Mesothelial cell apoptosis is confirmed in vivo by morphological change in cytokeratin distribution

Our plain-English summary of Mesothelial cell apoptosis is confirmed in vivo by morphological change in cytokeratin distribution, by E. Marchi, W. Liu, V.C. Broaddus (American Journal of Physiology. Lung Cellular and Molecular Physiology, 2000).

The study

• Authors: E. Marchi, W. Liu, V.C. Broaddus
• Journal: American Journal of Physiology. Lung Cellular and Molecular Physiology
• Published: 2000;278(3):L528-35
• Cited by: 21 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Apoptosis of mesothelial cells has been demonstrated in vitro but not in vivo. To identify apoptotic pleural cells as mesothelial, we used cytokeratin as a marker and found a striking spheroid, aggregated appearance of cytokeratin in apparently apoptotic mesothelial cells. In in vitro studies, we found that the aggregated cytokeratin pattern correlated with apoptosis in primary mesothelial cells from mice, rabbits, and humans and was not seen with necrosis. In in vivo studies in mice, we then used this cytokeratin pattern to identify and quantitate apoptotic mesothelial cells. Apoptotic mesothelial cells were best harvested by pleural lavage, indicating that they were loosely adherent or nonadherent. Instillation of RPMI 1640 medium or wollastonite for 24 h induced apoptosis in 0.1 +/- 0. 1 (SE) and 1.0 +/- 0.7%, respectively, of all mesothelial cells recovered, whereas instillation of known apoptotic stimuli, crocidolite asbestos (25 microg) for 24 h or actinomycin D plus murine tumor necrosis factor-alpha for 12 h, induced apoptosis in 5. 1 +/- 0.5 and 22.4 +/- 4.5%, respectively (significantly greater than in control experiments, P < 0.05). By analysis of cytokeratin staining, mesothelial cell apoptosis has been confirmed in vivo.

In plain English

This laboratory study in mice and cultured cells asked whether mesothelial cells, which line the chest cavity, undergo apoptosis (programmed cell death) inside the living body. In cultured mesothelial cells from mice, rabbits and humans, the authors first showed that a clumped pattern of the protein cytokeratin matched standard signs of apoptosis. This pattern was not seen in cells killed by necrosis. They then injected substances into the chest lining space of male mice and recovered cells by washing the space out. Apoptotic mesothelial cells were loosely attached or detached and were best collected in these washings. The share of recovered mesothelial cells that were apoptotic was 0.1% with culture medium alone and 1.0% with wollastonite, a control fiber. It was 5.1% with 25 µg of crocidolite asbestos for 24 hours and 22.4% with actinomycin D plus tumor necrosis factor for 12 hours. The last two were significantly higher than both controls. The authors concluded that mesothelial cell apoptosis was confirmed in vivo. They said they could not tell whether asbestos acted directly on the cells, and that it is not known whether the same would be found after inhaling asbestos.

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: Laboratory study combining in vitro experiments on cultured primary mesothelial cells with an animal experiment (substances injected into the pleural space of mice)
• Who was studied: Healthy male C57BL/6 mice aged 6–8 weeks; in vitro, primary pleural mesothelial cells from mice, rabbits and humans (human cells from pleural effusions or ascites); work done at the Lung Biology Center, San Francisco General Hospital. Group sizes in the main in vivo comparison were 6 mice (RPMI medium), 3 (wollastonite), 12 (crocidolite) and 6 (actinomycin D plus mTNF-α).
• Exposure: Pleural instillation of crocidolite asbestos (25 µg; mean fiber length 19 µm, width 0.25 µm) for 24 hours. Comparison groups were wollastonite (50 µg, a control fiber) or RPMI 1640 medium alone for 24 hours, and actinomycin D (1.0 µM) plus murine tumor necrosis factor-alpha (100 ng/ml) for 12 hours, all in a total volume of 0.5 ml.
• Main results:
  • In cultured mesothelial cells, almost all cells with aggregated cytokeratin had a condensed nucleus or an extremely pyknotic or absent nucleus. Among cells with aggregated cytokeratin, 76.7% had a condensed nucleus, 21.4% an extremely pyknotic or absent nucleus, and 2.0% a normal nucleus (n = 4 animals).
  • Of mesothelial cells with TUNEL-positive nuclei (a DNA-breakage sign of apoptosis), most but not all had cytokeratin aggregation: 69.0% (SD 13.5%; n = 3 animals).
  • Cells killed by necrosis with high concentrations of hydrogen peroxide showed absent or faint fragmented, linear cytokeratin, distinguishable from the aggregated pattern of apoptosis.
  • In mice, apoptotic mesothelial cells were loosely adherent or detached from the pleural surface and were best harvested by washing out (lavage) the pleural space.
  • In mice, the parietal pleura exposed to crocidolite asbestos for 24 hours showed gaps in the mesothelial cell layer. After RPMI medium, and on the uninjected side, the layer was continuous.
  • Control instillations in mice: RPMI medium alone or wollastonite for 24 hours produced apoptosis in 0.1% (SE 0.1) and 1.0% (SE 0.7) of recovered mesothelial cells, respectively.
  • Crocidolite asbestos (25 µg, 24 hours) produced apoptosis in 5.1% (SE 0.5) of recovered mesothelial cells in mice. Actinomycin D plus mTNF-α (12 hours) produced apoptosis in 22.4% (SE 4.5).
  • The crocidolite and actinomycin D plus mTNF-α results were statistically significantly different from both the RPMI and wollastonite control groups (p less than 0.05).
  • The instillations caused a variable inflammatory response in mice. The percentage of neutrophils was 49% after wollastonite and 62% after crocidolite, compared with 13% after RPMI and 19% after actinomycin D plus mTNF-α (n = 2 animals).
• The authors' conclusion: The authors concluded that, by analysis of cytokeratin staining, mesothelial cell apoptosis was confirmed in vivo.
• Limitations the authors state:
  • The apoptosis percentages are of recovered mesothelial cells, which are only a fraction of the total mesothelial cell population.
  • Measuring apoptosis at a single time point may underestimate its total effect over time.
  • Used alone, cytokeratin aggregation may miss some cells in early apoptosis.
  • The study could not determine whether asbestos caused apoptosis directly or through secondary effects such as inflammation.
  • It is not known whether mesothelial cell apoptosis would occur after inhaled asbestos exposure.
  • Injecting asbestos directly into the pleural space gives a higher acute exposure than inhalation.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Apoptosis, Necrosis, Wollastonite, Crocidolite, Tumor Necrosis Factor-alpha (TNF-α).

Related studies

• Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos — J.L. Goldberg, C.L. Zanella, et al. (1997)
• Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells — W. Liu, J.D. Ernst, et al. (2000)
• Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers — C.R. Timblin, G.D. Guthrie, et al. (1998)
• Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents — V. Levresse, S. Moritz, et al. (1998)
• Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos — K.A. BéruBé, T.R. Quinlan, et al. (1996)
• Potential role of ferritin heavy chain in oxidative stress and apoptosis in human mesothelial and mesothelioma cells: implications for asbestos-induced oncogenesis — W. Aung, S. Hasegawa, et al. (2007)

Browse more research in American Journal of Physiology. Lung Cellular and Molecular Physiology, or return to medical research.