Correspondence: Yoshiki Tokura, M.D., Ph.D., Department of Dermatology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan. Email: email@example.com
Titanium dioxide (TiO2) nanoparticles, widely used for daily products, are believed to be biologically inert, but they may cause adverse effects on cells, presumably depending on the particle size and shape. One of the critical targets of TiO2 particles is epidermal keratinocytes, and their initial response to TiO2 may be production of pro-inflammatory cytokines. We therefore investigated the effects of four types of TiO2 particles on cytokine expression/production by real-time reverse transcription polymerase chain reaction and enzyme-linked immunoassay. The TiO2 particles included three acicular types, FTL-100 (length, 1.68 μm; diameter, 130 nm), FTL-200 (2.86, 210) and FTL-300 (5.15, 270), and one globular type, PT-301 (diameter, 270 nm). Normal human epidermal keratinocytes (NHEK) were cultured with each of the TiO2 particles. During cultivation, the acicular forms of TiO2 were seen by scanning electron microscopy to be internalized by NHEK. The three acicular particles increased the mRNA expressions and supernatant productions of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor-α and IL-8 in particle number-dependent manners, whereas globular PT-301 had very weak activity. Thus, TiO2 particles may induce skin inflammation depending on the size and shape, providing knowledge on their health usage.
Recent advances in nanotechnology raise concerns about development, production route, diffusion and biological effects in industrial and consumer products of nanoparticles. Titanium dioxide (TiO2) nanoparticles are a representative nanomaterial and have been widely used for various daily products such as sunscreens, whose size is 35–145 nm. Because there has been no evidence for significant penetration of TiO2 nano-sized particles beyond the stratum corneum,[3, 4] fine TiO2 particles are estimated to be harmless for the skin. However, a number of studies have shown that TiO2 nanoparticles can cause several adverse effects on mammalian cells, including production of reactive oxygen species,[6, 7] reduction of cell viability and proliferation,[6, 7] and induction of apoptosis and genotoxicity. In these studies, the cytotoxic effects of TiO2 particles were extensively investigated, but the inflammation-provoking effects were poorly addressed. In this context, the finding that TiO2 nanoparticles stimulate innate immunity-provoking inflammasome, consisting of NOD-like receptor pyrin domain containing 3 (NLRP3), is important.
Because nanoparticles have unique physicochemical properties, some characteristic particles likely exhibit biological activities significantly different from fine-sized particles of the same chemical composition. Intriguingly, interactions of nanoparticles with biological systems are thought to depend on the particle size and shape. In fact, the toxic effects of the size and shape have been investigated on the respiratory tract cells, including alveolar macrophages and lung epithelial cells.
Relatively large TiO2 nanoparticles are divided into mainly two forms, globular and acicular types. The globular type (35–145 nm in diameter) is used for sunscreen photoprotective agents or others.[2, 3] On the other hand, the acicular type has been used recently for reinforcement of plastics and ceramics, paint, paper, rubber and catalysts, and friction and filtration materials.[12, 13] The skin is an immunological organ, and dermatitis is feasibly induced by exposure to external substances. TiO2 particles may be applied to the skin for cosmetic use or accidentally in industrial workplaces. Because keratinocytes are the major constituent of the epidermis and deeply involved in cutaneous immunity, it is assumed that they are primarily affected by TiO2 particles. To study the effect of TiO2 on keratinocytes, the immunobiological changes are thus considered to be important rather than the cytotoxic changes. Although TiO2 particles have been reported not to pass through the stratum corneum, barrier-disrupted conditions, such as atopic dermatitis,[16, 17] may allow TiO2 to permeate via this barrier. Therefore, it should be clarified whether keratinocytes are stimulated with TiO2 nanoparticles and whether the stimulation is dependent on the shape and size of nanoparticles.
In this study, we sought to investigate the effects of TiO2 nanoparticles with different shapes and sizes on the biological activities of human epidermal keratinocytes. In cultured keratinocytes, we observed the morphological changes induced by the addition of TiO2 particles. Because one of the initial responses of keratinocytes to the nanoparticles seems to be the release of pro-inflammatory cytokines,[14, 15] we then examined the effects of TiO2 particles on the production of various cytokines.
TiO2 particles and chemicals
Four types of TiO2 crystal particles, three globular and one acicular, were purchased from Ishihara Sangyo Kaisha (Osaka, Japan). The acicular types included FTL-100 (length, 1.68 μm; diameter, 130 nm), FTL-200 (length, 2.86 μm; diameter, 210 nm) and FTL-300 (length, 5.15 μm; diameter, 270 nm). The globular type was PT-301 (diameter, 270 nm). Because each of the four TiO2 particles has its own shape and weight, their biological activities can be compared on the basis of either the particle number or concentration. In consideration of the possible shape-dependent effects, we compared them mostly on the basis of particle number, and if necessary, compared them on the concentration basis for analyses. At the same number of needles, the ratios of concentration of TiO2 for PT-301 : FTL-100 : FTL-200 : FTL-300 were 1:2.23:9.90:29.5. For example, 1 × 107/cm2 of TiO2 number corresponds to 0.84 μg/mL in PT-301, 1.87 μg/mL in FTL-100, 8.32 μg/mL in FTL-200 and 24.76 μg/mL in FTL-300.
Z-YVAD, an inhibitor of caspase-1, was purchased from R&D Systems (Minneapolis, MN, USA). Polyinosinic–polycytidylic acid (poly[I:C]) was obtained from Sigma Chemical (St Louis, MO, USA).
Normal human epidermal keratinocytes (NHEK) were purchased from Cascade Biologics (Portland, OR, USA). They were grown in the serum-free keratinocyte growth medium Epilife (Cascade) and used at third passage in all experiments. Growth supplement was omitted 24 h before experiments.
Cells were counted by a hemocytometer and equally distributed in 24-well plates (Corning Glass Works, Corning, NY, USA) at a density of 3 × 104 cells/well and incubated with various concentrations of TiO2 for 48 h. To determine cell viability, medium was removed and cells were incubated with 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT; Wako, Osaka, Japan) at a final concentration of 5 mg/mL in phosphate-buffered saline (pH 7.4) for 4 h. Then, 1 mL dimethylsulfoxide (Wako) was added to the wells. After the formazan dye was totally dissolved, the solution was transferred to a 96-well plate (Corning) and read at 540 nm by iMark microplate reader (Bio-Rad, Hercules, CA, USA).
Scanning electron microscope (SEM)
Titanium dioxide particles were sputter-coated with platinum in a JFC-1600 auto fine coater (JEOL, Tokyo, Japan). TiO2 particles were then examined with a JSM-6390LA analytical SEM (JEOL). To observe morphology, NHEK were grown on two-well culture slides (BD, Franklin Lakes, NJ, USA) at a density of 3 × 104 cells/well and incubated with TiO2 particles for 24 h. Culture slides were fixed in 2% glutaraldehyde/0.1 mol/L sodium cacodylate buffer (pH 7.4) at 4°C for 1 h, rinsed by 0.1 mol/L sodium cacodylate buffer (pH 7.4), and post-fixed in 1% osmium tetraoxide/0.1 mol/L sodium cacodylate buffer at room temperature for 1 h. Samples were then dehydrated in graded ethanols from 50% to 99%. Ethanol was substituted to tert-buthl alcohol in the samples. Samples were freeze-dried with a JFD-300 freeze-dry machine (JEOL), sputter-coated with osmium in a Plasma Multi Coater PMC-5000 (Meiwafosis, Tokyo, Japan), and then examined with a S-4800 scanning electron microscope (Hitachi High-Technologies, Tokyo, Japan).
Titanium dioxide particles were added or not added as control at the starting of experimental NHEK culture of six-well plates (Corning). Cells were harvested 24 h later and subjected to real-time RT–PCR as described previously. Total mRNA was extracted from NHEK with the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Target gene expression was quantified with a two-step RT–PCR. cDNA was reverse transcribed from total RNA samples using the TaqMan RT reagents (Applied Biosystems, Foster City, CA, USA). The expression of human interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α was quantified using SYBR GreenER qPCR SuperMix (Invitrogen, Carlsbad, CA, USA) in an ABI PRISM 7000 sequence detection system (Applied Biosystems). As an endogenous reference for these PCR quantification studies, β-actin gene expression was measured. The following primer sets were used for real-time PCR assays: IL-1α sense, 5′-GTGAAATTTGACATGGGTGC-3′, anti-sense, 5′-CAGGCATCTCCTTCAGCAG-3′; IL-1β sense, 5′-ACAGATGAAGTGCTCCTTCCAG-3′, anti-sense, 5′-TGGAGAACACCACTTGTTGC -3′; IL-6 sense, 5′-GTGTGAAAGCAGCAAAGAGG-3′, anti-sense, 5′-GGTCAGGGGTGGTTATTGC-3′, IL-8 sense, 5′-TGCAGCTCTGTGTGAAGGTG-3′, anti-sense, 5′-GGTCCACTCTCAATCACTCTCAG-3′; TNF-α sense, 5′-TCCTTCAGACACCCTCAACC-3′, anti-sense, 5′-AGGCCCCAGTTTGAATTCTT-3′; and β-actin sense, 5′-CTTCTACAATGAGCTGCGTG-3′, anti-sense, 5′-TCATGAGGTAGTCAGTCAGG-3′. The relative expression was calculated using the 2−ΔΔCT method. The expression of the target gene normalized to an endogenous reference and relative to calibrator is given by the formula 2−ΔΔCT.
Two-day culture supernatants were measured for IL-1α, IL-1β and IL-8 using enzyme-linked immunosorbent assay (ELISA) using kits (R&D Systems) according to the manufacturer's protocol.
Student's t-test (unpaired) was employed to determine statistical differences between means. Correlations were studied by Pearson's product–moment correlation coefficient.
Viability of NHEK cultured with TiO2 particles
Because each of the four TiO2 particles has its own shape and weight, their biological activities can be compared on the basis of either the particle number or TiO2 concentration. At the same number of particles, the ratios of concentration of TiO2 in the four nanoparticles PT-301 : FTL-100 : FTL-200 : FTL-300 are 1:2.23:9.90: 29.5. To evaluate the possible shape-dependent effects of the TiO2 particles, we used both particle number and TiO2 concentration throughout the following studies.
First, we evaluated the toxicity of TiO2 particles toward NHEK as assessed by cell viability. All four TiO2 particles at concentrations of 30 μg/mL or less exhibited more than 85% viability of NHEK (Fig. 1a). Among the three acicular particles (FTL-100, FTL-200 and FTL-300), FTL-100 had the lowest viability on the concentration basis, because its particle number was highest. On the particle number basis, however, FTL-300 showed the lowest viability (Fig. 1b). We used the number or concentration of TiO2 particles giving higher than 70% viability of NHEK in the following experiments.
Morphological changes of NHEK cultured with TiO2 particles
The configurations of acicular and globular forms of TiO2 were observed under SEM (Fig. 2a–d). The acicular forms were added to NHEK culture and the morphological changes of NHEK were monitored. As typically seen in FTL-300 at 2.5 × 107 particle number/cm2, corresponding to 61.9 μg/mL (viability, 91%), TiO2 acicular particles spread on the surface of the cultured keratinocytes (Fig. 2e). Notably, close inspection revealed that some of the particles were internalized by keratinocytes at 24 h incubation (Fig. 2f).
Effects of acicular and globular nanoparticles on the expression/production of IL-8 by NHEK
Normal human epidermal keratinocytes were cultured with each form of the nanoparticles. The expression of cytokine mRNA was measured in the 24-h cultured cells by real-time RT–PCR, and the protein levels were monitored in the 48-h culture supernatants by ELISA. We first compared acicular FTL-100 and globular PT-301 in their abilities to enhance IL-8 expression on the TiO2 concentration basis. FTL-100 at 10 μg/mL augmented the expression of IL-8 mRNA, whereas the ability of PT-301 at the same concentration was not substantial (Fig. 3a). Accordingly, the concentration of IL-8 in the culture supernatants was increased by FTL-100 more markedly than by PT-301 (Fig. 3b). Thus, the acicular form had a higher ability to enhance IL-8 production than the globular form.
Effects of acicular particles on the expression/production of various cytokines by NHEK
Next, NHEK were cultured with each of the three TiO2 acicular particles, FTL-100, FTL-200 and FTL-300, and mRNA expressions of IL-1α, IL-1β, IL-6, TNF-α and IL-8 were examined. All forms of acicular particles increased the expression levels of all cytokines in particle number-dependent manners (Fig. 4). Notably, FTL-300 had the strongest ability to induce the cytokine expression.
When the cytokine amounts were measured in the culture supernatants, the three acicular forms again increased the IL-8 production in the order of FTL-300, FTL-200 and FTL-100 on the particle number basis (Fig. 5a). However, when analyzed on the TiO2 concentration basis, the stimulatory order was FLT-100, FLT-200 and FLT-300 (data not shown). The ability of FTL-100 to increase IL-1α (Fig. 5b) and IL-1β (Fig. 5c) were also confirmed at protein levels by ELISA.
No participation of inflammasome in the TiO2-augmented production of IL-1β by NHEK
Finally, we sought to address the mechanisms. Recently, crystals such as uric acid have been identified as a danger signal that triggers a cytosolic sensor, the inflammasome, where pro-IL-1β is processed to IL-1β with activated caspase-1. The stimulatory ability of the TiO2 particles for the cytokine production raised the possibility that TiO2 particles activate the crystal-mediated innate immunity in NHEK. We therefore investigated the effect of Z-YVAD, an inhibitor of caspase-1, on the production of IL-1β. When NHEK were cultured for 2 days with poly(I:C) at 30 μg/mL, they produced an increased amount of IL-1β (12.5 pg/mL) as compared with no addition control (0.7 pg/mL). This enhanced IL-1β production was inhibited by Z-YVAD at 50 μmol/L by 39%, indicating the participation of caspase-1 in IL-1β production. However, no inhibition by Z-YVAD was found in the IL-1β production induced by FL-300 instead of poly(I:C). This suggests that acicular TiO2 particles stimulate NHEK via an inflammasome-independent pathway.
Epidermal keratinocyte-derived cytokines, IL-1α, IL-1β, TNF-α, IL-6 and IL-8 are involved in both cutaneous innate and acquired immune responses by stimulating various immunocompetent cells, such as dendritic cells, mast cells and vascular endothelial cells. TNF-α is a powerful cytokine for the development of skin diseases such as psoriasis. IL-6 has various inflammatory effects on skin constituents and may exaggerate dermatitis. IL-8 serves as a chemokine for neutrophils so that it induces the infiltration of neutrophils in several skin diseases. Moreover, IL-1α, IL-1β and TNF-α collectively stimulate cutaneous dendritic cells, including Langerhans cells and dermal dendritic cells, to mature dendritic cells and allow them to migrate to the draining lymph nodes. Thus, the increased production of these cytokines leads to cutaneous inflammatory responses, as typically seen in contact dermatitis.
The present study demonstrated that TiO2 nanoparticles stimulate epidermal keratinocytes to produce these cytokines. We compared three acicular particles and one globular in their abilities to elaborate the cytokines and found that the acicular particles remarkably activate NHEK, but the ability of the globular one (diameter, 270 nm) is only limited. Among the three acicular forms, FTL-300 with the highest length and diameter had the highest activity on the particle number basis. Because the weight ratios per particle are 1:4.4: 3.2 in FTL-100, FTL-200 and FTL-300, and their IL-8 production activity ratios (particle number, 0.5–2.5 × 107/cm2) were 1: 2.5–4.7:1.8–2.8 (see Fig. 5a), it is likely that the activities are not only proportional to the weight, but also dependent on the shape.
The acicular TiO2 particles were internalized by NHEK during culture, implying that the particles are not simply cytotoxic, but can biologically stimulate keratinocytes. The nanomaterial uptake by keratinocytes has been reported with quantum dots, which are smaller ellipsoid materials with cadmium selenide core and a zinc sulfide shell. Quantum dots are recognized by lipid rafts and are internalized into early endosomes. However, because the size of TiO2 particles used in our study is much larger than that of quantum dots, the occurrence of intracellular endosome event is unlikely. Alternatively, the TiO2 particles may activate inflammasome by acting as crystal in keratinocytes, as reported in the responses to long needle-like carbon nanotubes and carbon black nanoparticles. Accordingly, it has recently been shown that TiO2 nanoparticles stimulate inflammasome.[9, 29] The nano-TiO2-dependent NLRP3 activity induces IL-1α/β secretion in keratinocytes, but unlike other particulate NLRP3 agonists, it does not require cytoskeleton-dependent phagocytosis. Upon activation, NLRP3 inflammasome yields caspase-1, which cleaves pro-IL-1β to form IL-1β, thereby exerting innate immune responses.[19, 20] We therefore tested the ability of FL-300 to activate caspase-1. However, the increased IL-1β production by FL-300 was not affected by Z-YVAD, an inhibitor of caspase-1, suggesting that acicular TiO2 particles stimulate NHEK via an inflammasome-independent pathway. In the TiO2-induced NLRP3 inflammasome study, Yazdi et al. used anatase or rutile TiO2 nanoparticles of 20 or 80 nm size, which are smaller than the TiO2 particles used herein. A study using monosodium urate crystals as a stimulant has recently shown an inflammasome-independent pathway, where P2Y6 receptor mediates the production of IL-1α, IL-8 and IL-6 by human keratinocytes. Although the mechanism underlying stimulation by the acicular TiO2 particles remains unclear, a shape-dependent recognition system may operate in epidermal keratinocytes.
Titanium dioxide is widely used in cosmetic reagents such as sunscreens. It has been shown that topical application of neither silicone-coated nor non-coated rutile type TiO2 have a promoting effect on skin carcinogenesis in ultraviolet B-irradiated rats or human c-Ha-ras proto-oncogene transgenic mice, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.[31, 32] However, even when TiO2 is unable to penetrate the epidermis, keratinocytes may be exposed to TiO2. Given this scenario, some inflammation possibly takes place in the skin. Our study suggests that the fine globular forms of TiO2 may be safe for clinical use through the skin, even if epidermal keratinocytes are exposed to the applied TiO2 through the stratum corneum. However, the acicular form is potentially harmful because of its ability to promote keratinocyte release of pro-inflammatory cytokines. While acicular TiO2 is not included in the cosmetic products, it is used as a material to reinforce plastics and ceramics and as friction and filtration materials. The occupational use of globular TiO2 may yield harmful effects on certain workers. It is considered that protection from the globular form is an issue to be kept in mind.