Promotion of allergic immune responses by intranasally-administrated nanosilica particles in mice
- Tokuyuki Yoshida†1, 2,
- Yasuo Yoshioka†1, 2, 3Email author,
- Maho Fujimura1, 2,
- Kohei Yamashita1, 2,
- Kazuma Higashisaka1, 2,
- Yuki Morishita1, 2,
- Hiroyuki Kayamuro1, 2,
- Hiromi Nabeshi1, 2,
- Kazuya Nagano2,
- Yasuhiro Abe2,
- Haruhiko Kamada2, 3,
- Shin-ichi Tsunoda2, 3, 4,
- Norio Itoh1,
- Tomoaki Yoshikawa1, 2 and
- Yasuo Tsutsumi1, 2, 3Email author
© Yoshida et al; licensee Springer. 2011
Received: 7 October 2010
Accepted: 4 March 2011
Published: 4 March 2011
With the increase in use of nanomaterials, there is growing concern regarding their potential health risks. However, few studies have assessed the role of the different physical characteristics of nanomaterials in allergic responses. Here, we examined whether intranasally administered silica particles of various sizes have the capacity to promote allergic immune responses in mice. We used nanosilica particles with diameters of 30 or 70 nm (nSP30 or nSP70, respectively), and conventional micro-sized silica particles with diameters of 300 or 1000 nm (nSP300 or mSP1000, respectively). Mice were intranasally exposed to ovalbumin (OVA) plus each silica particle, and the levels of OVA-specific antibodies (Abs) in the plasma were determined. Intranasal exposure to OVA plus smaller nanosilica particles tended to induce a higher level of OVA-specific immunoglobulin (Ig) E, IgG and IgG1 Abs than did exposure to OVA plus larger silica particles. Splenocytes from mice exposed to OVA plus nSP30 secreted higher levels of Th2-type cytokines than mice exposed to OVA alone. Taken together, these results indicate that nanosilica particles can induce allergen-specific Th2-type allergic immune responses in vivo. This study provides the foundations for the establishment of safe and effective forms of nanosilica particles.
With the recent development of nanotechnology, many nanomaterials with innovative functions have been developed. For example, nanoparticles of titanium dioxide and silica have been used in commercial applications related to medicine, cosmetics and food . In particular, amorphous (noncrystalline) nanosilica particles possess extraordinary advantages, including straightforward synthesis, relatively low cost, and easy surface modification [1, 2]. Nanosilica particles are increasingly being used for many applications, including cosmetics, food technology, medical diagnosis, cancer therapy, and drug delivery [1–4].
As the use of nanomaterials increases, there is rising concern regarding their potential health risks because there is preliminary evidence that the unique electrical and mechanical properties of nanomaterials is associated with undesirable biological interactions [5, 6]. In addition, it has recently become evident that particle characteristics, including particle size and surface properties, are important factors in pathologic alterations and cellular responses [7–10]. For instance, Nishimori et al have previously demonstrated that nanosilica particles with relatively small particle size induce a greater level of toxicity, including liver injury, than do silica particles with larger particle size . To create safe and effective forms of nanomaterials, studies which provide basic information regarding biological responses to nanomaterials are essential.
Numerous studies have shown that several types of nanomaterials increase the incidence of allergic immune diseases [12–14]. Activation of the Th2 response, including production of interleukin (IL)-4, IL-5, and IL-13 from Th2 cells (a subset of CD4+ T cells) and immunoglobulin (Ig) G1 or IgE from B cells, is responsible for many of the pathologic features of allergic immune diseases . Some reports have shown that intranasal or airway exposure to nanomaterials promotes allergic immune responses, indicating the immune-activating potential of nanomaterials [12, 13]. However, the role of the different physical characteristics of nanomaterials in the production of allergic responses has not been elucidated.
Here, we examined whether intranasal exposure to nanosilica particles has the capacity to promote allergic immune responses in mice. In addition, we investigated the relationship between the size of silica particles and allergic immune responses.
Materials and methods
Amorphous silica particles with a diameter of 30, 70, 300 and 1,000 nm (Micromod Partikeltechnologie, Rostock/Warnemünde, Germany, designated nSP30, nSP70, nSP300 and mSP1000, respectively) were used in this study. The particle numbers of silica particles were 3.5 × 1013, 2.8 × 1012, 3.5 × 1010, or 9.5 × 108 particles/mg (nSP30, nSP70, nSP300, or mSP1000, respectively). Silica particles were sonicated for 5 min and vortexed for 1 min before use. The size of particles was measured using a Zetasizer Nano-ZS (Malvern Instruments, UK). The mean size and the size distribution of particles were measured by means of dynamic light scattering. We confirmed that the particle size distributions of these silica particles were narrow.
Female BALB/c mice were purchased from Nippon SLC (Hamamatsu, Japan) and used at 6 to 8 weeks of age. All of the animal experimental procedures in this study were performed in accordance with the National Institute of Biomedical Innovation Guidelines for the Welfare of Animals.
Exposure protocols and detection of antigen-specific antibody responses by enzyme-linked immunosorbent assay
Female BALB/c mice were intranasally exposed to a 20 μL aliquot (10 μL per nostril) containing 10 μg of ovalbumin (OVA; Sigma Chemical Co, St. Louis, MO, USA) as antigen, plus nSP30, nSP70, nSP300, or mSP1000 at concentrations of 10, 50 or 250 μg/mouse, on days 0, 1, and 2. On day 21, plasma was collected to assess antigen-specific antibody (Ab) responses. Antigen-specific IgG and subclass IgG1 Ab levels were determined by enzyme-linked immunosorbent assay (ELISA). The ELISA plates (Maxisorp, type 96F; Nalge Nunc International, Naperville, IL, USA) were coated with 10 μg/ml OVA and incubated overnight at 4°C. Non-specific Ab binding was minimized by incubating the plates with 4% blocking solution (Block Ace; Dainippon Sumitomo Pharmaceuticals, Osaka, Japan) at 37°C for 2 h. Plasma dilutions were added to the antigen-coated plates and incubated at 37°C for a further 2 h. The coated plates were then washed with PBS containing 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG solution (Southern Biotechnology Associates, Birmingham, AL, USA) at 37°C for 2 h. The color reaction was developed with tetramethylbenzidine (MOSS, Inc., Pasadena, MD, USA), stopped with 2N H2SO4, and quantitated by measuring OD450 minus OD655 using a microplate reader. OVA-specific IgE Ab levels in plasma were determined using commercial ELISA kits (Dainippon Sumitomo Pharma, Osaka, Japan).
Isolation of splenocytes
Spleens were aseptically removed and placed in RPMI 1640 medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum, 50 mM 2-mercaptoethanol and 1% antibiotic cocktail (Nacalai Tesque, Kyoto, Japan). The single-cell suspension of splenocytes was treated with ammonium chloride to lyse the red blood cells, and the splenocytes were washed, counted, and suspended in RPMI medium supplemented with 10% fetal bovine serum, 50 mM 2-mercaptoethanol, 1% antibiotic cocktail, 10 mL/L of 100 × nonessential amino acids solution, 1 mM sodium pyruvate, and 10 mM HEPES to a final concentration of 1 × 107 cells/mL.
Antigen-specific cytokine responses
Antigen-specific cytokine responses were evaluated by culturing the splenocytes (5 × 106 cells/well) in the presence of OVA (1 mg/mL) in vitro. Cells were incubated at 37°C for 72 h. Culture supernatants from in vitro unstimulated and OVA-stimulated cells were analyzed by the Bio-Plex Multiplex Cytokine Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. The assay results were read on a Luminex 100 Multiplex Bio-Assay Analyzer (Luminex, Austin, TX, USA). The difference between the mean concentration of cytokines in supernatants from in vitro OVA-stimulated cells and unstimulated cells (background) was then calculated.
All values are expressed as mean ± SEM. Differences between groups were assessed using analysis of variance followed by Turkey's method.
Results and discussion
Antigen-specific IgE Ab responses to silica particles
Antigen-specific IgG Abs subclass responses of silica particles
Antigen-specific cytokine responses of silica particles
It is not clear why nanosilica particles such as nSP30 would induce Th2-polarized allergic immunity. Our results support previous reports showing that the immune-activating effect of nanomaterials increases with decreasing particle size [12, 17]. The mechanisms behind the immune-activating effect of nanomaterials have not been fully elucidated. Nygaard et al  showed that the higher specific surface area of nanomaterials as compared to micro-sized particles allows more antigen to be adsorbed per particle. We consider that one possible mechanism by which allergic immune responses induced by nanosilica particles is that many antigen-captured nanomaterials might be taken up by professional antigen presenting cells, such as dendritic cells. Another possible mechanism is that the nanomaterials induce oxidative stress [18, 19]. We have observed that nanosilica particles such as nSP30 are stronger inducers of oxidative stress than larger silica particles (unpublished data). Because there is accumulating evidence that oxidative stress plays a role in pro-inflammatory and immune-activating effects [20, 21], dendritic cells might be activated more efficiently by nSP30 than by larger silica particles. Furthermore, we also observed that induction of oxidative stress by nanosilica particles is decreased by surface modification of nanosilica particles (unpublished data). Therefore, surface modification might be one approach to decrease allergic immune responses induced by nanosilica particles.
Here, we show that nanosilica particles have the potential to induce allergic immune responses after intranasal exposure. We consider that further studies of the relationship between the characteristics of nanomaterials and allergic immune responses will facilitate the development of safe and effective nanomaterials.
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Japan Society for the Promotion of Science. This study was also supported in part by Health Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan; by Health Sciences Research Grants for Research on Publicly Essential Drugs and Medical Devices from the Japan Health Sciences Foundation; by a Global Environment Research Fund from Minister of the Environment; and by a the Knowledge Cluster Initiative; and by Food Safety Commission; and by The Nagai Foundation Tokyo; and by The Cosmetology Research Foundation; and by The Smoking Research Foundation.
- Fadeel B, Garcia-Bennett AE: Better safe than sorry: Understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug Deliv Rev 2010, 62: 362–374. 10.1016/j.addr.2009.11.008View Article
- Barik TK, Sahu B, Swain V: Nanosilica-from medicine to pest control. Parasitol Res 2008, 103: 253–258. 10.1007/s00436-008-0975-7View Article
- Bharali DJ, Klejbor I, Stachowiak EK, Dutta P, Roy I, Kaur N, Bergey EJ, Prasad PN, Stachowiak MK: Organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain. Proc Natl Acad Sci USA 2005, 102: 11539–11544. 10.1073/pnas.0504926102View Article
- Bottini M, D'Annibale F, Magrini A, Cerignoli F, Arimura Y, Dawson MI, Bergamaschi E, Rosato N, Bergamaschi A, Mustelin T: Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes. Int J Nanomedicine 2007, 2: 227–233.
- Kagan VE, Bayir H, Shvedova AA: Nanomedicine and nanotoxicology: two sides of the same coin. Nanomedicine 2005, 1: 313–316.View Article
- Nel A, Xia T, Madler L, Li N: Toxic potential of materials at the nanolevel. Science 2006, 311: 622–627. 10.1126/science.1114397View Article
- Albrecht C, Schins RP, Hohr D, Becker A, Shi T, Knaapen AM, Borm PJ: Inflammatory time course after quartz instillation: role of tumor necrosis factor-alpha and particle surface. Am J Respir Cell Mol Biol 2004, 31: 292–301. 10.1165/rcmb.2003-0300OCView Article
- He X, Nie H, Wang K, Tan W, Wu X, Zhang P: In vivo study of biodistribution and urinary excretion of surface-modified silica nanoparticles. Anal Chem 2008, 80: 9597–9603. 10.1021/ac801882gView Article
- Morishige T, Yoshioka Y, Inakura H, Tanabe A, Yao X, Narimatsu S, Monobe Y, Imazawa T, Tsunoda S, Tsutsumi Y, Mukai Y, Okada N, Nakagawa S: The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1beta production, ROS production and endosomal rupture. Biomaterials 2010, 31: 6833–6842. 10.1016/j.biomaterials.2010.05.036View Article
- Morishige T, Yoshioka Y, Tanabe A, Yao X, Tsunoda S, Tsutsumi Y, Mukai Y, Okada N, Nakagawa S: Titanium dioxide induces different levels of IL-1beta production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B. Biochem Biophys Res Commun 2010, 392: 160–165. 10.1016/j.bbrc.2009.12.178View Article
- Nishimori H, Kondoh M, Isoda K, Tsunoda S, Tsutsumi Y, Yagi K: Silica nanoparticles as hepatotoxicants. Eur J Pharm Biopharm 2009, 72: 496–501. 10.1016/j.ejpb.2009.02.005View Article
- de Haar C, Hassing I, Bol M, Bleumink R, Pieters R: Ultrafine but not fine particulate matter causes airway inflammation and allergic airway sensitization to co-administered antigen in mice. Clin Exp Allergy 2006, 36: 1469–1479. 10.1111/j.1365-2222.2006.02586.xView Article
- Nygaard UC, Hansen JS, Samuelsen M, Alberg T, Marioara CD, Lovik M: Single-walled and multi-walled carbon nanotubes promote allergic immune responses in mice. Toxicol Sci 2009, 109: 113–123. 10.1093/toxsci/kfp057View Article
- Bonner JC: Nanoparticles as a potential cause of pleural and interstitial lung disease. Proc Am Thorac Soc 2010, 7: 138–141. 10.1513/pats.200907-061RMView Article
- Finkelman FD, Hogan SP, Hershey GK, Rothenberg ME, Wills-Karp M: Importance of cytokines in murine allergic airway disease and human asthma. J Immunol 2010, 184: 1663–1674. 10.4049/jimmunol.0902185View Article
- Yoshida T, Yoshioka Y, Fujimura M, Yamashita K, Higashisaka K, Nakanishi R, Morishita Y, Kayamuro H, Nabeshi H, Nagano K, Abe Y, Kamada H, Tsunoda S, Yoshikawa T, Itoh N, Tsutsumi Y: Potential adjuvant effect of intranasal urban aerosols in mice through induction of dendritic cell maturation. Toxicol Lett 2010, 199: 383–388. 10.1016/j.toxlet.2010.10.002View Article
- Nygaard UC, Samuelsen M, Aase A, Lovik M: The capacity of particles to increase allergic sensitization is predicted by particle number and surface area, not by particle mass. Toxicol Sci 2004, 82: 515–524. 10.1093/toxsci/kfh287View Article
- Brown DM, Wilson MR, MacNee W, Stone V, Donaldson K: Size-dependent proinflammatory effects of ultrafine polystyrene particles: a role for surface area and oxidative stress in the enhanced activity of ultrafines. Toxicol Appl Pharmacol 2001, 175: 191–199. 10.1006/taap.2001.9240View Article
- Shvedova AA, Kagan VE, Fadeel B: Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems. Annu Rev Pharmacol Toxicol 2010, 50: 63–88. 10.1146/annurev.pharmtox.010909.105819View Article
- Swindle EJ, Metcalfe DD: The role of reactive oxygen species and nitric oxide in mast cell-dependent inflammatory processes. Immunol Rev 2007, 217: 186–205. 10.1111/j.1600-065X.2007.00513.xView Article
- Martinon F: Signaling by ROS drives inflammasome activation. Eur J Immunol 2010, 40: 616–619. 10.1002/eji.200940168View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.