Mechanisms of TNF-alpha- and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis

Abstract

Psoriatic arthritis (PsA) is an inflammatory joint disease characterized by extensive bone resorption. The mechanisms underlying this matrix loss have not been elucidated. We report here that blood samples from PsA patients, particularly those with bone erosions visible on plain radiographs, exhibit a marked increase in osteoclast precursors (OCPs) compared with those from healthy controls. Moreover, PsA PBMCs readily formed osteoclasts in vitro without exogenous receptor activator of NF-kappaB ligand (RANKL) or MCSF. Both osteoprotegerin (OPG) and anti-TNF antibodies inhibited osteoclast formation. Additionally, cultured PsA PBMCs spontaneously secreted higher levels of TNF-alpha than did healthy controls. In vivo, OCP frequency declined substantially in PsA patients following treatment with anti-TNF agents. Immunohistochemical analysis of subchondral bone and synovium revealed RANK-positive perivascular mononuclear cells and osteoclasts in PsA specimens. RANKL expression was dramatically upregulated in the synovial lining layer, while OPG immunostaining was restricted to the endothelium. These results suggest a model for understanding the pathogenesis of aggressive bone erosions in PsA. OCPs arise from TNF-alpha-activated PBMCs that migrate to the inflamed synovium and subchondral bone, where they are exposed to unopposed RANKL and TNF-alpha. This leads to osteoclastogenesis at the erosion front and in subchondral bone, resulting in a bidirectional assault on psoriatic bone.

Figure 1

Osteoclasts are prominent in the psoriatic joint. A representative example of a large multinucleated osteoclast in Howship’s lacuna is shown photographed with a ×40 objective.

Figure 2

OCPs in PBMCs of PsA patients. PBMCs were obtained from PsA patients and healthy controls and cultured in the absence (a and b) or presence (c and d) of MCSF (25 ng/ml) and RANKL (100 ng/ml) for 14 days, fixed and stained for TRAP, and photographed at ×10 magnification. Numbers in parentheses represent the total number of osteoclasts per 10⁶ PBMCs for each sample.

Figure 3

Large numbers of osteoclasts arise from unstimulated PsA PBMCs. PBMCs were obtained from 24 PsA patients and 12 healthy controls, cultured in the absence of MCSF and RANKL for 14 days, fixed, and stained for TRAP. The number of TRAP-positive multinucleated cells (osteoclasts) was counted and is presented as osteoclasts per million PBMCs plated.

Figure 4

Cultured PsA PBMCs erode bone in a wafer assay. PBMCs were cultured with or without MCSF and RANKL on cortical bovine bone wafers. (a) After 21 days the wafers were stained with toluidine blue to identify resorption lacunae, shown photographed at ×10. Representative PsA and control samples are shown. (b) The percentages of eroded surface area on the wafers from unstimulated cultures from PsA patients (n = 6) and healthy controls (n = 6) were quantified as described in Methods.

Figure 5

OCP frequency in peripheral blood is greater in PsA patients with erosive arthritis. PBMCs were obtained from ten PsA patients with and ten PsA patients without erosions on plain radiographs. The cells were cultured, fixed, and stained for TRAP, and osteoclast numbers were determined as described in Methods. Data are expressed as median osteoclasts per 10⁶ PBMCs.

Figure 6

RANK expression in the psoriatic joint. Tissues from PsA patients containing synovium and bone were obtained following surgery and processed for immunohistochemistry as described in Methods. Antibodies specific for RANK stain red-brown. RANK-positive osteoclasts (arrows) are prominently located at the synovial edge of the pannus-bone interface, shown photographed at ×20 (a), and RANK-positive mononuclear cells increase in number moving from the endothelium (asterisks) to the erosion front (×20) (b). In subchondral bone, RANK-positive osteoclasts were observed in cutting cones that are void of other cell types, as shown photographed at ×20 magnification (c). RANK-positive mononuclear cells and osteoclasts (arrows) surround an endothelial cell traversing subchondral bone (d).

Figure 7

RANKL and OPG expression in the PsA synovium. Retrieval tissues containing synovium from PsA patients were processed for immunohistochemistry with antibodies specific for RANKL and OPG as described in Methods. A representative synovial membrane from a PsA patient stained with anti-RANKL antibody (a), anti-OPG antibody (b), secondary antibody only (c), and H&E (d) are shown photographed at ×20. Of note is the very specific and intense RANKL staining by synovial lining cells and the OPG staining restricted to the endothelial cells below the synovial lining.

Figure 8

RANK, RANKL, and OPG expression in PsA synovium. Total RNA was isolated from PsA and OA synovium and used as the template to determine RANK, RANKL, and OPG mRNA expression by RT-PCR as described in Methods. The sizes of the specific PCR products are indicated. Lanes 1–5 show PsA patients with radiographic bone erosions, lane 6 shows a patient without erosions, and lanes 7 and 8 show OA patients.

Figure 9

Anti–TNF-α therapy reduces OCP frequency in patients with PsA. (a) PBMCs from five PsA patients were cultured to determine the OCP frequency before and after 12 weeks of anti–TNF-α therapy (four patients with etanercept and one with infliximab). The data are expressed as osteoclasts per 10⁶ PBMCs. The number of OCPs in peripheral blood was significantly reduced in all of the patients (P < 0.001). (b) The percentage of CD11b⁺ PBMCs significantly declined in four patients with erosive PsA, following 2 weeks of etanercept therapy, as determined by FACS (P < 0.026). (c and d) Representative histograms of CD14/CD11b staining from a PsA patient before (c) and after (d) etanercept therapy.

Figure 10

TNF-α produced by PsA PBMCs enhances osteoclastogenesis. (a) Supernatants from cultured PsA PBMCs were added to PBMCs from healthy donors to stimulate osteoclastogenesis as described in Methods. The mean number of osteoclasts that arose from three unstimulated healthy control PBMC cultures (white bar), cultures incubated with MCSF and RANKL (light gray bar), and cultures to which PsA and PBMC supernatants (black, medium gray, and dark gray bars) were added are presented as the mean ± SD (*P < 0.02). (b) To determine the effect of TNF-α in the cocultures, PsA PBMC supernatant 2 (Sup 2) from a was added to two different healthy control PBMC cultures, and osteoclasts were counted with or without anti-TNF antibodies.

Figure 11

OPG inhibits osteoclast formation in unstimulated PsA PBMCs. TRAP osteoclastogenesis assays were performed on PsA PBMCs cultured without RANKL or MCSF in the continuous presence of OPG-Fc (1 μg/ml). Etanercept (1 μg/ml) was added as indicated. One representative experiment out of three is shown. Data are expressed as the mean ± SEM of four independent wells (*P < 0.05).

Figure 12

Schematic model of osteolysis in the psoriatic joint. Extensive erosions observed in the PsA joint are mediated by a bidirectional attack on bone. We propose that circulating OCPs enter the synovium and are induced to become osteoclasts by RANKL expressed by synoviocytes (outside-in). In parallel, OCPs traverse endothelial cells in the subchondral bone and undergo osteoclastogenesis following RANKL stimulation from osteoblasts and stromal cells (inside-out).