Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis.

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

doi:10.1186/s13075-014-0496-y

Abstract

IntroductionBone 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.MethodsWe 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.ResultsOur 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.ConclusionsIn conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.

Highlights

Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts
We first demonstrated that exogenous addition of IL-23 in in vitro human Peripheral blood mononuclear cell (PBMC) cultured in the presence of macrophage colonystimulating factor (M-CSF) for 3 days was sufficient to elevate the levels of leukotriene B4 (LTB4) in the conditioned medium as compared to control cultures treated with MCSF alone (M-CSF + IL-23: 35.79 ? 3.7 pg/ml, M-CSF: 5.1 ? 2.0 pg/ml, P < 0.01) as detected by enzyme-linked immunoassay (EIA) (Figure 1a)
To investigate whether this expression is maintained in terminally differentiated multinucleated osteoclasts, PBMC adherent cells were cultured for 8 days in the presence of M-CSF and further differentiated by exogenous addition of Receptor activator of nuclear factor κB ligand (RANKL) for 6 days

Summary

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)? Coupled receptors (GPCRs) possess the ability to transmit intracellular signals within milliseconds of activation, whereas growth factor and cytokine receptors lack this rapidity and specificity in signaling [7,8]. This rapid induction of inflammation observed during IL-23 gene transfer prompted us to investigate, alternate inflammatory pathways associated with GPCRs. One pathway that has been associated with rapid inflammation and osteoclast formation is the leukotriene activation pathway [9]

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Conclusion