Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells

Abstract

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14⁺ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1β, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14⁺ cells are responsible, at least in part, for the production of IL-1β, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14⁺ cells play an essential role in the occurrence of APRs following N-BP administration.

Keywords: acute-phase reaction; bisphosphonate; inflammatory cytokines; monocytes; zoledronate; γδ T cells.

Figure 1

Increased expression of inflammatory cytokines in PBMCs following exposure to nitrogen‐containing bisphosphonates. (A‐D) Human PBMCs were incubated for 8 h in the absence (Cont) or presence of 5 µm of etidronate (ETI), pamidronate (PAM) or zoledronate (ZOL). Bisphosphonates classified as second‐ and third‐generation agents contain nitrogen atoms. Expression of mRNAs for TNF‐α (A), IL‐1β (B), IL‐6 (C) and IFN‐γ (D) was normalized against that of GAPDH and expressed as a value relative to that obtained in cells without treatment with bisphosphonates (Cont). **P < 0.01, *P < 0.05. (E‐H) Human PBMCs were incubated for 24 h in the absence or the presence of etidronate (square, ETI), pamidronate (triangle, PAM) or zoledronate (circle, ZOL) at the concentrations of 5, 25 or 125 mm. TNF‐α (E), IL‐6 (F), IFN‐γ (G) and IL‐1β (H) in the culture supernatants were determined by ELISA.

Figure 2

The requirement of γδ T cells for induced expression of mRNAs for inflammatory cytokines in PBMCs. γδ T cells were removed from human PBMCs using magnetic beads coupled with the antibody against γδ TCR. (A, B) Expressions of γδ TCR and CD3 by PBMCs before (A) and after (B) removal of γδ T cells. Cells expressing both γδ TCR and CD3 were considered to be γδT cells. (C‐F) PBMCs before [γδ T(+)PBMC] and after [γδ T(−)PBMC] removal of γδ T cells were incubated for 8 h in the absence (−) or presence (+) of 5 µm of zoledronate (ZOL). The expression of mRNAs for TNF‐α (C), IL‐6 (D), IFN‐γ (E) and IL‐1β (F) was normalized against that of GAPDH and expressed as a value relative to that obtained with γδ T(+)PBMC without treatment with zoledronate (far left column in each panel). *P < 0.05 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Increased expression of mRNAs for inflammatory cytokines in CD14⁺ cells by zoledronate in the presence of PBMCs. CD14⁺ cells were separated from human PBMCs using magnetic beads coupled with an antibody against CD14. (A) Levels of expression of CD14 by separated CD14⁺ cells (solid line) and the remaining PBMCs (dotted line) are shown. (B‐E) CD14⁺ cells and PBMCs were separately cultured in Transwell^® Permeable Support (Corning Inc.) dishes. CD14⁺ cells were placed in the lower plates and PBMCs in the upper inserts, then incubated for 8 h in the absence (−) or presence (+) of 5 µm of zoledronate (ZOL), after which total RNA was isolated from CD14⁺ cells cultured in the lower plates. The expression of mRNAs for TNF‐α (B), IL‐6 (C), IFN‐γ (D) and IL‐1β (E) was normalized against that of GAPDH and expressed as a value relative to that obtained in CD14⁺ cells without treatment with zoledronate (unfilled columns). **P < 0.01, *P < 0.05.