ARTHRITIS & RHEUMATISM
Vol. 58, No. 7, July 2008, pp 2030–2040
© 2008, American College of Rheumatology
CXCR2-Specific Chemokines Mediate
Leukotriene B4–Dependent Recruitment of Neutrophils to
Inflamed Joints in Mice With Antigen-Induced Arthritis
Renata Grespan,1Sandra Y. Fukada,1Henrique P. Lemos,1Silvio M. Vieira,1
Marcelo H. Napimoga,1Mauro M. Teixeira,2Alasdair R. Fraser,3Foo Y. Liew,3
Iain B. McInnes,3and Fernando Q. Cunha1
Objective. To investigate the mechanism underly-
ing neutrophil migration into the articular cavity in
experimental arthritis and, by extension, human inflam-
Methods. Antigen-induced arthritis (AIA) was
generated in mice with methylated bovine serum albu-
min (mBSA). Migration assays and histologic analysis
were used to evaluate neutrophil recruitment to knee
joints. Levels of inflammatory mediators were measured
by enzyme-linked immunosorbent assay. Antibodies and
pharmacologic inhibitors were used in vivo to determine
the role of specific disease mediators. Samples of syno-
vial tissue and synovial fluid from rheumatoid arthritis
(RA) or osteoarthritis patients were evaluated for
CXCL1 and CXCL5 expression.
Results. High levels of CXCL1, CXCL5, and leu-
kotriene B4(LTB4) were expressed in the joints of
arthritic mice. Confirming their respective functional
roles, repertaxin (a CXCR1/CXCR2 receptor antago-
nist), anti-CXCL1 antibody, anti-CXCL5 antibody, and
MK886 (a leukotriene synthesis inhibitor) reduced
mBSA-induced neutrophil migration to knee joints.
Repertaxin reduced LTB4production in joint tissue,
and neutrophil recruitment induced by CXCL1 or
CXCL5 was inhibited by MK886, suggesting a sequen-
tial mechanism. Levels of both CXCL1 and CXCL5 were
elevated in synovial fluid and were released in vitro by
RA synovial tissues. Moreover, RA synovial fluid neu-
trophils stimulated with CXCL1 or CXCL5 released
significant amounts of LTB4.
Conclusion. Our data implicate CXCL1, CXCL5,
and LTB4, acting sequentially, in neutrophil migration
in AIA. Elevated levels of CXCL1 and CXCL5 in the
synovial compartment of RA patients provide robust
comparative data indicating that this mechanism plays
a role in inflammatory joint disease. Together, these
results suggest that inhibition of CXCL1, CXCL5, or
LTB4may represent a potential therapeutic strategy
Rheumatoid arthritis (RA) is a chronic inflam-
matory disease of autoimmune pathogenesis that results
in the destruction of cartilage and bone. It is character-
ized by the infiltration of neutrophils, T and B lympho-
cytes, and macrophages into the synovial membrane and
fluid compartment (1,2). Neutrophils play an important
role in RA pathogenesis, contributing directly to acute
and chronic inflammation and immune dysregulation
(3). Similar histopathologic and immune features may be
observed in models of antigen-induced arthritis (AIA),
making this a useful model in which to investigate the
relationship between cellular and molecular regulatory
Previous studies suggest that neutrophil recruit-
ment to the RA joint is mediated by a variety of factors,
including cytokines, leukotriene B4(LTB4), and chemo-
kines (6–8). LTB4is one of the most potent chemoattrac-
Supported by grants from the Conselho Nacional de Pesquisa,
the Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo, and the
Coordenadoria de Aperfeic ¸oamento de Pessoal de Nı ´vel Superior.
1Renata Grespan, MS, Sandra Y. Fukada, PhD, Henrique P.
Lemos, MS, Silvio M. Vieira, MS, Marcelo H. Napimoga, PhD,
Fernando Q. Cunha, PhD: University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil;
2Mauro M. Teixeira, PhD: Federal University of Minas Gerais, Belo
Horizonte, Brazil;3Alasdair R. Fraser, PhD, Foo Y. Liew, PhD, Iain B.
McInnes, MD: University of Glasgow, Glasgow, UK.
Address correspondence and reprint requests to Fernando Q.
Cunha, PhD: Department of Pharmacology, Faculty of Medicine of
Ribeira ˜o Preto, University of Sa ˜o Paulo, Avenida Bandeirantes 3900,
Ribeira ˜o Preto, Sa ˜o Paulo 14049-900, Brazil. E-mail: fdqcunha@
Submitted for publication June 12, 2007; accepted in revised
form March 27, 2008.
tants of leukocytes and is implicated in the pathogenesis
of RA. Neutrophils may participate directly in inflam-
mation via production of LTB4, thus increasing cellular
recruitment and activation (9). Genetic deletion of
BLT1 (a receptor for LTB4) is sufficient to abrogate
collagen-induced arthritis (CIA) in mice (10,11), and
leukotriene receptor antagonists can reduce clinical
symptoms and histologic changes in murine CIA (12,13).
A subclass of CXC chemokines that contains the
ELR sequence (ELR?) preceding the first cysteine
residue of the CXC motif plays a major role in the
recruitment of neutrophils to inflamed tissue following
activation (14,15). Both CXCL1 (keratinocyte-derived
chemokine [KC]) and CXCL5 (lipopolysaccharide-
induced CXC chemokine [LIX]) belong to the ELR?
subgroup and are considered to be murine homologs of
human CXCL1 (growth-related oncogene ? [GRO?])
and CXCL5 (epithelial neutrophil–activating peptide
78), respectively (16,17). Recent data have shown that
both CXCR1 and CXCR2 are expressed in mice (18,19).
Though murine CXCR1 is a functional receptor for
CXCL6 (GCP-2) and CXCL8 (interleukin-8), it is not
bound or activated by murine or human CXCL1, human
CXCL2/3 (GRO??), CXCL5, or CXCL7 (neutrophil-
activating peptide 2) (20).
A number of studies have investigated the in-
volvement of ELR? CXC chemokines in the pathogen-
esis of RA. Chemokine receptor inhibitors such as
repertaxin have been shown to reduce neutrophil re-
cruitment and activation in vivo (21). Podolin and
coworkers (22) demonstrated that an antagonist of
CXCR2 inhibited neutrophil migration, which resulted
in diminished lesions in AIA in rabbits. Furthermore,
treatment with antibodies to ELR? CXC chemokines
ameliorated inflammation in monosodium urate crystal–
induced arthritis and adjuvant-induced arthritis (23,24).
Although chemokines and LTB4 have been
shown to contribute to neutrophil recruitment in RA,
the mechanism by which these chemoattractants elicit
neutrophil migration is not fully understood. There is
evidence that LTB4, acting through the BLT1 receptor,
triggers subsequent chemokine production, which in-
duces joint inflammation in the K/BxN serum transfer
model (11). Conversely, at least in the peritoneal cavity,
chemokines stimulate neutrophil migration via LTB4
release (25). In the present study, we assessed whether
CXC chemokine ligands of the CXCR2 receptor
(CXCL1 and CXCL5) and LTB4were involved in
neutrophil recruitment to the articular cavity of mice
with methylated bovine serum albumin (mBSA)–
induced arthritis, and we investigated the mechanism
underlying this recruitment. We subsequently explored
homologous chemokine expression in tissues and fluids
derived from the synovial compartment of patients with
RA and osteoarthritis (OA), in order to demonstrate an
analogous mechanism in human arthritis.
MATERIALS AND METHODS
Animals. Male BALB/c, 129/Sv (wild-type [WT]), and
5-lipoxygenase–knockout mice (129/Sv 5-LO?/?) weighing
18–22 gm each were housed in temperature-controlled rooms
(22–25°C) in the animal facility of the School of Medicine of
Ribeira ˜o Preto, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil, and
received water and food ad libitum. The male and female
5-LO?/?(129-Alox5tmlFun) mice (26) and strain-matched WT
mice were bred in the animal facility of the School of Medicine
of Ribeira ˜o Preto using breeding mice obtained from The
Jackson Laboratory (Bar Harbor, ME).
Induction of experimental arthritis. BALB/c mice
were sensitized with 500 ?g of mBSA (Sigma, St. Louis, MO)
in 0.2 ml of an emulsion containing 0.1 ml phosphate buffered
saline (PBS) and 0.1 ml Freund’s complete adjuvant (Sigma)
administered by subcutaneous injection. Booster injections of
mBSA dissolved in Freund’s incomplete adjuvant (Sigma)
were given 7 and 14 days after the first immunization. Nonim-
munized mice received similar injections but without the
antigen (mBSA). Twenty-one days after the initial injection,
arthritis was induced in the immunized animals by intraartic-
ular injection of mBSA (1, 3, or 10 ?g/cavity) dissolved in 10 ?l
PBS. Nonimmunized and immunized mice were challenged
with mBSA or with PBS. Mice were killed 6, 12, 24, 48, or 96
hours after intraarticular injections, and neutrophil migration
was determined as described below.
In vivo neutrophil migration analysis. Immunized or
nonimmunized mice had mBSA or PBS injected directly into
the articular cavity. Another group of mice were injected
intraarticularly with 10 ?l of LTB4(5 or 50 ng/cavity) (Sigma,
St. Louis, MO), CXCL1 (1 or 10 ng/cavity), or CXCL5 (1 or 10
ng/cavity) (both from PeproTech, London, UK). At the end of
the experimental period, the mice were killed, and the articular
cavities were washed twice with 5 ?l PBS containing 1 mM
EDTA, then diluted to a final volume of 100 ?l with PBS/
EDTA to evaluate leukocyte migration at the indicated times.
Total cell counts were performed in a cell counter (ACT;
Beckman Coulter, Miami, FL), and differential cell counts
(100 cells total) were performed on cytocentrifuge slides
(Cytospin 3; Shandon, Pittsburgh, PA) with Rosenfeld stain.
Differential cell counts were performed with a light micro-
scope (Zeiss, Wetzlar, Germany), and the results were ex-
pressed as the number of neutrophils per cavity.
Histologic assessment. Mice were killed and articular
joints were removed 24 hours after challenge with mBSA. The
joints were immediately fixed in 10% buffered formalin, then
decalcified for 24 hours. The tissues were then processed and
embedded in paraffin. Tissue sections (5 ?m) were prepared
and stained with hematoxylin and eosin using standard meth-
ods. The slices were examined with a Nikon microscope and
were photographed with a digital camera (Coolpix 4500;
Nikon, Melville, NY) coupled to an imaging system.
CHEMOKINE-MEDIATED NEUTROPHIL RECRUITMENT IN MURINE AIA 2031
Enzyme-linked immunosorbent assay (ELISA). Levels
of CXCL1 and CXCL5 at 3 and 12 hours after injection of
mBSA were quantified in the joint tissues of immunized or
nonimmunized mice and PBS-injected immunized mice by
ELISA. Briefly, joints were dissected out, frozen with liquid
nitrogen, crushed in a mortar and pestle, then solubilized in
PBS. Synovial tissue extracts were then measured using ELISA
(27), with the results expressed as picograms per milliliter.
LTB4concentrations at 3 and 12 hours after injection of mBSA
were assessed in the joint tissue of immunized or nonimmu-
nized mice and PBS-injected immunized mice as above using a
commercial enzyme immunoassay kit (Cayman Chemical, Ann
Arbor, MI) according to the manufacturer’s instructions.
Modulation of neutrophil migration. Immunized mice
were treated either 1 hour before mBSA challenge (10 ?g/
cavity) with dexamethasone (1 mg/kg subcutaneously) or with
MK886 (1 mg/kg orally) or 30 minutes before challenge with
repertaxin (30 mg/kg subcutaneously). Dexamethasone and
repertaxin were both dissolved in PBS, and MK886 was
dissolved in 0.1% methylcellulose in PBS. Anti-CXCL1 and
anti-CXCL5 antibodies were administered simultaneously with
mBSA. Neutrophil migration was measured 24 hours after
mBSA was administered.
To determine the effects of repertaxin on LTB4in the
exudates harvested from the knee joint, mice were treated with
repertaxin (30 mg/kg subcutaneously) and, 30 minutes later,
were challenged intraarticularly with mBSA. The concentra-
tion of LTB4was determined by ELISA 3 hours after challenge
To determine the effects of MK886 on CXCL1 and
CXCL5 in the exudates harvested from the knee joint, mice
were treated with MK886 (1 mg/kg orally) and, 1 hour later,
were challenged intraarticularly with mBSA. The concentra-
tions of CXCL1 and CXCL5 were determined by ELISA 3
hours after challenge with mBSA.
To determine the effects of MK886 on neutrophil
migration induced by CXCL1 or CXCL5, BALB/c mice re-
ceived MK886 (1 mg/kg orally) and, 1 hour later, were again
administered MK886 (intraarticularly) with CXCL1 (10 ng/
cavity) or with CXCL5 (10 ng/cavity). Neutrophil migration
was measured 6 hours after chemokine administration.
To determine the effects of repertaxin on neutrophil
migration induced by LTB4, repertaxin (30 mg/kg subcutane-
ously) was injected into immunized BALB/c mice 30 minutes
before the administration of LTB4(50 ng/cavity). Neutrophil
migration was measured 6 hours after LTB4administration.
Detection of CXCL1 and CXCL5 in the human syno-
vial compartment. Eight matched synovial tissue and synovial
fluid samples were collected from patients undergoing arthro-
plasty as part of their treatment (4 OA patients, 4 RA
patients). Four more synovial fluid samples were collected by
aspiration of the joints of additional OA and RA patients.
Patients received information about the study and gave in-
formed consent to provide tissue samples for the study, in
accordance with West Glasgow Ethics Committee protocol.
The synovial tissues were prepared for culture by maceration
and enzyme digestion (Liberase Blendzyme; Roche Diagnos-
tics, Mannheim, Germany) for 2 hours at 37°C. The cell
preparation was washed, resuspended at 2 ? 106cells/ml, and
plated onto 96-well plates. Synovial cultures were incubated
for 24 hours at 37°C, and the supernatants were collected and
frozen at ?20°C until testing. Synovial fluid samples were
centrifuged at 10,000g for 10 minutes to remove particulate
material, diluted 1:1 with PBS, and stored at ?20°C until used.
Supernatants and synovial fluid samples were examined for
CXCL1 and CXCL5 concentrations using a Luminex multiplex
assay (Invitrogen, Paisley, UK). The samples were run in
duplicate according to the manufacturer’s instructions, and
chemokine concentrations were calculated from standard
curves as recommended.
To assess chemokine-mediated LTB4expression in
synovial cells, 3 more synovial fluid samples were collected
from RA patients, and mononuclear cells and neutrophils were
isolated using density centrifugation. The purity of cell frac-
Figure 1. Neutrophil migration after methylated bovine serum albu-
min (mBSA) challenge in immunized mice. A, Injection of mBSA into
the articular cavity of nonimmunized (NI) or immunized (IM) mice at
the indicated doses; neutrophil migration was determined 24 hours
later. Phosphate buffered saline (PBS) injection into immunized mice
was used as a control. Values are the mean and SEM of 5 mice per
group. ? ? P ? 0.05 versus control mice, by analysis of variance with
Bonferroni adjustment. B, Time dependence of neutrophil migration
in immunized mice challenged with PBS or mBSA. Values are the
mean ? SEM of 5 mice. ? ? P ? 0.05 versus control mice, by analysis
of variance with Bonferroni adjustment. C and D, Histologic analysis of
knee joints from immunized mice (top). Knee joints from immunized
mice after challenge with PBS (control) showed a normal joint space
(C). Knee joints from immunized mice after challenge with mBSA,
showed neutrophil infiltration into the joint space and synovium (D).
Boxed areas in the top panels are shown at higher magnification in the
bottom panels. (Original magnification ? 50 top; ? 1,000 bottom.)
2032 GRESPAN ET AL
tions was assessed using fluorescence-activated cell sorting and
were confirmed to be ?95%. Additionally, 3 primary RA
fibroblast cultures (at passage 3) were assessed. Cells were
cultured at a density of 2 ? 106, treated with 0, 10, and 100
ng/ml CXCL1 or CXCL5 overnight, and the supernatants were
collected and stored at ?20°C until used. The supernatants
were then tested with appropriate controls in a commercial
LTB4competitive ELISA (Endogen, Cambridge, MA).
Statistical analysis. Data are reported as the mean ?
SEM and are representative of 2 or 3 separate experiments.
The means of different treatments were compared by analysis
of variance with Bonferroni adjustment for multiple compari-
sons or by Student’s t-test. P values less than 0.05 were
Induction of neutrophil migration with mBSA in
immunized mice. The intraarticular injection of mBSA,
but not PBS (control), in immunized mice induced dose-
and time-dependent neutrophil migration (Figures 1A
and B). The mBSA challenge induced significant neu-
trophil migration at 6 hours, which peaked at 24 hours,
declined 48 hours later, and returned to basal levels 96
hours after challenge (Figure 1B). Twenty-four hours
after mBSA challenge, the knee joints exhibited promi-
nent neutrophil infiltration (Figure 1D) as compared
with knee joints of mice challenged with PBS (Figure
1C). Furthermore, an elevated titer of serum IgG against
mBSA was detected in preimmunized mice but not in
nonimmune littermate controls (data not shown).
Involvement of LTB4and CXC chemokines in
neutrophil migration induced by mBSA. The concen-
trations of CXCL1, CXCL5, and LTB4in the synovial
exudates of mice challenged with mBSA (10 ?g/
cavity) were determined 3 and 12 hours postinocula-
tion. Both CXCL1 (Figure 2A) and LTB4(Figure 2C)
Figure 2. Concentrations of CXCL1, CXCL5, and leukotriene B4
(LTB4) in articular exudates from immunized mice. The concentra-
tions of CXCL1 (A), CXCL5 (B), and LTB4(C) were determined 3
and 12 hours after challenge with mBSA in immunized mice and in
nonimmunized mice or with PBS in immunized mice (control). Values
are the mean and SEM of 3 mice. ? ? P ? 0.05 versus control mice,
by analysis of variance with Bonferroni adjustment. See Figure 1 for
CHEMOKINE-MEDIATED NEUTROPHIL RECRUITMENT IN MURINE AIA2033
were significantly increased at 3 and 12 hours, whereas
CXCL5 (Figure 2B) was increased mainly at 3 hours
after mBSA challenge in immunized mice. We next
investigated whether CXCL1, CXCL5, and LTB4in-
duced neutrophil migration in mice. We observed
significant neutrophil migration into the articular
exudates 6 hours after injection of CXCL1 (10 ng/
cavity), CXCL5 (10 ng/cavity), or LTB4(50 ng/cavity)
(Figures 3A, B, and C, respectively). Together, these
results suggest that mBSA-induced neutrophil migra-
tion in immunized mice is mediated by CXCL1,
CXCL5, and LTB4. Neutrophil migration induced by
mBSA in immunized mice was inhibited by treatment
with MK886, repertaxin, anti-CXCL5 and anti-CXCL1
antibodies, and dexamethasone (Figure 4A). The role of
LTB4in neutrophil migration in sensitized animals was
confirmed in 5-LO?/?mice. This analysis showed that
mBSA-induced neutrophil migration into the articular
cavity was significantly lower in the 5-LO?/?mice than
in the syngeneic WT mice (Figure 4B).
Necessity of LTB4for induction of neutrophil
migration by CXCL1 or CXCL5. We tested the hypothe-
sis that CXCL1 and CXCL5 could induce neutrophil
migration via an LTB4-dependent mechanism. Adminis-
tration of the same dosage of mBSA that caused neu-
trophil migration into the knee joint of sensitized mice
was also able to promote a significant increase in LTB4
concentration in articular exudates when compared with
control mice (Figure 5A). However, LTB4production in
the knee joint was significantly inhibited by treatment
with repertaxin (Figure 5A). Conversely, production of
CXCL1 and CXCL5 in the knee joint was not inhibited
by treatment with the LTB4inhibitor MK886 (Figure
5B). We then demonstrated that intraarticular adminis-
tration of CXCL1 (10 ng/cavity) or CXCL5 (10 ng/
cavity) (Figure 5C) in animals treated with MK886 failed
to induce neutrophil recruitment. These results suggest
that CXCL1- and CXCL5-induced neutrophil migration
depends upon LTB4production. We confirmed that
LTB4production occurs downstream of the release of
Figure 3. Induction of neutrophil migration into the articular cavity of
mice by CXCL1, CXCL5, and leukotriene B4(LTB4). Phosphate
buffered saline (PBS) (control), CXCL1 (1 or 10 ng/cavity) (A),
CXCL5 (1 or 10 ng/cavity) (B), or LTB4(5 or 50 ng/cavity) (C) was
injected into the articular cavities of mice, and 6 hours later, neutrophil
migration was evaluated. Values are the mean and SEM of 5 mice. ?
? P ? 0.05 versus PBS, by analysis of variance with Bonferroni
2034 GRESPAN ET AL
CXCL1 and CXCL5, since LTB4-induced neutrophil
migration was not inhibited by treatment of the animals
with repertaxin, an antagonist of CXCR1 and CXCR2
(Figure 5D). Thus, these results suggest that mBSA
induces neutrophil migration into the articular cavity via
the sequential release of CXCL1 and CXCL5, followed
Expression of CXCL1 and CXCL5 in the human
synovial compartment. To determine whether these
observations might have relevance in RA, we investi-
gated the expression of human CXCL1 and CXCL5 in
synovial tissues from RA patients as compared with OA
patients. Synovial fluid from RA patients showed ele-
vated levels of CXCL1 and CXCL5 compared with those
in OA patients; CXCL1 was particularly evident (Figure
6A). We then investigated the tissue-specific expression
of CXCL1 and CXCL5 by culturing primary synovial
membrane cells overnight and assessing chemokine se-
cretion. Synovial membrane from RA patients showed
significantly increased secretion of CXCL1 and CXCL5
as compared with tissue from OA patients (Figure 6B).
Therefore, the synovial membrane was confirmed as the
likely origin of the elevated expression in synovial fluid.
To determine which of the synovial tissue components
was responsible for LTB4secretion in response to
CXCL1 or CXCL5, we obtained purified neutrophils
and mononuclear cells from the synovial fluid of RA
patients and fibroblasts cultured from RA synovium.
These cells were then stimulated with CXCL1 or CXCL5.
Only neutrophils demonstrated a significant increase in
LTB4production when stimulated with CXCL1 (10 or
100 ng/ml) or CXCL5 (10 ng/ml) (Figure 6C).
Many data support the concept that neutrophil
infiltration into RA synovial tissues, with the subsequent
release of proteolytic enzymes and free radicals, contrib-
utes significantly to the tissue damage associated with
RA (28,29). Studies investigating the mechanism in-
volved in the migration of these cells could facilitate the
development of new therapies for RA. In the present
study, we used a murine model of mBSA-induced arthri-
tis, which stimulates a delayed-type hypersensitivity re-
sponse with histopathologic features similar to those
observed in human RA (4,5), to investigate the partici-
pation of CXCL1 and CXCL5 and LTB4in neutrophil
migration to the knee joint of immunized mice. We also
evaluated the expression of these chemokines in the
synovial compartment of RA patients as compared with
patients with noninflammatory OA. We observed that
CXCL1, CXCL5, and LTB4were produced and played a
key role in mBSA-induced neutrophil recruitment in
immunized mice. The ELR? chemokines CXCL1 and
CXCL5 acted on CXCR2 to induce LTB4release, which
Figure 4. Inhibition of mBSA-induced neutrophil migration by inhib-
itors and in 5-LO?/?mice. A, Prior to intraarticular challenge with
mBSA, immunized animals were treated with 1 mg/kg of dexametha-
sone (Dex; 1 hour before) or with 30 mg/kg of repertaxin (RPT; 30
minutes before). Anti-IgG isotype control (?-CTE), anti-CXCL1, and
anti-CXCL5 were injected at the same time as the mBSA. Neutrophil
migration was evaluated 24 hours after mBSA challenge in treated
groups and in nonimmunized and immunized mice challenged with
PBS. B, Immunized animals were treated with MK886 (MK; 1 mg/kg)
or with vehicle control (?-CTE; 0.1% methylcellulose in PBS) 1 hour
before challenge with mBSA. Neutrophil migration was evaluated 24
hours after PBS or mBSA challenge. Wild-type 129/Sv and 5-LO?/?
immunized and nonimmunized mice were challenged with mBSA, and
24 hours later, neutrophil migration was calculated and compared with
that in PBS-challenged immunized mice (control). Values are the
mean and SEM of 5 mice. ? ? P ? 0.05 versus control mice; # ? P ?
0.05 versus immunized mice after mBSA challenge, by analysis of
variance with Bonferroni adjustment. See Figure 1 for other defini-
CHEMOKINE-MEDIATED NEUTROPHIL RECRUITMENT IN MURINE AIA2035
then mediated neutrophil migration to the joint cavity.
Moreover, we confirmed that the production and secre-
tion of CXCL1 and CXCL5 within the synovial compart-
ment of RA patients was elevated in comparison with
OA patients and that these chemokines stimulated LTB4
production by RA synovial fluid neutrophils specifically.
The last decade has seen particular advances in
the expression and functional targeting of cytokines,
exemplified best by the advent of tumor necrosis factor
? blockade in the clinical setting (30). Similarly, there
has been intense interest in the use of chemokines as
therapeutic targets, although without the prominent
success seen in the cytokine field. It is therefore imper-
ative to better understand the mechanisms of the key
chemokine activities detected in RA tissues. The CXC
chemokines with an ELR motif (ELR? chemokines)
are crucial for neutrophil recruitment, via activation of
CXCR1 and CXCR2 receptors (14,15).
In the present study, we observed that mBSA
administered into the femorotibial joints of immunized
mice induced a dose- and time-dependent articular
neutrophil accumulation that was dependent upon the
endogenous release of CXCL1 and CXCL5, since coad-
ministration of anti-CXCL1 or anti-CXCL5 antibodies
with mBSA significantly reduced the degree of neutro-
phil infiltration. Significantly elevated concentrations of
these chemokines were observed in the joint exudates of
mBSA-challenged mice compared with controls.
In addition, our results showed that these chemo-
kines act via CXCR2 to induce neutrophil migration,
since repertaxin, a CXCR1/R2 antagonist (21), inhibited
neutrophil migration in mBSA-administered mice. Once
the mechanism of neutrophil recruitment was clarified in
our murine model, we investigated the expression of the
corresponding chemokines in the synovial compartment
of OA and RA patients. We confirmed that the synovial
fluid of RA patients contained significantly elevated
levels of CXCL1 and CXCL5 and that synovial tissue
from RA patients produced significantly higher quanti-
ties of both chemokines as compared with synovial tissue
Figure 5. Inhibition of CXCL1/CXCL5-mediated neutrophil migration by repertaxin and MK886.
Prior to intraarticular (IA) challenge with mBSA, immunized mice were treated with 30 mg/kg of
repertaxin (RPT; 30 minutes before) or with 1 mg/kg of MK886 (MK; 1 hour before). A and B, Levels
of leukotriene B4(LTB4) (A) and CXCL1/CXCL5 (B) were quantified 3 hours after mBSA challenge
in immunized and nonimmunized mice, compared with that in PBS-challenged immunized mice
(control). C, Mice were treated with PBS or MK886 (1 mg/kg) 1 hour before IA injection with CXCL1
or CXCL5 (10 ng/cavity). D, Mice were treated with PBS or repertaxin (30 mg/kg), and 30 minutes
later, LTB4(50 ng/cavity) was injected IA, and neutrophil migration was evaluated 6 hours later.
Values are the mean and SEM of 5 mice. ? ? P ? 0.05 versus control mice; # ? P ? 0.05 versus
immunized mice after mBSA challenge; ## ? P ? 0.05 versus mice injected with chemokines, by
analysis of variance with Bonferroni adjustment. See Figure 1 for other definitions.
2036 GRESPAN ET AL
from OA patients. Our data are consistent with those of
Fujiwara et al (23), who demonstrated that CXCL1
contributes to neutrophil recruitment in monosodium
urate crystal–induced arthritis in rabbits, and those of
Koch et al (31), who showed increased expression of
CXCL1 in the synovial fluid of RA patients and dem-
onstrated that the enhancement correlated with the
accumulation of neutrophils.
Furthermore, it has been also demonstrated that
CXCL5 is involved in the progression of arthritis mod-
els, since anti-CXCL5 treatment before the establish-
ment of disease led to a decrease in the severity of knee
joint inflammation in a rat model of adjuvant-induced
arthritis (24). Activation and influx of neutrophils into
the pulmonary interstitium induced by CXCL5 resulted
in neutrophil-mediated pulmonary microvascular injury
(32). These studies concur to show that CXCL1 and
CXCL5 are important in the pathogenesis of RA, induc-
ing migration of neutrophils to synovial joints, where
they contribute to tissue damage. However, in contrast
with previously published data, we critically investigated
the mechanism by which CXCL1 and CXCL5 control
neutrophil migration in an arthritis model.
Several studies have suggested that LTB4pro-
Figure 6. Increase in CXCL1 and CXCL5 levels in synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA). Matched synovial
fluid and synovial tissue samples from osteoarthritis (OA) and RA patients were collected, tissues were disaggregated, synovial cells were cultured
overnight, and supernatants were collected. Synovial fluid samples (6 OA and 6 RA) and synovial cell supernatants (4 OA and 4 RA) were assayed
for CXCL1 and CXCL5 expression. A, CXCL1 was significantly elevated in RA synovial fluid (RASF) compared with OA synovial fluid (OASF).
CXCL5 demonstrated a trend toward higher levels in RA synovial fluid (P ? 0.111 by Student’s t-test). Bars indicate the mean. B, Levels of both
CXCL1 and CXCL5 were significantly increased in supernatants from RA synovial membrane (RASM) compared with OA synovial membrane
(OASM) (P ? 0.05). Values are the mean and SEM of 3 mice. ? ? P ? 0.05 versus OASM, by Student’s t-test. C, Synovial fluid neutrophils (?95%
purity) were cultured at 2 ? 106cells/ml and treated with varying doses of CXCL1 and CXCL5, then cultured for 24 hours. Supernatants were
collected and tested in a competitive leukotriene B4enzyme-linked immunosorbent assay. Values are the mean and SEM of 3 mice. ? ? P ? 0.05
versus untreated control, by Student’s t-test.
CHEMOKINE-MEDIATED NEUTROPHIL RECRUITMENT IN MURINE AIA2037
vokes neutrophil activation and is involved in RA patho-
genesis (9,12,33). We showed that neutrophil recruit-
ment is dependent upon endogenous release of LTB4,
since MK886 (a leukotriene synthesis inhibitor) reduced
neutrophil migration in the joint cavity of mBSA-
challenged mice, and in mBSA-challenged 5-LO?/?
immunized mice, the degree of neutrophil accumulation
was reduced compared with that in WT immunized
mice. Moreover, significantly elevated levels of LTB4
were observed in the joint cavity in this model. Other
studies have shown that the LTB4concentration is also
increased in both synovial fluid (34,35) and serum (36)
from RA patients. The BLT2 receptor is thought to be
the main receptor mediating the effects of LTB4in the
synovial tissues (12,37,38). LTB4antagonists are effec-
tive in reducing collagen- and cytokine-induced arthritis,
and in other studies, 5-LO?/?mice have been shown to
exhibit reduced levels of CIA (12,38).
These findings prompted us to test the possible
interdependence of CXCL1, CXCL5, and LTB4in the
mediation of neutrophil migration to the joint cavity in
AIA. In contrast with PBS treatment alone, mice treated
with repertaxin had low levels of LTB4in articular
exudates, indicating a need for CXCR2 activation to
induce LTB4production and subsequent neutrophil
recruitment. To confirm this hypothesis, pretreating
mice with MK886 led to a marked inhibition of CXCL1-
and CXCL5-induced neutrophil migration. We dis-
counted the possibility that LTB4induced chemokine
release, since MK886 did not alter the production of
CXCL1 and CXCL5 in the articular exudates when
compared with PBS-treated animals. Moreover, reper-
taxin did not reduce the neutrophil recruitment induced
Recently, we showed that LTB4mediates CCL3-
induced neutrophil migration in a peritoneal immune
inflammation model (25). Although the precise mecha-
nisms by which CXCL1 and CXCL5 signaling through
the CXCR2 receptor induce LTB4production was not
fully elucidated, we demonstrated that in RA patients,
these chemokines stimulate the release of this potent
mediator, predominantly from neutrophils. It was ob-
served that neutrophils, but not synovial fibroblasts or
mononuclear cells isolated from the synovial fluid of RA
patients, release significant amounts of LTB4after being
challenged with CXCL1 or CXCL5. In contrast with our
results, Kim and coworkers (10) showed that BLT1 is
needed for subsequent chemokine production and joint
inflammation in the K/BxN serum–transfer model. In
fact, the authors found that the levels of multiple
chemokines and their receptors, including CXCL5 and
CXCR2, were consistently lower or absent in BLT1-
knockout mice. An exception was CXCL1 (KC), which
was not detectable in the synovial tissue of either WT or
BLT1-knockout mice. Moreover, adoptive transfer of
WT neutrophils restored the up-regulation of inflamma-
tory chemokines in the joints. A difference in experi-
mental models may explain these apparent contradic-
Our data do not preclude the participation of
other mediators in neutrophil migration in this and
other immune models of RA. However, the data ob-
tained from OA and RA patients lend compelling
support to the recruitment mechanism identified in this
study. Synovial tissue and synovial fluid from RA pa-
tients showed significantly increased levels of CXCL1
and CXCL5 compared with samples from OA patients,
a trend reported by other researchers (31,39). The level
of CXCL5 in the synovial fluid of RA patients in our
study was much lower than has previously been reported
(39), but this may be due to the different assay protocols
used. RA is generally characterized by significant infil-
tration of neutrophils into the synovial space from the
joint tissue, and the confirmed presence of CXCL1 and
CXCL5 in both tissue and fluid would suggest that they
can act as key mediators in the recruitment of neutro-
phils. It has previously been shown that LTB4is in-
creased in the synovial fluid of RA patients (9,40). In
this study, we showed that CXCL1 and CXCL5 induced
the production of LTB4by neutrophils in the synovial
fluid of RA patients, providing robust evidence for a
mechanism that links this potent neutrophil chemotactic
factor with chemokine mediators. It would suggest that
CXCL1 and CXCL5 released by synovial cells stimulate
neutrophil migration as well as LTB4release by the
migrating neutrophils. LTB4acts to amplify the neutro-
phil recruitment process through autocrine secretion.
These data contribute to a better understanding
of the mechanisms involved in promoting synovitis and
suggest that CXCL1 and CXCL5 may be effective
targets for therapeutic intervention in RA itself. Target-
ing LTB4alone has recently been investigated using a
novel LTB4receptor antagonist (41). Though the antag-
onist was effective at ameliorating arthritis in a murine
model, a clinical trial of the drug showed only marginal
benefit in the treatment of RA when used as mono-
therapy. Further studies using this inhibitor in conjunc-
tion with other disease-modifying antirheumatic drugs
or CXCR2-targeted inhibitors are needed to determine
any synergistic effects. Results from these investigations
could then lead to future therapies for RA.
2038 GRESPAN ET AL
We are grateful to Giuliana Bertozi, Ju ´lio Anselmo
Siqueira, Fabı ´ola Leslie Mestriner, Ana Ka ´tia dos Santos, and
Diva Amabile Montanha de Sousa for technical assistance.
Dr. Cunha 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
Study design. Grespan, Fukada, Lemos, Vieira, Liew, McInnes,
Acquisition of data. Grespan, Fukada, Lemos, Vieira, Fraser.
Analysis and interpretation of data. Grespan, Fukada, Lemos, Vieira,
Teixeira, Fraser, Liew, McInnes, Cunha.
Manuscript preparation. Grespan, Fukada, Napimoga, Teixeira,
Fraser, Liew, McInnes, Cunha.
Statistical analysis. Grespan, Fraser, Liew.
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