The effect of Lipoxin A4 on the interaction between macrophage and osteoblast: possible role in the treatment of aseptic loosening

Abstract

Background: Aseptic loosening (AL) is the main problem of total joints replacement (TJR) by the implantation of permanently prosthetic components. In vitro and in vivo studies have clearly demonstrated that wear debris and its byproducts could trigger inflammation in the peri-implant tissue. Lipoxins (LXs) are endogenous eicosanoids synthesized locally from arachidonate acid (AA) at sites of inflammation and mediate pro-resolving activity. A number of studies have demonstrated the effect of LXA4 to counteract inflammation in different cell and animal models, but till now, no relative report about the role of LXs in progress or prevention of AL.

Methods: Murine RAW264.7 macrophage cell line and MC3T3-E1 osteoblasts (OB) cell line were purchased. Co-cultured model of these two cell lines was established. To explore the effect of exogenous Lipoxin A4 (LXA4) on polymethylmethacrylate (PMMA) induced inflammation, pro-inflammatory cytokines including TNF-alpha, IL-1beta, PGE2 and GM-CSF were measured by ELISA kits and bone resorption was quantified by measuring calcium release from 5-day-old mice calvaria in vitro. To determine further the endogenous effect of LXA4, cells were co-cultured and with or without 15-lipoxygease (15-LO) blocking by 15-LO siRNA. Both real-time PCR and western blotting were applied to confirm the inhibitory efficiency of 15-LO by siRNA.

Results: 0.1 mg/ml, 0.5 mg/ml and 1.0 mg/ml PMMA showed a time-dependent manner to trigger production of all the pro-inflammatory cytokines studied. Exogenous 0-100 nM LXA4 presented an inhibitory effect on both generation of above cytokines and PMMA stimulated calvarial bone resorption with a dose-dependent manner. LXA4 in supernatant from neither rest macrophages nor macrophages cultured alone exposing to PMMA was detectable. In co-cultured cells challenged by PMMA, LXA4 was increased significantly, while, this enhance could be partly inhibited by 15-LO siRNA. When LXA4 generation was blocked with 15-LO siRNA, the PMMA induced pro-inflammatory cytokines were elevated and bone resorption was accelerated.

Conclusion: In the present study, we demonstrated that LXA4 had a favorable inhibitory effect on PMMA-induced inflammation in a macrophage and OB co-culture system.

Figure 1

Changes of pro-inflammatory cytokines in culture media of macrophages exposed to different concentration of PMMA. All the cytokines were measured with correspondent ELISA kits. **, P < 0.001 compared to 0 hour group.

Figure 2

LXA₄ inhibited the production of pro-inflammatory cytokines in culture media of macrophages stimulated with PMMA. Cells were treated with 1.0 mg/ml PMMA and different dose of LXA₄ for 24 hours. **, P < 0.001 compared to control group; #, P < 0.05 compared to cells treated with PMMA only.

Figure 3

LXA₄ blocked PMMA stimulated calvarial bone resorption. Each point represented the mean ± standard error of mean of 5-calvarial halves. **, P < 0.001 compared to control group; #, P < 0.05 compared to cells treated with PMMA only.

Figure 4

Effect of PMMA on endogenous LXA₄ production. A, LXA₄ content in culture media was monitored with ELISA kit. **, P < 0.01 compared to macrophages+PMMA; #, P < 0.05 compared with macrophage+OB+PMMA. B, Inhibition of 15-LO siRNA on 15-LO mRNA expression measured by RT-QPCR. Results were normalized to GAPDH and expressed as fold induction over cells co-cultured with OB without 15-LO siRNA. C, Inhibition of 15-LO siRNA on 15-LO protein expression measured by western blotting. GAPDH was applied as internal control.

Figure 5

Effect of endogenous LXA₄ on the production of pro-inflammatory cytokines. Culture media were collected 24 hours after treatment. **, P < 0.01 compared to control Mφ cells; #, P < 0.05 compared to Mφ+OB+PMMA cells.

Figure 6

Effect of endogenous LXA₄ on PMMA induced bone resorption. **, P < 0.01 compared to Mφ group; #, P < 0.05 compared to Mφ+OB+PMMA group; ##, P < 0.05 compared to Mφ+PMMA group.