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Ascorbic acid pre-treated quartz stimulates TNF-alpha release in RAW 264.7 murine macrophages through ROS production and membrane lipid peroxidation.

Scarfì S, Magnone M, Ferraris C, Pozzolini M, Benvenuto F, Benatti U, Giovine M - Respir. Res. (2009)

Bottom Line: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways.Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS.Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, Section of Biochemistry, University of Genova, Genova, Italy. soniascarfi@unige.it

ABSTRACT

Background: Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2) by ascorbic acid pre-treated quartz (QA) compared to untreated quartz (Q) in the murine macrophage cell line RAW 264.7.

Methods: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways.

Results: Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-alpha production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself.

Conclusion: Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

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Related in: MedlinePlus

Lipid peroxidation and ROS production in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of lipid peroxidation of RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 8 ×) using the specific fluorescent probe BODIPY 581/591 in time-lapse experiments, with acquisitions of 1 slide/min. A, C, E show the decrease of red fluorescence during 35 min, while B, D, F show the contemporary increase of green fluorescence in the same cells. A, B control, untreated cells; C, D cells challenged with 100 μg/ml quartz particles; E, F cells challenged with quartz particles in the presence of 100 μg/ml DXS. (B) Fluorimetric quantitation of lipid peroxidation in RAW 264.7 cells challenged with 100 μg/ml Q or QA (white bars) particles in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or of 2 μg/ml Cytocalasin B for 1 h. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q-challenged cells (T test, p < 0.005), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.005). (C) Fluorimetric measurement of ROS production in RAW 264.7 macrophages in the same conditions of (B) after 1 h incubation. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q challenged cells (T test, p < 0.0025), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.0005).
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Figure 5: Lipid peroxidation and ROS production in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of lipid peroxidation of RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 8 ×) using the specific fluorescent probe BODIPY 581/591 in time-lapse experiments, with acquisitions of 1 slide/min. A, C, E show the decrease of red fluorescence during 35 min, while B, D, F show the contemporary increase of green fluorescence in the same cells. A, B control, untreated cells; C, D cells challenged with 100 μg/ml quartz particles; E, F cells challenged with quartz particles in the presence of 100 μg/ml DXS. (B) Fluorimetric quantitation of lipid peroxidation in RAW 264.7 cells challenged with 100 μg/ml Q or QA (white bars) particles in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or of 2 μg/ml Cytocalasin B for 1 h. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q-challenged cells (T test, p < 0.005), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.005). (C) Fluorimetric measurement of ROS production in RAW 264.7 macrophages in the same conditions of (B) after 1 h incubation. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q challenged cells (T test, p < 0.0025), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.0005).

Mentions: Qualitative confocal microscopy studies were performed using a membrane intercalating fluorescent probe, BODIPY 581/591 C11 [31-33], which reveals lipid peroxidation through a specific fluorescence emission shift from red to green. Figure 5A shows the result of time-course experiments in which probe-loaded RAW 264.7 macrophages were monitored for lipid peroxidation in the presence of various stimuli. Control, untreated cells (panels A-B) showed no fluorescence shift during an observation time of 35 min (1 slide/min), while a clear shift from red to green was observed when cells were challenged either with quartz alone (panels C-D) or with quartz in the presence of DXS (panels E-F). Fluorimetric measurment of the green fluorescence of the same samples, allowed a quantification of the lipid peroxidation rate under these conditions. Results are shown in Figure 5B. As expected Q and QA particles increased lipid peroxidation in RAW 264.7 macrophages as compared to untreated, control cells, with QA showing significantly higher values than Q (p < 0.0005). The peroxidation values measured on RAW 264.7 cells incubated with DXS or Cytocalasin B, in the presence of Q (Q+D and Q+Cyto bars, respectively) or QA particles (QA+D and QA+Cyto bars) showed a further increase as compared to Q and QA particles alone (Q+D vs Q, p < 0.005; QA+D vs QA, p < 0.0005).


Ascorbic acid pre-treated quartz stimulates TNF-alpha release in RAW 264.7 murine macrophages through ROS production and membrane lipid peroxidation.

Scarfì S, Magnone M, Ferraris C, Pozzolini M, Benvenuto F, Benatti U, Giovine M - Respir. Res. (2009)

Lipid peroxidation and ROS production in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of lipid peroxidation of RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 8 ×) using the specific fluorescent probe BODIPY 581/591 in time-lapse experiments, with acquisitions of 1 slide/min. A, C, E show the decrease of red fluorescence during 35 min, while B, D, F show the contemporary increase of green fluorescence in the same cells. A, B control, untreated cells; C, D cells challenged with 100 μg/ml quartz particles; E, F cells challenged with quartz particles in the presence of 100 μg/ml DXS. (B) Fluorimetric quantitation of lipid peroxidation in RAW 264.7 cells challenged with 100 μg/ml Q or QA (white bars) particles in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or of 2 μg/ml Cytocalasin B for 1 h. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q-challenged cells (T test, p < 0.005), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.005). (C) Fluorimetric measurement of ROS production in RAW 264.7 macrophages in the same conditions of (B) after 1 h incubation. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q challenged cells (T test, p < 0.0025), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.0005).
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Figure 5: Lipid peroxidation and ROS production in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of lipid peroxidation of RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 8 ×) using the specific fluorescent probe BODIPY 581/591 in time-lapse experiments, with acquisitions of 1 slide/min. A, C, E show the decrease of red fluorescence during 35 min, while B, D, F show the contemporary increase of green fluorescence in the same cells. A, B control, untreated cells; C, D cells challenged with 100 μg/ml quartz particles; E, F cells challenged with quartz particles in the presence of 100 μg/ml DXS. (B) Fluorimetric quantitation of lipid peroxidation in RAW 264.7 cells challenged with 100 μg/ml Q or QA (white bars) particles in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or of 2 μg/ml Cytocalasin B for 1 h. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q-challenged cells (T test, p < 0.005), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.005). (C) Fluorimetric measurement of ROS production in RAW 264.7 macrophages in the same conditions of (B) after 1 h incubation. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and QA challenged cells (T test, p < 0.0005); the asterisk indicates a statistically significant difference between Q+D or Q+Cyto and Q challenged cells (T test, p < 0.0025), while the symbol # indicates a statistically significant difference between QA+D or QA+Cyto and QA-challenged cells (T test, p < 0.0005).
Mentions: Qualitative confocal microscopy studies were performed using a membrane intercalating fluorescent probe, BODIPY 581/591 C11 [31-33], which reveals lipid peroxidation through a specific fluorescence emission shift from red to green. Figure 5A shows the result of time-course experiments in which probe-loaded RAW 264.7 macrophages were monitored for lipid peroxidation in the presence of various stimuli. Control, untreated cells (panels A-B) showed no fluorescence shift during an observation time of 35 min (1 slide/min), while a clear shift from red to green was observed when cells were challenged either with quartz alone (panels C-D) or with quartz in the presence of DXS (panels E-F). Fluorimetric measurment of the green fluorescence of the same samples, allowed a quantification of the lipid peroxidation rate under these conditions. Results are shown in Figure 5B. As expected Q and QA particles increased lipid peroxidation in RAW 264.7 macrophages as compared to untreated, control cells, with QA showing significantly higher values than Q (p < 0.0005). The peroxidation values measured on RAW 264.7 cells incubated with DXS or Cytocalasin B, in the presence of Q (Q+D and Q+Cyto bars, respectively) or QA particles (QA+D and QA+Cyto bars) showed a further increase as compared to Q and QA particles alone (Q+D vs Q, p < 0.005; QA+D vs QA, p < 0.0005).

Bottom Line: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways.Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS.Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, Section of Biochemistry, University of Genova, Genova, Italy. soniascarfi@unige.it

ABSTRACT

Background: Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2) by ascorbic acid pre-treated quartz (QA) compared to untreated quartz (Q) in the murine macrophage cell line RAW 264.7.

Methods: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways.

Results: Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-alpha production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself.

Conclusion: Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

Show MeSH
Related in: MedlinePlus