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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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Related in: MedlinePlus

Hemolytic activity of Qz-3 (pure quartz) and Qz-4 (pure quartz etched with HF). Qz-3 and Qz-4 were incubated at increasing concentrations expressed as surface area doses (cm2/ml) in the presence of human red blood cells. Values are mean ± SD from five independent experiments. *p < 0.05 and ***p < 0.001 vs control not exposed to silica particles.
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Fig1: Hemolytic activity of Qz-3 (pure quartz) and Qz-4 (pure quartz etched with HF). Qz-3 and Qz-4 were incubated at increasing concentrations expressed as surface area doses (cm2/ml) in the presence of human red blood cells. Values are mean ± SD from five independent experiments. *p < 0.05 and ***p < 0.001 vs control not exposed to silica particles.

Mentions: We previously reported the hemolytic potential of Qz-1, Qz-2 and VS [20] (Qz-1 ≅ VS > Qz-2). The RBC lysis activity of Qz-3 (originating from a different batch than that previously tested in [20]) and Qz-4 (not tested before) was examined here (Figure 1) and compared in Table 1 with the hemolytic activity of the other three silica samples. Since RBC membranolysis is a surface-driven process, doses were expressed per surface area unit (Table 1). Both silica samples showed a dose-dependent hemolytic activity from 6.25 up to 200 cm2/ml. The hemolytic potential of Qz-4 was higher than that of Qz-3 at any of the doses investigated.Figure 1


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)

Hemolytic activity of Qz-3 (pure quartz) and Qz-4 (pure quartz etched with HF). Qz-3 and Qz-4 were incubated at increasing concentrations expressed as surface area doses (cm2/ml) in the presence of human red blood cells. Values are mean ± SD from five independent experiments. *p < 0.05 and ***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

Fig1: Hemolytic activity of Qz-3 (pure quartz) and Qz-4 (pure quartz etched with HF). Qz-3 and Qz-4 were incubated at increasing concentrations expressed as surface area doses (cm2/ml) in the presence of human red blood cells. Values are mean ± SD from five independent experiments. *p < 0.05 and ***p < 0.001 vs control not exposed to silica particles.
Mentions: We previously reported the hemolytic potential of Qz-1, Qz-2 and VS [20] (Qz-1 ≅ VS > Qz-2). The RBC lysis activity of Qz-3 (originating from a different batch than that previously tested in [20]) and Qz-4 (not tested before) was examined here (Figure 1) and compared in Table 1 with the hemolytic activity of the other three silica samples. Since RBC membranolysis is a surface-driven process, doses were expressed per surface area unit (Table 1). Both silica samples showed a dose-dependent hemolytic activity from 6.25 up to 200 cm2/ml. The hemolytic potential of Qz-4 was higher than that of Qz-3 at any of the doses investigated.Figure 1

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