Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models

Our plain-English summary of Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models, by T.R. Quinlan, K.A. BeruBe, M.P. Hacker, et al (Free Radical Biology & Medicine, 1998).

The study

• Authors: T.R. Quinlan, K.A. BeruBe, M.P. Hacker, D.J. Taatjes, C.R. Timblin, J. Goldberg, P. Kimberley, P. O'Shaughnessy, D. Hemenway, J. Torino, L.A. Jimenez, B.T. Mossman
• Journal: Free Radical Biology & Medicine
• Published: 1998;24(5):778-88
• Cited by: 52 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

In plain English

This was a laboratory study in rats and cultured cells. Male Fischer 344 rats breathed crocidolite or chrysotile asbestos (7–10 mg/m3 air, 6 hours a day, 5 days a week) and were compared with sham-exposed rats. Further rats received the drug bleomycin, a second lung-scarring model. Alveolar macrophages (lung immune cells) washed from asbestos-exposed rats released significantly more nitrite and nitrate, breakdown products of nitric oxide. Output peaked roughly 3-fold above sham rats at 3 days; by day 20 only chrysotile still raised it. An inhibitor of the nitric oxide-making enzyme significantly reduced this output in macrophages from crocidolite- and bleomycin-exposed rats. In asbestos-exposed rats, lung-washing neutrophil percentages were significantly raised at all time points. Crocidolite raised iNOS messenger RNA in cultured cells, but Western blots and immunostaining found no measurable iNOS protein increase in these cells or in macrophages washed from exposed lungs. Crocidolite and its non-fibrous chemical relative riebeckite increased iNOS gene promoter activity 2–3 fold; cristobalite silica did not do so significantly. The authors concluded that inhaled asbestos raised macrophage nitric oxide production by a NOS-dependent pathway.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal inhalation experiment in rats (with a bleomycin instillation comparison model) plus in vitro cell culture experiments
• Who was studied: Male Fischer 344 rats weighing 200–250 g, 4 per group, lavaged at 3, 9 and 20 days of asbestos exposure (bleomycin groups at 1, 3 and 7 days). Also RAW 264.7 cells (a mouse macrophage-like cell line) and alveolar macrophages from control rats, studied in vitro
• Exposure: Inhalation of National Institute of Environmental Health Science reference samples of crocidolite or chrysotile asbestos at 7–10 mg/m3 air, 6 hours/day, 5 days/week. A single intratracheal dose of bleomycin (3.6 mg/kg) in other rats. In vitro exposure of cells to crocidolite, riebeckite (non-fibrous analog of crocidolite), cristobalite silica, LPS and interferon-gamma
• Main results:
  • Alveolar macrophages from rats inhaling crocidolite or chrysotile produced significantly more nitrites and nitrates. Peak increases were roughly 3-fold compared with sham rats after 3 days of exposure to either fiber type.
  • By day 20, only chrysotile still raised nitrite/nitrate levels. Crocidolite-exposed rats had levels roughly comparable to sham controls.
  • Bleomycin-exposed rats had greater nitrite/nitrate production than either asbestos group, with peak increases 8-fold higher than controls at 3 days.
  • Adding the NOS inhibitor NMMA significantly reduced nitric oxide generation at all time points by macrophages from rats exposed to bleomycin or crocidolite.
  • Asbestos inhalation did not significantly increase total cell numbers in lung lavage. In asbestos-exposed rats, neutrophil percentages were significantly raised at all time points, peaking at 20 days. In bleomycin-exposed rats, neutrophils declined to control levels by day 7.
  • iNOS mRNA rose in RAW 264.7 cells and in rat macrophages exposed to crocidolite in vitro, with larger rises after LPS. No increase in iNOS mRNA was seen in macrophages lavaged from rats after 3 days of asbestos inhalation.
  • iNOS protein was not detected by Western blot in asbestos-exposed RAW 264.7 cells or macrophages. Macrophages exposed to asbestos in vitro, or lavaged from asbestos-exposed rats, showed very low immunostaining, at levels comparable to sham controls.
  • In lung tissue from asbestos-exposed rats, immunostaining (data not shown) indicated 3- to 4-fold more iNOS-positive macrophages than in sham controls. These cells were few in number and mainly interstitial.
  • In RAW 264.7 cells, crocidolite at all concentrations gave statistically significant 2–3 fold increases in iNOS promoter activity, and riebeckite gave similar results. Cristobalite silica caused non-significant increases. Interferon-gamma and LPS gave 10- and 30-fold increases respectively.
• The authors' conclusion: The authors concluded that their in vivo and in vitro evidence supports the hypothesis that inhaled asbestos raises nitric oxide production from lung macrophages by a NOS-dependent pathway. They suggested this release likely also depends on cytokine release from inflammatory and other lung cells.
• Limitations the authors state:
  • Small increases in iNOS protein, seen mainly in interstitial macrophages, may not be reflected in macrophages recovered by lavage. The Western blot may also be less sensitive than the Northern blot.
  • In vitro methods were used to test specificity across mineral dusts because large inhalation experiments were of limited feasibility and too expensive.
  • The relative dip at 9 days may reflect rats being removed from exposure chambers over the weekend, giving a brief recovery period.

Topics

Medical terms in this study, explained in our Lexicon: Alveolar Macrophage, Reactive Nitrogen Species (RNS), Inflammation, Crocidolite, Chrysotile, Inducible Nitric Oxide Synthase (iNOS), Bronchoalveolar Lavage (BAL), Pulmonary Fibrosis.

Related studies

• Asbestos fibers and interferon-gamma up-regulate nitric oxide production in rat alveolar macrophages — G. Thomas, T. Ando, et al. (1994)
• Intrapleural interleukin-2 induces nitric oxide production in pleural effusions from malignant mesothelioma: a possible mechanism of interleukin-2-mediated cytotoxicity? — C. Porta, V. Rizzo, et al. (2002)
• Nitric oxide (NO) synthase activity in the lung and NO synthesis in alveolar macrophages of rats increased on exposure to asbestos — H. Iguchi, S. Kojo, et al. (1996)
• Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis — K.E. Driscoll, J.K. Maurer, et al. (1995)
• Iron inhibits the nitric oxide synthesis elicited by asbestos in murine macrophages — E. Aldieri, D. Ghigo, et al. (2001)
• Asbestos redirects nitric oxide signaling through rapid catalytic conversion to nitrite — D.D. Thomas, M.G. Espey, et al. (2006)

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