Immunostimulatory Effects of Silver Nanoparticles in Immune Cells and their Relevance to Atopic Dermatitis

Abstract

Engineered nanomaterials (NMs) are increasingly used in numerous consumer products and biomedical applications that affect humans directly and indirectly. However, in vitro and in vivo studies have revealed that nanomaterials can result in toxicity. The immunostimulatory effect of silver nanoparticles (AgNPs) is dependent on particle size and the production of reactive oxygen species (ROS). Although these products have many opportunities to contact with the skin, the toxicity of AgNPs has not been clearly defined. Mast cells are potent effectors of the allergic response. They play an important role in asthma, atopic dermatitis (AD) and other allergic reactions. In this study, RBL2H3 mast cell viability was dependent on the particles size. Granule release, Ca2+ influx, the production of hydrogen peroxide and superoxide and mitochondrial membrane damage were used to evaluate cytotoxicity of AgNPs. To evaluate the synergistic effect of AgNPs on the induction of allergic reactions in animals, NC/Nga mice were used in this study. NC/Nga mice spontaneously develop an eczematous AD-like skin lesion when kept under conventional conditions, but not when maintained under specific pathogen-free (SPF) conditions. They are considered an appropriate animal model for AD. Macrophages are active phagocytic cells present in many tissues. The internalization of nanoparticles by phagocytic cells can occur through phagocytosis/macropinocytosis, receptor-mediated endocytosis and passive penetration. Phagocytosis and macropinocytosis are mediated by actin polymerization, which causes cell membrane ruffling. The process is inhibited by cytochalasin D. Endocytosis can occur as clathrin-dependent endocytosis and caveolae-dependent endocytosis. Clathrin-dependent endocytosis is inhibited by chlorpromazine, a cationic amphiphilic drug that prevents the recycling of clathrin. Caveolae-dependent endocytosis is inhibited by nystatin, an antibiotic and sterol-binding agent that removes membrane cholesterol, which is important for maintaining and sealing the membrane of caveolae. In this study, the ability of AgNPs to evoke allergic immune responses was investigated in mast cells and macrophages (in vitro) and atopic animal model (in vivo) to assess. Cytotoxicity in mast cells was evaluated using Cell Counting Kit-8 (CCK8). Cell death was induced by 5 nm AgNPs, but not by 100 nm AgNPs. Granule release was evaluated with the β-hexosaminidase assay. Granule release in RBL2H3 cells was induced by 5 nm AgNPs, but not by 100 nm AgNPs. N-acetylcystein (NAC) at 5 mM inhibited granule release in RBL2H3 cells. To assess intracellular Ca2+ levels, RBL2H3 cells were prestained with Fluo4-AM and then treated with AgNPs in HBSS with and without Ca2+. In both groups with and without Ca2+, 5 nm AgNPs but not 100 nm AgNPs increased the fluorescence intensity. To evaluate the effect of intracellular Ca2+ levels on granule release, RBL2H3 cells were pretreated with thapsigargin and then treated with AgNPs. Thapsigargin inhibited the granule release induced by 5 nm AgNPs. In addition, the production of hydrogen peroxide and superoxide was assessed by staining RBL2H3 cells with CM-H2DCFDA or MitoSOX, respectively. Treatment with 5 nm AgNPs increased the fluorescence intensity of CM-H2DCFDA and MitoSOX in a dose-dependent manner. In addition, JC-1 staining was performed to assess mitochondrial depolarization. JC-1 fluorescence intensity decreased in cells treated with 5 nm AgNPs. To investigate the synergistic effect of AgNPs on the induction of AD, ear thickness was measured once a week and the severity score of the AD-like skin lesions was evaluated once a week. Ear swelling was more severe in mice treated with 5 nm AgNPs + Ad Biostir when compared with that in the AD control group. The severity score of the skin also increased in mice treated with 5 nm AgNPs. Epidermal thickness in the group treated with 5 nm AgNPs + Ad Biostir was thicker when compared to that of the Ad Biostir-only group. The number of infiltrated mast cells in skin lesions was higher in the group treated with 5 nm AgNPs + Ad Biostir than in the Ad Biostir-only group. Total IgE levels in the serum increased in the group treated with 5 nm AgNPs + Ad Biostir. The viability of U937 cells declined abruptly when cells were treated with 5 nm AgNPs. The effects of AgNP endocytosis or phagocytosis on cell death were then evaluated. After treatment of U937 cells with AgNPs, neither chlorpromazine, cytochalasin D, nor nystatin prevented cell death. At relatively high concentrations, chlorpromazine, cytochalasin D and nystatin partially inhibited the production of IL-8 following exposure to AgNPs. These findings were confirmed with real-time RT-PCR assays. Collectively, it was suggested that these immunostimulatory effects has contributed to causing AD. These inflammatory effects should be considered and evaluated in addition to cytotoxicity when studying the effects of AgNPs exposure in human physiology.