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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

Involvement of the inflammasome, phagocytosis and cathepsin B in inducing the release of mature IL-1β by different silica particles. LPS-primed primary murine macrophages were incubated with silica at equal surface dose (20 cm2/ml) and with ATP (5 mM) as positive control. Culture supernatants were collected after 6 h (1 h for ATP) and cell viability by WST-1 (B, D, F) and IL-1β release (pg/ml) by ELISA (C, E, G) were measured. (A) A representative Western blot analysis conducted on culture supernatants or cell extracts to detect pro-IL-1β (17 kDa) or mature IL-1β (36 kDa) is shown. (B, C) WT mice and mice deficient in the adaptor molecule ASC (ASC−/−) are compared. Determinations were performed in six replicates (B) or quadruplicate (C) in a single experiment and expressed as the mean ± SD. Cells pre-treated for 1 h with cytochalasin D (5 μM) (D, E) or CA-074-Me (10 μM) (F, G) are compared to untreated cells. Determinations were performed in quadruplicate and expressed as the mean ± SD. Data from one representative experiment out of two (D, E) or one single experiment (F, G) are depicted. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control not exposed to silica particles in each group; #p < 0.05, ##p < 0.01 and ###p < 0.001 WT vs ASC−/− or non-treated vs treated with inhibitors, for each sample.
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Fig4: Involvement of the inflammasome, phagocytosis and cathepsin B in inducing the release of mature IL-1β by different silica particles. LPS-primed primary murine macrophages were incubated with silica at equal surface dose (20 cm2/ml) and with ATP (5 mM) as positive control. Culture supernatants were collected after 6 h (1 h for ATP) and cell viability by WST-1 (B, D, F) and IL-1β release (pg/ml) by ELISA (C, E, G) were measured. (A) A representative Western blot analysis conducted on culture supernatants or cell extracts to detect pro-IL-1β (17 kDa) or mature IL-1β (36 kDa) is shown. (B, C) WT mice and mice deficient in the adaptor molecule ASC (ASC−/−) are compared. Determinations were performed in six replicates (B) or quadruplicate (C) in a single experiment and expressed as the mean ± SD. Cells pre-treated for 1 h with cytochalasin D (5 μM) (D, E) or CA-074-Me (10 μM) (F, G) are compared to untreated cells. Determinations were performed in quadruplicate and expressed as the mean ± SD. Data from one representative experiment out of two (D, E) or one single experiment (F, G) are depicted. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control not exposed to silica particles in each group; #p < 0.05, ##p < 0.01 and ###p < 0.001 WT vs ASC−/− or non-treated vs treated with inhibitors, for each sample.

Mentions: To discriminate between mature IL-1β released into the supernatant after inflammasome activation and immature pro-IL-1β potentially due to silica cytotoxicity, we performed a Western blot analysis to separate the two proteins on the basis of their different molecular weight. LPS-primed macrophages were incubated for 6 h with 20 cm2/ml of silica or with ATP (5 mM) used as positive control. As shown in Figure 4A, significant differences in the expression levels of mature IL-1β among cells treated with various types of silica were observed. VS showed the highest amount of mature IL-1β, even more than the ATP positive control. Qz-1 induced a lower amount of mature IL-1β compared to VS, whereas IL-1β could not be detected for Qz-2 and Qz-3. Detection of mature/pro-IL-1β in the supernatant or cell extract not exposed to silica (Ctl) showed that priming with LPS was effective because only the pro-IL-1β form was present in cell extract, and no release of mature IL-1β was found in the supernatant. These results are consistent with the data obtained by ELISA. In order to evaluate the involvement of inflammasome in the release of IL-1β by several silica types and to confirm the mechanism leading to its activation, ASC-deficient cells, a phagocytosis inhibitor and an inhibitor of cathepsin B were used. Peritoneal macrophages from mice knock-out for the apoptosis-associated speck-like protein (ASC−/−), which is one of the three main components of the inflammasome complex, were compared with macrophages from wild type mice (WT, C57BL/6). No significant difference in cytotoxic activity between WT and ASC−/− macrophages was found (Figure 4B), indicating that silica-induced cytotoxicity is independent from ASC, as reported by Cassel et al. for Nalp3 [22]. LPS-primed ASC−/− macrophages displayed an evident defect in their ability to induce IL-1β compared with macrophages from WT (Figure 4C). The relative order of cytotoxicity and IL-1β production of the silica samples reflected the one already reported in Figures 2 and 3.Figure 4


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)

Involvement of the inflammasome, phagocytosis and cathepsin B in inducing the release of mature IL-1β by different silica particles. LPS-primed primary murine macrophages were incubated with silica at equal surface dose (20 cm2/ml) and with ATP (5 mM) as positive control. Culture supernatants were collected after 6 h (1 h for ATP) and cell viability by WST-1 (B, D, F) and IL-1β release (pg/ml) by ELISA (C, E, G) were measured. (A) A representative Western blot analysis conducted on culture supernatants or cell extracts to detect pro-IL-1β (17 kDa) or mature IL-1β (36 kDa) is shown. (B, C) WT mice and mice deficient in the adaptor molecule ASC (ASC−/−) are compared. Determinations were performed in six replicates (B) or quadruplicate (C) in a single experiment and expressed as the mean ± SD. Cells pre-treated for 1 h with cytochalasin D (5 μM) (D, E) or CA-074-Me (10 μM) (F, G) are compared to untreated cells. Determinations were performed in quadruplicate and expressed as the mean ± SD. Data from one representative experiment out of two (D, E) or one single experiment (F, G) are depicted. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control not exposed to silica particles in each group; #p < 0.05, ##p < 0.01 and ###p < 0.001 WT vs ASC−/− or non-treated vs treated with inhibitors, for each sample.
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Fig4: Involvement of the inflammasome, phagocytosis and cathepsin B in inducing the release of mature IL-1β by different silica particles. LPS-primed primary murine macrophages were incubated with silica at equal surface dose (20 cm2/ml) and with ATP (5 mM) as positive control. Culture supernatants were collected after 6 h (1 h for ATP) and cell viability by WST-1 (B, D, F) and IL-1β release (pg/ml) by ELISA (C, E, G) were measured. (A) A representative Western blot analysis conducted on culture supernatants or cell extracts to detect pro-IL-1β (17 kDa) or mature IL-1β (36 kDa) is shown. (B, C) WT mice and mice deficient in the adaptor molecule ASC (ASC−/−) are compared. Determinations were performed in six replicates (B) or quadruplicate (C) in a single experiment and expressed as the mean ± SD. Cells pre-treated for 1 h with cytochalasin D (5 μM) (D, E) or CA-074-Me (10 μM) (F, G) are compared to untreated cells. Determinations were performed in quadruplicate and expressed as the mean ± SD. Data from one representative experiment out of two (D, E) or one single experiment (F, G) are depicted. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control not exposed to silica particles in each group; #p < 0.05, ##p < 0.01 and ###p < 0.001 WT vs ASC−/− or non-treated vs treated with inhibitors, for each sample.
Mentions: To discriminate between mature IL-1β released into the supernatant after inflammasome activation and immature pro-IL-1β potentially due to silica cytotoxicity, we performed a Western blot analysis to separate the two proteins on the basis of their different molecular weight. LPS-primed macrophages were incubated for 6 h with 20 cm2/ml of silica or with ATP (5 mM) used as positive control. As shown in Figure 4A, significant differences in the expression levels of mature IL-1β among cells treated with various types of silica were observed. VS showed the highest amount of mature IL-1β, even more than the ATP positive control. Qz-1 induced a lower amount of mature IL-1β compared to VS, whereas IL-1β could not be detected for Qz-2 and Qz-3. Detection of mature/pro-IL-1β in the supernatant or cell extract not exposed to silica (Ctl) showed that priming with LPS was effective because only the pro-IL-1β form was present in cell extract, and no release of mature IL-1β was found in the supernatant. These results are consistent with the data obtained by ELISA. In order to evaluate the involvement of inflammasome in the release of IL-1β by several silica types and to confirm the mechanism leading to its activation, ASC-deficient cells, a phagocytosis inhibitor and an inhibitor of cathepsin B were used. Peritoneal macrophages from mice knock-out for the apoptosis-associated speck-like protein (ASC−/−), which is one of the three main components of the inflammasome complex, were compared with macrophages from wild type mice (WT, C57BL/6). No significant difference in cytotoxic activity between WT and ASC−/− macrophages was found (Figure 4B), indicating that silica-induced cytotoxicity is independent from ASC, as reported by Cassel et al. for Nalp3 [22]. LPS-primed ASC−/− macrophages displayed an evident defect in their ability to induce IL-1β compared with macrophages from WT (Figure 4C). The relative order of cytotoxicity and IL-1β production of the silica samples reflected the one already reported in Figures 2 and 3.Figure 4

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