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Why does the hemolytic activity of silica predict its pro-inflammatory activity?

Pavan C, Rabolli V, Tomatis M, Fubini B, Lison D - Part Fibre Toxicol (2014)

Bottom Line: A significant correlation between IL-1β release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis.IL-1β release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation.These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, "G. Scansetti" Interdepartmental Center for Studies on Asbestos and Other Toxic Particulates, University of Torino, Via P. Giuria 7, 10125, Turin, Italy. cristina.pavan@unito.it.

ABSTRACT

Background: The hemolytic activity of inhaled particles such as silica has been widely investigated in the past and represents a usual toxicological endpoint to characterize particle reactivity despite the fact that red blood cells (RBCs) are not involved in the pathogenesis of pulmonary inflammation or fibrosis caused by some inhaled particles. The inflammatory process induced by silica starts with the activation of the inflammasome, which leads to the release of mature IL-1β. One of the upstream mechanisms causing activation of the inflammasome is the labilization of the phagolysosomal membrane after particle phagocytosis. Considering RBC lysis as a model of membrane damage, we evaluated the relationship between hemolytic activity and inflammasome-dependent release of IL-1β for a panel of selected silica particles, in search of the toxicological significance of the hemolytic activity of an inhaled particle.

Methods: Well-characterized silica particles, including four quartz samples and a vitreous silica, with different surface properties and hemolytic potential were tested for their capacity to induce inflammasome-dependent release of IL-1β in LPS-primed primary murine peritoneal macrophages by ELISA and Western blot analysis. The mechanisms of IL-1β maturation and release were clarified by using ASC-deficient cells and inhibitors of phagocytosis and cathepsin B.

Results: The silica samples induced dose-dependent hemolysis and IL-1β release of different amplitudes. A significant correlation between IL-1β release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis. IL-1β release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation.

Conclusions: The same physico-chemical properties of silica particles which are relevant for the lysis of the RBC membrane also appear implicated in the labilization of the phagolysosome, leading to inflammasome activation and release of the pro-inflammatory cytokine IL-1β. These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.

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Cytotoxic activity of different silica particles in primary murine macrophages. LPS-primed primary murine macrophages were incubated with 20 cm2/ml of silica for 6 h and then evaluated for cell viability by means of the WST-1 assay. The silica samples included a commercial quartz (Qz-1), the same quartz heated at 800°C (Qz-2), a vitreous silica (VS), a pure quartz (Qz-3) and the pure quartz etched with HF (Qz-4). ATP (5 mM) was used as positive control. Results are expressed as percentage of the control (macrophages not exposed to silica particles -Ctl). Values are mean ± SD from at least three independent experiments. ***p < 0.001 vs control not exposed to silica particles.
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Fig2: Cytotoxic activity of different silica particles in primary murine macrophages. LPS-primed primary murine macrophages were incubated with 20 cm2/ml of silica for 6 h and then evaluated for cell viability by means of the WST-1 assay. The silica samples included a commercial quartz (Qz-1), the same quartz heated at 800°C (Qz-2), a vitreous silica (VS), a pure quartz (Qz-3) and the pure quartz etched with HF (Qz-4). ATP (5 mM) was used as positive control. Results are expressed as percentage of the control (macrophages not exposed to silica particles -Ctl). Values are mean ± SD from at least three independent experiments. ***p < 0.001 vs control not exposed to silica particles.

Mentions: Cytotoxic activity was determined in preliminary experiments over a range of concentrations (Additional file 1: Figure S1) in order to avoid the use of cytotoxic doses and irrelevant release of immature pro-IL-1β in the culture supernatant in subsequent experiments. Primary murine peritoneal macrophages were primed with LPS [22] and incubated 6 h with LPS-free silica particles. Figure 2 shows the cell viability determined by WST-1 assay at the concentration of 20 cm2/ml of silica sample. VS was the most cytotoxic, followed by the commercial quartz Qz-1 and Qz-2. Qz-3 was not statistically different compared to the control, while Qz-4 showed an intermediate cytotoxic activity. Only VS was already cytotoxic at the lowest dose of 10 cm2/ml. Qz-2 was less cytotoxic than Qz-1 at the highest dose of 40 cm2/ml, while Qz-3 was fully inactive at all the doses investigated (Additional file 1: Figure S1). Based on these results, we selected a low (20 cm2/ml) and a moderately cytotoxic dose (40 cm2/ml) as appropriate to evaluate silica-induced mature IL-1β release.Figure 2


Why does the hemolytic activity of silica predict its pro-inflammatory activity?

Pavan C, Rabolli V, Tomatis M, Fubini B, Lison D - Part Fibre Toxicol (2014)

Cytotoxic activity of different silica particles in primary murine macrophages. LPS-primed primary murine macrophages were incubated with 20 cm2/ml of silica for 6 h and then evaluated for cell viability by means of the WST-1 assay. The silica samples included a commercial quartz (Qz-1), the same quartz heated at 800°C (Qz-2), a vitreous silica (VS), a pure quartz (Qz-3) and the pure quartz etched with HF (Qz-4). ATP (5 mM) was used as positive control. Results are expressed as percentage of the control (macrophages not exposed to silica particles -Ctl). Values are mean ± SD from at least three independent experiments. ***p < 0.001 vs control not exposed to silica particles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318150&req=5

Fig2: Cytotoxic activity of different silica particles in primary murine macrophages. LPS-primed primary murine macrophages were incubated with 20 cm2/ml of silica for 6 h and then evaluated for cell viability by means of the WST-1 assay. The silica samples included a commercial quartz (Qz-1), the same quartz heated at 800°C (Qz-2), a vitreous silica (VS), a pure quartz (Qz-3) and the pure quartz etched with HF (Qz-4). ATP (5 mM) was used as positive control. Results are expressed as percentage of the control (macrophages not exposed to silica particles -Ctl). Values are mean ± SD from at least three independent experiments. ***p < 0.001 vs control not exposed to silica particles.
Mentions: Cytotoxic activity was determined in preliminary experiments over a range of concentrations (Additional file 1: Figure S1) in order to avoid the use of cytotoxic doses and irrelevant release of immature pro-IL-1β in the culture supernatant in subsequent experiments. Primary murine peritoneal macrophages were primed with LPS [22] and incubated 6 h with LPS-free silica particles. Figure 2 shows the cell viability determined by WST-1 assay at the concentration of 20 cm2/ml of silica sample. VS was the most cytotoxic, followed by the commercial quartz Qz-1 and Qz-2. Qz-3 was not statistically different compared to the control, while Qz-4 showed an intermediate cytotoxic activity. Only VS was already cytotoxic at the lowest dose of 10 cm2/ml. Qz-2 was less cytotoxic than Qz-1 at the highest dose of 40 cm2/ml, while Qz-3 was fully inactive at all the doses investigated (Additional file 1: Figure S1). Based on these results, we selected a low (20 cm2/ml) and a moderately cytotoxic dose (40 cm2/ml) as appropriate to evaluate silica-induced mature IL-1β release.Figure 2

Bottom Line: A significant correlation between IL-1β release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis.IL-1β release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation.These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, "G. Scansetti" Interdepartmental Center for Studies on Asbestos and Other Toxic Particulates, University of Torino, Via P. Giuria 7, 10125, Turin, Italy. cristina.pavan@unito.it.

ABSTRACT

Background: The hemolytic activity of inhaled particles such as silica has been widely investigated in the past and represents a usual toxicological endpoint to characterize particle reactivity despite the fact that red blood cells (RBCs) are not involved in the pathogenesis of pulmonary inflammation or fibrosis caused by some inhaled particles. The inflammatory process induced by silica starts with the activation of the inflammasome, which leads to the release of mature IL-1β. One of the upstream mechanisms causing activation of the inflammasome is the labilization of the phagolysosomal membrane after particle phagocytosis. Considering RBC lysis as a model of membrane damage, we evaluated the relationship between hemolytic activity and inflammasome-dependent release of IL-1β for a panel of selected silica particles, in search of the toxicological significance of the hemolytic activity of an inhaled particle.

Methods: Well-characterized silica particles, including four quartz samples and a vitreous silica, with different surface properties and hemolytic potential were tested for their capacity to induce inflammasome-dependent release of IL-1β in LPS-primed primary murine peritoneal macrophages by ELISA and Western blot analysis. The mechanisms of IL-1β maturation and release were clarified by using ASC-deficient cells and inhibitors of phagocytosis and cathepsin B.

Results: The silica samples induced dose-dependent hemolysis and IL-1β release of different amplitudes. A significant correlation between IL-1β release and hemolytic activity was evidenced (r = 0.827) by linear regression analysis. IL-1β release was completely abolished in ASC-deficient cells and reduced by inhibitors, confirming the involvement of the inflammasome and the requirement of phagocytosis and cathepsin B for activation.

Conclusions: The same physico-chemical properties of silica particles which are relevant for the lysis of the RBC membrane also appear implicated in the labilization of the phagolysosome, leading to inflammasome activation and release of the pro-inflammatory cytokine IL-1β. These findings strengthen the relevance of the hemolysis assay to predict the pro-inflammatory activity of silica dusts.

Show MeSH
Related in: MedlinePlus