine epiilumination was achieved with a 150W variable HBO mercury lamp
in conjunction with a Zeiss filter set 15 (546/12-nm band-pass
CXCR1/CXCR2 CHEMOKINE RECEPTOR BLOCKADE IN AIA
filter, 580-nm Fourier transforms, 590-nm late potentials;
Zeiss, Wetzlar, Germany). The microscopic images were captured with a video camera (5100 HS; Panasonic, Secaucus, NJ)
and recorded on an S-VHS videotape, using both filter blocks
consecutively. Data analysis was performed off-line.
Rolling leukocytes were defined as those cells moving
slower than the cells moving at a regular flux in a given vessel.
The flux of rolling cells was measured as the number of rolling
cells passing by a given point in the venule per minute, with
results expressed as cells/minute. A leukocyte was considered
to be adherent if it remained stationary for at least 30 seconds,
and total leukocyte adhesion was quantified as the number of
adherent cells within a 100-␮m length of venule, with results
expressed as cells/mm2.
Evaluation of hypernociception. Mice were placed in a
quiet room in acrylic cages (12 ⫻ 10 ⫻ 17 cm in height) with
a wire-grid floor for 15–30 minutes, before testing for environmental adaptation. Stimulations were performed only when
the mice were in quiet conditions, i.e., without exploratory
movements or defecation and not resting on their paws. In
these experiments, an electronic pressure meter was used. This
apparatus consisted of a hand-held force transducer fitted with
a polypropylene tip (Insight Instruments, Ribeirão Preto, San
Paulo, Brazil). For the present model, a large tip (4.15 mm2)
was adapted to the probe (19,20). An increasing perpendicular
force was applied to the central area of the plantar surface of
the hind paw to induce dorsal flexion of the femorotibial joint,
followed by withdrawal of the paw. A tilted mirror below the
grid provided a clear view of each animal’s hind paw. The
electronic pressure meter automatically recorded the intensity
of the force applied when the paw was withdrawn, with results
expressed as the flexion-elicited withdrawal threshold (in
grams). The test was repeated until 3 measurements yielded
consistent results (i.e., the variation among these measurements was lower than 0.5 gm).
Hypernociception was tested before and after injection
of saline or antigen, with results expressed as the change in the
withdrawal threshold. This was calculated by subtracting the
zero-time mean measurements from the time-interval mean
measurements. The mean ⫾ SEM withdrawal threshold was
12.8 ⫾ 0.5 gm (n ⫽ 30) at the zero-time measurement (i.e.,
before injection of hypernociceptive agents)
Histology. The knee joint was removed and fixed for 12
hours with 8% paraformaldehyde (pH 7.2). The joints were
then incubated in 20% EDTA at pH 7.2 for 3 days at room
temperature to decalcify the bone. Samples were washed with
PBS and dehydrated. After being embedded in paraffin, the
joints were sliced into 3-␮m–thick sections that were stained
with hematoxylin and eosin (H&E). To eliminate potential
bias, the slides were scored by 2 independent observers (FMC
and VP). The sections were graded subjectively using various
parameters, as follows: severity of synovial hyperplasia (pannus formation), cellular exudate, and cartilage depletion/bone
erosion (each scored 0 [normal] to 3 [severe]), and extent of
synovial infiltrate (scored 0–5, with higher scores indicating
greater infiltration). The grades for all parameters were subsequently summed to obtain an arthritis index, with results
expressed as the median arthritis score (21).
Quantification of neutrophil accumulation in the tissue. The extent of neutrophil accumulation in the mouse tissue
was measured by assaying MPO activity, using a technique
2331
routinely performed in our laboratory (10,22). Briefly, the
knee joint was removed and frozen at ⫺70°C. Upon thawing of
the sample, the tissue (0.1 gm of tissue per 1.9 ml of buffer) was
homogenized and processed for determination of MPO activity. The assay included 25 ␮l of 3,3⬘-5,5⬘-tetramethylbenzidine
(Sigma) in PBS (pH 5.4) as the color reagent. The number of
neutrophils in each sample was calculated with reference to a
standard curve of the number of neutrophils obtained from the
peritoneal cavity of 5% casein–treated mice processed in the
same manner, with results in the synovial tissue expressed as
the relative number of neutrophils per milligram of tissue wet
weight. Using this method allows the test to be specific for
neutrophils, as opposed to macrophages and lymphocytes
(results not shown).
Measurement of cytokines and chemokines in periarticular tissue. The concentrations of TNF␣ and the chemokines CXCL1 (also known as keratinocyte-derived chemokine)
and CXCL2 (also known as macrophage inflammatory protein
2) were measured in the periarticular tissue using a commercially available enzyme-linked immunosorbent assay (ELISA),
following the instructions supplied by the manufacturer (DuoSet kits; R&D Systems, Minneapolis, MN). Briefly, 100 mg of
tissue was homogenized in 1 ml of PBS (0.4M NaCl and 10 mM
NaPO4) containing antiproteases (0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA,
and 20 kallikrein inhibitor units of aprotinin A) and 0.05%
Tween 20. The samples were then centrifuged for 10 minutes
at 3,000g, and the supernatant was immediately assessed by
ELISA at a 1:3 dilution in PBS. All samples were assayed in
duplicate.
Figure 1. Kinetics of tissue inflammation and cytokine/chemokine
expression in a mouse model of antigen-induced arthritis. The numbers of neutrophils in the synovial cavity (A) and relative units of
neutrophils in the periarticular tissue, as determined with a myeloperoxidase assay (B), were assessed at various times after injection of 10
␮g of methylated bovine serum albumin (mBSA) or 10 ␮l sterile saline
(control) into the knee joint of immunized animals. The concentrations of CXCL1 (C) and tumor necrosis factor ␣ (TNF␣) (D) in the
periarticular tissue were assessed by enzyme-linked immunosorbent
assay after induction of arthritis. Bars show the mean and SEM results
from 5 mice per group. ⴱ ⫽ P ⬍ 0.01 versus control mice.
2332
COELHO ET AL
parixin was dissolved in saline, and DF2162 was dissolved in
0.05% carboxymethylcellulose.
Statistical analysis. Results are expressed as the
mean ⫾ SEM. Differences between groups were evaluated by
analysis of variance followed by Student’s t-tests and NewmanKeuls post hoc tests. P values less than 0.05 were considered
significant.
Figure 2. Kinetics of the interaction between leukocytes and endothelial cells in the synovial microvasculature. Rolling (A) and adhesion
(B) of leukocytes to the synovial endothelium were assessed at various
times after injection of 10 ␮g of methylated bovine serum albumin
(mBSA) or 10 ␮l sterile saline (control) into the knee joint of
immunized mice. The flux of rolling cells was measured as the number
of rolling cells passing by a given point in the venule per minute. A
leukocyte was considered to be adherent if it remained stationary for
at least 30 seconds, and total leukocyte adhesion was quantified as the
number of adherent cells within a 100-␮m length of venule. Bars show
the mean and SEM results from 5 mice per group. ⴱ ⫽ P ⬍ 0.01 versus
control mice.
Drug preparations. Reparixin (R-2-[4-isobutylphenyl]propionyl methylsulfonamide) and DF2162 were synthesized in the Department of Chemistry at Dompé Pharma.
Methylated BSA and CFA were purchased from Sigma. Re-
RESULTS
Kinetics of joint inflammation in the AIA model.
Initial experiments evaluated the kinetics of joint inflammation after intraarticular challenge with antigen in
immunized mice. There was an increase in the number
of neutrophils in the synovial cavity (Figure 1A) and in
the relative number of neutrophils in the periarticular
tissue (Figure 1B), which was first detected 6 hours after
challenge and peaked at 24 hours after challenge. Neutrophil accumulation in the joint was still observed after
48 hours and subsided by 7 days after antigen challenge
(Figure 1A). In the periarticular tissue, neutrophil accumulation returned to baseline levels by 48 hours after
challenge (Figure 1B).
Neutrophil influx was preceded by an increase in
Figure 3. Effects of treatment with reparixin on neutrophil recruitment and joint inflammation after induction of arthritis in mice. Reparixin (30
mg/kg subcutaneously [SC]) or vehicle (200 ␮l saline SC) was administered 40 minutes before and 3 and 6 hours after induction of arthritis. The
numbers of neutrophils in the synovial cavity (A) and relative units of neutrophils in the periarticular tissue, as determined with a myeloperoxidase
assay (B), were assessed 24 hours after arthritis induction with 10 ␮g of methylated bovine serum albumin (mBSA) or injection of 10 ␮l sterile saline
(control) into the knee joints of immunized mice. Bars show the mean and SEM results from 5 mice per group. The arthritis score in the knee joints
of vehicle-treated and reparixin-treated mice (C) was graded in a blinded manner, as described in Materials and Methods. Results are the median
of 4–5 mice per group. ⴱ ⫽ P ⬍ 0.05 versus control mice; # ⫽ P ⬍ 0.05 versus vehicle-treated arthritic mice. Sections of the knee joints were stained
with hematoxylin and eosin (D) to assess histopathologic features in the control mice (i), vehicle-treated arthritic mice (ii), and reparixin-treated
arthritic mice (iii) at 24 hours after induction of arthritis or injection of sterile saline as control (original magnification ⫻ 40). Insets,
Higher-magnification (original magnification ⫻ 400) views of the regions indicated by arrows in panels i, ii, and iii. Representative results are shown.
CXCR1/CXCR2 CHEMOKINE RECEPTOR BLOCKADE IN AIA
the levels of CXCL1 in the periarticular tissue (Figure
1C). Levels of CXCL1 peaked at 6 hours and dropped to
baseline levels by 48 hours after challenge. Levels of
CXCL2 in the periarticular tissue followed a similar
expression pattern (mean ⫾ SEM 343 ⫾ 44 pg per 100
mg tissue after PBS challenge versus 1,107 ⫾ 207 pg per
100 mg tissue at 6 hours, 774 ⫾ 42 pg per 100 mg tissue
at 24 hours, 831 ⫾ 203 pg per 100 mg tissue at 48 hours,
and 862 ⫾ 188 pg per 100 mg tissue at 7 days after
antigen challenge [n ⫽ 5 per group]; P ⬍ 0.05). The
concentration of TNF␣ in the periarticular tissue peaked
at 48 hours and was still detectable by 7 days after
antigen challenge (Figure 1D).
Leukocyte–endothelial cell interactions in the
synovial microcirculation were also investigated. As seen
in Figures 2A and B, intraarticular antigen challenge in
immunized mice was accompanied by an increase in
leukocyte rolling and adhesion, which was mostly seen
within the first 48 hours after challenge. Histologic
analysis of H&E-stained joint sections from the mice
with AIA demonstrated a dense infiltration of neutrophils in the synovium, as well as synovial hyperplasia at
24 hours after challenge (results not shown). Further
experiments evaluating the effects of CXCR2 receptor
antagonists were conducted in the joints primarily at 24
hours after administration of antigen.
Inhibition of joint inflammation by CXCR2 antagonists in the AIA model. Treatment with reparixin
greatly reduced antigen-induced recruitment of neutrophils into the synovial cavity (Figure 3A) and into the
periarticular tissue (Figure 3B). Results of quantification of the histologic features concurred with the qualitative aspects of the synovial tissue, and there was a
significant reduction in the arthritis score in reparixintreated animals (Figure 3C). Histopathologic assessment
of the tissue sections showed that treatment with reparixin reduced the inflammatory exudate, especially in
perivascular regions (Figure 3D).
Treatment with reparixin did not modify the
levels of antigen-induced CXCL1 in the joints of mice
with AIA (Figure 4A) but did decrease the levels of
CXCL2 (mean ⫾ SEM 645 ⫾ 108 pg per 100 mg tissue
in control [untreated] mice versus 386 ⫾ 58 pg per 100
mg tissue in vehicle [PBS]–treated mice and 398 ⫾ 38 pg
per 100 mg tissue in reparixin-treated mice [n ⫽ 5
animals per group]; P ⬍ 0.05). The compound also
reduced the antigen-induced release of TNF␣ in the
periarticular tissue at 24 hours after challenge (Figure
4B).
In addition to the local production of cytokines
and influx of neutrophils, there was significant hyperno-
2333
Figure 4. Effects of treatment with reparixin on the levels of CXCL1,
levels of tumor necrosis factor ␣ (TNF␣), and intensity of hypernociception after induction of arthritis in mice. Reparixin (30 mg/kg
subcutaneously [SC]) or vehicle (200 ␮l saline SC) was administered 40
minutes before and 3 and 6 hours after injection of 10 ␮g of
methylated bovine serum albumin (mBSA) or 10 ␮l sterile saline
(control). The concentrations of CXCL1 (A) and TNF␣ (B) in the
periarticular tissue were assessed by enzyme-linked immunosorbent
assay at 24 hours after induction of arthritis. The time–response curve
of hypernociception after induction of arthritis was evaluated using an
electronic pressure meter test at 1–144 hours after intraarticular (IA)
injection of mBSA or saline vehicle (C). Mice were treated with
reparixin (30 mg/kg SC), DF2162 (15 mg/kg orally), or vehicle 40
minutes before and 3 and 6 hours after induction of arthritis, and the
intensity of hypernociception was evaluated at 24 hours after arthritis
induction (D). In C and D, hypernociception is presented as the change
(⌬) in withdrawal threshold (in grams), calculated by subtracting the
zero-time mean measurements from the time-interval mean measurements. The mean ⫾ SEM withdrawal threshold at zero-time was
12.8 ⫾ 0.5 gm (n ⫽ 30). Bars show the mean and SEM results from 5
mice per group. ⴱ ⫽ P ⬍ 0.05 versus control mice; # ⫽ P ⬍ 0.05 versus
vehicle-treated arthritic mice.
ciception after intraarticular antigen challenge in immunized mice (Figure 4C). Inflammation-related hypernociception was first noticeable at 3 hours after challenge,
became more intense by 6 hours after challenge, peaked
at 24 hours after challenge, and subsided by 7 days after
challenge. Treatment with reparixin diminished the extent of inflammation-related hypernociception observed
at 6 hours and 24 hours after antigen challenge in
immunized mice (Figure 4D).
In order to confirm the relevance of CXCR1/
CXCR2 in this mouse model of AIA, further experiments were conducted with an orally active derivative of
reparixin, DF2162 (10). DF2162 was administered by
oral gavage at a dose of 15 mg/kg. At this dose,
treatment with DF2162 was associated with a significant
2334
COELHO ET AL
reduction in neutrophil recruitment into the synovial
cavity and the periarticular tissue at 24 hours after AIA
induction (Table 1). In addition, this dose of the compound also reduced local production of TNF␣ (Table 1)
and the inflammation-related hypernociception (Figure
4D) observed at 24 hours in this model of AIA.
Inhibition of leukocyte–endothelial cell interactions by reparixin in the AIA model. A series of experiments were then conducted using intravital microscopy
to evaluate whether the effects of reparixin in AIA
correlated with its ability to prevent interactions between leukocytes and synovial microvessels. In order to
avoid any possible confounding effect of the compound
in the local production of cytokines (see Figure 4B),
reparixin was administered only 30 minutes prior to the
intravital microscopy procedure. As seen in Figure 5,
treatment of the mice with reparixin reduced leukocyte
rolling (60% inhibition) and abrogated leukocyte adhesion (100% inhibition) to the synovial microvessels at 24
hours after challenge in immunized mice. The majority
of leukocytes interacting with endothelial cells, as assessed in histologic sections of the tissue, were neutrophils (Figure 3D and results not shown).
Table 1. Effects of treatment with DF2162 on neutrophil recruitment and on the levels of TNF␣, CXCL1, and CXCL2 after initiation
of antigen-induced arthritis in mice*
Arthritic
Neutrophils in synovial cavity, ⫻
104/knee
Neutrophils in
periarticular
tissue, relative
units
CXCL1, pg/100
mg tissue
CXCL2, pg/100
mg tissue
TNF␣, pg/100 mg
tissue
Control
Vehicle
DF2162
0.10 ⫾ 0.07
7.68 ⫾ 1.91†
2.68 ⫾ 1.02‡
0.08 ⫾ 0.01
0.38 ⫾ 0.14†
0.13 ⫾ 0.05‡
137 ⫾ 32
962 ⫾ 104†
456 ⫾ 137‡
112 ⫾ 10
306 ⫾ 33†
130 ⫾ 31‡
9⫾4
147 ⫾ 39†
68 ⫾ 24‡
* Values are the mean ⫾ SEM resuts from 5–6 mice per group.
DF2162 (15 mg/kg) or vehicle (0.05% carboxymethylcellulose) was
administered orally 40 minutes before and 3 and 6 hours after arthritis
induction. The number of neutrophils in the synovial cavity was
assessed 24 hours after injection of 10 ␮g of methylated bovine serum
albumin or 10 ␮l sterile saline (control) into the knee joint of
immunized mice. The number of neutrophils in the periarticular tissue
was assessed at 24 hours using a myeloperoxidase assay. The concentrations of CXCL1, CXCL2, and tumor necrosis factor ␣ (TNF␣) in
the periarticular tissue were assessed by enzyme-linked immunosorbent assay at 24 hours after arthritis induction.
† P ⬍ 0.05 versus control mice.
‡ P ⬍ 0.05 versus vehicle-treated arthritic mice.
Figure 5. Effects of treatment with reparixin on the interaction
between leukocytes and endothelial cells in the synovial microvasculature. Reparixin (30 mg/kg intravenously [IV]) or vehicle (100 ␮l
saline IV) was administered 30 minutes before intravital microscopy.
Rolling (A) and adhesion (B) of leukocytes to the synovial endothelium were assessed at 24 hours after injection of 10 ␮g of methylated
bovine serum albumin (mBSA) or 10 ␮l sterile saline (control) into the
knee joint of immunized mice. The flux of rolling cells was measured
as the number of rolling cells passing by a given point in the venule per
minute. A leukocyte was considered to be adherent if it remained
stationary for at least 30 seconds, and total leukocyte adhesion was
quantified as the number of adherent cells within a 100-␮m length of
venule. Bars show the mean and SEM results from 5 mice per group.
ⴱ ⫽ P ⬍ 0.01 versus control mice; # ⫽ P ⬍ 0.01 versus vehicle-treated
arthritic mice.
DISCUSSION
In the present study, we identified a significant
role for CXCR1/CXCR2 in modulating tissue inflammation in a mouse model of experimental arthritis. Of note,
treatment with CXCR1/CXCR2 allosteric inhibitors
prevented local neutrophil influx, reduced local production of TNF␣, and diminished hypernociception, an
index of pain. Inhibition of leukocyte–endothelial cell
interactions, especially the effects on firm adhesion of
leukocytes, appeared to be a major mechanism of action
of the inhibitors in this model of antigen-induced inflammation in the knee joint.
Neutrophils are thought to play an important role
in the development of inflammatory joint disease, as has
been evidenced in several studies involving experimental
models of arthritis (9–11). Treatment with reparixin or
DF2162 greatly decreased the influx of neutrophils into
the knee joint and the periarticular tissue after antigen
challenge in immunized mice. Inhibition of neutrophil
influx was associated with significant amelioration of
overall disease in the tissue. These results are consistent
with the known role of CXCR2 in mediating the influx of
neutrophils in several models of inflammation in vivo,
including a model of AIA in rats (for example, see refs.
10, 12–15, and 23).
More recently, it has become clear that, in addition to CXCR2, murine leukocytes, especially neutrophils, also possess CXCR1 (24). The role of this receptor
CXCR1/CXCR2 CHEMOKINE RECEPTOR BLOCKADE IN AIA
in mediating neutrophil recruitment in vivo is not
known, and the tools to investigate the receptor are not,
as yet, readily available. Of note, the compounds used in
the present experiments, reparixin and DF2162, inhibited the function of both CXCR1 and CXCR2 (10,13),
and therefore any speculation regarding the relative
roles of each receptor in this system is not possible. The
compounds had no significant effect on the expression of
CXCR2 on circulating neutrophils in our experiments
(results not shown). Together with the findings from
other published studies (10,15,25), our results demonstrate that inhibition of CXCR1/CXCR2 appears to be
an effective means of preventing neutrophil influx in
models of arthritis.
We also evaluated the mechanisms by which
reparixin could be preventing neutrophil influx in the
system. To this end, we used intravital microscopy to
study the interaction between leukocytes and endothelial cells in the synovial microvasculature (18). Our
results showed that firm adhesion of leukocytes to
endothelial cells was suppressed in reparixin-treated
mice. This is consistent with the role of CXCR2 in
triggering integrin-dependent adhesion of neutrophils to
endothelial cells (26,27). There was also partial inhibition of rolling of leukocytes. This was surprising, inasmuch as leukocyte rolling is dependent on selectins and
their ligands but not on chemoattractant receptors such
as CXCR1/CXCR2 (27).
Expression of selectins or selectin ligands on
endothelial cells in a particular site of inflammation is
modulated by the expression of cytokines such as TNF␣
(27,28). Our results showed that reparixin treatment
prevented TNF␣ production when the treatment was
administered before antigen challenge. However, in the
intravital microscopy experiments, reparixin was administered just prior to the microscopy procedure and did
not modify local levels of cytokines (results not shown).
Hence, prevention of the expression of cell adhesion
molecules that mediate rolling does not appear to be a
mechanism that would explain the short-term effects of
reparixin treatment.
One alternative explanation for the effects of
reparixin is that adhered neutrophils could be relevant in
the rolling of further leukocytes, as has been shown in
other systems (29). Although this was a phenomenon
that was observed in our system, there was not enough
resolution to quantify rolling on adhered cells. Taken
together, our data suggest that short-term inhibition of
neutrophil–endothelial cell interactions is a major mechanism by which reparixin may interfere with AIA in
mice.
2335
Cytokine-based therapies, especially strategies
that block or antagonize TNF␣, have been used for the
treatment of RA and found to be useful in preventing
progression of disease in groups of patients (30). In the
present study, blockade of CXCR1/CXCR2 with reparixin or DF2162 significantly inhibited the local production of TNF␣. Although a role of TNF␣ in facilitating
the influx of neutrophils in vivo is well known, many
studies have also clearly demonstrated that the influx of
neutrophils facilitates the local production of TNF␣
(31,32). Therefore, inhibition of neutrophil influx could
potentially explain the lower levels of TNF␣ in the joints
of immunized mice after antigen challenge and subsequent treatment with reparixin.
It is not clear whether the neutrophils themselves
produce TNF␣ or whether these cells release other
intermediate mediators that induce the release of TNF␣
by resident cells, including macrophages. Regardless of
the TNF␣-producing cell type, the present results and
those from other studies are consistent with the notion
of a positive cooperative loop between TNF␣ and neutrophil influx, which appears to be relevant in joint
inflammation and injury. The ability of reparixin to
prevent neutrophil adhesion and the consequent influx
of neutrophils may lead to suppression of this positive
loop and have beneficial effects in arthritis.
Pain is the most frequent and disabling symptom
in patients with arthritis. In experimental models, pain
related to joint inflammation is better described as
hypernociception (33). Several effects of reparixin could
explain its ability to ameliorate inflammation-related
hypernociception induced by AIA. Our previous study
(34) and those from other investigators (35,36) have
clearly shown an essential role for neutrophils in mediating inflammation-related hypernociception induced by
antigen challenge in mice. Moreover, blockade of TNF␣
prevents inflammation-related hypernociception induced by a range of stimuli, including antigen injection
(for review, see ref. 33). Thus, inhibition of both neutrophil influx and local TNF␣ production could account
for the inhibitory effects of reparixin in AIA-associated
hypernociception related to inflammation.
In conclusion, this study shows that treatment
with allosteric inhibitors of CXCR1/CXCR2 prevented 3
major aspects of arthritis, namely neutrophil recruitment, TNF␣ production, and inflammation-related hypernociception. Importantly, this study shows that the
compounds appear to act via blockade of leukocyte
adhesion, with consequent inhibition of neutrophil migration to the site of joint inflammation. These beneficial effects of allosteric inhibitors of CXCR1/CXCR2
2336
COELHO ET AL
suggest that these compounds deserve further evaluation
for the treatment of arthritis.
ACKNOWLEDGMENT
We are grateful to Dr. Rosa Arantes (Departamento
de Patologia Geral, UFMG) for help with histopathologic
analysis.
AUTHOR CONTRIBUTIONS
Dr. Mauro Teixeira had full access to all of the data in the
study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Coelho, Pinho, Bertini, A. L. Teixeira, M. M. Teixeira.
Acquisition of data. Coelho, Amaral, Sachs, Costa, Rodrigues, Vieira,
Silva, Bertini.
Analysis and interpretation of data. Coelho, Pinho, Souza, M. M.
Teixeira.
Manuscript preparation. Coelho, A. L. Teixeira, M. M. Teixeira.
Statistical analysis. Coelho, A. L. Teixeira, M. M. Teixeira.
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