TLR2 Signaling in Chondrocytes Drives Calcium Pyrophosphate Dihydrate and Monosodium Urate Crystal-Induced Nitric Oxide Generation

Abstract

Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate (MSU) deposited in synovium and articular cartilage initiate joint inflammation and cartilage degradation in large part by binding and directly activating resident cells. TLRs trigger innate host defense responses to infectious pathogens, and the expression of certain TLRs by synovial fibroblasts has revealed the potential for innate immune responses to be triggered by mesenchymally derived resident cells in the joint. In this study we tested the hypothesis that chondrocytes also express TLRs and that one or more TLRs centrally mediate chondrocyte responsiveness to CPPD and MSU crystals in vitro. We detected TLR2 expression in normal articular chondrocytes and up-regulation of TLR2 in osteoarthritic cartilage chondrocytes in situ. We demonstrated that transient transfection of TLR2 signaling-negative regulator Toll-interacting protein or treatment with TLR2-blocking Ab suppressed CPPD and MSU crystal-induced chondrocyte release of NO, an inflammatory mediator that promotes cartilage degeneration. Conversely, gain-of-function of TLR2 in normal chondrocytes via transfection was associated with increased CPPD and MSU crystal-induced NO release. Canonical TLR signaling by parallel pathways involving MyD88, IL-1R-associated kinase 1, TNF receptor-associated factor 6, and IκB kinase and Rac1, PI3K, and Akt critically mediated NO release in chondrocytes stimulated by both CPPD and MSU crystals. We conclude that CPPD and MSU crystals critically use TLR2-mediated signaling in chondrocytes to trigger NO generation. Our results indicate the potential for innate immunity at the level of the articular chondrocyte to directly contribute to inflammatory and degenerative tissue reactions associated with both gout and pseudogout.

Innate immunity conferred by toll-like receptors 2 and 4 and myeloid differentiation factor 88 expression is pivotal to monosodium urate monohydrate crystal-induced inflammation

“…We speculate that TLR-2 and TLR-4 or closely interacting proteins are among the plasma membrane proteins directly engaged by MSU crystals. Articular chondrocytes have been particularly advantageous in testing this notion, because we observed that normal articular chondrocytes constitutively expressed TLR-2 but not several other TLRs, including TLR-4, in vitro (25). Moreover, direct up-regulation and down-regulation of MSU crystalinduced chondrocyte nitric oxide production was inducible by specific "gain-of-function" of TLR-2 expression versus "loss-of-function" of TLR-2-dependent signaling (25).…”

“…These findings implicated the complement arm of innate immunity as one of the factors that controls acute gouty inflammation. Furthermore, we observed that MSU crystals activate the chondrocyte, a cell of mesenchymal origin, in a manner that requires canonical signal transduction via Toll-like receptor 2 (TLR-2) (25). TLRs play a vital role in host defense by initiating the innate immune response against pathogens (26).…”

“…Recently, we observed that TLR-2-mediated and MyD88-dependent signaling in chondrocytes played critical roles in NF-B activation and nitric oxide generation in response to MSU crystals (25). However, the direct exposure of MSU crystals to chondrocytes is generally limited in the joint, unlike the case with fibroblast-like and macrophage-like synovium lining cells (1).…”

Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals

“…In addition, binding of agonistic mAb to TREM-1 on monocytes in combination with the ligands for TLR-2, TLR-3, or TLR-4 synergistically amplified the cellular production of proinflammatory cytokines (8,10,11). Liu-Bryan et al recently demonstrated an essential role of TLR-2 in MSU crystal-induced acute inflammation (14). This indicates that MSU crystals are potent ligands for TLR-2, and recognition of the crystals by TLR-2-expressing inflammatory cells promotes rapid induction of various inflammation mediators.…”

“…However, the mechanisms that regulate TREM-1 expression in various inflammatory diseases remain unknown. An essential role of TLR-mediated signaling in acute gout was recently demonstrated (14). Because TREM-1 may potentially amplify the inflammatory responses initiated by TLRs, we hypothesized that cooperation between TREM-1 and TLRs may occur during acute attacks of gout.…”