Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR-2 and TLR-4 activation

Abstract

The interplay between the innate immune system and inflammatory bone destruction in the joints of individuals with rheumatoid arthritis (RA) remains unclear. This study was undertaken to explore the effect of toll-like receptor (TLR) signaling in fibroblast-like synoviocytes (FLS) on the expression of RANKL and induction of osteoclastogenic activity. The levels of RANKL mRNA and protein were measured using RT–PCR, real-time PCR, and immunostaining. Monocytes were cocultured with RA -FLS that had been stimulated with TLR ligands in fresh media and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. Osteoclast molecule markers were measured using real-time PCR. Expression of TLR-2 and TLR-4 was higher in RA-FLS than in OA-FLS and normal skin fibroblasts. TLR-2 and TLR-4 ligands induced RANKL expression in RA-FLS. TLR stimulation of RA-FLS also induced the production of IL-1β and TNF-α to a lesser extent; however, it had no effect on IL-17 production. Inhibition of TLR induced IL-1β production, which partially reversed the upregulation of RANKL induced by TLR ligands. RA-FLS stimulated by TLR-2 and TLR-4 ligands and cocultured with human monocytes induced high levels of expression of TRAP, RANK, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9, suggesting that RA-FLS promote osteoclast differentiation. Our results suggest that the TLR signaling pathway, through TLR-2 and TLR-4, induces RANKL expression in RA-FLS and the expression of RANKL promotes the differentiation of osteoclasts in RA synovium. Targeting specific TLRs may be a promising approach to prevent inflammatory bone destruction in the pathogenesis of RA.

Introduction

The innate immune system plays a decisive role in host defense and self-tolerance [1]. Cells of the innate immune system express pattern-recognition receptors, such as the toll-like receptors (TLRs) that recognize certain highly conserved molecules shared by a broad range of bacteria and viruses. The recognition of specific microbial molecules, such as lipopolysaccharides (LPSs) result in the upregulation of costimulatory molecules in antigen-presenting cells, providing the second signal necessary for generating efficient T-cell responses against invading pathogens [2]. The innate immune system controls the subsequent adaptive immune response. Because of this important regulatory role of TLRs, it has been speculated that aberrant TLR signaling may be involved in the generation of autoimmunity.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease and the most frequent autoimmune form of arthritis. The destruction of cartilage and bone in RA is mediated by a variety of proinflammatory cytokines. Although the exact mechanisms responsible for this destruction remain obscure, the complex interactions among lymphocytes, macrophages, and synovial fibroblasts may elaborate proinflammatory cytokines that activate matrix metalloproteinase (MMP) and osteoclast activity [3]. RA is characterized by chronic synovial inflammation and hyperplasia culminating in joint destruction, which is a major cause of disability in this disease [4], [5], [6].

Osteoclasts are of macrophage–monocyte origin and are giant multinucleated cells with the capacity to degrade bone matrix. Under normal conditions, bone homeostasis depends on the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. However, in inflammatory bone disorders characterized by local bone loss, such as RA, osteoclastogenesis is accelerated relative to osteoblastic activity at diseased sites outside the marrow cavity, resulting in abnormal bone resorption and causing bony destruction [7], [8]. The receptor activator of nuclear factor-κB ligand (RANKL), a member of the tumor necrosis factor (TNF) family of cytokines, is a key molecule involved in the differentiation of osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF). Because activated synoviocytes and T lymphocytes also express a large amount of RANKL, the immune system and skeletal system may be closely interrelated [9], [10]. Page and Miossec [18] reported that treatment of synoviocytes with TNF-α or IL-1β in combination with IL-17 was particularly potent at inducing RANKL expression.

Activation/proliferation of synoviocytes, infiltration and retention of leucocytes and angiogenesis lead to the formation of an invasive pannus that destroys articular cartilage and bone [46]. The present study was designed to examine whether synovial fibroblasts can cause monocytes to differentiate into osteoclasts without the help of T cells. A number of TLR ligands are present in RA joints and these ligands stimulate the RA fibroblast-like synoviocytes (RA-FLS). TLRs are involved in the regulation of the inflammatory responses in the synovial compartment. TLRs recognize many exogenous and host-derived molecules; the latter are present in RA serum and synovial fluid. Evidence for a role of TLRs in RA pathogenesis originates from findings that inducible Hsp70, which is a TLR-4 ligand, is increased in RA synovial fluid and in dendritic cells (DCs) isolated from RA synovial fluid [47]. Proinflammatory cytokines increase and regulate the expression of TLR-2 and TLR-4. However, it is not clear whether human synovial fibroblasts activate osteoclastogenesis by producing RANKL-stimulated TLR ligation. Furthermore, we tested the hypothesis that stimulation of TLR-2 and TLR-4 has a synergistic effect on RANKL production by RA-FLS.

CpG DNA also interacts with osteoblastic TLR-9 and elicits intracellular events that lead to the increased expression of molecules regulating osteoclastogenesis [16]. A number of lines of evidence demonstrate the importance of TLR signaling in RA pathogenesis; however, the direct involvement of the TLR pathway in osteoclastogenesis induced bone destruction remains obscure. Because of the importance of TLR signaling in the development of arthritis and the expression of RANKL in RA-FLS, we hypothesized that activation of the innate immune system through TLRs may affect the osteoclastogenic activity of RA-FLS by modulating RANKL expression.

To explore the functional consequences of TLR signaling in RA joints, we assessed the effect of TLR-2 and TLR-4 ligation on the expression of RANKL in RA-FLS. We also evaluated the effect of proinflammatory cytokines on RANKL expression induced by TLR ligation. Here, we show that ligation of TLR-2 and TLR-4 induces RANKL expression in RA-FLS, and that this is mediated partly by IL-1β. We also show that TLR-mediated RANKL expression in RA-FLS may facilitate the differentiation of osteoclasts from monocytes in RA.