Comparison of Inflammatory Effects in THP-1 Monocytes and Macrophages after Exposure to Metal Ions

Abstract

Monocytes and macrophages are the first barrier of the innate immune system, which interact with abrasion and corrosion products, leading to the release of proinflammatory mediators and free reactive molecules. The aim of this study was to understand inflammation-relevant changes in monocytes and macrophages after exposure to corrosion products. To do this, the THP-1 cell line was used to analyze the effects of metal ions simultaneously in monocytes and differentiated macrophages. Cells were stimulated with several concentrations of metal salts (CoCl₂, NiCl₂, CrCl₃ × 6H₂O) to analyze viability, gene expression, protein release and ROS production. Untreated cells served as negative controls. While exposure to Cr(3+) did not influence cell viability in both cell types, the highest concentration (500 µM) of Co(2+) and Ni(2+) showed cytotoxic effects mirrored by significantly reduced metabolism, cell number and a concomitant increase of ROS. The release of IL-1β, IL-8, MCP-1 and M-CSF proteins was mainly affected in macrophages after metal ion exposure (100 µM), indicating a higher impact on pro-inflammatory activity. Our results prove that monocytes and macrophages react very sensitively to corrosion products. High concentrations of bivalent ions lead to cell death, while lower concentrations trigger the release of inflammatory mediators, mainly in macrophages.

Keywords: aseptic loosening; corrosion; inflammation; macrophages; metal ions; monocytes.

Figure 1

Cell viability of THP-1 monocytes (a–c) and macrophages (d–f) after exposure to metal salts. WST-1 assay (a,b) and CyQUANT NF Cell Proliferation Assay (c,d) were carried out after 48 h of stimulation with different concentrations of cobalt, chromium and nickel ions. Figure 1e,f show the WST-1 activity per 1,000,000 cells. All values were normalized to untreated cells and shown as a percentage, with negative control set as 100% (dotted line). Data (n = 12) are shown as box plots with minimum, 25th percentile, median, 75th percentile and maximum values. Significant differences were calculated by repeated measures two-way ANOVA and Bonferroni’s multiple comparisons test as post hoc test from the original values (non-normalized). Significantly different between different concentrations: * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from untreated control: # p < 0.05, ## p < 0.01, ### p < 0.001; significantly different from cobalt at the same concentration: §; significantly different from nickel at the same concentration: °.

Figure 2

Light microscopic pictures of THP-1 monocytes exposed to metal salts and untreated cells. Monocytes were treated with 100 µM (a–c) and 500 µM (d–f) of cobalt, chromium and nickel ions. Untreated cells are depicted in Figure 2g. Pictures were taken after 48 h of exposure in 20× magnification. Scale bar = 50 µm.

Figure 3

Light microscopic pictures of THP-1 macrophages exposed to metal salts and untreated cells. Macrophages were treated with 100 µM (a–c) and 500 µM (e–g) of cobalt, chromium and nickel ions). Untreated cells are depicted in Figure 3g. Pictures were taken after 48 h of exposure in 20× magnification. Scale bar = 50 µm.

Figure 4

Reactive oxygen species (ROS) quantification in cell culture supernatants. THP-1 monocytes and macrophages were treated with different concentrations of cobalt, chromium and nickel ions over 48 h. Untreated cells served as negative control. ROS formation was determined in cell culture supernatants using OxiSelect™ In vitro ROS/RNS Assay. Data are shown as percentage in relation to control cells (100%, dotted line) as single datapoints with mean ± SD (n = 3). Significances between groups were calculated with Two-way ANOVA and Bonferroni’s multiple comparisons test as a post hoc test. Significantly different between concentrations or different metal salt treatments at the same concentration: * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from untreated control: # p < 0.05, ## p < 0.01, ### p < 0.001.

Figure 5

Gene expression and protein release of interleukin (IL-) 1β following exposure to metal salts. Gene expression (a) and protein release into cell culture supernatants (b) was determined for THP-1 monocytes and macrophages after 48 h of treatment with the concentration of 100 µM of cobalt, chromium and nickel salts. Untreated cells served as negative controls. Data (n = 3) are depicted as single datapoints with mean ± SD. Gene expression data are shown as percentage of untreated cells (2^{(−∆∆Ct)}, 100%, dotted line), while protein release data represent values of the specific protein amount normalized to total protein content in cell culture supernatants. Significances between groups were calculated with two-way ANOVA with Bonferroni post hoc test using ∆CT values for gene expression and specific protein amount normalized to total protein content for protein release in cell culture supernatants. Significantly different from untreated control: # p < 0.05, ## p < 0.01, ### p < 0.001. Abbreviations: n.d. = not detectable; NC = negative control (untreated cells).

Figure 6

Gene expression and protein release of monocyte chemotactic protein (MCP-) 1 (a,b) and Interleukin (IL-) 8 (c,d) following exposure to metal salts. Gene expression (a,c) and protein release into cell culture supernatants (b,d) were determined for THP-1 monocytes and macrophages after 48 h of treatment with the concentration of 100 µM of cobalt, chromium and nickel salts. Untreated cells served as negative controls. Data (n = 3) are depicted as single datapoints with mean ± SD. Gene expression data are shown as percentage of untreated cells (2^{(−∆∆Ct)}, 100%, dotted line) while protein release data represent values of the specific protein amount normalized to total protein content in cell culture supernatants. Significances between groups were calculated with two-way ANOVA and Bonferroni’s multiple comparison test as post hoc test using ∆CT values for gene expression and specific protein amount normalized to total protein content for protein release in cell culture supernatants. Significantly different between stimulation groups: * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from untreated control: # p < 0.05, ## p < 0.01, ### p < 0.001. Abbreviations: n.d. = not detectable; NC = negative control (untreated cells).

Figure 7

Gene expression and protein release of macrophage colony-stimulating factor (M-CSF) (a,b) and receptor activator of NF-κB (RANK) (c) following exposure to metal salts. Gene expression (a,c) and protein release into cell culture supernatants (b) was determined for THP-1 monocytes and macrophages after 48 h of treatment with the concentration of 100 µM of cobalt, chromium and nickel salts. Untreated cells served as negative controls. Data (n = 3) are depicted as single datapoints with mean ± SD. Gene expression data are shown as percentage of untreated cells (2^{(−∆∆Ct)}, 100%, dotted line) while protein release data represent values of the specific protein amount normalized to total protein content in cell culture supernatants. Significances between groups were calculated with two-way ANOVA and Bonferroni’s multiple comparison test as post hoc test using ∆CT values for gene expression and specific protein amount normalized to total protein content for protein release in cell culture supernatants. Significantly different between stimulation groups: * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from untreated control: # p < 0.05, ## p < 0.01, ### p < 0.001. Abbreviations: n.d. = not detectable; NC = negative control (untreated cells).