Novel Nanoparticulate and Ionic Titanium Antigens for Hypersensitivity Testing

Abstract

Titanium is used in a wide variety of materials ranging from medical devices to materials used in everyday life. Adverse biological reactions that could occur in patients, consumers, and workers should be monitored and prevented. There is a lack of available agents to test and predict titanium-related hypersensitivity. The aim of this study was to develop two bioavailable titanium substances in ionic and nanoparticulate form to serve as antigens for hypersensitivity testing in vitro. Peripheral blood mononuclear cells from 20 test subjects were stimulated with the antigens and secretion of monocytic and lymphatic cytokines and chemokines were measured by a multiplex bead assay. Lymphocyte stimulation indices were also determined in a subset of test subjects by measuring CD69 and HLA-DR expression by flow cytometry. Cytokine profiling revealed that both antigens increased production of typical monocyte and macrophage secreted cytokines after 24 h, with significant increases in IL-1β, IL-7, IL-10, IL-12, IL-2R, IL-6, GM-CSF, TNF-α, IL-1RA, MIP-1α, MIP-1β, IFN-α, and IL-15. Lymphatic cytokines and chemokines were not significantly induced by activation. After seven days of stimulation, ionic-Ti (2.5 μg/mL) caused proliferation (stimulation index > 2) of CD4+ cells and CD8+ cells in all persons tested (N = 6), while titanium dioxide nanoparticles (50 μg/mL) only caused significant proliferation of CD4+ cells. Our preliminary results show that the experimental titanium antigens, especially the ionic form, induce a general inflammatory response in vitro. A relevant cohort of test subjects is required to further elucidate their potential for predictive hypersensitivity testing.

Keywords: adverse effects of nanomaterials on immune system; allergy; cytokine; flow cytometry; hypersensitivity; implant; lymphoproliferation; multiplex bead assay; titanium.

Figure A1

Flow charts showing gating of negative controls. (A) Gating on CD3+ lymphoid cells. (B) CD4+ cells (green), CD8+ cells red). (C) Activated effector T helper cells (CD45RO+/HLA-DR+) in upper right quadrant. (D) Activated effector T cytotoxic cells (CD45RO+/HLA-DR+). (E) CFSE-staining of CD4+ T cells showing no cell division. (F) CFSE-staining of CD8+ T cells showing no cell division.

Figure A2

Flow chart example of the gating for the stimulated cell populations (2.5 ug/L ionic-Ti after seven days). (A) Gating on CD3+ lymphoid cells. (B) CD4+ cells (green), CD8+ cells (red). (C) Activated effector T helper cells (CD45RO+/HLA-DR+) in upper right quadrant. (D) Activated effector T cytotoxic cells (CD45RO+/HLA-DR+) in upper right quadrant. (E) CFSE-staining of CD4+ T cells showing cell division in several generations. (F) CFSE-staining of CD8+ T cells showing cell division in several generations.

Figure 1

Lymphocyte proliferation (stimulation index (SI) = % of stimulated CFSE positive cells relative to the mean % of unstimulated control cells) of CD4+ cells and CD8+ cells after stimulation with ionic-Ti (a (CD4+), b (CD8+)) and nano-TiO₂ (c (CD4+)). (N = 6).

Figure 2

Expression of CD69 after 48 h (a,b) and HLA-DR after seven days (c,d) on CD4+ cells (a,c) and CD8+ cells (b,d) after stimulation with ionic-Ti (N = 6). * Wilcoxon matched pair, p < 0.05.

Figure 3

Statistical significance and median concentration (pg/mL) levels for cytokines and chemokines after PBMC stimulation in 24 h with low and high concentrations of nano-TiO₂, ionic-Ti and positive controls (PHA and PMA) versus unstimulated cells. * p < 0.05, ** p < 0.001 compared to control using Wilcoxon signed rank test. Abbreviations: interleukin (IL), receptor (R), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), interferon (IFN), macrophage inflammatory protein (MIP), monocyte chemoattractant protein (MCP), interferon gamma induced protein-10 (IP-10), monocyte induced by gamma interferon (MIG).

Figure 4

Expression levels of IL-1β (a,b), IL-6 (c,d), and GM-CSF (e,f) were increased after stimulation with either nano-Ti or ionic-Ti for 24 h. Graphs show median and interquartile range (N = 20). *** p < 0.0001, ** p < 0.01, * p < 0.05.