Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles
Introduction
The use of nanoparticles (NPs) in various fields, including biotechnology, has gained increasing attention in the past few years (Moghimi et al., 2005). For example, NPs have great potential as tools for drug delivery in medicine (Laroui et al., 2009). However, the rapid development of nanotechnology has resulted in a surge of interest in nanotoxicology, since exposure of humans to NPs resulting from industrial activity is unavoidable.
Titanium dioxide (TiO2) is used extensively as an industrial nanomaterial. TiO2 NPs are used in a variety of products including anti-fouling paints, coatings, ceramics, sunscreens, and also as additives in pharmaceuticals and food colorants (Jin et al., 2008, Vamanu et al., 2008). Titanium (pure or in alloys) is widely used in several types of implanted medical devices, including cardiovascular stents, joint replacements and dental implants (Brien et al., 1992, Buly et al., 1992, Arys et al., 1998). Several studies evaluating potential health risks of TiO2 NPs to humans have received increasing attention (Puccetti and Leblanc, 2000, Radnai et al., 2000, Vamanu et al., 2008, Schanen et al., 2009). It appears that, TiO2 NPs possess pro-inflammatory properties similar to those exhibited by other nanomaterials (Blackford et al., 1997, Park et al., 2009, Schanen et al., 2009). Analysis of the pertinent scientific literature indicates that the reported pro-inflammatory effects of TiO2 NPs were observed in vitro tests conducted with pulmonary cells, or from in vivo studies in animals following lung instillation.
Polymorphonuclear neutrophils (PMNs) are primordial players in innate immunity and provide an effective defense against bacterial and fungal infections (Whyte et al., 1997, Akgul et al., 2001). They are terminally mature non-dividing phagocytic cells which develop in the bone marrow from CD34+ stem cells. It has been estimated that ∼5 × 1010 cells per day, in a normal adult, are released from the bone marrow, representing one of the fastest rates of cell turnover in the human body. Therefore, PMN cell turnover must be under strict control. Interestingly, PMNs are known to spontaneously undergo apoptosis, without apparent stimulation, explaining, in part, why the number of PMNs remains relatively stable in healthy individuals. In contrast, during inflammation, the number of PMNs markedly increases but, under normal circumstances, resolution of inflammation is largely achieved by elimination of apoptotic PMNs by professional phagocytes, including macrophages.
Curiously, despite the fact that neutrophils play a key role during inflammation, and that increased PMNs have been observed in TiO2-induced lung inflammation in vivo, there is a lack of literature concerning the role of TiO2 NPs on neutrophil cell physiology, particularly human PMNs. One study conducted more than 20 years ago reported that pure rutile or anatase TiO2 preparations elicited only a weak chemiluminescent response, indicating that these NPs did not significantly increase reactive oxygen species production by these cells (Hedenborg, 1988).
In the present study, we investigated the potential agonistic effect of TiO2 NPs on several human neutrophil responses/functions, some known to occur rapidly and others necessitating a prolonged exposure to NPs for measurement. We found that TiO2-induced changes in cellular morphology and caused the rapid and vigorous tyrosine phosphorylation of several proteins, including p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases-1/2 (Erk-1/2). TiO2, also induced suppression of PMN apoptosis and increased the production of several cytokines/chemokines, including IL-8, the predominant chemokine produced by these cells.
Section snippets
Chemicals, agonists and antibodies
The chemicals agonist and antibodies used in this were Titanium dioxide (TiO2) nanoparticles (anatase crystals) (Vivenano, Toronto, ONT); PD98059 and SB203580 inhibitors (Sigma–Aldrich, Saint-Louis, Missouri); anti-phosphospecific p38 (pTpY180/182) and Erk-1/2 (pTpY185/187) (BioSource, Camarillo, CA); antibodies against the non-phosphorylated form of p38 (sc-535) (Santa Cruz, Biotechnology, Santa Cruz, CA); polyclonal Erk-1/2 (Upstate Biotechnology, Lake Placid, NY); anti-phosphotyrosine (Cell
IL-4 induced morphological changes in neutrophils
The induction of morphological changes in PMNs is a rapid and simple assay used as a reflection of neutrophil activation (Girard et al., 1997). PMNs were incubated with increasing concentrations of TiO2 (0–100 μg/ml) and morphology was observed, overtime, under light microscopy. As shown in Fig. 1, untreated neutrophils remained spherical (panel A), whereas cells incubated with an increasing concentration of TiO2 exhibited morphological changes. Results illustrated in Fig. 1 were obtained after 1
Discussion
Different cellular responses and functions were investigated in order to clearly establish that TiO2 is a human PMN agonist. We initially eliminated the possibility that TiO2-induced cell necrosis in human PMNs. Our results agree with those of a previous study which reported that a concentration of 200 μg/ml TiO2 did not affect cell viability of human PMNs, as assessed by lysozyme release and trypan blue exclusion (Hedenborg, 1988). Although the maximum concentration of TiO2 used in our study is
Acknowledgments
This study was partly supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Environmental Health Research Network (RRSE) from Fonds de la Recherche en Santé du Québec (FRSQ). DMGG holds a Foundation Armand-Frappier M.Sc. award and DG is a Scholar from FRSQ. We thank Mary Gregory for reading this manuscript.
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