Effect of silica particle size on macrophage inflammatory responses

Abstract

Amorphous silica particles, such as nanoparticles (<100 nm diameter particles), are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area) and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1β secretion, cell death and lung inflammation). Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1β secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.

Figure 1. IL-1β secretion from bone marrow-derived macrophages (BMDMs) in response to different sizes of silica particles.

C57BL/6 mouse BMDMs primed with LPS [10 ng/mL] were stimulated with silica particles of the indicated size for 4 hours. The amount of IL-1β in culture supernatants was measured by ELISA. The relationship between the amount of IL-1β secretion and (A) the dose, (B) the number, or (C) the surface area of the indicated silica particles is shown. S.D. was less than 10% of the mean of triplicates (not shown). Similar results were obtained in at least three independent experiments.

Figure 2. Silica particles of varying sizes are internalized by BMDMs via an actin cytoskeleton-dependent pathway.

(A) BMDMs were incubated with (lower panels) or without (upper panels) cytochalasin D (Cyto D) [20 μM] for 1 hour, and were then stimulated with FITC-labeled silica particles [0.3 mg/mL] for 1 hour. After staining with PE-labeled anti-CD11b mAb, internalization of silica particles by BMDMs was analyzed by confocal microscopy. White bars indicate 10 microns. (B) LPS [10 ng/mL]-primed BMDMs were incubated with (black columns) or without (white columns) Cyto D [20 μM] for 1 hour, and then were stimulated with the indicated size silica particles [0.3 mg/mL] or ATP [1 mM] for 4 hours. The amount of IL-1β in culture supernatant was measured by ELISA. Data are indicated as the mean + S.D. Similar results were obtained in at least three independent experiments.

Figure 3. Lysosomal destabilization by silica particles of varying sizes.

(A) LPS [10 ng/mL]-primed BMDMs were stimulated with FITC-dextran [1 mg/ml] and the indicated size of silica particles [0.3 mg/ml] or ATP [1 mM] for 2 hours. Intracellular distribution of FITC-dextran in BMDMs was analyzed by confocal microscopy. White bars indicate 10 microns. (B) LPS [10 ng/mL]-primed BMDMs were treated with LysoTracker-Red [200 nM], followed by silica particles of the indicated size [0.3 mg/ml] or ATP [1 mM] for 2 hours. Percentage of LysoTracker-negative population was quantified by flow cytometry. Columns represent mean + S.D. of triplicates. *P<0.05, **P<0.01 compared with (–) control. (C) LPS [10 ng/mL]-primed BMDMs were treated with (black columns) or without (white columns) bafilomycin A1 [200 nM] for 1 hour, and then were stimulated with the indicated size of silica particles [0.3 mg/mL] or ATP [1 mM] for 4 hours. The amount of IL-1β in culture supernatant was measured by ELISA. N.D., not detected. Data are indicated as the mean + S.D. Similar results were obtained in two (A) or three (B, C) independent experiments.

Figure 4. Inflammasome activation in BMDMs stimulated with silica particles of different sizes.

LPS [10 ng/mL]-primed BMDMs were stimulated with the indicated sizes of silica [0.3 mg/mL] for 2 hours. The leftmost lane indicates LPS-unprimed and silica-untreated BMDMs. Maturation of caspase-1, IL-1β and caspase-11 in pooled supernatants and in cell extracts were analyzed by immunoblot. Anti-mouse β-actin antibody was used as a loading control. Similar results were obtained in at least three independent experiments.

Figure 5. Cytotoxicity of different sizes of silica against BMDMs.

BMDMs were stimulated with the indicated sizes of silica particles for 2(LDH) release. The percentage of LDH release was calculated as follows: 100 × experimental release/maximal (1% Triton-X-treated) release. Data are shown as the mean + S.D. of quadruplicate. Similar results were obtained in at least three independent experiments.

Figure 6. Silica-induced pulmonary inflammation.

(A) C57BL/6 mice were injected intra-tracheally (i.t.) with saline (n = 3), 30 nm silica (n = 4) or 3000 nm silica (n = 4) [25 mg/kg]. Six hours later, bronchoalveolar lavage fluid (BALF) was harvested, and the amounts of IL-1β, TNF-α, and IL-6 were measured by ELISA. Data are indicated as the mean + S.D. *P < 0.05, **P < 0.01, two-tailed Student's t-test. Similar results were obtained in at least two independent experiments. (B) Cells in BALF harvested as described in panel (A) were stained with anti-Gr-1 mAb and anti-CD45 mAb, and analyzed by flow cytometry. Upper dot plots indicate Gr-1⁺ CD45⁺ cells and Gr-1⁻ CD45⁺ cells. Lower histograms indicate Gr-1 expression on CD45-positive cells. (C) Mice were treated as described in panel (A). Six or 24 hours later, lung inflammation was analyzed by micro-computed tomography. Similar results were obtained in at least two (A, B, C) independent experiments.