Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis

Neha Dixit, Dennis J. Wu, Yesser H. Belgacem, Laura N Borodinsky, M. Eric Gershwin, Iannis Adamopoulos

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

INTRODUCTION: Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)-producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.

METHODS: We utilized whole blood-derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C (U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.

RESULTS: Our data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release-activated channel-mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. These effects were dependent on Ca2+ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.

CONCLUSIONS: In conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.

Original languageEnglish (US)
Pages (from-to)496
Number of pages1
JournalArthritis Research & Therapy
Volume16
Issue number6
DOIs
StatePublished - 2014

ASJC Scopus subject areas

  • Medicine(all)

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