The Journal of Immunology Basic Calcium Phosphate Crystals Induce Monocyte/ Macrophage IL-1b Secretion through the NLRP3 Inflammasome In Vitro Borba ´la Paza ´r,* Hang-Korng Ea, †,‡ Sharmal Narayan,* Laeticia Kolly,* Nathalie Bagnoud,* Ve ´ronique Chobaz,* Thierry Roger, x Fre ´de ´ric Liote ´, †,‡ Alexander So,* and Nathalie Busso* Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3–inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1b in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1b after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1b after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1b secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human dis- ease. The Journal of Immunology, 2011, 186: 2495–2502. D eposition of basic calcium phosphate (BCP) crystals, which include octacalcium phosphate (OCP), carbonate- substituted (CA), hydroxyapatite (HA), tricalcium phos- phate, and whitlockite, can occur in any tissues but s.c., periarticular, and intra-articular locations are the most frequent sites. Articular cartilage calcification is associated with severe osteoarthritis (OA) and destructive arthropathies, such as Milwaukee shoulder syn- drome. Interestingly, BCP crystals are detected in 100% of knee and hip osteoarthritic cartilages harvested at the time of total joint arthroplasty (1, 2). BCP crystal deposition in knee articular carti- lage is correlated with cartilage destruction and more severe lesions (1). Their presence in synovial fluid predicts radiological OA progression (3). Extra-articular depositions of BCP crystals are mostly asymptomatic but can give rise to acute inflammatory attacks. Typically, acute calcific periarticular attacks, like other in- flammatory responses secondary to microcrystal deposition, such as monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, is characterized by a self-limiting reaction with a rapid onset of pain, swelling, erythema, and transient joint lim- itation (4, 5). This acute attack when occurring at the first meta- tarsophalangeal joint is similar to gout, which is secondary to MSU crystal deposition, and is qualified as pseudopodagra by some authors (5). However, although recent fundamental and clinical research clearly demonstrates the implication of IL-1b and its maturation by the inflammasome in gout and pseudogout (because of CPPD crystal deposition) attacks (6–9), little is known about BCP crystal-induced inflammation, in particular the mechanism of IL-1b production and the role of the inflammasome. In vitro, BCP crystals stimulate cells through two main mech- anisms. They can first activate cells after being endocytosed or phagocytosed, leading to intralysosomal crystal dissolution with subsequent elevation of intracellular Ca 2+ levels and release of inflammatory cytokines. Crystal phagocytosis can be enhanced by IgG or complement component opsonisation. The other mech- anism of crystal activation involves a direct crystal–cell membrane interaction due either to electrostatic bonds with naked crystal surface or through membrane receptor stimulation by naked or protein-coated crystals (10). Thus, BCP crystals activate TNF-a production through TLR-4 (11), and crystal–cell interactions in- duce rapid calcium influx (12), cytoplasmic membrane perme- ability modification (13), and chondrocyte apoptosis, which is enhanced by annexin V coating (14). They can induce several cellular functions including human foreskin fibroblast prolif- eration, proto-oncogene stimulation, inflammatory cytokine (IL- 1b and TNF-a) and NO production, metalloprotease production and activation, cyclooxygenase-1 and cyclooxygenase-2, and PGE 2 *Department of Rheumatology, Centre Hospitalier Universitaire Vaudois, 1011 Lau- sanne, Switzerland; x Infectious Diseases Service, Department of Medicine, Univer- sity of Lausanne, 1011 Lausanne, Switzerland; † Unite ´ Mixte de Recherche S606, INSERM, Lariboisie `re Hospital, 75010 Paris, France; and ‡ Institut Claude Bernard- IFR2, Universite ´ Paris–Diderot, Faculte ´ de Medicine, 75890 Paris Cedex 18, France Received for publication April 21, 2010. Accepted for publication December 1, 2010. This work was supported by a grants from the Fonds National Suisse de La Re- cherche Scientifique (310030-130085/1 and K-32K1-116460), the Jean and Linette Warnery Foundation, Association pour Rhumatisme et Travail, the Association pour la Recherche en Pathologie Synoviale, and the Association pour Cristaux et Carti- lage. Address correspondence and reprint requests to Dr. Nathalie Busso, Department of Rheumatology, Centre Hospitalier Universitaire Vaudois, Nestle ´ 05-5029, 1011 Lau- sanne, Switzerland. E-mail address: Nathalie.Busso@chuv.ch Abbreviations used in this article: ASC, apoptosis-associated speck-like protein; BCP, basic calcium phosphate; BMDM, bone marrow-derived macrophage; CA, carbonate- substituted apatite; CAS-1, caspase-1; CPPD, calcium pyrophosphate dihydrate; DAMP, danger-associated molecular pattern; HA, hydroxyapatite; monosodium urate; KCl, potassium chloride; MSU, monosodium urate; NAC, N-acetylcysteine; OCP, octacalcium phosphate; PI, propidium iodide; ROS, reactive oxygen species; sh, short hairpin. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001284