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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

ROS and TNF-α production in quartz-stimulated cells in the presence of ROS scavengers. (A) Fluorimetric measurement of ROS production in RAW 264.7 macrophages challenged with 15, 50 and 100 μg/ml Q or QA particles. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference of QA-challenged cells compared to control, untreated cells (C), (T test, p < 0.0005); the asterisk indicates a statistically significant difference of Q-challenged cells compared to C (T test, p < 0.0005). (B) TNF-α production was evaluated in RAW 264.7 cells after 6 hrs incubation with 100 μg/ml Q or QA particles (black bars: Q samples; white bars: QA samples) in the presence of 4000 U/ml catalase (Qc, QAc bars), 50 mM mannitol (Qm, QAm bars) and 2 mM desferoxamine (Qd, QAd bars). Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and Qc bars and between QA and QAc bars (T test, p < 0.0005). The asterisks indicate a statistically significant difference between Q and Qm, and between QA and QAm bars (T test, p < 0.0005).
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Figure 3: ROS and TNF-α production in quartz-stimulated cells in the presence of ROS scavengers. (A) Fluorimetric measurement of ROS production in RAW 264.7 macrophages challenged with 15, 50 and 100 μg/ml Q or QA particles. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference of QA-challenged cells compared to control, untreated cells (C), (T test, p < 0.0005); the asterisk indicates a statistically significant difference of Q-challenged cells compared to C (T test, p < 0.0005). (B) TNF-α production was evaluated in RAW 264.7 cells after 6 hrs incubation with 100 μg/ml Q or QA particles (black bars: Q samples; white bars: QA samples) in the presence of 4000 U/ml catalase (Qc, QAc bars), 50 mM mannitol (Qm, QAm bars) and 2 mM desferoxamine (Qd, QAd bars). Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and Qc bars and between QA and QAc bars (T test, p < 0.0005). The asterisks indicate a statistically significant difference between Q and Qm, and between QA and QAm bars (T test, p < 0.0005).

Mentions: ROS production was evaluated with a ROS-specific fluorescent probe after 1 hour incubation of RAW 264.7 cells in the presence of Q or QA particles (Figure 3A). For both stimuli ROS production was dose- and time-dependent, with QA inducing a significantly higher increase compared to Q particles at all concentrations tested. The most relevant differences were recorded at 100 μg/ml particles concentration with a 2.6-fold increase in ROS production in QA-stimulated cells as compared to control, untreated cells, and 1.8-fold increase as compared to Q-stimulated cells.


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)

ROS and TNF-α production in quartz-stimulated cells in the presence of ROS scavengers. (A) Fluorimetric measurement of ROS production in RAW 264.7 macrophages challenged with 15, 50 and 100 μg/ml Q or QA particles. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference of QA-challenged cells compared to control, untreated cells (C), (T test, p < 0.0005); the asterisk indicates a statistically significant difference of Q-challenged cells compared to C (T test, p < 0.0005). (B) TNF-α production was evaluated in RAW 264.7 cells after 6 hrs incubation with 100 μg/ml Q or QA particles (black bars: Q samples; white bars: QA samples) in the presence of 4000 U/ml catalase (Qc, QAc bars), 50 mM mannitol (Qm, QAm bars) and 2 mM desferoxamine (Qd, QAd bars). Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and Qc bars and between QA and QAc bars (T test, p < 0.0005). The asterisks indicate a statistically significant difference between Q and Qm, and between QA and QAm bars (T test, p < 0.0005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: ROS and TNF-α production in quartz-stimulated cells in the presence of ROS scavengers. (A) Fluorimetric measurement of ROS production in RAW 264.7 macrophages challenged with 15, 50 and 100 μg/ml Q or QA particles. Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference of QA-challenged cells compared to control, untreated cells (C), (T test, p < 0.0005); the asterisk indicates a statistically significant difference of Q-challenged cells compared to C (T test, p < 0.0005). (B) TNF-α production was evaluated in RAW 264.7 cells after 6 hrs incubation with 100 μg/ml Q or QA particles (black bars: Q samples; white bars: QA samples) in the presence of 4000 U/ml catalase (Qc, QAc bars), 50 mM mannitol (Qm, QAm bars) and 2 mM desferoxamine (Qd, QAd bars). Values are the mean ± SD from 4 experiments. The symbol § indicates a statistically significant difference between Q and Qc bars and between QA and QAc bars (T test, p < 0.0005). The asterisks indicate a statistically significant difference between Q and Qm, and between QA and QAm bars (T test, p < 0.0005).
Mentions: ROS production was evaluated with a ROS-specific fluorescent probe after 1 hour incubation of RAW 264.7 cells in the presence of Q or QA particles (Figure 3A). For both stimuli ROS production was dose- and time-dependent, with QA inducing a significantly higher increase compared to Q particles at all concentrations tested. The most relevant differences were recorded at 100 μg/ml particles concentration with a 2.6-fold increase in ROS production in QA-stimulated cells as compared to control, untreated cells, and 1.8-fold increase as compared to Q-stimulated cells.

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