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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

RT-PCR of TNF-α mRNA in quartz-treated RAW 264.7 cells. TNF-α mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis from 0.5 to 24 h following cell stimulation with 100 μg/ml of QA (white bars) or Q particles (black bars). Results are the mean of three independent experiments performed in triplicate, and are expressed as TNF-α mRNA, normalized on the GAPDH transcription, relative to control cells at time-zero. The asterisks indicate a significant difference between samples challenged with Q and QA particles (T Test, p < 0.025).
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Figure 2: RT-PCR of TNF-α mRNA in quartz-treated RAW 264.7 cells. TNF-α mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis from 0.5 to 24 h following cell stimulation with 100 μg/ml of QA (white bars) or Q particles (black bars). Results are the mean of three independent experiments performed in triplicate, and are expressed as TNF-α mRNA, normalized on the GAPDH transcription, relative to control cells at time-zero. The asterisks indicate a significant difference between samples challenged with Q and QA particles (T Test, p < 0.025).

Mentions: TNF-α mRNA synthesis was measured by quantitative RT-PCR analysis in RAW 264.7 macrophages incubated with 100 μg/ml Q or QA particles for 30 min, 3 hours or 24 hours and compared to values recorded at the same time points on untreated, control cells (Figure 2).


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)

RT-PCR of TNF-α mRNA in quartz-treated RAW 264.7 cells. TNF-α mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis from 0.5 to 24 h following cell stimulation with 100 μg/ml of QA (white bars) or Q particles (black bars). Results are the mean of three independent experiments performed in triplicate, and are expressed as TNF-α mRNA, normalized on the GAPDH transcription, relative to control cells at time-zero. The asterisks indicate a significant difference between samples challenged with Q and QA particles (T Test, p < 0.025).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: RT-PCR of TNF-α mRNA in quartz-treated RAW 264.7 cells. TNF-α mRNA transcription was monitored in RAW 264.7 macrophages by RT-PCR analysis from 0.5 to 24 h following cell stimulation with 100 μg/ml of QA (white bars) or Q particles (black bars). Results are the mean of three independent experiments performed in triplicate, and are expressed as TNF-α mRNA, normalized on the GAPDH transcription, relative to control cells at time-zero. The asterisks indicate a significant difference between samples challenged with Q and QA particles (T Test, p < 0.025).
Mentions: TNF-α mRNA synthesis was measured by quantitative RT-PCR analysis in RAW 264.7 macrophages incubated with 100 μg/ml Q or QA particles for 30 min, 3 hours or 24 hours and compared to values recorded at the same time points on untreated, control cells (Figure 2).

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