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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

TNF-α production in quartz-incubated RAW 264.7 cells. (A) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 hrs (black bars) and 18 hrs (white bars) stimulation with quartz, as determined using the L929 bio-assay. Cells were challenged with untreated (Q) or AA-treated (QA) quartz particles at 15, 50 and 100 μg/ml. Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values, at all particle concentrations at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (B) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 h (black bars) and 18 hrs (white bars) stimulation with QA or Q particles in the presence of murine Interferon-γ (1 ng/ml). Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values at all particle concentrations, at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (C) TNF-α production in rat alveolar macrophages stimulated with 100 μg/ml QA or Q particles was evaluated after 6 h incubation. Values are the mean ± SD from 3 experiments. The asterisk indicates a statistically significant increase of TNF-α expression between Q100 and C samples (T Test, p < 0.0005), while the symbol § indicate a statistically significant difference between Q100 and QA100 TNF-α values (T Test, p < 0.0005).
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Figure 1: TNF-α production in quartz-incubated RAW 264.7 cells. (A) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 hrs (black bars) and 18 hrs (white bars) stimulation with quartz, as determined using the L929 bio-assay. Cells were challenged with untreated (Q) or AA-treated (QA) quartz particles at 15, 50 and 100 μg/ml. Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values, at all particle concentrations at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (B) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 h (black bars) and 18 hrs (white bars) stimulation with QA or Q particles in the presence of murine Interferon-γ (1 ng/ml). Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values at all particle concentrations, at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (C) TNF-α production in rat alveolar macrophages stimulated with 100 μg/ml QA or Q particles was evaluated after 6 h incubation. Values are the mean ± SD from 3 experiments. The asterisk indicates a statistically significant increase of TNF-α expression between Q100 and C samples (T Test, p < 0.0005), while the symbol § indicate a statistically significant difference between Q100 and QA100 TNF-α values (T Test, p < 0.0005).

Mentions: Two procedures of TNF-α assay were followed, using a Mouse TNF-α (Mono/Poly) OptEIA kit (BD Biosciences, Pharmingen, San Diego, CA, USA) and a cytotoxicity-based assay using the murine fibroblast L929 cell line [21,22]. The two procedures were applied to the first TNF-α quantification in RAW 264.7 cell supernatants after incubation with 15, 50 and 100 μg/ml Q or QA particles (see Figure 1A) yielding quite comparable results, therefore for all the following quantifications only the second method was used.


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)

TNF-α production in quartz-incubated RAW 264.7 cells. (A) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 hrs (black bars) and 18 hrs (white bars) stimulation with quartz, as determined using the L929 bio-assay. Cells were challenged with untreated (Q) or AA-treated (QA) quartz particles at 15, 50 and 100 μg/ml. Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values, at all particle concentrations at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (B) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 h (black bars) and 18 hrs (white bars) stimulation with QA or Q particles in the presence of murine Interferon-γ (1 ng/ml). Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values at all particle concentrations, at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (C) TNF-α production in rat alveolar macrophages stimulated with 100 μg/ml QA or Q particles was evaluated after 6 h incubation. Values are the mean ± SD from 3 experiments. The asterisk indicates a statistically significant increase of TNF-α expression between Q100 and C samples (T Test, p < 0.0005), while the symbol § indicate a statistically significant difference between Q100 and QA100 TNF-α values (T Test, p < 0.0005).
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Figure 1: TNF-α production in quartz-incubated RAW 264.7 cells. (A) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 hrs (black bars) and 18 hrs (white bars) stimulation with quartz, as determined using the L929 bio-assay. Cells were challenged with untreated (Q) or AA-treated (QA) quartz particles at 15, 50 and 100 μg/ml. Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values, at all particle concentrations at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (B) TNF-α concentration detected in RAW 264.7 cell supernatants after 6 h (black bars) and 18 hrs (white bars) stimulation with QA or Q particles in the presence of murine Interferon-γ (1 ng/ml). Values are the mean ± SD from 4 experiments. The asterisks indicate a statistically significant difference between Q and QA values at all particle concentrations, at 6 h (T test, p < 0.0005). The symbols § indicate a statistically significant difference between Q and QA values at all particle concentrations, at 18 h (T test, p < 0.0005). (C) TNF-α production in rat alveolar macrophages stimulated with 100 μg/ml QA or Q particles was evaluated after 6 h incubation. Values are the mean ± SD from 3 experiments. The asterisk indicates a statistically significant increase of TNF-α expression between Q100 and C samples (T Test, p < 0.0005), while the symbol § indicate a statistically significant difference between Q100 and QA100 TNF-α values (T Test, p < 0.0005).
Mentions: Two procedures of TNF-α assay were followed, using a Mouse TNF-α (Mono/Poly) OptEIA kit (BD Biosciences, Pharmingen, San Diego, CA, USA) and a cytotoxicity-based assay using the murine fibroblast L929 cell line [21,22]. The two procedures were applied to the first TNF-α quantification in RAW 264.7 cell supernatants after incubation with 15, 50 and 100 μg/ml Q or QA particles (see Figure 1A) yielding quite comparable results, therefore for all the following quantifications only the second method was used.

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