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Characterisation of cartilage intermediate layer protein (CILP)-induced arthropathy in mice

Z Yao, H Nakamura, K Masuko-Hongo, M Suzuki-Kurokawa, K Nishioka, T Kato

Arthritis Research Centre, Institute of Medical Science, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan

Correspondence to:
Correspondence to:
Professor T Kato
Arthritis Research Centre, Institute of Medical Science, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8512, Japan; t3kato{at}marianna-u.ac.jp

Objectives: To characterise cartilage intermediate layer protein (CILP)-induced arthropathy in mice.

Methods: The first and second halves of the nucleotide triphosphate pyrophosphohydrolase (NTPPHase) non-homologous region of human CILP were prepared as recombinant proteins (C1 and C2, respectively), including three overlapping fragments of C2 (C2F1, C2F2, and C2F3). C57BL/6 mice were immunised with these proteins to induce arthritis. In addition, a separate group of mice were immunised repeatedly with the mixture of C1 and C2 to see the effect of chronic immunisation. Arthritis developed in the mice, and cellular and humoral immune responses against CILP were analysed.

Results: Immunisation with C2 and with the mixture C2F1/C2F2/C2F3 caused the severest arthritis to develop in mice. Immunisation with one of C1, C2F1, C2F2, or C2F3 caused milder arthritis, even though each of the fragments carried T cell epitopes. Immunisation either with C1 or C2 alone evoked cellular and humoral immune responses to both the C1 and C2 proteins. Further, the repeated immunisation with the C1/C2 mixture caused tendon calcification and bone irregularity, together with decreased NTPPH activity.

Conclusions: The results show that multiple T cell epitopes are needed for the development of CILP-induced arthritis, and present the characteristic new model of mild arthropathy accompanied by extra-articular calcifications. An immune response to putative murine CILP/NTPPH may be involved in the ectopic calcifications in the arthritic mice.

Keywords: arthritis; cartilage intermediate layer protein (CILP); chondrocyte; animal model; NTPPH

Abbreviations: BSA, bovine serum albumin; CIA, collagen-induced arthritis; CILP, cartilage intermediate layer protein; ELISA, enzyme linked immunosorbent assay; FCA, Freund’s complete adjuvant; FIA, Freund’s incomplete adjuvant; HC gp, human cartilage glycoprotein; MBP, maltose binding protein; NTPPHase, nucleotide triphosphate pyrophosphohydrolase; OD, optical density; PBST, phosphate buffered saline-Tween-20; PPi, inorganic pyrophosphate; RA, rheumatoid arthritis; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SI, stimulation index

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