Libby amphibole-induced mesothelial cell autoantibodies bind to surface plasminogen and alter collagen matrix remodeling

Our plain-English summary of Libby amphibole-induced mesothelial cell autoantibodies bind to surface plasminogen and alter collagen matrix remodeling, by R. Hanson, C. Evilia, J. Gilmer, et al (Physiological Reports, 2016).

The study

• Authors: R. Hanson, C. Evilia, J. Gilmer, L. Woods, B. Black, R. Flores, J.C. Pfau
• Journal: Physiological Reports
• Published: 2016;4(15)
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Lamellar pleural thickening (LPT) is a fibrotic disease induced by exposure to Libby amphibole (LA) asbestos that causes widespread scarring around the lung, resulting in deterioration of pulmonary function. Investigating the effects of autoantibodies to mesothelial cells (MCAA) present in the study populations has been a major part of the effort to understand the mechanism of pathogenesis. It has been shown in vitro that human mesothelial cells (Met5a) exposed to MCAA increase collagen deposition into the extracellular matrix (ECM). In this study, we sought to further elucidate how MCAA drive increased collagen deposition by identifying the protein targets bound by MCAA on the cellular surface using biotinylation to label and isolate surface proteins. Isolated surface protein fractions were identified as containing MCAA targets using ELISA The fractions that demonstrated binding by MCAA were then analyzed by tandem mass spectrometry (MS/MS) and MASCOT analysis. The most promising result from the MASCOT analysis, plasminogen (PLG), was tested for MCAA binding using purified human PLG in an ELISA We report that serum containing MCAA bound at an optical density (OD) 3 times greater than that of controls, and LA-exposed subjects had a high frequency of positive tests for anti-PLG autoantibodies. This work implicates the involvement of the plasminogen/plasmin system in the mechanism of excess collagen deposition in Met5a cells exposed to MCAA Elucidating this mechanism could contribute to the understanding of LPT.

© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

In plain English

This laboratory study looked for the cell-surface proteins recognized by mesothelial cell autoantibodies (MCAA). These are antibodies found in blood from some people exposed to Libby amphibole (LA) asbestos. The authors used cultured human mesothelial cells (Met-5A) and serum from LA-exposed people seen at a clinic in Libby, Montana, with commercial normal human serum for comparison. Mass spectrometry of the surface-protein fractions that MCAA bound most strongly pointed to plasminogen as the top candidate. Pooled MCAA-positive serum bound purified plasminogen three times more than MCAA-negative or normal serum, and 88% of MCAA-positive samples showed binding to plasminogen. In cell experiments, removing anti-plasminogen antibodies from MCAA-positive serum significantly reduced the extra collagen deposited by the cells, virtually to control levels, though some antibody binding to the cells remained. The authors concluded that a subset of MCAA binds plasminogen and likely contributes to a sizable portion of the excess collagen deposition in these cells. They noted that not all fractions could be analyzed, so other targets may have been missed.

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) study using proteomics on cultured human mesothelial cells, plus antibody-binding tests on human serum samples
• Who was studied: Serum samples from LA-exposed subjects at the Center for Asbestos Related Diseases (CARD) clinic in Libby, Montana. Samples were grouped as MCAA-positive (33 men, 23 women, mean age 61.0) or MCAA-negative (30 men, 21 women, mean age 59.2). Normal human serum from a commercial supplier (Precision Med; 25 men, 15 women, mean age 58.7) was matched for age and sex. The figure for the plasminogen binding test gives N = 11 to 20 per group. Cells were the nonmalignant, transformed human mesothelial cell line Met-5A. Collection dates are not given.
• Exposure: Libby amphibole (LA) asbestos, through the serum donors' prior exposure; no exposure measurements are reported. In the experiments, the cells were exposed to pooled serum (MCAA-positive, cleared of anti-plasminogen antibodies, or cleared of all IgG) and to a commercial inhibitory anti-plasminogen antibody, not to asbestos fibers.
• Main results:
  • Of the 143 size fractions of mesothelial cell surface proteins, MCAA binding was statistically highest in fractions 27 and 42 (P < 0.05) compared with all other fractions.
  • Proteins found in both high-binding fractions but not in the non-binding fraction 30 were compared. Among these, plasminogen (PLG) had the highest MASCOT score.
  • Mean binding of pooled MCAA-positive serum to purified plasminogen was three times higher (P < 0.01) than that of MCAA-negative samples or normal human serum.
  • 88% of the MCAA-positive samples showed binding to plasminogen. The authors reported this percentage was significantly higher than in MCAA-negative or normal human serum.
  • In Met-5A cells, both a commercial plasminogen-inhibiting antibody and MCAA increased collagen deposition above cells treated with cleared serum (P values 3.4E-4 and 1.1E-5, respectively).
  • MCAA-positive serum cleared of anti-plasminogen antibodies bound Met-5A cells less than whole MCAA-positive serum (P = 3.6E-7) but more than serum cleared of all IgG (P = 4.1E-8). The authors took this to indicate that other MCAA target other cell proteins.
  • Collagen deposition induced by MCAA-positive serum was significantly reduced (P = 0.001), virtually to control levels, when anti-plasminogen antibodies were removed from the serum.
• The authors' conclusion: The authors concluded that a subset of MCAA preferentially binds plasminogen, and that this binding likely contributes to a sizable portion of the excess collagen deposition seen in Met-5A cells exposed to asbestos-induced MCAA.
• Limitations the authors state:
  • Not all fractions could be analyzed, so the efficiency of surface-protein recovery could not be validated. Some potential MCAA targets may not have been extracted or detected.
  • The commercial anti-plasminogen antibody used for comparison was a mouse monoclonal antibody, quite different in isotype and epitope specificities from the human autoantibodies.
  • The work was done in cultured cells. The authors stated that the role of the plasminogen–plasmin system in excess collagen deposition still needs to be confirmed in vivo in a mouse model.

Topics

Medical terms in this study, explained in our Lexicon: Libby amphiboles, Mesothelial Cells, Collagen, Pleural Thickening, Deposition.

Related studies

• Libby amphibole-induced mesothelial cell autoantibodies promote collagen deposition in mice — J. Gilmer, K. Serve, et al. (2016)
• Asbestos-associated mesothelial cell autoantibodies promote collagen deposition in vitro — K.M. Serve, B. Black, et al. (2013)
• Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages — D.J. Blake, S.A. Wetzel, et al. (2008)
• Mesothelial cell autoantibodies upregulate transcription factors associated with fibrosis — J. Gilmer, T. Harding, et al. (2017)
• Collagen accumulation over time in the murine lung after exposure to crocidolite asbestos or Libby amphibole — A.M. Smartt, M. Brezinski, et al. (2010)
• Mesothelial cell and anti-nuclear autoantibodies associated with pleural abnormalities in an asbestos exposed population of Libby MT — L.S. Marchand, S. St-Hilaire, et al. (2012)

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