RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFalpha or anti-IL-1 therapies

Abstract

Introduction: Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFalpha, IL-1beta, and receptor activator of NF-kappaB ligand (RANKL). Anti-IL-1 or anti-TNFalpha therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.

Methods: Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFalpha inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (alpha1AGP), multiple cytokines) were measured in serum (day 14 post onset).

Results: Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFalpha reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFalpha and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFalpha and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum alpha1AGP, IL-1beta, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFalpha and IL-17. Anti-TNFalpha reduced systemic alpha1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic alpha1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.

Conclusions: Anti-TNFalpha or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.

Figure 1

Effect of anti-TNFα, anti-IL-1, or anti-RANKL therapy on hind paw swelling. Effect of PEGylated soluble TNF receptor type I (TNFRI), IL-1 receptor antagonist (IL-1Ra) or osteoprotegerin (OPG)-Fc on hind paw swelling. (a) Hind paw swelling was assessed on day 4 post onset, prior to the beginning of therapies. Swelling was assessed in adjuvant-induced arthritis (AIA) rats by measuring average hind paw volume via water plethysmography, and in collagen-induced arthritis (CIA) rats by precision caliper measurements of paw diameter. (b), (c) Percentage changes in paw swelling in AIA and CIA rats, as measured from the time of treatment initiation (day 4) to day 14. Data represent means ± standard error of the means, n = 8/group. ^cSignificantly different from control (nonarthritic) rats, P < 0.05. ^vSignificantly different from vehicle (Veh)-treated arthritic rats, P < 0.05. ^oSignificantly different from osteoprotegerin-treated arthritic rats, P < 0.05.

Figure 2

Lumbar vertebrae from nonarthritic control and vehicle-treated adjuvant-induced arthritis and collagen-induced arthritis rats. Representative photomicrographs of lumbar vertebrae from nonarthritic control and vehicle (Veh) (PBS)-treated adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) rats. The trabeculae beneath the physeal plate (pale vertical column at the left margin) were attenuated in arthritic animals but the bone marrow composition and density - including the population of subphyseal osteoclasts (brown cells, cathepsin K-positive) - were equivalent among nonarthritic and arthritic animals. Stain: immunohistochemistry for cathepsin K with H & E counterstain. Magnification: ×100.

Figure 3

Effect of anti-TNFα, anti-IL-1, or anti-RANKL therapy on bone mineral density. Effects of PEGylated soluble TNF receptor type I (TNFRI), IL-1 receptor antagonist (IL-1Ra) or osteoprotegerin (OPG)-Fc on areal bone mineral density (BMD) of the (a), (b) ankle and (c), (d) lumbar vertebrae. Baseline BMD measures were obtained by dual X-ray absorptiometry on the day of onset for clinical arthritis (day 0). Treatments were initiated on day 4, and final BMD was measured on day 14 post onset. Data represent means ± standard error of the means, n = 8/group. ^cSignificantly different from control (nonarthritic) rats, P < 0.05. ^vSignificantly different from vehicle (Veh)-treated arthritic rats, P < 0.05. ^oSignificantly different from OPG-treated arthritic rats, P < 0.05. AIA, adjuvant-induced arthritis; CIA, collagen-induced arthritis.

Figure 4

Effect of anti-TNFα, anti-IL-1, or anti-RANKL therapy on bone resorption markers. Effects of PEGylated soluble TNF receptor type I (TNFRI), IL-1 receptor antagonist (IL-1Ra) or osteoprotegerin (OPG)-Fc on serum levels of the bone resorption markers (a), (b) receptor activator of NF-κB ligand (RANKL) and (c), (d) tartrate-resistant acid phosphatase 5b (TRACP-5B) in (a), (c) adjuvant-induced arthritis (AIA) rats and in (b), (d) collagen-induced arthritis (CIA) rats. Values were determined on day 14 post onset, 10 days after the initiation of treatment. Data represent means ± standard error of the means, n = 8/group. ^cSignificantly different from control (nonarthritic) rats, P < 0.05. ^vSignificantly different from vehicle (Veh)-treated arthritic rats, P < 0.05. ^oSignificantly different from OPG-treated arthritic rats, P < 0.05.

Figure 5

Serum markers and mediators of inflammation in adjuvant-induced arthritis (AIA) rats. Values were determined on day 14 post onset, 10 days after the initiation of treatment. Data represent means ± standard error of the means, n = 8/group. ^cSignificantly different from control (nonarthritic) rats, P < 0.05. ^vSignificantly different from vehicle (Veh)-treated arthritic rats, P < 0.05. ^oSignificantly different from osteoprotegerin (OPG)-treated arthritic rats, P < 0.05. α₁AGP, acute-phase protein alpha-1-acid glycoprotein; AIA, adjuvant-induced arthritis; CCL2, chemokine (C-C motif) ligand 2; CRP, C-reactive protein; PGE₂, prostaglandin E₂.

Figure 6

Serum markers and mediators of inflammation in collagen-induced arthritis (CIA) rats. Values were determined on day 14 post onset, 10 days after the initiation of treatment. Data represent means ± standard error of the means, n = 8/group. ^cSignificantly different from control (non-arthritic) rats, P < 0.05. ^vSignificantly different from vehicle (Veh)-treated arthritic rats, P < 0.05. ^oSignificantly different from osteoprotegerin (OPG)-treated arthritic rats, P < 0.05. α₁AGP, acute-phase protein alpha-1-acid glycoprotein; CCL2, chemokine (C-C motif) ligand 2; CIA, collagen-induced arthritis; CRP, C-reactive protein; PGE₂, prostaglandin E₂.

Figure 7

Linear regression analyses of serum cytokines versus local bone loss or local inflammation. (a) to (d) Bone loss was quantified as the percentage change in areal bone mineral density (BMD) from the day of onset for clinical arthritis (day 0) to day 14 post onset. (e) and (f) Paw swelling was quantified as the percentage change from day 4 post onset (treatment initiation) to day 14 post onset. Serum receptor activator of NF-κB ligand (RANKL) and IL-1β were evaluated on day 14 post onset. Open circles, nonarthritic controls + vehicle; black circles, arthritis + vehicle; open triangles, arthritis + IL-1 receptor antagonist; grey diamonds, arthritis + PEGylated soluble TNF receptor type I; grey circles, arthritis + OPG-Fc. n = 8/group. AIA, adjuvant-induced arthritis; CIA, collagen-induced arthritis.