ARTHRITIS & RHEUMATISMVol. 63, No. 2, February 2011, pp 422–433DOI 10.1002/art.30147© 2011, American College of Rheumatology

Octacalcium Phosphate Crystals Induce Inflammation In VivoThrough 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 Veronique Chobaz,1 Frederic Liote,2 Thomas Vogl,3 Dirk Holzinger,3

Alexander Kai-Lik So,1 and Nathalie Busso1

Objective. To determine the mechanisms involvedin inflammatory responses to octacalcium phosphate(OCP) crystals in vivo.

Methods. OCP crystal–induced inflammation wasmonitored using a peritoneal model of inflammation inmice with different deficiencies affecting interleukin-1(IL-1) secretion (IL-1�–/–, IL-1�–/–, ASC–/–, andNLRP3–/– mice) or in mice pretreated with IL-1 inhibi-tors (anakinra [recombinant IL-1 receptor antagonist]and anti–IL-1�). The production of IL-1�, IL-1�, andmyeloid-related protein 8 (MRP-8)–MRP-14 complexwas determined by enzyme-linked immunosorbent as-say. Peritoneal neutrophil recruitment and cell viabilitywere determined by flow cytometry. Depletion of mastcells or resident macrophages was performed by pre-

treatment with compound 48/80 or clodronate lipo-somes, respectively.

Results. OCP crystals induced peritoneal inflam-mation, as demonstrated by neutrophil recruitment andup-modulation of IL-1�, IL-1�, and MRP-8–MRP-14complex, to levels comparable with those induced bymonosodium urate monohydrate crystals. This OCPcrystal–induced inflammation was both IL-1�– andIL-1�–dependent, as shown by the inhibitory effects ofanakinra and anti–IL-1� antibody treatment. Accord-ingly, OCP crystal stimulation resulted in milder in-flammation in IL-1�–/– and IL-1�–/– mice. Interestingly,ASC–/– and NLRP3–/– mice did not show any alterationin their inflammation status in response to OCP crys-tals. Depletion of the resident macrophage populationresulted in a significant decrease in crystal-inducedneutrophil infiltration and proinflammatory cytokineproduction in vivo, whereas mast cell depletion had noeffect. Finally, OCP crystals induced apoptosis/necrosisof peritoneal cells in vivo.

Conclusion. These data indicate that macro-phages, rather than mast cells, are important for initi-ating and driving OCP crystal–induced inflammation.Additionally, OCP crystals induce IL-1–dependent peri-toneal inflammation without requiring the NLRP3 in-flammasome.

Basic calcium phosphate (BCP) crystals includinghydroxyapatite, carbonated apatite, tricalcium phos-phate, and octacalcium phosphate (OCP) have longbeen associated with rheumatic syndromes. BCP crystaldeposition occurs most frequently in soft tissue, muscle,and articular sites and can manifest with acute inflam-mation and tissue degradation. Indeed, the presence ofBCP crystals in synovial fluid is more common in

Supported by the Fonds National Suisse de la RechercheScientifique (grant 310030-130085/1) and the Jean and Linette Warn-ery Foundation. Drs. Ea and Liote’s work was supported by grantsfrom the Fondation pour la Recherche Medicale, the Association pourla Recherche en Pathologie Synoviale, and the Association Rhuma-tisme et Travail. Dr. Holzinger’s work was supported by a grant fromthe German Ministry of Education and Research (BMBF projectAID-NET).

1Sharmal Narayan, PhD, Borbola Pazar, MD, PhD, LaeticiaKolly, PhD, Nathaliane Bagnoud, MSc, Veronique Chobaz, AlexanderKai-Lik So, PhD, FRCP, Nathalie Busso, PhD: Centre HospitalierUniversitaire Vaudois and University of Lausanne, Lausanne, Swit-zerland; 2Hang-Korng Ea, MD, PhD, Frederic Liote, MD, PhD:INSERM UMR-S606, Hopital Lariboisiere, Assistance Publique-Hopitaux de Paris, and Universite Paris Denis Diderot, Paris, France;3Thomas Vogl, PhD, Dirk Holzinger, MD: University of Munster,Munster, Germany.

Drs. Narayan and Pazar contributed equally to this work.Address correspondence to Nathalie Busso, PhD, Division of

Rheumatology, DAL, Laboratory of Rheumatology, CHUV, Nestle05-5029, 1011 Lausanne, Switzerland. E-mail: Nathalie.Busso@chuv.ch.

Submitted for publication May 3, 2010; accepted in revisedform November 4, 2010.

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patients with more severe osteoarthritis (OA) (1). Fur-thermore, it has recently been reported that BCP crystaldeposition in knee and hip cartilage is associated withend-stage OA (2,3). In the study concerning hip OA (3),the amount of calcification (predominantly BCP crys-tals) correlated with clinical symptoms and histologicOA grade. The role of inflammation itself in OA diseaseprogression is still uncertain. BCP crystals have alsobeen associated with destructive arthropathies such asthe Milwaukee shoulder syndrome (4). However, themechanisms that underlie the inflammatory reactioninduced by BCP crystals remain unclear.

In vitro, BCP crystals induce fibroblast prolifer-ation, protooncogene stimulation, production of inflam-matory cytokines (interleukin-1 [IL-1] and tumor necro-sis factor �), metalloproteinase production andactivation, cyclooxygenase 1 (COX-1), COX-2, and pros-taglandin E2 production (5,6), and chondrocyte pro-duction of nitric oxide and apoptosis (7,8). In vivo, BCPcrystals 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 inmonosodium urate monohydrate (MSU) crystal– andcalcium pyrophosphate dihydrate (CPPD) crystal–induced inflammation; MSU and CPPD crystals areassociated with acute gout and pseudogout, respectively.It is not known whether BCP crystals induce inflamma-tion through this pathway.

IL-1� is a potent inflammatory cytokine, theproduction of which is tightly controlled at the level ofgene expression, proteolytic processing, and secretion(10). Thus, proIL-1� protein (protein of 35 kd molarmass) is converted to active IL-1� (protein of 17 kdmolar mass) mainly by caspase 1, but other leukocyteproteinases such as proteinase 3, elastase, chymase, andgranzyme A may also be involved during inflammation(11–16). The activity of caspase 1 is regulated by theinflammasome, an intracellular multicomponent com-plex that is assembled following cellular stimuli frompathogens and danger signals (17). Several inflamma-some complexes have been described, and activation ofthe inflammasome has been linked to infectious andautoinflammatory diseases (for review, see ref. 17).

NLRP3 is thus far the best characterized inflam-masome and is formed by the adaptor protein ASC,caspase 1, and NLRP3 (18). NLRP3 gain-of-functionmutations are responsible for one of the hereditaryautoinflammatory syndromes, cryopyrin-associated peri-odic syndrome, that responds dramatically to IL-1 inhi-bition (19). Similarly, NLRP3 is needed for monocyteIL-1� production upon stimulation with MSU and

CPPD crystals (20), and studies have demonstrated theclinical efficacy of IL-1� blockade in both acute gout andpseudogout attacks (21–23). However, the situation invivo could be different, as has been suggested by studiesin which a role of the inflammasome was not demon-strated in murine models of arthritis that are well knownto be IL-1� dependent (15). Possible explanations forthis discrepancy include the contribution of multiple celltypes to the inflammatory state in vivo which is not thecase in vitro, and the possibility that crystals exert othereffects on tissues to provoke an inflammatory responseindependent of IL-1� production. Finally, crystals mayinteract with host proteins in vivo to modify theirphlogistic effects, as has been demonstrated in MSUcrystal–induced inflammation (24,25). This prompted usto assess in vivo the inflammatory effect of OCP crystalsusing the murine peritonitis model, and to dissect themechanisms involved in IL-1� production. Furthermore,the contribution of the NLRP3 inflammasome in OCPcrystal–induced inflammation and peritonitis was inves-tigated.

MATERIALS AND METHODS

Mice. C57BL/6J mice were purchased from Harlan.IL-1�–/– and IL-1�–/– mice were a gift from Dr. YoichiroIwakura (University of Tokyo, Tokyo, Japan) (26). ASC–/–

mice (27) and NLRP3–/– mice (20) were backcrossed into theC57BL/6J background for at least 9 generations and werecompared with wild-type (WT) littermates in this study. Micewere bred under conventional, non–specific pathogen–freeconditions. 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 aspreviously described (9,20). Crystals were suspended in sterilephosphate buffered saline (PBS) and dispersed by brief soni-cation. All crystals were determined to be endotoxin free(�0.01 endotoxin units/10 mg of crystal) by Limulus amebo-cyte cell lysate assay.

Crystal-induced peritonitis. Mice were injected intra-peritoneally (IP) with 1 mg of MSU or OCP crystals in 0.5 mlsterile PBS. To analyze the involvement of IL-1, mice wereinjected IP with either 10 �g of neutralizing rabbit polyclonalanti–IL-1� antibody (in 0.5 ml PBS) (Novartis) or 200 �g ofanakinra (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 weretreated with 1 mg of the neutrophil elastase inhibitormethoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone(AAPV; Calbiochem) 1 hour before crystal administration.(AAPV was initially resuspended in DMSO at 10 mg/ml anddiluted in PBS at 1 mg/ml for injections.) Control mice wereinjected with the vehicle alone. After 6 hours, blood wascollected, mice were euthanized by CO2 administration, and

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peritoneal exudate cells were subsequently harvested by per-forming lavage with 3 ml of PBS. Total numbers of viableperitoneal exudate cells were determined by trypan blueexclusion. Lavage fluids were centrifuged at 450g for 10minutes. Supernatants were used for analysis of cytokinesand myeloid-related protein 8 (MRP-8)–MRP-14 complex.Cells were subjected to cytospin staining and flow cytometricanalysis. Neutrophil numbers in the peritoneal exudate cellswere determined by multiplying the total cell numbers by thepercentage of lymphocyte antigen 6 complex, locus G (Ly-6G)–positive CD11b� cells in individual mice.

Flow cytometric analysis. Peritoneal exudate cells wereresuspended 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 allophycocyanin-conjugated anti-F4/80 (clone BM8) (all from eBioscience).Peritoneal cells (1 � 106) were incubated with appropriateconjugated antibodies for 30 minutes at 4°C in the dark.Stained cells were subsequently washed twice in FACS bufferand fixed in BD CellFIX solution (BD Biosciences). All dataacquisition 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). Cyto-kine levels in harvested lavage fluid supernatant were analyzedby IL-1� ELISA (BioLegend) and IL-1� ELISA (eBioscience)according to the manufacturers’ instructions. Concentrationsof MRP-8–MRP-14 complex in serum and peritoneal super-natants were determined by ELISA as previously described (28).

Isolation of thioglycolate-elicited peritoneal macro-phages. Macrophages were isolated from the peritoneal cavityof C57BL/6J mice as described previously (29). Briefly, naivemice were given IP injections of 4% sterile thioglycolate (0.5ml). After 4 days, peritoneal cells collected by lavage wereseeded at 1 � 106/ml in RPMI 1640 medium supplementedwith 10% calf serum and antibiotics for 4 hours to allow themacrophages to adhere to the plates. Nonadherent cells weresubsequently removed and adherent macrophages were usedfor experiments.

Isolation of bone marrow–derived mast cells. Bonemarrow–derived mast cells were generated from bone marrowof C57BL/6J mice as described previously (30). Briefly, naivemice were killed and intact femurs and tibias were harvested.Sterile RPMI 1640 medium was repeatedly flushed throughthe bone shaft using a syringe with a 25-G needle. After lysis ofred blood cells, cells were washed and cultured at a concen-tration 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 addedweekly to the cultures. Nonadherent cells were transferred tofresh medium at least once per week. Cells were used after 8weeks of culture, when a mast cell purity of �95% wasachieved as assessed by toluidine blue staining and FACSanalysis of CD117 (c-Kit) expression using FITC-conjugatedanti-CD117 mAb (clone 2B8; eBioscience).

Depletion of mast cells. Treatment with compound48/80 (Sigma) was based on slight modifications to a previouslyreported protocol (31). Briefly, mice were treated IP withcompound 48/80 (2 daily injections of 10 �g) for 3 days beforecrystal administration. Control mice received sterile PBS. Mastcell depletion was confirmed by identification of toluidineblue–stained cells in the peritoneal fluid, following which micewere immediately challenged with an IP dose of 1 mg of OCPcrystals.

Depletion of resident macrophages. Clodronate lipo-somes were kindly provided by Dr. Nico van Rooijen (VUUniversity Medical Center, Amsterdam, The Netherlands) andwere prepared as previously described (32). Mice were givenan IP injection of 200 �l of clodronate liposomes. Control micereceived liposomes containing PBS. Three days later, macro-phage depletion in liposome-treated mice was confirmed byflow cytometry, following which mice were immediately chal-lenged with an IP dose of 1 mg of OCP crystals.

Peritoneal cell viability. Cell viability was assessed byflow cytometric analysis using the FITC Annexin V ApoptosisDetection Kit I (BD Biosciences) according to the manufac-turer’s instructions. Briefly, 6 hours after IP injection of 1 mgof OCP crystals, peritoneal cells were recovered and stainedwith FITC-conjugated annexin V and propidium iodide (PI).Viable, early apoptotic, and late apoptotic and/or necrotic cellswere identified as annexin V negative/PI negative, annexin Vpositive/PI negative, and annexin V positive/PI positive, re-spectively.

Statistical analysis. All values are expressed as themean � SEM. Variation between data sets was evaluated usingStudent’s t-test or one-way analysis of variance where appro-priate. P values less than 0.05 were considered significant. Datawere analyzed using GraphPad Prism software.

RESULTS

OCP crystal stimulation induces acute inflam-mation. To study the inflammatory response to OCPcrystals in vivo, we used an established model of crystal-induced neutrophil infiltration into the peritoneal cavity.In previous experiments we determined that IP admin-istration of OCP crystals induces a dose-dependentaccumulation of neutrophils at the site of crystal depo-sition, with a plateau effect observed between 0.5 mgand 1 mg (data not shown). Therefore, for subsequent invivo experiments, we used a dose of 1 mg of OCPcrystals/mouse. MSU crystals injected at the same dosewere used as a positive control. We initially assessed thein vivo kinetics of neutrophils and macrophages duringOCP crystal–induced peritonitis. Compared with base-line levels (PBS-injected negative controls), absolutenumbers of neutrophils (Ly-6G�CD11b�) in the peri-toneal lavage fluid increased gradually following IPinjection of OCP crystals, reaching a plateau by 6 hours(Figure 1A). In contrast, absolute numbers of macro-

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phages (Ly-6G–CD11b�) decreased substantially frombaseline values 3 hours after IP injection of OCP crystals(Figure 1B). After 6 hours, the numbers of macrophageswithin the peritoneal lavage fluid began to increaseslowly, although this increase was not statistically signif-icant. Since 6 hours after OCP crystal administrationappears to be a good time point for showing the acutereaction of both neutrophils and macrophages in re-sponse to OCP crystal injection into the peritonealcavity, the 6-hour time point was used as the end pointfor peritoneal lavage fluid harvest for subsequent in vivoexperiments.

We next compared the inflammatory response toOCP 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 (Figure1C). As expected, the absolute numbers of peritonealLy-6G–CD11b� macrophages decreased significantlyafter crystal injection (mean � SEM 706,000 � 128,000in 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 neu-trophils was associated with elevated levels of bothIL-1� and IL-1� in the peritoneal lavage fluid of crystal-injected mice compared with levels in PBS-injectedcontrols (Figures 1D and E, respectively). These resultsalso correlated with a significant increase in the level ofMRP-8–MRP-14 complex, which is considered a reliablemarker of inflammation (33,34), both in peritoneallavage fluid (Figure 1F) and in serum samples (Figure1G). Taken together, these observations demonstrate

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 killed3, 6, 9, 12, or 24 hours following OCP crystal administration. A and B, Neutrophils (A) and macrophages (B) in peritoneal lavage fluid werequantified by flow cytometry. Values are the mean � SEM from at least 5 mice per group. ��� � P � 0.001 versus baseline. C, NaiveC57BL/6J mice were injected IP with 1 mg of either OCP crystals or monosodium urate monohydrate (MSU) crystals. Mice injected withPBS were used as negative controls. Six hours following crystal administration, neutrophil recruitment into the peritoneal cavity wasquantified by flow cytometry. D and E, Levels of interleukin-1� (IL-1�) (D) and IL-1� (E) in the peritoneal lavage fluid were assessed byenzyme-linked immunosorbent assay (ELISA). F and G, Levels of myeloid-related protein 8 (MRP-8)–MRP-14 complex in the peritoneallavage fluid (F) or in serum (G) were assessed by ELISA. Values are the mean � SEM from at least 6 mice per group. � � P � 0.05;�� � P � 0.01; ��� � P � 0.001. Ly-6G � lymphocyte antigen 6 complex, locus G.

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that in vivo OCP crystal stimulation is able to inducestrong inflammation both locally and systemically.

Blockade or deficiency of IL-1 strongly reducesOCP crystal–induced inflammation. We next assessedwhether OCP crystal–triggered inflammation acts viaIL-1–dependent pathways. Naive mice were treated withanakinra (recombinant IL-1Ra), which binds tightly toIL-1R type I, blocking the activity of either IL-1� orIL-1� (35). Anakinra-treated mice displayed a signifi-cant reduction in both OCP crystal– and MSU crystal–induced neutrophil recruitment (Figure 2A). DecreasedOCP crystal– and MSU crystal–induced neutrophil in-filtration into the peritoneal cavity was also observed inmice treated with IL-1�–neutralizing antibodies. Collec-tively, these results demonstrate that IL-1R activationand IL-1 production play essential roles in OCP crystal–triggered inflammation.

Since we showed that both IL-1� and IL-1� levelswere increased in peritoneal lavage fluid samples fromcrystal-injected mice (Figures 1D and E, respectively)and that anti–IL-1� antibodies and recombinant IL-1Rawere efficient in blocking neutrophil recruitment, wenext assessed the relative contributions of IL-1� andIL-1� in OCP crystal–induced inflammation. Mice defi-

cient in either IL-1� or IL-1� were injected with OCPcrystals, and peritoneal neutrophil recruitment was as-sessed 6 hours following crystal administration (Figure2B). In the absence of IL-1�, there was a striking andsignificant decrease in neutrophil recruitment uponOCP crystal injection. A similar decrease was observedin IL-1�–deficient mice, although this was not statisti-cally significant. As expected, IL-1�–/– and IL-1�–/– miceindeed did not produce IL-1� and IL-1�, respectively,after OCP crystal administration, and the absence of oneof 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 incrystal-induced neutrophil recruitment. However, these2 soluble factors when functioning separately may not besolely responsible for facilitating crystal-induced neutro-phil accumulation in the peritoneal cavity.

OCP crystal–stimulated inflammation is inde-pendent of ASC and NLRP3. Since we have shown thatIL-1� plays a prominent role in OCP crystal–inducedinflammation, we next investigated the contribution ofthe inflammasome, the multiprotein complex able toconvert proIL-1� into biologically active IL-1�, to OCP

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 aftercrystal injection, mice were killed and neutrophil accumulation in the peritoneal cavity was assessed by flow cytometry. Values are themean � 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 ofIL-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 pergroup.� � P � 0.05; �� � P � 0.01; ��� � P � 0.001. See Figure 1 for definitions.

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crystal–induced inflammation. We investigated the rolesof the adaptor protein ASC and of the inflammasomesensor NLRP3. ASC–/– and NLRP3–/– mice were in-jected IP with OCP crystals. Compared with WT mice,these deficient mice did not show any alteration in theirinflammatory response, as demonstrated by similarnumbers of neutrophils in the peritoneal cavity (Figure3A) along with comparable levels of MRP-8–MRP-14complex (Figure 3B) and IL-1� (Figure 3C) in theperitoneal lavage fluid. In addition, anakinra had similarinhibitory effects in WT, ASC–/–, and NLRP3–/– mice(Figure 3D). Taken together, these results indicate thatthese OCP crystal–associated inflammatory responsesare independent of the classic NLRP3 inflammasomeand suggest that other inflammasomes may play a role,or, alternatively, that OCP crystal–induced inflamma-tion involves a caspase 1–independent IL-1�–processingmechanism. In this context we have tested the involve-ment of neutrophil elastase, a proteinase able to convertproIL-1� into biologically active IL-1� (13,36). Indeed,when mice were treated prophylactically with AAPV, aninhibitor of neutrophil elastase, we found a nonsignifi-

cant trend toward a decrease (a 30% reduction) in bothneutrophil recruitment and IL-1� levels in peritonealfluid (data not shown).

Mast cell depletion does not attenuate OCPcrystal–induced inflammation. We first tested whethermast cells were able to respond to OCP crystals byreleasing IL-1� and IL-1�, thereby contributing to theonset of OCP crystal–induced inflammation. We there-fore 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–associatedinduction of IL-1 by mast cells, we next induced OCPcrystal–mediated peritonitis in mast cell–depleted mice.Mice locally injected with compound 48/80 had �95%depletion of peritoneal mast cells (Figure 4C). Weobserved comparable levels of neutrophil recruitmentbetween OCP crystal–injected mice pretreated withcompound 48/80 and those pretreated with PBS (Figure4D). These results suggest that resident mast cells do not

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 cavitywas quantified by flow cytometry. As expected, OCP crystal injection induced a significant increase in neutrophil recruitmentcompared with that in PBS-injected controls, and similar numbers of neutrophils were recruited in ASC–/– mice, NLRP3–/– mice, andWT 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 hoursafter crystal injection, neutrophil recruitment into the peritoneal cavity was quantified by flow cytometry. Anakinra significantlyreduced neutrophil recruitment in all mouse strains. Values are the mean � SEM from at least 10 mice per group. � � P � 0.05; ��

� P � 0.01; ��� � P � 0.001. See Figure 1 for other definitions.

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play an important role in the cellular response inducedfollowing crystal administration.

Macrophage depletion abolishes OCP crystal–induced inflammation. Peritoneal macrophages couldalso be likely candidates that respond to OCP crystalsby releasing IL-1� and IL-1� and thereby triggeringOCP crystal–induced inflammation. When purifiedperitoneal macrophages that were previously primedwith LPS (37) were stimulated with OCP crystals, cellswere able to release both IL-1� and IL-1� in super-natants (Figures 5A and B). We next analyzed theinflammatory response to OCP crystals in mice de-pleted of resident macrophages by pretreatment withclodronate liposomes (32). An IP injection of clodro-nate liposomes 3 days prior to peritoneal lavage fluidharvest resulted in �90% depletion of resident macro-phages (Figure 5C). Control mice received liposomescontaining PBS. We found that macrophage-depletedmice were not able to recruit neutrophils upon IPadministration of OCP crystals, whereas in non–macrophage-depleted mice the vast majority of peri-toneal cells were neutrophils (Figures 5C and D). Asexpected, IL-1� levels in peritoneal lavage fluid were

significantly decreased in mice pretreated with clo-dronate liposomes (Figure 5E).

OCP crystals induce apoptosis/necrosis of peri-toneal cells in vivo. We have found that OCP crystalshave a deleterious effect on the viability of murine bonemarrow–derived macrophages cultured in vitro (�50%cell death after 6-hour incubation with 500 �g/ml OCPcrystals) (37). In order to assess the relevance of such aphenomenon following in vivo OCP crystal administra-tion, peritoneal cells were costained with annexin V andPI. We found that �50% of cells recovered 6 hours afterIP injection of OCP crystals were dead (either in lateapoptosis or in necrosis). In contrast, the vast majority ofcells from PBS-injected mice were viable (Figures 6Aand B).

DISCUSSION

OCP crystals, members of the BCP crystal family,elicit joint as well as periarticular inflammation and mayhave a pathogenic role in OA. The mechanisms ofinflammation due to BCP crystals have not been exten-sively studied. Here we report that in the murine peri-

Figure 4. Mast cells are not required for OCP crystal–induced inflammation. A and B, Bone marrow–derived mast cells fromwild-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). Valuesare 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 hoursbefore OCP crystal administration. Control mice were similarly treated with PBS alone. C, Before crystal injection, mast cells fromperitoneal fluid of compound 48/80–injected mice or PBS-injected mice were stained with toluidine blue and counted. Values are themean � 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 micewas not significant. � � P � 0.05; ��� � P � 0.001. See Figure 1 for other definitions.

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tonitis model, OCP crystals cause a strong recruitmentof neutrophils that is comparable with levels achievedwith MSU crystals, whose proinflammatory propertieshave been well documented (20,38,39). These findingsprompted us to investigate the molecular mechanismsresponsible for neutrophil accumulation in an experi-mental model of OCP crystal–induced peritonitis.

The recent emergence of the inflammasome as acaspase 1 activator and its role in hereditary autoinflam-matory syndromes and in gout raises the question ofwhether this complex is equally relevant in IL-1� pro-

duction in BCP crystal– and, especially, OCP crystal–induced inflammatory situations. To address the role ofthe inflammasome in OCP crystal–induced pathogene-sis, we assessed the effect of genetic deletion of differentelements of the inflammasome on neutrophil recruit-ment into the peritoneal cavity upon OCP crystal ad-ministration. We found no effect of either ASC orNLRP3 deficiency on neutrophil recruitment, and simi-lar amounts of MRP were detected in the peritonealcavity. The absence of an obvious influence of NLRP3and ASC on OCP crystal–induced inflammation un-

Figure 5. Resident macrophages are crucial cells for OCP crystal–induced inflammation. A and B, Bone marrow–derivedmacrophages from wild-type mice were stimulated with 500 �g/ml of OCP crystals in vitro with or without prior priming withlipopolysaccharide (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. Threedays later, mice were challenged with an IP injection of 1 mg of OCP crystals. Peritoneal lavage fluid was collected 6 hours after crystalstimulation, 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 containingliposomes only; middle, OCP crystal–injected mice previously pretreated with PBS containing liposomes; bottom, OCP crystal–injectedmice previously pretreated with clodronate liposomes. IL-1� levels in the peritoneal lavage fluid from macrophage-depleted andnon–macrophage-depleted mice injected with crystals were assessed by ELISA (E). Values in A, B, D, and E are the mean � SEM from5 mice per group. Plots in C are representative of 3 mice per group. � � P � 0.05; ��� � 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 athttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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equivocally rules out a role for the NLRP3 inflamma-some in OCP-induced inflammatory responses in vivo.These findings contrast with observations that MSUcrystals (20), as well as inorganic particles such asasbestos fibers, silica particles, and alum crystals,activate the NLRP3 inflammasome to produce IL-1�(40–42).

The presence of IL-1� in the peritoneal exudateand the attenuation of inflammation by IL-1� blockadeand IL-1� deficiency suggest that there is an NLRP3inflammasome–independent mechanism. We and othershave recently demonstrated the existence ofinflammasome-independent pathways of IL-1� process-ing in IL-1�–mediated diseases such as antigen-inducedarthritis (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 distinctfrom caspase 1 are able to process proIL-1� (discussedin refs. 11 and 16). Neutrophil-, macrophage-, and mastcell–derived serine proteinases such as proteinase 3,elastase, cathepsin G, and chymase have been reportedto be able to convert proIL-1� into the 21-kd active form(10,12,13). A crucial role of chymase and elastase inproIL-1� activation was recently shown in the K/BxNarthritis model using specific proteinase inhibitors (14).Consistent with this latter finding, we tested the effectsof AAPV in OCP crystal–induced peritonitis and founda 30% reduction in neutrophil recruitment and perito-neal IL-1� (this difference did not reach significance

Figure 6. OCP crystals induce apoptosis/necrosis of peritoneal cells in vivo. A and B, Mice were injected with PBS as a control or with1 mg of OCP crystals. After 6 hours, cells were recovered from peritoneal lavage fluid and analyzed for viability by flow cytometry usingannexin V and propidium iodide (PI) staining. Viable, early apoptotic, and late apoptotic and/or necrotic cells were identified asannexin 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. ��� � P � 0.001. C, Model of OCP crystal–induced peritoneal inflammation. The major eventtriggering OCP crystal–induced peritoneal inflammation is mediated by OCP crystal–induced cell death. IL-1� is released fromnecrotic cells. IL-1� and IL-1� bind to IL-1 receptor type I (IL-1RI) on target cells. IL-1RI activation subsequently induces theproduction of CXCL1 by mesothelial cells and fibroblasts. CXCL1 in turn facilitates neutrophil recruitment, which can also bemediated by MRP and IL-8. ProIL-1� can also be released from dead cells. Proteinases derived from neutrophils (such as proteinase3, 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.

430 NARAYAN ET AL

[data not shown]), suggesting that a part of proIL-1�processing in OCP crystal–induced peritonitis is due toneutrophil elastase.

OCP crystal–induced inflammation in the perito-nitis model depends on both IL-1� and IL-1�. The firstline of evidence for this was the finding that both IL-1�and IL-1� were released into the peritoneal exudateafter crystal injection. A role for both cytokines washighlighted by the inhibitory effects of individual defi-ciency of IL-1� and IL-1�. Interestingly, their effects oninflammation seem to be linked, since IL-1� deficiencyhad an effect on peritoneal IL-1� levels and vice versa.Such a reciprocal regulation of IL-1� over the produc-tion of IL-1� has been previously reported in anotherexperimental model (26). IL-1� is biologically active inits precursor form and can be found on the surface ofseveral cells, particularly monocytes, where it is referredto as membrane IL-1� (10). Cleavage of the precursorby calpain, a membrane-associated calcium-activatedcysteine proteinase, releases mature IL-1�. It may alsobe released from dying cells (45).

We demonstrated that OCP crystals inducednecrosis of �50% of peritoneal cells, principally repre-sented by infiltrating neutrophils. In this context, it islikely that at least part of the peritoneal fluid IL-1� thatwe measured following OCP crystal injection was re-leased from dying cells. Interestingly, it has been shownthat IL-1� released from necrotic cells triggers CXCL1/cytokine-induced neutrophil chemoattractant (KC) se-cretion and recruitment of neutrophils via IL-1R/myeloid differentiation factor 88 signaling onneighboring mesothelial cells (46). Therefore, we cananticipate that the inflammatory properties of OCPcrystals include their ability to induce cellular necrosis.The subsequent passive release of IL-1� from dying cellswould in turn facilitate chemokine production (eventu-ally CXCL1) and neutrophil recruitment to the inflamedsite. Validation of such a mechanism would requirecrystal administration in mice with a targeted mutationin CXCL1/KC (47).

It has recently been shown that in addition topassive release of danger signals such as uric acid orATP, necrotic cells drive inflammatory cell infiltrationin vivo and induce the production of IL-1� in an NLRP3inflammasome–dependent manner (42,48). Our data donot support the influence of such a mechanism, sinceneutrophil recruitment and IL-1� production were notaltered in the absence of the NLRP3 inflammasome.Such a discrepancy could be explained by the dose ofnecrotic cells required to activate the NLRP3 inflam-masome. To trigger a sterile inflammatory response

through NLRP3, 107 necrotic cells were injected IP (48).In our experimental setting, cells were progressivelydying and we recovered �10-fold fewer necrotic cells6 hours following crystal administration. Thus, below acertain threshold of necrosis, the NLRP3 inflammasomeis probably not activated.

Mast cells and resident macrophages have beenimplicated in MSU crystal–induced peritonitis (36,38),and we investigated the contribution of these cells inOCP crystal–induced peritonitis. Depletion studies usingeither compound 48/80 or clodronate liposomes showedthat only peritoneal macrophage depletion led to areduction of neutrophil recruitment and IL-1� levels,demonstrating that resident macrophages play an essen-tial role in the production of IL-1� and in neutrophilrecruitment in OCP crystal–induced inflammation, eventhough in in vitro studies, both cell types secreted IL-1�when stimulated with OCP crystals. We recently foundthat OCP crystals stimulated IL-1� secretion in murinebone marrow–derived macrophages and peritonealmacrophages through an NLRP3 inflammasome–dependent pathway in vitro (37). The difference, interms of NLRP3 dependency, between the in vivo andin vitro results may be due to OCP crystal–inducedcell death and the release of non–caspase 1 protein-ases already discussed, or to factors that can modulatecrystal interactions with cells, such as protein coatingof crystals in vivo (24,25).

An interesting observation during crystal-inducedperitonitis was that peritoneal macrophages decreasedsignificantly in the peritoneal lavage fluid following IPadministration of both OCP and MSU crystals. Similardisappearance of macrophages has been previously ob-served in harvested peritoneal lavage fluid following IPinjection with MSU crystals (49). This disappearance ofmacrophages shortly following administration of inflam-matory stimuli has been termed “macrophage disap-pearance reaction” and has been highlighted in othermodels of acute inflammation (50–52).

Finally, we observed a significant release ofMRP-8–MRP-14 complex (S100A8/A9, calprotectin)during OCP crystal–induced peritonitis, which may fur-ther amplify the local inflammatory response. MRPs aresecreted by monocytes and neutrophils following cellularactivation or necrosis (for review, see ref. 53) andparticipate in a positive feedback loop of neutrophilrecruitment by up-regulating integrin expression andmediating chemotaxis (54,55). Not only peritoneal butalso serum levels of MRP-8–MRP-14 complex wereincreased upon OCP crystal injection. Similar resultswere reported in a murine air pouch model of MSU

OCP CRYSTALS ARE PROINFLAMMATORY IN VIVO 431

crystal–induced inflammation that was inhibited by anti-MRP antibodies (56). This suggests that in BCP crystal–related diseases such as the Milwaukee shoulder syn-drome, MRP levels will be increased and might play arole in pathogenesis.

In conclusion, we have demonstrated that in an invivo 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 bycrystals (Figure 6C). Furthermore, our data highlightthat macrophages play a crucial role in this inflamma-tory process but mast cells do not. These mechanismscan account for the acute inflammatory reaction seen inacute periarthritis and arthritis due to OCP crystals. InOA, in which BCP crystals are found in the cartilage aswell as in the joint fluid, these mechanisms do not seemto predominate, since inflammation is not a prominentfeature. These results have implications in the search foreffective therapies for BCP crystal–associated diseases.

ACKNOWLEDGMENTS

We are grateful to Dr. Nico van Rooijen (VU Univer-sity Medical Center, Amsterdam, The Netherlands) for kindlyproviding us with clodronate liposomes. We thank Dr.Yoichiro Iwakura (University of Tokyo, Tokyo, Japan) forproviding us with IL-1�–/– and IL-1�–/– mice and ProfessorJurg Tschopp (University of Lausanne, Lausanne, Switzer-land) for providing us with NLRP3–/– and ASC–/– mice.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Busso had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Narayan, Pazar, Ea, Kolly, Bagnoud,Chobaz, Liote, So, Busso.Acquisition of data. Narayan, Pazar, Ea, Kolly, Bagnoud, Chobaz,Vogl, Holzinger.Analysis and interpretation of data. Narayan, Pazar, Ea, Kolly,Bagnoud, Chobaz, Liote, Vogl, Holzinger, So, Busso.

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