Supplementary Material for: Calcitonin Gene-Related Peptide Modulates the Production of Pro-Inflammatory Cytokines Associated with Periprosthetic Osteolysis by THP-1 Macrophage-Like Cells

Objective: An anti-resorptive impact of the neuropeptide calcitonin gene-related peptide (CGRP) on periprosthetic osteolysis, the leading cause of early prosthesis loosening, has been shown previously. In this study, the impact of CGRP on pro-inflammatory cytokine production associated with periprosthetic osteolysis was analysed using THP-1 macrophage-like cells. Methods: Cells were stimulated with ultra-high-molecular-weight polyethylene (UHMWPE) particles (cell-to-particle ratios of 1:100 and 1:500) and lipopolysaccharides (LPS; 1 µg/ml) to establish osteolytic conditions, and simultaneously treated with CGRP (10^-8M). Receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNF)-a mRNA expression were measured by quantitative RT-PCR. RANK protein was detected by Western blot. Secreted protein levels of TNF-a as well as interleukin (IL)-1ß and IL-6 were quantified in cell culture supernatants by ELISA and Bio-Plex cytokine assay, respectively. Results: Activation of macrophage-like cells failed to enhance the production of RANK but led to a dose- and time-dependent increase of TNF-a mRNA and secreted protein levels of TNF-a, IL-1ß and IL-6. Application of CGRP time-dependently suppressed TNF-a mRNA expression induced by low-particle concentrations and LPS, while both particle- and LPS-induced secretion of TNF-a was inhibited. A pronounced inhibitory effect of CGRP on LPS-induced cytokine production at 24 h of incubation was also observed with IL-1ß and IL-6. Conclusions: CGRP shows a time-dependent inhibitory effect on the secretion of osteolysis-associated pro-inflammatory cytokines, indicating an indirect anti-resorptive influence of the neuropeptide on both aseptic prosthesis loosening and bacterially induced bone resorption which might enhance the life time of total joint replacements.