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In vivo visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption
  1. Yoshinobu Matsuura1,2,3,
  2. Junichi Kikuta1,3,
  3. Yuika Kishi1,
  4. Tetsuo Hasegawa1,
  5. Daisuke Okuzaki4,
  6. Toru Hirano2,
  7. Masafumi Minoshima5,
  8. Kazuya Kikuchi3,5,
  9. Atsushi Kumanogoh2,3,
  10. Masaru Ishii1,3
  1. 1 Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan
  2. 2 Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Osaka, Japan
  3. 3 WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan
  4. 4 Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
  5. 5 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan
  1. Correspondence to Assistant Professor Junichi Kikuta and Professor Masaru Ishii, Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka 565-0871, Japan; jkikuta{at}, mishii{at}


Objectives Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destructionin vivo has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted in vivo.

Methods Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry.

Results Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation.

Conclusions Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens.

  • dmards (biologic)
  • pharmacokinetics
  • inflammation

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  • Handling editor Josef S Smolen

  • Contributors MI conceived and designed the study. YM performed the imaging experiments and data analysis with the assistance of JK, YK and THa, DO performed the RNA sequence analysis. MM and KK provided the pH-sensing chemical fluorescent probe. THi and AK discussed the experiments and results. YM and JK co-wrote the initial draft. MI wrote the final draft.

  • Funding This research was supported by research grants from Ono Pharmaceutical, Bristol-Myers Squibb and Chugai Pharmaceutical.

  • Competing interests None declared.

  • Patient consent Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.