Immune Netw.  2012 Jun;12(3):104-112. 10.4110/in.2012.12.3.104.

Induction of Functional Changes of Dendritic Cells by Silica Nanoparticles

• Kang K
• Lim JS

• KMID: 2168008
• DOI: http://doi.org/10.4110/in.2012.12.3.104

Abstract

Silica is one of the most abundant compounds found in nature. Immoderate exposure to crystalline silica has been linked to pulmonary disease and crystalline silica has been classified as a Group I carcinogen. Ultrafine (diameter <100 nm) silica particles may have different toxicological properties compared to larger particles. We evaluated the effect of ultrafine silica nanoparticles on mouse bone marrow-derived dendritic cells (BMDC) and murine dendritic cell line, DC2.4. The exposure of dendritic cells (DCs) to ultrafine silica nanoparticles showed a decrease in cell viability and an induction of cell death in size- and concentration-dependent manners. In addition, in order to examine the phenotypic changes of DCs following co-culture with silica nanoparticles, we added each sized-silica nanoparticle along with GM-CSF and IL-4 during and after DC differentiation. Expression of CD11c, a typical DC marker, and multiple surface molecules such as CD54, CD80, CD86, MHC class II, was changed by silica nanoparticles in a size-dependent manner. We also found that silica nanoparticles affect inflammatory response in DCs in vitro and in vivo. Finally, we found that p38 and NF-kappaB activation may be critical for the inflammatory response by silica nanoparticles. Our data demonstrate that ultrafine silica nanoparticles have cytotoxic effects on dendritic cells and immune modulation effects in vitro and in vivo.

Keyword

Silica nanoparticles; Dendritic cells; Apoptosis; Inflammatory response

MeSH Terms

Animals
Apoptosis
Cell Death
Cell Survival
Coculture Techniques
Crystallins
Dendritic Cells
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-4
Lung Diseases
Mice
Nanoparticles
NF-kappa B
Silicon Dioxide
Silicones
Crystallins
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-4
NF-kappa B
Silicon Dioxide
Silicones

Figure

• 

  Figure 1 Induction of dendritic cell death by silica nanoparticles. For determining cell viability of DC2.4 cells after treatment with silica nanoparticles, cells were treated with silica nanoparticles for 24 h and 48 h. Cell viability was determined using MTT assay (A) and trypan blue staining (B). Cells were treated with silica nanoparticles (40 µg/ml) for 24 h and 7-AAD (PerCP) and annexin V-FITC staining was used to analyze the DC2.4 cell death. The percentages of stained/unstained cells are shown in each quadrant (C). The total sum of annexin V-positive cells was quantified (D). Results represent the mean±SD from triplicates. *p<0.05, **p<0.01, ***p<0.001 for one-way ANOVA.

• 

  Figure 2 Silica nanoparticles affect the expression of surface antigens on dendritic cells. Bone marrow-derived DCs were generated by culturing bone marrow cells with 10 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 6 days. Thereafter, cells were co-cultured with silica nanoparticles (40 µg/ml) for 24 h (A). On the other hand, bone marrow-derived cells were cultured with 10 ng/ml of GM-CSF, 10 ng/ml of IL-4, and silica nanoparticles (10 µg/ml) for 7 days (B). Cells were allowed to react with appropriate antibodies at 4℃ for 30 min for the detection of CD11c, CD54, CD80, CD86 and MHC class II. Cells were analyzed using a FACSCanto™II. The data are represented as relative Mean Fluorescence Intensity (MFI).

• 

  Figure 3 Silica nanoparticles induce TNF-α production in dendritic cells. DC2.4 cells (A) and bone marrow-derived dendritic cells (B, D, and F) were exposed to silica nanoparticles (40 µg/ml) for 24 h. Then, bone marrow-derived cells were cultured with 10 ng/ml of GM-CSF, 10 ng/ml of IL-4, and 10 µg/ml of silica nanoparticles for 7 days (C, E, and G). Cells were harvested for RNA preparation. Transcriptional levels of cytokines were detected using RT-PCR (A~C). The supernatants of BMDCs were collected for 24 h. The levels of TNF-α (D and E) and IL-12p70 (F and G) in culture supernatants were determined by ELISA. Data represent the mean±SD of duplicates. *p<0.05, **p<0.01 for one-way ANOVA.

• 

  Figure 4 Silica nanoparticles elicit inflammatory responses in vivo. C57BL/6 mice were injected subcutaneously with 700 µl liquid matrigel containing silica nanoparticles (10 mg/kg). After 10 days, gels were excised (A) and hemoglobin in matrigel plugs was determined using Drabkin's reagent (B). BMDCs were exposed to silica nanoparticles (40 µg/ml) for 24 h (C and D). In addition, bone marrow-derived cells were cultured with 10 ng/ml of GM-CSF, 10 ng/ml of IL-4, and 10 µg/ml of silica nanoparticles for 7 days (E and F). The culture supernatants were collected for last 24 h and concentrated with centricon centrifugal filter devices. A mixture of 500 µl liquid matrigel and 200 µl concentrated supernatant was injected to C57BL/6 mice subcutaneously. After 9~10 days, gels were excised (C and E) and hemoglobin in matrigel plugs was determined (D and F). DC 2.4 cells were treated with silica nanoparticles (40 µg/ml) for 24 h. DC2.4 cells treated with silica nanoparticles were isolated by a density gradient centrifugation on ficoll to remove contamination of silica nanoparticles. C57BL/6 mice were injected subcutaneously with 700 µl liquid matrigel containing 5×105 DC2.4 cells. After 10 days, gels were excised and matrigel plugs were stained with hematoxylin & eosin (H&E) (G). Results represent the mean±SD from duplicates. *p<0.05 for one-way ANOVA.

• 

  Figure 5 Phosphorylation of p38 MAPK is essentially associated with the induction of in vivo inflammatory responses by nanoparticles. DC2.4 cells were co-cultured with 40 µg/ml of silica nanoparticles for 6 h. Equal amounts of whole lysates were subjected to electrophoresis on a SDS-PAGE and Western blot analysis was performed using specific antibodies, respectively.

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