Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5

Our plain-English summary of Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5, by A.M. Boylan, D.A. Sanan, D. Sheppard, et al (The Journal of Clinical Investigation, 1995).

The study

• Authors: A.M. Boylan, D.A. Sanan, D. Sheppard, V.C. Broaddus
• Journal: The Journal of Clinical Investigation
• Published: 1995;96(4):1987-2001
• Cited by: 72 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects.

In plain English

This laboratory study exposed cultured pleural mesothelial cells from healthy New Zealand White rabbits for 2 hours to crocidolite asbestos coated with purified proteins or human serum, or to vitronectin-coated wollastonite as a control fiber. The authors developed a fluorescence confocal method in which the membrane dye DiI made internalized fibers fluoresce, and they counted internal and external fibers blinded. Coating crocidolite with vitronectin, but not with fibronectin, collagen I or albumin, increased the number of fibers internalized without changing the number adherent to the cell surface. Vitronectin coating did not increase uptake of wollastonite. Flow cytometry and immunoprecipitation showed the vitronectin receptors alpha v beta 5 and alpha v beta 3 on these cells. A vitronectin substrate, excess soluble vitronectin and a blocking antibody to alpha v beta 5 (but not to alpha v beta 3) each reduced internalization of vitronectin-coated crocidolite. The authors wrote that RGD-containing peptides cut this uptake by 70%, but the fiber counts they printed are listed in the opposite order. Serum coating of crocidolite also increased internalization, and removing vitronectin from the serum returned it to baseline.

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: In vitro experimental study using cultured rabbit pleural mesothelial cells, with a newly developed fluorescence confocal microscopy assay of fiber internalization
• Who was studied: Pleural mesothelial cells harvested from healthy New Zealand White rabbits, used in culture between passages three and six. At least 100 cells were examined per condition.
• Exposure: Crocidolite asbestos obtained from the National Institute of Environmental Health and Safety, with wollastonite as a control fiber. Crocidolite was coated with vitronectin, fibronectin, collagen I, laminin or albumin, or with human serum, bronchoalveolar lavage liquid or pleural liquid. Wollastonite was tested in uptake experiments coated with vitronectin.
• Main results:
  • Vitronectin coating increased internalization of crocidolite compared with other protein coats or no coat (figure legend: P = 0.03, n = 3 experiments, 125 cells counted per experiment)
  • The vitronectin effect was on internalization only. The number of fibers adhering to the cells did not change.
  • Vitronectin coating did not increase uptake of the control fiber wollastonite. The increase for crocidolite was significant at a vitronectin coating concentration of 30 micrograms per millilitre (P less than 0.02, n = 3).
  • Both vitronectin receptors, alpha v beta 5 and alpha v beta 3, were detected on the rabbit mesothelial cells by flow cytometry and confirmed by immunoprecipitation. No alpha v beta 1 was found.
  • Cells grown on a vitronectin substrate internalized fewer vitronectin-coated crocidolite fibers than cells on other substrates (P = 0.035, n = 6), with no change in external binding
  • Excess soluble vitronectin reduced uptake of vitronectin-coated crocidolite at all concentrations tested (P = 0.03, n = 4) without altering adherent fiber numbers
  • A blocking antibody to alpha v beta 5 reduced internalization of vitronectin-coated crocidolite. A blocking antibody to alpha v beta 3 and nonblocking antibodies had no effect (P = 0.04, n = 7).
  • The authors stated that RGD-containing peptides reduced internalization of vitronectin-coated crocidolite by 70% compared with control peptides (P less than 0.05). However, the counts they printed assign 0.39 plus or minus 0.07 fibers per cell to the RGD peptide and 0.13 plus or minus 0.04 to the control, which is the reverse order. The serum-coated fiber comparison shows the same reversal.
  • Serum coating of crocidolite raised internalization to about the same degree as vitronectin coating. Serum depleted of vitronectin gave baseline internalization, which was restored by adding back vitronectin at 300 micrograms per millilitre but not at 30 (P less than 0.01, n = 3). Uptake of serum-coated fibers was inhibited by blocking alpha v beta 5 (P less than 0.003, n = 3).
  • By immunofluorescence, alpha v beta 5 colocalized with many vitronectin-coated crocidolite fibers. Alpha v beta 3 was seen in focal contacts and did not colocalize.
  • Vitronectin could be eluted from crocidolite fibers incubated with human serum, concentrated bronchoalveolar lavage liquid and pleural liquid
  • At the fiber concentrations used, no acute toxicity was detected by lactate dehydrogenase or chromium release. By 6 hours, cells exposed to vitronectin-coated crocidolite had changed shape, and this change was inhibited by the blocking antibody to alpha v beta 5.
• The authors' conclusion: The authors concluded that pleural mesothelial cells recognize and internalize vitronectin-coated and serum-coated crocidolite via the integrin alpha v beta 5. They suggested this interaction with a cell surface receptor may offer insight into how asbestos affects cells.
• Limitations the authors state:
  • The confocal technique could not reliably detect small fibers or particles, which restricted it to fibers longer than about 2 to 3 micrometres
  • This work did not establish whether internalization of fibers matters for their pathogenicity
  • The specific role of internalization in producing the biological effects of asbestos was not addressed

Topics

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

Medical terms in this study, explained in our Lexicon: Vitronectin, Crocidolite, Mesothelial Cells, Mesothelioma.

Related studies

• Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells — J. Wu, W. Liu, et al. (2000)
• Role of alphavbeta5 integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells — P. Pande, T.A. Mosleh, et al. (2006)
• Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells — W. Liu, J.D. Ernst, et al. (2000)
• Evidence of a role for mesothelial cell-derived interleukin 8 in the pathogenesis of asbestos-induced pleurisy in rabbits — A.M. Boylan, C. Rüegg, et al. (1992)
• beta1-integrin mediates asbestos-induced phosphorylation of AKT and ERK1/2 in a rat pleural mesothelial cell line — A. Berken, J. Abel, et al. (2003)
• Crocidolite induces prostaglandin I(2) release mediated by vitronectin receptor and cyclooxygenase-2 in lung cells — F.J. Leyva, K. Roberts (2010)

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