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Receptor activator NF-B ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis

T N Crotti¹, M D Smith², H Weedon³, M J Ahern⁴, D M Findlay⁵, M Kraan⁶, P P Tak⁶, D R Haynes¹

¹ Department of Pathology, University of Adelaide, Adelaide, South Australia
² Department of Medicine, Flinders Medical Centre and Repatriation General Hospital, Adelaide, South Australia
³ Rheumatology Research Unit, Repatriation General Hospital, Daw Park, Adelaide, South Australia
⁴ Department of Medicine, Flinders University, South Australia
⁵ Department of Orthopaedics, University of Adelaide, Adelaide, South Australia
⁶ Amsterdam Medical Centre, Amsterdam, The Netherlands

Correspondence to:
Correspondence to:
Dr D R Haynes, Department of Pathology, University of Adelaide, Adelaide, South Australia 5005;david.haynes{at}adelaide.edu.au

Objectives: To compare receptor activator of NF-B ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues.

Methods: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL.

Results: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA).

Conclusions: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.

Keywords: bone; rheumatoid arthritis; receptor activator NF-B ligand; immunohistochemistry; spondyloarthropathies

Abbreviations: AEC, 3-amino-9-ethylcarbazole; DIA, digital image analysis; FLS, fibroblast-like synoviocytes; HRP, horseradish peroxidase; IL, interleukin; IOD, integrated optical density; mAb, monoclonal antibody; MOD, mean optical density; OA, osteoarthritis; OPG, osteoprotegerin; RA, rheumatoid arthritis; RANK, receptor activator of NF-B; RANKL, receptor activator of NF-B ligand; SpA, spondyloarthropathy; SQA, semiquantitative assessment; TNF, tumour necrosis factor

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