Methotrexate and low-dose prednisolone downregulate osteoclast function by decreasing receptor activator of nuclear factor-κβ expression in monocytes from patients with early rheumatoid arthritis

Abstract

Objective: Rheumatoid arthritis (RA) is a systemic, immune-mediated inflammatory disease that ultimately leads to bone erosions and joint destruction. Methotrexate (MTX) slows bone damage but the mechanism by which it acts is still unknown. In this study, we aimed to assess the effect of MTX and low-dose prednisolone (PDN) on circulating osteoclast (OC) precursors and OC differentiation in patients with RA.

Methods: Patients with RA before and at least 6 months after MTX therapy were analysed and compared with healthy donors. A blood sample was collected in order to assess receptor activator of NF-κβ (RANK) ligand surface expression on circulating leucocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines and OC differentiation assays were performed.

Results: Classical activation markers of monocytes and RANK increased in patients with RA at baseline, compared with control healthy donors, and after MTX and low-dose PDN (MTX+PDN) exposure they decreased to control levels. Although the number of OC was not different between groups, the percentage of resorbed area and the resorbed area per pit reduced after treatment. Serum soluble receptor activator of nuclear factor-kappa (RANKL) levels increased at baseline compared with healthy donors and normalised after therapy.

Conclusion: Our results suggest that MTX+PDN play an important role in downregulating OC function, which we believe occurs through the decrease in RANK surface expression in monocytes.

Keywords: Bone; DMARD; Monocytes; Osteoclast; Rheumatoid arthritis.

Figure 1

Frequency of neutrophils and RANKL neutrophils. (A) Neutrophil frequency is significantly higher in patients with RA at baseline when compared with healthy donors (both frequency p=0.0110 and absolute number p=0.0032). (B) RANKL+neutrophil count is also higher at baseline when compared with healthy donors (p=0.0060). Neutrophil number is decreased after MTX and low-dose prednisolone treatment in patients with RA (absolute number only, p=0.0155). Each dot represents a sample and the line in healthy represents median. RA, rheumatoid arthritis; MTX, methotrexate; RANKL, receptor activator of nuclear factor-κβ ligand.

Figure 2

Phenotype of monocyte subpopulations. (A) Classical subpopulation phenotype, (B) intermediate subpopulation, (C) non-classical subpopulation phenotype. Before treatment there was higher expression of CCR2 in classical and intermediate subpopulations, HLA-DR and CD86 in classical subpopulation, CD51/CD61 in the intermediate subpopulation, CD11b in non-classical subpopulation and RANK both in intermediate and non-classical subpopulations. HLA-DR expression remained higher in patients after treatment when compared with controls. CCR2, CD86, CD11b and RANK were decreased in the referred populations after treatment. *p<0.05, **p<0.01. Each dot represents a sample and the line in healthy represents median. MFI, median fluorescence intensity; CCR2, C-C chemokine receptor type 2; HLA, human leucocyte antigen; RANK, receptor activator of nuclear factor-κB; MTX, methotrexate.

Figure 3

Functional assays in differentiated OC. (A) No differences in OC number/mm² or in pit number were found. Percentage of resorbed area was higher in patients at baseline at culture day 14 and 21 when compared with controls. Resorbed area per pit was lower at culture day 14 when compared with patients at baseline. At culture day 21, patients with RA at baseline had higher resorbed area per pit than controls. Dots and lines represent median and IQR. (B) Representative images of resorption pit assay in all groups at culture day 21; scale bar represents 100 μm. d, day; OC, osteoclast; RA, rheumatoid arthritis.

Figure 4

Serum sRANKL and OPG levels in healthy donors and patients with RA at baseline and after MTX and low-dose prednisolone treatment. Both sRANKL and the sRANKL/OPG ratio increased in patients at baseline when compared with healthy donors. There were no differences in circulating OPG level before and after treatment or when compared with controls. The sRANKL/OPG ratio remained significantly higher after treatment. Each dot represents a sample and the line in healthy represents median. sRANKL, soluble receptor activator of nuclear factor-κB ligand; OPG, osteoprotegerin; RA, rheumatoid arthritis.