Elsevier

Toxicology in Vitro

Volume 24, Issue 3, April 2010, Pages 1002-1008
Toxicology in Vitro

Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles

https://doi.org/10.1016/j.tiv.2009.12.007Get rights and content

Abstract

This paper describes the in vitro effects of titanium dioxide (TiO2) nanoparticles (NPs) upon human neutrophils. Kinetic experiments revealed no cell necrosis after 24 h of treatment with TiO2 (0–100 μg/ml). In contrast, TiO2-induced change in cellular morphology in a concentration-dependent manner in neutrophils over time, indicating its potential to activate these cells. To further support this, we demonstrated that TiO2 markedly and rapidly induced tyrosine phosphorylation events, including phosphorylation of two key enzymes, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases-1/2 (Erk-1/2). We also determined the effects of TiO2 on two neutrophil functions requiring a longer exposure period between NPs and cells: apoptosis and cytokine production. Interestingly, at concentrations ⩾20 μg/ml, TiO2 inhibited neutrophil apoptosis in a concentration-dependent manner after 24 h of treatment. Supernatants from TiO2-induced neutrophils were harvested after 24 h and tested for the presence of 36 different analytes (cytokines, chemokines) using an antibody array assay. TiO2 treatment increased production of 13 (36%) analytes, including IL-8, which exhibited the greatest increase (∼16 × control cell levels). The increased production of IL-8 was confirmed by ELISA. We conclude that TiO2 exerts important neutrophil agonistic properties in vitro.

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.

References (45)

  • M. Pelletier et al.

    Activation of human neutrophils by the air pollutant sodium sulfite (Na2SO3): comparison with immature promyelocytic HL-60 and DMSO-differentiated HL-60 cells reveals that Na2SO3 is a neutrophil but not a HL-60 cell agonist

    Clinical Immunology

    (2000)
  • C.I. Vamanu et al.

    Induction of cell death by TiO2 nanoparticles: studies on a human monoblastoid cell line

    Toxicology in Vitro

    (2008)
  • R. Alam et al.

    Macrophage inflammatory protein-1 alpha and monocyte chemoattractant peptide-1 elicit immediate and late cutaneous reactions and activate murine mast cells in vivo

    Journal of Immunology

    (1994)
  • A. Arys et al.

    Analysis of titanium dental implants after failure of osseointegration: combined histological, electron microscopy, and X-ray photoelectron spectroscopy approach

    Journal of Biomedical Materials Research

    (1998)
  • P.A. Bennett et al.

    The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes

    Journal of Cell Science

    (1993)
  • E. Bermudez et al.

    Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles

    Toxicological Sciences

    (2004)
  • F. Binet et al.

    Arsenic trioxide (AT) is a novel human neutrophil pro-apoptotic agent: effects of catalase on AT-induced apoptosis, degradation of cytoskeletal proteins and de novo protein synthesis

    British Journal of Haematology

    (2006)
  • F. Binet et al.

    Arsenic trioxide induces de novo protein synthesis of annexin-1 in neutrophils: association with a heat shock-like response and not apoptosis

    British Journal of Haematology

    (2008)
  • F. Binet et al.

    Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2

    Journal of Leukocyte Biology

    (2008)
  • J.A. Blackford et al.

    Comparison of inducible nitric oxide synthase gene expression and lung inflammation following intratracheal instillation of silica, coal, carbonyl iron, or titanium dioxide in rats

    Journal of Toxicology and Environmental Health

    (1997)
  • W.W. Brien et al.

    Metal levels in cemented total hip arthroplasty. A comparison of well-fixed and loose implants

    Clinical Orthopaedics and Related Research

    (1992)
  • J. Chen et al.

    In vivo acute toxicity of titanium dioxide nanoparticles to mice after intraperitoneal injection

    Journal of Applied Toxicology

    (2009)
  • Cited by (115)

    • Impact of ultra-small silver nanoparticles of 2 nm (AgNP<inf>2</inf>) on neutrophil biology: AgNP<inf>2</inf> alter the actin cytoskeleton and induce karyorrhexis by a mitogen-activated protein kinase-dependent mechanism in vitro and transitorily attract neutrophils in vivo

      2022, Chemico-Biological Interactions
      Citation Excerpt :

      In this study, if not specified, AgNP2 were used at a final concentration of 100 μg/ml. This is based on preliminary data and on previous experiments performed in granulocytes with other NPs [18–21], including AgNPs of larger diameters [15–17]. The NP suspension obtained from the manufacturer was examined using a Hitachi H-7100 transmission electron microscope to validate the ultra-small size of these NP.

    View all citing articles on Scopus
    View full text