Live-cell imaging of macrophage phagocytosis of asbestos fibers under fluorescence microscopy

Our plain-English summary of Live-cell imaging of macrophage phagocytosis of asbestos fibers under fluorescence microscopy, by T. Ishida, N. Fujihara, T. Nishimura, et al (Genes and Environment, 2019).

The study

• Authors: T. Ishida, N. Fujihara, T. Nishimura, H. Funabashi, R. Hirota, T. Ikeda, A. Kuroda
• Journal: Genes and Environment
• Published: 2019;41:14
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Frustrated phagocytosis occurs when an asbestos fiber > 10 μm in length is engulfed imperfectly by a macrophage, and it is believed to be associated with chromosomal instability. Few studies have focused on dynamic cellular imaging to assess the toxicity of hazardous inorganic materials such as asbestos. One reason for this is the relative lack of fluorescent probes available to facilitate experimental visualization of inorganic materials. We recently developed asbestos-specific fluorescent probes based on asbestos-binding proteins, and achieved efficient fluorescent labeling of asbestos.

Results: Live-cell imaging with fluorescent asbestos probes was successfully utilized to dynamically analyze asbestos phagocytosis. The fluorescently labeled asbestos fibers were phagocytosed by RAW 264.7 macrophages. Internalized fibers of < 5 μm in length were visualized clearly via overlaid phase contrast and fluorescence microscopy images, but they were not clearly depicted using phase contrast images alone. Approximately 60% of the cells had phagocytosed asbestos fibers after 2 h, but over 96% of cells remained alive even 24 h after the addition of asbestos fibers. Immediate cell death was only observed when an asbestos fiber was physically pulled from a cell by an external force. Notably, at 24 h after the addition of asbestos fibers an approximately 4-fold increase in the number of binucleated cells was observed. Monitoring of individual cell divisions of cells that had phagocytosed asbestos suggested that binucleated cells were formed via the inhibition of cell separation, by asbestos fibers of > 10 μm in length that were localized in the proximity of the intercellular bridge.

Conclusions: Fluorescently labeled asbestos facilitated visualization of the dynamic biological processes that occur during and after the internalization of asbestos fibers, and indicated that (i) frustrated phagocytosis itself does not lead to immediate cell death unless the asbestos fiber is physically pulled from the cell by an external force, and (ii) macrophages that have phagocytosed asbestos can divide but sometimes the resulting daughter cells fuse, leading to the formation of a binucleated cell. This fusion only seemed to occur when a comparatively long asbestos fiber (> 10 μm) was shared by two daughter cells.

In plain English

This laboratory study used cells in dishes, not people. The authors coated amosite asbestos (crocidolite in some images) with a fluorescent probe they had previously developed. They added fibers to RAW 264.7 macrophages, particle-engulfing cells, and filmed them. By 2 hours, 40 of 68 cells (58.8%) had taken up fibers; 11 of 68 (16.2%) had taken up fibers over 10 μm long. Over 96% of cells were alive 24 hours after fibers were added. Of 153 fiber-containing cells followed, two died suddenly with leaking contents, only when a fiber was pulled from a cell by an outside force. In one filmed case, the fiber was apparently pulled from one macrophage by forces exerted by another holding its other end. At 24 hours, two-nucleus cells were about four times as common as without asbestos. In 4 of 53 filmed divisions, all in cells holding long fibers, the daughter cells failed to separate and fused. The authors concluded that incomplete engulfment of long fibers did not itself cause immediate cell death, and that daughter-cell fusion seemed to occur only with long fibers aligned with the bridge between them.

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 (in vitro) cell-culture experiment using fluorescently labeled asbestos, live-cell time-lapse imaging and confocal microscopy
• Who was studied: RAW 264.7 macrophage cell line grown in culture dishes; for example, 68 cells assessed for fiber uptake at 2 h, 153 fiber-containing cells followed for leakage, 53 cell divisions monitored
• Exposure: Amosite asbestos (used unless otherwise noted) and crocidolite asbestos, unlabeled or fluorescently labeled, added at 50 μg/mL for continuous live imaging and 25 μg/mL in other experiments; mean fiber length of the preparation 3.6 ± 0.4 μm
• Main results:
  • By 2 h, 58.8% of cells (40/68) had phagocytosed asbestos fibers; 16.2% (11/68) had phagocytosed fibers longer than 10 μm
  • Over 96% of cells remained alive 24 h after addition of either unlabeled or fluorescently labeled asbestos fibers
  • Leakage of cell contents and cell death were seen only when a fiber was physically pulled from a cell by an external force; this occurred in two of 153 cells observed
  • In the filmed case, each end of one long fiber was engulfed by a different macrophage, and the fiber was apparently pulled from one by forces exerted by the other
  • Mean length of fibers inside cells (5.6 ± 0.9 μm) was longer than that of the total fiber preparation (3.6 ± 0.4 μm); fibers on the cell surface averaged 2.4 ± 0.3 μm
  • At 24 h after asbestos was added, binucleated cells were approximately four times more numerous than in cultures without asbestos; cells with more nuclei were about 10-fold more frequent
  • Of 53 monitored divisions of fiber-containing cells (9 with long fibers, 44 with short fibers), 4 divisions of cells with long fibers did not complete separation and the daughter cells fused into a binucleated cell
  • In those four cells, mean internalized fiber length was 13.9 ± 1.9 μm (mean ± SE); short fibers in the intercellular bridge (21 instances) did not appear to affect separation
  • Labeling did not significantly change fiber surface charge: mean zeta potential −25.47 ± 0.83 mV for unlabeled vs −24.48 ± 0.55 mV for labeled asbestos (n = 4)
• The authors' conclusion: The authors concluded that frustrated phagocytosis itself does not lead to immediate cell death unless the fiber is pulled from the cell by an external force, and that macrophages containing asbestos can divide but the daughter cells sometimes fuse into a binucleated cell, which only seemed to occur with a long fiber (> 10 μm) near and parallel to the intercellular bridge
• Limitations the authors state:
  • The authors state that further research is needed to explain why long fibers tend to remain inside cells
  • The authors qualify that pulled-fiber cell death may not be a major initial toxic event, at least in vitro
  • The resolution of conventional fluorescence microscopy is limited by optical diffraction

Topics

Medical terms in this study, explained in our Lexicon: Macrophage, Phagocytosis, Frustrated Phagocytosis.

Related studies

• Degradation of cartilage proteoglycan and collagen by synovial cells. Stimulation by macrophages under activation by phagocytosis, lymphocyte factors, bacterial products or other inflammatory stimuli — C. Peeters-Joris, G. Vaes (1984)
• Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing — A. Kuroda, M. Alexandrov, et al. (2016)
• Development of an automated asbestos counting software based on fluorescence microscopy — M. Alexandrov, E. Ichida, et al. (2015)
• Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy — T. Nishimura, M. Alexandrov, et al. (2016)
• Evaluation of sensitivity of fluorescence-based asbestos detection by correlative microscopy — T. Ishida, M. Alexandrov, et al. (2012)
• Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues — L. Pascolo, A. Gianoncelli, et al. (2011)

Browse more research in Genes and Environment, or return to medical research.