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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

Phagocytosis in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of quartz phagocytosis in RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 4 ×), red staining marking quartz autofluorescence. I, cells challenged for 1 h with 100 μg/ml quartz particles; II, the same in the presence of 100 μg/ml DXS, and III in the presence of 2 μg/ml Cytocalasin B. The white bar spans 8 μm. (B) Analysis of free intacellular silicates in RAW 264.7 cells challenged with or without (C) 100 μg/ml Q or QA in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or 2 μg/ml Cytocalasin B, after 6 h (black bars) or 24 h (white bars). Values are the mean ± SD from 4 experiments. The asterisk indicates a statistically significant difference between Q or QA and C at 6 h (T test, p < 0.0025), while the symbol § indicates a statistically significant difference between Q or QA and C at 24 h (T test, p < 0.0005).
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Figure 4: Phagocytosis in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of quartz phagocytosis in RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 4 ×), red staining marking quartz autofluorescence. I, cells challenged for 1 h with 100 μg/ml quartz particles; II, the same in the presence of 100 μg/ml DXS, and III in the presence of 2 μg/ml Cytocalasin B. The white bar spans 8 μm. (B) Analysis of free intacellular silicates in RAW 264.7 cells challenged with or without (C) 100 μg/ml Q or QA in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or 2 μg/ml Cytocalasin B, after 6 h (black bars) or 24 h (white bars). Values are the mean ± SD from 4 experiments. The asterisk indicates a statistically significant difference between Q or QA and C at 6 h (T test, p < 0.0025), while the symbol § indicates a statistically significant difference between Q or QA and C at 24 h (T test, p < 0.0005).

Mentions: It is known that macrophage scavenger receptors are the main route of internalization of Q particles in phagocytes [27]. In order to investigate whether the scavenger receptor activation triggered by contact with quartz particles is a necessary step to initiate quartz internalization in our cell model, RAW 264.7 cells were incubated with the scavenger receptor inhibitor DXS [28-30] or a well known inhibitor of phagocytosis, Cytocalasin B, at a concentration of 2 μg/ml, and then challenged with 100 μg/ml Q particles. Phagocytosis was then evaluated by confocal microscopy observation in time-course experiments while the concentration of intracellular soluble free silicates was quantified after 6 and 24 hours of incubation (Figure 4).


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)

Phagocytosis in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of quartz phagocytosis in RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 4 ×), red staining marking quartz autofluorescence. I, cells challenged for 1 h with 100 μg/ml quartz particles; II, the same in the presence of 100 μg/ml DXS, and III in the presence of 2 μg/ml Cytocalasin B. The white bar spans 8 μm. (B) Analysis of free intacellular silicates in RAW 264.7 cells challenged with or without (C) 100 μg/ml Q or QA in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or 2 μg/ml Cytocalasin B, after 6 h (black bars) or 24 h (white bars). Values are the mean ± SD from 4 experiments. The asterisk indicates a statistically significant difference between Q or QA and C at 6 h (T test, p < 0.0025), while the symbol § indicates a statistically significant difference between Q or QA and C at 24 h (T test, p < 0.0005).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662810&req=5

Figure 4: Phagocytosis in quartz-stimulated cells in the presence of DXS and Cytocalasin B. (A) Confocal microscopy analysis of quartz phagocytosis in RAW 264.7 cells (single stacks acquired with oil objective 63.0 ×, digital zoom 4 ×), red staining marking quartz autofluorescence. I, cells challenged for 1 h with 100 μg/ml quartz particles; II, the same in the presence of 100 μg/ml DXS, and III in the presence of 2 μg/ml Cytocalasin B. The white bar spans 8 μm. (B) Analysis of free intacellular silicates in RAW 264.7 cells challenged with or without (C) 100 μg/ml Q or QA in the presence (Q+D, QA+D and Q+Cyto, QA+Cyto) or absence of 100 μg/ml DXS or 2 μg/ml Cytocalasin B, after 6 h (black bars) or 24 h (white bars). Values are the mean ± SD from 4 experiments. The asterisk indicates a statistically significant difference between Q or QA and C at 6 h (T test, p < 0.0025), while the symbol § indicates a statistically significant difference between Q or QA and C at 24 h (T test, p < 0.0005).
Mentions: It is known that macrophage scavenger receptors are the main route of internalization of Q particles in phagocytes [27]. In order to investigate whether the scavenger receptor activation triggered by contact with quartz particles is a necessary step to initiate quartz internalization in our cell model, RAW 264.7 cells were incubated with the scavenger receptor inhibitor DXS [28-30] or a well known inhibitor of phagocytosis, Cytocalasin B, at a concentration of 2 μg/ml, and then challenged with 100 μg/ml Q particles. Phagocytosis was then evaluated by confocal microscopy observation in time-course experiments while the concentration of intracellular soluble free silicates was quantified after 6 and 24 hours of incubation (Figure 4).

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