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

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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

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Leukotriene B4
Osteoclasts
Osteogenesis
Interleukin-23
Calcium
Type C Phospholipases
Bone and Bones
Actins
Blood Cells
Leukotriene B4 Receptors
Phosphatidylinositol 3-Kinase
Arachidonate 5-Lipoxygenase
Calcium Signaling
Leukotrienes
Phospholipases A2
Calcium Channels
Cytoplasmic and Nuclear Receptors
Immunoenzyme Techniques
Ion Channels
Arthritis

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis. / Dixit, Neha; Wu, Dennis J.; Belgacem, Yesser H.; Borodinsky, Laura N; Gershwin, M. Eric; Adamopoulos, Iannis.

In: Arthritis Research & Therapy, Vol. 16, No. 6, 2014, p. 496.

Research output: Contribution to journalArticle

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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.",
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T1 - Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis

AU - Dixit, Neha

AU - Wu, Dennis J.

AU - Belgacem, Yesser H.

AU - Borodinsky, Laura N

AU - Gershwin, M. Eric

AU - Adamopoulos, Iannis

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

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