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Octacalcium phosphate crystals induce inflammation in vivo through interleukin-1 but independent of the NLRP3 inflammasome in mice.

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Vol. 63, No. 2, February 2011, pp 422–433
DOI 10.1002/art.30147
© 2011, American College of Rheumatology
Octacalcium Phosphate Crystals Induce Inflammation In Vivo
Through Interleukin-1 but Independent of the
NLRP3 Inflammasome in Mice
Sharmal Narayan,1 Borbola Pazar,1 Hang-Korng Ea,2 Laeticia Kolly,1
Nathaliane Bagnoud,1 Véronique Chobaz,1 Frédéric Lioté,2 Thomas Vogl,3 Dirk Holzinger,3
Alexander Kai-Lik So,1 and Nathalie Busso1
Objective. To determine the mechanisms involved
in inflammatory responses to octacalcium phosphate
(OCP) crystals in vivo.
Methods. OCP crystal–induced inflammation was
monitored using a peritoneal model of inflammation in
mice with different deficiencies affecting interleukin-1
(IL-1) secretion (IL-1 ␣ –/– , IL-1 ␤ –/– , ASC –/– , and
NLRP3–/– mice) or in mice pretreated with IL-1 inhibitors (anakinra [recombinant IL-1 receptor antagonist]
and anti–IL-1␤). The production of IL-1␣, IL-1␤, and
myeloid-related protein 8 (MRP-8)–MRP-14 complex
was determined by enzyme-linked immunosorbent assay. Peritoneal neutrophil recruitment and cell viability
were determined by flow cytometry. Depletion of mast
cells or resident macrophages was performed by pre-
treatment with compound 48/80 or clodronate liposomes, respectively.
Results. OCP crystals induced peritoneal inflammation, as demonstrated by neutrophil recruitment and
up-modulation of IL-1␣, IL-1␤, and MRP-8–MRP-14
complex, to levels comparable with those induced by
monosodium urate monohydrate crystals. This OCP
crystal–induced inflammation was both IL-1␣– and
IL-1␤–dependent, as shown by the inhibitory effects of
anakinra and anti–IL-1␤ antibody treatment. Accordingly, OCP crystal stimulation resulted in milder inflammation in IL-1␣–/– and IL-1␤–/– mice. Interestingly,
ASC–/– and NLRP3–/– mice did not show any alteration
in their inflammation status in response to OCP crystals. Depletion of the resident macrophage population
resulted in a significant decrease in crystal-induced
neutrophil infiltration and proinflammatory cytokine
production in vivo, whereas mast cell depletion had no
effect. Finally, OCP crystals induced apoptosis/necrosis
of peritoneal cells in vivo.
Conclusion. These data indicate that macrophages, rather than mast cells, are important for initiating and driving OCP crystal–induced inflammation.
Additionally, OCP crystals induce IL-1–dependent peritoneal inflammation without requiring the NLRP3 inflammasome.
Supported by the Fonds National Suisse de la Recherche
Scientifique (grant 310030-130085/1) and the Jean and Linette Warnery Foundation. Drs. Ea and Lioté’s work was supported by grants
from the Fondation pour la Recherche Médicale, the Association pour
la Recherche en Pathologie Synoviale, and the Association Rhumatisme et Travail. Dr. Holzinger’s work was supported by a grant from
the German Ministry of Education and Research (BMBF project
Sharmal Narayan, PhD, Borbola Pazar, MD, PhD, Laeticia
Kolly, PhD, Nathaliane Bagnoud, MSc, Véronique Chobaz, Alexander
Kai-Lik So, PhD, FRCP, Nathalie Busso, PhD: Centre Hospitalier
Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland; 2Hang-Korng Ea, MD, PhD, Frédéric Lioté, MD, PhD:
INSERM UMR-S606, Hôpital Lariboisière, Assistance PubliqueHôpitaux de Paris, and Université Paris Denis Diderot, Paris, France;
Thomas Vogl, PhD, Dirk Holzinger, MD: University of Münster,
Münster, Germany.
Drs. Narayan and Pazar contributed equally to this work.
Address correspondence to Nathalie Busso, PhD, Division of
Rheumatology, DAL, Laboratory of Rheumatology, CHUV, Nestlé
05-5029, 1011 Lausanne, Switzerland. E-mail: Nathalie.Busso@
Submitted for publication May 3, 2010; accepted in revised
form November 4, 2010.
Basic calcium phosphate (BCP) crystals including
hydroxyapatite, carbonated apatite, tricalcium phosphate, and octacalcium phosphate (OCP) have long
been associated with rheumatic syndromes. BCP crystal
deposition occurs most frequently in soft tissue, muscle,
and articular sites and can manifest with acute inflammation and tissue degradation. Indeed, the presence of
BCP crystals in synovial fluid is more common in
patients with more severe osteoarthritis (OA) (1). Furthermore, it has recently been reported that BCP crystal
deposition in knee and hip cartilage is associated with
end-stage OA (2,3). In the study concerning hip OA (3),
the amount of calcification (predominantly BCP crystals) correlated with clinical symptoms and histologic
OA grade. The role of inflammation itself in OA disease
progression is still uncertain. BCP crystals have also
been associated with destructive arthropathies such as
the Milwaukee shoulder syndrome (4). However, the
mechanisms that underlie the inflammatory reaction
induced by BCP crystals remain unclear.
In vitro, BCP crystals induce fibroblast proliferation, protooncogene stimulation, production of inflammatory cytokines (interleukin-1 [IL-1] and tumor necrosis factor ␤ ), metalloproteinase production and
activation, cyclooxygenase 1 (COX-1), COX-2, and prostaglandin E2 production (5,6), and chondrocyte production of nitric oxide and apoptosis (7,8). In vivo, BCP
crystals have been reported to be proinflammatory,
inducing neutrophil influx in the rat air pouch model (9).
Recently, a role for IL-1␤ has been demonstrated in
monosodium urate monohydrate (MSU) crystal– and
calcium pyrophosphate dihydrate (CPPD) crystal–
induced inflammation; MSU and CPPD crystals are
associated with acute gout and pseudogout, respectively.
It is not known whether BCP crystals induce inflammation through this pathway.
IL-1␤ is a potent inflammatory cytokine, the
production of which is tightly controlled at the level of
gene expression, proteolytic processing, and secretion
(10). Thus, proIL-1␤ protein (protein of 35 kd molar
mass) is converted to active IL-1␤ (protein of 17 kd
molar mass) mainly by caspase 1, but other leukocyte
proteinases such as proteinase 3, elastase, chymase, and
granzyme A may also be involved during inflammation
(11–16). The activity of caspase 1 is regulated by the
inflammasome, an intracellular multicomponent complex that is assembled following cellular stimuli from
pathogens and danger signals (17). Several inflammasome complexes have been described, and activation of
the inflammasome has been linked to infectious and
autoinflammatory diseases (for review, see ref. 17).
NLRP3 is thus far the best characterized inflammasome and is formed by the adaptor protein ASC,
caspase 1, and NLRP3 (18). NLRP3 gain-of-function
mutations are responsible for one of the hereditary
autoinflammatory syndromes, cryopyrin-associated periodic syndrome, that responds dramatically to IL-1 inhibition (19). Similarly, NLRP3 is needed for monocyte
IL-1␤ production upon stimulation with MSU and
CPPD crystals (20), and studies have demonstrated the
clinical efficacy of IL-1␤ blockade in both acute gout and
pseudogout attacks (21–23). However, the situation in
vivo could be different, as has been suggested by studies
in which a role of the inflammasome was not demonstrated in murine models of arthritis that are well known
to be IL-1␤ dependent (15). Possible explanations for
this discrepancy include the contribution of multiple cell
types to the inflammatory state in vivo which is not the
case in vitro, and the possibility that crystals exert other
effects on tissues to provoke an inflammatory response
independent of IL-1␤ production. Finally, crystals may
interact with host proteins in vivo to modify their
phlogistic effects, as has been demonstrated in MSU
crystal–induced inflammation (24,25). This prompted us
to assess in vivo the inflammatory effect of OCP crystals
using the murine peritonitis model, and to dissect the
mechanisms involved in IL-1␤ production. Furthermore,
the contribution of the NLRP3 inflammasome in OCP
crystal–induced inflammation and peritonitis was investigated.
Mice. C57BL/6J mice were purchased from Harlan.
IL-1␣–/– and IL-1␤–/– mice were a gift from Dr. Yoichiro
Iwakura (University of Tokyo, Tokyo, Japan) (26). ASC–/–
mice (27) and NLRP3–/– mice (20) were backcrossed into the
C57BL/6J background for at least 9 generations and were
compared with wild-type (WT) littermates in this study. Mice
were bred under conventional, non–specific pathogen–free
conditions. Mice ages 8–12 weeks were used for experiments.
Institutional approval was obtained for these experiments.
Preparation of MSU and OCP crystals. Sterile,
pyrogen-free MSU and BCP crystals were synthesized as
previously described (9,20). Crystals were suspended in sterile
phosphate buffered saline (PBS) and dispersed by brief sonication. All crystals were determined to be endotoxin free
(⬍0.01 endotoxin units/10 mg of crystal) by Limulus amebocyte cell lysate assay.
Crystal-induced peritonitis. Mice were injected intraperitoneally (IP) with 1 mg of MSU or OCP crystals in 0.5 ml
sterile PBS. To analyze the involvement of IL-1, mice were
injected IP with either 10 ␮g of neutralizing rabbit polyclonal
anti–IL-1␤ antibody (in 0.5 ml PBS) (Novartis) or 200 ␮g of
anakinra (recombinant IL-1 receptor antagonist [IL-1Ra])
(Kineret; Amgen) 30 minutes prior to crystal administration.
An equal volume of sterile PBS was injected into control mice.
To test the involvement of neutrophil proteinases, mice were
treated with 1 mg of the neutrophil elastase inhibitor
(AAPV; Calbiochem) 1 hour before crystal administration.
(AAPV was initially resuspended in DMSO at 10 mg/ml and
diluted in PBS at 1 mg/ml for injections.) Control mice were
injected with the vehicle alone. After 6 hours, blood was
collected, mice were euthanized by CO2 administration, and
peritoneal exudate cells were subsequently harvested by performing lavage with 3 ml of PBS. Total numbers of viable
peritoneal exudate cells were determined by trypan blue
exclusion. Lavage fluids were centrifuged at 450g for 10
minutes. Supernatants were used for analysis of cytokines
and myeloid-related protein 8 (MRP-8)–MRP-14 complex.
Cells were subjected to cytospin staining and flow cytometric
analysis. Neutrophil numbers in the peritoneal exudate cells
were determined by multiplying the total cell numbers by the
percentage of lymphocyte antigen 6 complex, locus G (Ly6G)–positive CD11b⫹ cells in individual mice.
Flow cytometric analysis. Peritoneal exudate cells were
resuspended in fluorescence-activated cell sorting (FACS)
buffer (5% fetal calf serum [FCS] plus 5 mM EDTA in PBS)
and incubated with conjugated monoclonal antibodies (mAb).
The mAb used were phycoerythrin-conjugated anti–Ly-6G
(clone RB6-8C5), fluorescein isothiocyanate (FITC)–
conjugated anti-CD11b (clone M1/70), and allophycocyaninconjugated anti-F4/80 (clone BM8) (all from eBioscience).
Peritoneal cells (1 ⫻ 106) were incubated with appropriate
conjugated antibodies for 30 minutes at 4°C in the dark.
Stained cells were subsequently washed twice in FACS buffer
and fixed in BD CellFIX solution (BD Biosciences). All data
acquisition was performed on a FACSCalibur flow cytometer
(BD Biosciences) using CellQuest software (BD Biosciences).
Data analysis was performed using FlowJo software (Tree Star).
Enzyme-linked immunosorbent assay (ELISA). Cytokine levels in harvested lavage fluid supernatant were analyzed
by IL-1␣ ELISA (BioLegend) and IL-1␤ ELISA (eBioscience)
according to the manufacturers’ instructions. Concentrations
of MRP-8–MRP-14 complex in serum and peritoneal supernatants were determined by ELISA as previously described (28).
Isolation of thioglycolate-elicited peritoneal macrophages. Macrophages were isolated from the peritoneal cavity
of C57BL/6J mice as described previously (29). Briefly, naive
mice were given IP injections of 4% sterile thioglycolate (0.5
ml). After 4 days, peritoneal cells collected by lavage were
seeded at 1 ⫻ 106/ml in RPMI 1640 medium supplemented
with 10% calf serum and antibiotics for 4 hours to allow the
macrophages to adhere to the plates. Nonadherent cells were
subsequently removed and adherent macrophages were used
for experiments.
Isolation of bone marrow–derived mast cells. Bone
marrow–derived mast cells were generated from bone marrow
of C57BL/6J mice as described previously (30). Briefly, naive
mice were killed and intact femurs and tibias were harvested.
Sterile RPMI 1640 medium was repeatedly flushed through
the bone shaft using a syringe with a 25-G needle. After lysis of
red blood cells, cells were washed and cultured at a concentration of 1 ⫻ 106/ml in RPMI 1640 supplemented with 10%
FCS, 100 units/ml penicillin, and 100 ␮g/ml streptomycin.
Recombinant mouse IL-3 (5 ng/ml; R&D Systems) was added
weekly to the cultures. Nonadherent cells were transferred to
fresh medium at least once per week. Cells were used after 8
weeks of culture, when a mast cell purity of ⬎95% was
achieved as assessed by toluidine blue staining and FACS
analysis of CD117 (c-Kit) expression using FITC-conjugated
anti-CD117 mAb (clone 2B8; eBioscience).
Depletion of mast cells. Treatment with compound
48/80 (Sigma) was based on slight modifications to a previously
reported protocol (31). Briefly, mice were treated IP with
compound 48/80 (2 daily injections of 10 ␮g) for 3 days before
crystal administration. Control mice received sterile PBS. Mast
cell depletion was confirmed by identification of toluidine
blue–stained cells in the peritoneal fluid, following which mice
were immediately challenged with an IP dose of 1 mg of OCP
Depletion of resident macrophages. Clodronate liposomes were kindly provided by Dr. Nico van Rooijen (VU
University Medical Center, Amsterdam, The Netherlands) and
were prepared as previously described (32). Mice were given
an IP injection of 200 ␮l of clodronate liposomes. Control mice
received liposomes containing PBS. Three days later, macrophage depletion in liposome-treated mice was confirmed by
flow cytometry, following which mice were immediately challenged with an IP dose of 1 mg of OCP crystals.
Peritoneal cell viability. Cell viability was assessed by
flow cytometric analysis using the FITC Annexin V Apoptosis
Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. Briefly, 6 hours after IP injection of 1 mg
of OCP crystals, peritoneal cells were recovered and stained
with FITC-conjugated annexin V and propidium iodide (PI).
Viable, early apoptotic, and late apoptotic and/or necrotic cells
were identified as annexin V negative/PI negative, annexin V
positive/PI negative, and annexin V positive/PI positive, respectively.
Statistical analysis. All values are expressed as the
mean ⫾ SEM. Variation between data sets was evaluated using
Student’s t-test or one-way analysis of variance where appropriate. P values less than 0.05 were considered significant. Data
were analyzed using GraphPad Prism software.
OCP crystal stimulation induces acute inflammation. To study the inflammatory response to OCP
crystals in vivo, we used an established model of crystalinduced neutrophil infiltration into the peritoneal cavity.
In previous experiments we determined that IP administration of OCP crystals induces a dose-dependent
accumulation of neutrophils at the site of crystal deposition, with a plateau effect observed between 0.5 mg
and 1 mg (data not shown). Therefore, for subsequent in
vivo experiments, we used a dose of 1 mg of OCP
crystals/mouse. MSU crystals injected at the same dose
were used as a positive control. We initially assessed the
in vivo kinetics of neutrophils and macrophages during
OCP crystal–induced peritonitis. Compared with baseline levels (PBS-injected negative controls), absolute
numbers of neutrophils (Ly-6G⫹CD11b⫹) in the peritoneal lavage fluid increased gradually following IP
injection of OCP crystals, reaching a plateau by 6 hours
(Figure 1A). In contrast, absolute numbers of macro-
Figure 1. Octacalcium phosphate (OCP) crystals induce peritoneal inflammation. Naive C57BL/6J mice were injected intraperitoneally
(IP) with 1 mg of OCP crystals. Mice injected with phosphate buffered saline (PBS) were used as negative controls. Injected mice were killed
3, 6, 9, 12, or 24 hours following OCP crystal administration. A and B, Neutrophils (A) and macrophages (B) in peritoneal lavage fluid were
quantified by flow cytometry. Values are the mean ⫾ SEM from at least 5 mice per group. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus baseline. C, Naive
C57BL/6J mice were injected IP with 1 mg of either OCP crystals or monosodium urate monohydrate (MSU) crystals. Mice injected with
PBS were used as negative controls. Six hours following crystal administration, neutrophil recruitment into the peritoneal cavity was
quantified by flow cytometry. D and E, Levels of interleukin-1␣ (IL-1␣) (D) and IL-1␤ (E) in the peritoneal lavage fluid were assessed by
enzyme-linked immunosorbent assay (ELISA). F and G, Levels of myeloid-related protein 8 (MRP-8)–MRP-14 complex in the peritoneal
lavage fluid (F) or in serum (G) were assessed by ELISA. Values are the mean ⫾ SEM from at least 6 mice per group. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. Ly-6G ⫽ lymphocyte antigen 6 complex, locus G.
phages (Ly-6G–CD11b⫹) decreased substantially from
baseline values 3 hours after IP injection of OCP crystals
(Figure 1B). After 6 hours, the numbers of macrophages
within the peritoneal lavage fluid began to increase
slowly, although this increase was not statistically significant. Since 6 hours after OCP crystal administration
appears to be a good time point for showing the acute
reaction of both neutrophils and macrophages in response to OCP crystal injection into the peritoneal
cavity, the 6-hour time point was used as the end point
for peritoneal lavage fluid harvest for subsequent in vivo
We next compared the inflammatory response to
OCP and MSU crystals in vivo. Upon IP administration,
OCP crystals were able to induce Ly-6G⫹CD11b⫹
neutrophil influx at levels comparable with those
achieved following injection of MSU crystals (Figure
1C). As expected, the absolute numbers of peritoneal
Ly-6G–CD11b⫹ macrophages decreased significantly
after crystal injection (mean ⫾ SEM 706,000 ⫾ 128,000
in PBS-injected mice, 396,000 ⫾ 230,000 in OCP crystal–
injected mice, and 121,000 ⫾ 38,000 in MSU crystal–
injected mice) (data not shown). The recruitment of neutrophils was associated with elevated levels of both
IL-1␣ and IL-1␤ in the peritoneal lavage fluid of crystalinjected mice compared with levels in PBS-injected
controls (Figures 1D and E, respectively). These results
also correlated with a significant increase in the level of
MRP-8–MRP-14 complex, which is considered a reliable
marker of inflammation (33,34), both in peritoneal
lavage fluid (Figure 1F) and in serum samples (Figure
1G). Taken together, these observations demonstrate
Figure 2. IL-1 blockade or deficiency prevents OCP crystal–induced inflammation. A, Naive mice or mice pretreated with anti–IL-1␤
monoclonal antibodies or anakinra were administrated an IP dose of 1 mg of either OCP crystals or MSU crystals. Six hours after
crystal injection, mice were killed and neutrophil accumulation in the peritoneal cavity was assessed by flow cytometry. Values are the
mean ⫾ SEM from at least 10 mice per group. B–D, Wild-type, IL-1␣–/–, and IL-1␤–/– mice were injected IP with 1 mg OCP crystals.
Six hours after crystal injection, neutrophil accumulation in the peritoneal cavity was quantified by flow cytometry (B), and levels of
IL-1␣ (C) and IL-1␤ (D) in the peritoneal lavage fluid were assessed by ELISA. Values are the mean ⫾ SEM from at least 4 mice per
group.ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for definitions.
that in vivo OCP crystal stimulation is able to induce
strong inflammation both locally and systemically.
Blockade or deficiency of IL-1 strongly reduces
OCP crystal–induced inflammation. We next assessed
whether OCP crystal–triggered inflammation acts via
IL-1–dependent pathways. Naive mice were treated with
anakinra (recombinant IL-1Ra), which binds tightly to
IL-1R type I, blocking the activity of either IL-1␣ or
IL-1␤ (35). Anakinra-treated mice displayed a significant reduction in both OCP crystal– and MSU crystal–
induced neutrophil recruitment (Figure 2A). Decreased
OCP crystal– and MSU crystal–induced neutrophil infiltration into the peritoneal cavity was also observed in
mice treated with IL-1␤–neutralizing antibodies. Collectively, these results demonstrate that IL-1R activation
and IL-1 production play essential roles in OCP crystal–
triggered inflammation.
Since we showed that both IL-1␣ and IL-1␤ levels
were increased in peritoneal lavage fluid samples from
crystal-injected mice (Figures 1D and E, respectively)
and that anti–IL-1␤ antibodies and recombinant IL-1Ra
were efficient in blocking neutrophil recruitment, we
next assessed the relative contributions of IL-1␣ and
IL-1␤ in OCP crystal–induced inflammation. Mice defi-
cient in either IL-1␣ or IL-1␤ were injected with OCP
crystals, and peritoneal neutrophil recruitment was assessed 6 hours following crystal administration (Figure
2B). In the absence of IL-1␣, there was a striking and
significant decrease in neutrophil recruitment upon
OCP crystal injection. A similar decrease was observed
in IL-1␤–deficient mice, although this was not statistically significant. As expected, IL-1␣–/– and IL-1␤–/– mice
indeed did not produce IL-1␣ and IL-1␤, respectively,
after OCP crystal administration, and the absence of one
of these cytokines affected the production of the other
(Figures 2C and D). These results indicate that IL-1␣
and IL-1␤ as independent cytokines are important in
crystal-induced neutrophil recruitment. However, these
2 soluble factors when functioning separately may not be
solely responsible for facilitating crystal-induced neutrophil accumulation in the peritoneal cavity.
OCP crystal–stimulated inflammation is independent of ASC and NLRP3. Since we have shown that
IL-1␤ plays a prominent role in OCP crystal–induced
inflammation, we next investigated the contribution of
the inflammasome, the multiprotein complex able to
convert proIL-1␤ into biologically active IL-1␤, to OCP
Figure 3. ASC or NLRP3 deficiency does not affect OCP crystal–induced inflammation. A, Wild-type (WT), ASC–/–, and NLRP3–/–
mice were injected IP with 1 mg of OCP crystals. Six hours following crystal challenge, neutrophil accumulation in the peritoneal cavity
was quantified by flow cytometry. As expected, OCP crystal injection induced a significant increase in neutrophil recruitment
compared with that in PBS-injected controls, and similar numbers of neutrophils were recruited in ASC–/– mice, NLRP3–/– mice, and
WT mice. B and C, Levels of MRP-8–MRP-14 complex (B) and IL-1␤ (C) in the peritoneal lavage fluid were assessed by ELISA. D,
WT, ASC–/–, and NLRP3–/– mice were treated with 200 ␮g of anakinra 30 minutes prior to injection with 1 mg OCP crystals. Six hours
after crystal injection, neutrophil recruitment into the peritoneal cavity was quantified by flow cytometry. Anakinra significantly
reduced neutrophil recruitment in all mouse strains. Values are the mean ⫾ SEM from at least 10 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ
⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
crystal–induced inflammation. We investigated the roles
of the adaptor protein ASC and of the inflammasome
sensor NLRP3. ASC–/– and NLRP3–/– mice were injected IP with OCP crystals. Compared with WT mice,
these deficient mice did not show any alteration in their
inflammatory response, as demonstrated by similar
numbers of neutrophils in the peritoneal cavity (Figure
3A) along with comparable levels of MRP-8–MRP-14
complex (Figure 3B) and IL-1␤ (Figure 3C) in the
peritoneal lavage fluid. In addition, anakinra had similar
inhibitory effects in WT, ASC–/–, and NLRP3–/– mice
(Figure 3D). Taken together, these results indicate that
these OCP crystal–associated inflammatory responses
are independent of the classic NLRP3 inflammasome
and suggest that other inflammasomes may play a role,
or, alternatively, that OCP crystal–induced inflammation involves a caspase 1–independent IL-1␤–processing
mechanism. In this context we have tested the involvement of neutrophil elastase, a proteinase able to convert
proIL-1␤ into biologically active IL-1␤ (13,36). Indeed,
when mice were treated prophylactically with AAPV, an
inhibitor of neutrophil elastase, we found a nonsignifi-
cant trend toward a decrease (a 30% reduction) in both
neutrophil recruitment and IL-1␤ levels in peritoneal
fluid (data not shown).
Mast cell depletion does not attenuate OCP
crystal–induced inflammation. We first tested whether
mast cells were able to respond to OCP crystals by
releasing IL-1␣ and IL-1␤, thereby contributing to the
onset of OCP crystal–induced inflammation. We therefore generated bone marrow–derived mast cells of
⬎95% purity. OCP crystal stimulation of mast cells,
either previously primed with lipopolysaccharide (LPS)
or not, resulted in a massive release of IL-1␣ and IL-1␤
into the supernatant (Figures 4A and B, respectively).
To study in vivo the effect of this OCP crystal–associated
induction of IL-1 by mast cells, we next induced OCP
crystal–mediated peritonitis in mast cell–depleted mice.
Mice locally injected with compound 48/80 had ⬎95%
depletion of peritoneal mast cells (Figure 4C). We
observed comparable levels of neutrophil recruitment
between OCP crystal–injected mice pretreated with
compound 48/80 and those pretreated with PBS (Figure
4D). These results suggest that resident mast cells do not
Figure 4. Mast cells are not required for OCP crystal–induced inflammation. A and B, Bone marrow–derived mast cells from
wild-type mice were stimulated with 500 ␮g/ml of OCP crystals in vitro with or without prior priming with lipopolysaccharide (LPS).
Six hours following stimulation, culture supernatants were harvested and analyzed for secretion of IL-1␣ (A) and IL-1␤ (B). Values
are the mean ⫾ SEM of triplicate cultures. C and D, Mice received 10 ␮g of compound 48/80 in 0.5 ml PBS IP 72, 48, and 24 hours
before OCP crystal administration. Control mice were similarly treated with PBS alone. C, Before crystal injection, mast cells from
peritoneal fluid of compound 48/80–injected mice or PBS-injected mice were stained with toluidine blue and counted. Values are the
mean ⫾ SEM from 3 mice per group. D, Neutrophil accumulation was quantified 6 hours after crystal injection. Values are the mean
⫾ SEM from 5 mice per group. The difference in neutrophil recruitment between non–mast cell–depleted and mast cell–depleted mice
was not significant. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
play an important role in the cellular response induced
following crystal administration.
Macrophage depletion abolishes OCP crystal–
induced inflammation. Peritoneal macrophages could
also be likely candidates that respond to OCP crystals
by releasing IL-1␣ and IL-1␤ and thereby triggering
OCP crystal–induced inflammation. When purified
peritoneal macrophages that were previously primed
with LPS (37) were stimulated with OCP crystals, cells
were able to release both IL-1␣ and IL-1␤ in supernatants (Figures 5A and B). We next analyzed the
inflammatory response to OCP crystals in mice depleted of resident macrophages by pretreatment with
clodronate liposomes (32). An IP injection of clodronate liposomes 3 days prior to peritoneal lavage fluid
harvest resulted in ⬎90% depletion of resident macrophages (Figure 5C). Control mice received liposomes
containing PBS. We found that macrophage-depleted
mice were not able to recruit neutrophils upon IP
administration of OCP crystals, whereas in non–
macrophage-depleted mice the vast majority of peritoneal cells were neutrophils (Figures 5C and D). As
expected, IL-1␤ levels in peritoneal lavage fluid were
significantly decreased in mice pretreated with clodronate liposomes (Figure 5E).
OCP crystals induce apoptosis/necrosis of peritoneal cells in vivo. We have found that OCP crystals
have a deleterious effect on the viability of murine bone
marrow–derived macrophages cultured in vitro (⬃50%
cell death after 6-hour incubation with 500 ␮g/ml OCP
crystals) (37). In order to assess the relevance of such a
phenomenon following in vivo OCP crystal administration, peritoneal cells were costained with annexin V and
PI. We found that ⬃50% of cells recovered 6 hours after
IP injection of OCP crystals were dead (either in late
apoptosis or in necrosis). In contrast, the vast majority of
cells from PBS-injected mice were viable (Figures 6A
and B).
OCP crystals, members of the BCP crystal family,
elicit joint as well as periarticular inflammation and may
have a pathogenic role in OA. The mechanisms of
inflammation due to BCP crystals have not been extensively studied. Here we report that in the murine peri-
Figure 5. Resident macrophages are crucial cells for OCP crystal–induced inflammation. A and B, Bone marrow–derived
macrophages from wild-type mice were stimulated with 500 ␮g/ml of OCP crystals in vitro with or without prior priming with
lipopolysaccharide (LPS). Six hours following stimulation, culture supernatants were harvested and analyzed for secretion of IL-1␣ (A)
and IL-1␤ (B). C–E, Mice were treated IP with 200 ␮l of clodronate liposomes. Control mice received liposomes containing PBS. Three
days later, mice were challenged with an IP injection of 1 mg of OCP crystals. Peritoneal lavage fluid was collected 6 hours after crystal
stimulation, and the recruitment of peritoneal neutrophils was assessed by flow cytometry (C and D). In C, neutrophils
(Ly-6G⫹F4/80–) are represented as R2; macrophages (Ly-6G⫺F4/80⫹) are represented as R3. Top, Mice treated with PBS containing
liposomes only; middle, OCP crystal–injected mice previously pretreated with PBS containing liposomes; bottom, OCP crystal–injected
mice previously pretreated with clodronate liposomes. IL-1␤ levels in the peritoneal lavage fluid from macrophage-depleted and
non–macrophage-depleted mice injected with crystals were assessed by ELISA (E). Values in A, B, D, and E are the mean ⫾ SEM from
5 mice per group. Plots in C are representative of 3 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001. PE ⫽ phycoerythrin; APC ⫽
allophycocyanin (see Figure 1 for other definitions). Color figure can be viewed in the online issue, which is available at
tonitis model, OCP crystals cause a strong recruitment
of neutrophils that is comparable with levels achieved
with MSU crystals, whose proinflammatory properties
have been well documented (20,38,39). These findings
prompted us to investigate the molecular mechanisms
responsible for neutrophil accumulation in an experimental model of OCP crystal–induced peritonitis.
The recent emergence of the inflammasome as a
caspase 1 activator and its role in hereditary autoinflammatory syndromes and in gout raises the question of
whether this complex is equally relevant in IL-1␤ pro-
duction in BCP crystal– and, especially, OCP crystal–
induced inflammatory situations. To address the role of
the inflammasome in OCP crystal–induced pathogenesis, we assessed the effect of genetic deletion of different
elements of the inflammasome on neutrophil recruitment into the peritoneal cavity upon OCP crystal administration. We found no effect of either ASC or
NLRP3 deficiency on neutrophil recruitment, and similar amounts of MRP were detected in the peritoneal
cavity. The absence of an obvious influence of NLRP3
and ASC on OCP crystal–induced inflammation un-
Figure 6. OCP crystals induce apoptosis/necrosis of peritoneal cells in vivo. A and B, Mice were injected with PBS as a control or with
1 mg of OCP crystals. After 6 hours, cells were recovered from peritoneal lavage fluid and analyzed for viability by flow cytometry using
annexin V and propidium iodide (PI) staining. Viable, early apoptotic, and late apoptotic and/or necrotic cells were identified as
annexin V negative/PI negative, annexin V positive/PI negative, and annexin V positive/PI positive, respectively. Values are the mean
⫾ SEM from 4 mice per group. ⴱⴱⴱ ⫽ P ⬍ 0.001. C, Model of OCP crystal–induced peritoneal inflammation. The major event
triggering OCP crystal–induced peritoneal inflammation is mediated by OCP crystal–induced cell death. IL-1␣ is released from
necrotic cells. IL-1␣ and IL-1␤ bind to IL-1 receptor type I (IL-1RI) on target cells. IL-1RI activation subsequently induces the
production of CXCL1 by mesothelial cells and fibroblasts. CXCL1 in turn facilitates neutrophil recruitment, which can also be
mediated by MRP and IL-8. ProIL-1␤ can also be released from dead cells. Proteinases derived from neutrophils (such as proteinase
3, elastase, and cathepsin G) or mast cells (such as chymase and cathepsin G) can activate proIL-1␤ independent of the inflammasome.
See Figure 1 for other definitions.
equivocally rules out a role for the NLRP3 inflammasome in OCP-induced inflammatory responses in vivo.
These findings contrast with observations that MSU
crystals (20), as well as inorganic particles such as
asbestos fibers, silica particles, and alum crystals,
activate the NLRP3 inflammasome to produce IL-1␤
The presence of IL-1␤ in the peritoneal exudate
and the attenuation of inflammation by IL-1␤ blockade
and IL-1␤ deficiency suggest that there is an NLRP3
inflammasome–independent mechanism. We and others
have recently demonstrated the existence of
inflammasome-independent pathways of IL-1␤ processing in IL-1␤–mediated diseases such as antigen-induced
arthritis (43), collagen-induced arthritis (44), K/BxN
serum transfer–induced arthritis (14), acute arthritis
(15), and experimental models of infections (for review,
see ref. 11). In these disease models, enzymes distinct
from caspase 1 are able to process proIL-1␤ (discussed
in refs. 11 and 16). Neutrophil-, macrophage-, and mast
cell–derived serine proteinases such as proteinase 3,
elastase, cathepsin G, and chymase have been reported
to be able to convert proIL-1␤ into the 21-kd active form
(10,12,13). A crucial role of chymase and elastase in
proIL-1␤ activation was recently shown in the K/BxN
arthritis model using specific proteinase inhibitors (14).
Consistent with this latter finding, we tested the effects
of AAPV in OCP crystal–induced peritonitis and found
a 30% reduction in neutrophil recruitment and peritoneal IL-1␤ (this difference did not reach significance
[data not shown]), suggesting that a part of proIL-1␤
processing in OCP crystal–induced peritonitis is due to
neutrophil elastase.
OCP crystal–induced inflammation in the peritonitis model depends on both IL-1␣ and IL-1␤. The first
line of evidence for this was the finding that both IL-1␣
and IL-1␤ were released into the peritoneal exudate
after crystal injection. A role for both cytokines was
highlighted by the inhibitory effects of individual deficiency of IL-1␣ and IL-1␤. Interestingly, their effects on
inflammation seem to be linked, since IL-1␣ deficiency
had an effect on peritoneal IL-1␤ levels and vice versa.
Such a reciprocal regulation of IL-1␣ over the production of IL-1␤ has been previously reported in another
experimental model (26). IL-1␣ is biologically active in
its precursor form and can be found on the surface of
several cells, particularly monocytes, where it is referred
to as membrane IL-1␣ (10). Cleavage of the precursor
by calpain, a membrane-associated calcium-activated
cysteine proteinase, releases mature IL-1␣. It may also
be released from dying cells (45).
We demonstrated that OCP crystals induced
necrosis of ⬃50% of peritoneal cells, principally represented by infiltrating neutrophils. In this context, it is
likely that at least part of the peritoneal fluid IL-1␣ that
we measured following OCP crystal injection was released from dying cells. Interestingly, it has been shown
that IL-1␣ released from necrotic cells triggers CXCL1/
cytokine-induced neutrophil chemoattractant (KC) secretion and recruitment of neutrophils via IL-1R/
myeloid differentiation factor 88 signaling on
neighboring mesothelial cells (46). Therefore, we can
anticipate that the inflammatory properties of OCP
crystals include their ability to induce cellular necrosis.
The subsequent passive release of IL-1␣ from dying cells
would in turn facilitate chemokine production (eventually CXCL1) and neutrophil recruitment to the inflamed
site. Validation of such a mechanism would require
crystal administration in mice with a targeted mutation
in CXCL1/KC (47).
It has recently been shown that in addition to
passive release of danger signals such as uric acid or
ATP, necrotic cells drive inflammatory cell infiltration
in vivo and induce the production of IL-1␤ in an NLRP3
inflammasome–dependent manner (42,48). Our data do
not support the influence of such a mechanism, since
neutrophil recruitment and IL-1␤ production were not
altered in the absence of the NLRP3 inflammasome.
Such a discrepancy could be explained by the dose of
necrotic cells required to activate the NLRP3 inflammasome. To trigger a sterile inflammatory response
through NLRP3, 107 necrotic cells were injected IP (48).
In our experimental setting, cells were progressively
dying and we recovered ⬃10-fold fewer necrotic cells
6 hours following crystal administration. Thus, below a
certain threshold of necrosis, the NLRP3 inflammasome
is probably not activated.
Mast cells and resident macrophages have been
implicated in MSU crystal–induced peritonitis (36,38),
and we investigated the contribution of these cells in
OCP crystal–induced peritonitis. Depletion studies using
either compound 48/80 or clodronate liposomes showed
that only peritoneal macrophage depletion led to a
reduction of neutrophil recruitment and IL-1␤ levels,
demonstrating that resident macrophages play an essential role in the production of IL-1␤ and in neutrophil
recruitment in OCP crystal–induced inflammation, even
though in in vitro studies, both cell types secreted IL-1␤
when stimulated with OCP crystals. We recently found
that OCP crystals stimulated IL-1␤ secretion in murine
bone marrow–derived macrophages and peritoneal
macrophages through an NLRP3 inflammasome–
dependent pathway in vitro (37). The difference, in
terms of NLRP3 dependency, between the in vivo and
in vitro results may be due to OCP crystal–induced
cell death and the release of non–caspase 1 proteinases already discussed, or to factors that can modulate
crystal interactions with cells, such as protein coating
of crystals in vivo (24,25).
An interesting observation during crystal-induced
peritonitis was that peritoneal macrophages decreased
significantly in the peritoneal lavage fluid following IP
administration of both OCP and MSU crystals. Similar
disappearance of macrophages has been previously observed in harvested peritoneal lavage fluid following IP
injection with MSU crystals (49). This disappearance of
macrophages shortly following administration of inflammatory stimuli has been termed “macrophage disappearance reaction” and has been highlighted in other
models of acute inflammation (50–52).
Finally, we observed a significant release of
MRP-8–MRP-14 complex (S100A8/A9, calprotectin)
during OCP crystal–induced peritonitis, which may further amplify the local inflammatory response. MRPs are
secreted by monocytes and neutrophils following cellular
activation or necrosis (for review, see ref. 53) and
participate in a positive feedback loop of neutrophil
recruitment by up-regulating integrin expression and
mediating chemotaxis (54,55). Not only peritoneal but
also serum levels of MRP-8–MRP-14 complex were
increased upon OCP crystal injection. Similar results
were reported in a murine air pouch model of MSU
crystal–induced inflammation that was inhibited by antiMRP antibodies (56). This suggests that in BCP crystal–
related diseases such as the Milwaukee shoulder syndrome, MRP levels will be increased and might play a
role in pathogenesis.
In conclusion, we have demonstrated that in an in
vivo model, OCP crystals induce inflammation via IL-1␣
and IL-1␤, independent of the NLRP3 inflammasome,
and this process is linked to cell death induced by
crystals (Figure 6C). Furthermore, our data highlight
that macrophages play a crucial role in this inflammatory process but mast cells do not. These mechanisms
can account for the acute inflammatory reaction seen in
acute periarthritis and arthritis due to OCP crystals. In
OA, in which BCP crystals are found in the cartilage as
well as in the joint fluid, these mechanisms do not seem
to predominate, since inflammation is not a prominent
feature. These results have implications in the search for
effective therapies for BCP crystal–associated diseases.
We are grateful to Dr. Nico van Rooijen (VU University Medical Center, Amsterdam, The Netherlands) for kindly
providing us with clodronate liposomes. We thank Dr.
Yoichiro Iwakura (University of Tokyo, Tokyo, Japan) for
providing us with IL-1␣–/– and IL-1␤–/– mice and Professor
Jurg Tschopp (University of Lausanne, Lausanne, Switzerland) for providing us with NLRP3–/– and ASC–/– mice.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Busso 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 conception and design. Narayan, Pazar, Ea, Kolly, Bagnoud,
Chobaz, Lioté, So, Busso.
Acquisition of data. Narayan, Pazar, Ea, Kolly, Bagnoud, Chobaz,
Vogl, Holzinger.
Analysis and interpretation of data. Narayan, Pazar, Ea, Kolly,
Bagnoud, Chobaz, Lioté, Vogl, Holzinger, So, Busso.
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