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

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.

Keywords: Asbestos; Libby amphibole; autoimmunity; collagen; plasminogen; pleural fibrosis; proteomics.

Figure 1

Protein fraction ELISA for MCAA binding. The fractions with sufficient protein were then analyzed using an ELISA protocol as described in the Materials and Methods. Samples were prepared in triplicate and analyzed by absorbance on a plate reader set to 450 nm. Standard error of the mean was calculated; and samples were considered of interest if they had an absorbance significantly higher than sample 78, which had a mean absorbance of 1 (*P < 0.01, n = 3 wells per fraction) by unpaired two‐tailed t‐test. This graph is representative of three separate experiments.

Figure 2

Binding of plasminogen by MCAA via ELISA. Plasminogen was coated in a 96 well plate and various treatment groups were added as described in the ELISA protocol. Normal Human Serum was obtained from Precision Med (Solana Beach, CA). The other two groups were sera taken from the population of Libby Montana, either positive or negative for MCAA. The plate was read at 450 nm. Top: Data indicate the average number of standard deviations above the mean absorbance for known negative control sera. N = 11–20 in each subset, *P < 0.01 by one‐way ANOVA and by two‐tailed t‐tests comparing to Normal Human Sera. Bottom: The percent positive for anti‐plasminogen among the subsets of serum samples. N = 11–20 in each subset, *P < 0.01 by Fisher's exact test.

Figure 3

STRING summary networks. A brief search was performed using the STRING database to test the validity of the tandem mass spectrometry data. This search generated several networks that indicated proteins of interest that had functional relationships that could provide rationale for a mechanism by which PLG could affect COL1A1 expression. (A) Network view based on various comparison methods. Green correlates to genes located within 300 base pairs from each other on the genome; Dark blue relates genes by the frequency of occurrence together in various organisms; Black indicates instances of coexpression; Pink to standard molecular biology experiments; Sky blue to relationships detected in curated databases; Yellow shows relationships indicated by text‐mining of PubMed abstracts; and purple correlates to Homology. (B) Network view generated from a composite of all the scores evaluating the data supporting the connections observed in network A (Serve et al. 2013). Confidence scores were determined by calculating the probability of a functional interaction between two proteins based, off of the multiple lines of evidence. The method was validated against a baseline probability calculated from proteins with well‐known interactions from the KEGG database (Serve et al. 2013; Szklarczyk et al. 2015).

Figure 4

ELISA demonstrating the potential role of MCAA in collagen deposition. The collagen deposition induced by pooled MCAA+ serum was compared to the collagen deposition induced by a commercial antihuman plasminogen antibody. Error bars indicate SEM. Unpaired, two‐tailed t‐tests comparing the mean of treatment with No Treatment indicated that differences were statistically significant, *P < 0.05. N = 6 ELISA wells in each treatment group.

Figure 5

Binding of serum cleared of plasminogen‐targeting antibodies. Pooled serum that had been cleared of anti‐PLG antibodies was tested for MCAA+ binding to surface proteins on mesothelial cells. The binding of two samples that had been cleared for different times (12 and 24 h) were compared to binding of pooled serum cleared of all IgG (IgG Cleared) and un‐manipulated pooled MCAA+ serum. The binding of PLG‐cleared serum was higher than IgG cleared, but less than that of pooled MCAA+. The difference between groups was confirmed via unpaired, two‐tailed t‐test with P‐values <0.05 when compared with IgG Cleared (a), compared with pooled MCAA (b), and 12 h versus 24 h (c). N = 6 wells in each treatment group.

Figure 6

Collagen deposition by cells treated with serum cleared of PLG targeting MCAA. To test how much of the collagen deposition induced by MCAA could be attributed to the presence of the PLG‐targeting MCAA subset, the collagen deposition induced by pooled MCAA+ serum was compared to that induced by pooled MCAA+ serum that had been cleared of anti‐PLG antibodies. The observed difference between MCCA+ and No treatment was statistically significant when analyzed via unpaired, two‐tailed t‐test P < 0.05 (a). Clearing the anti‐PLG antibodies using affinity chromatography significantly reduced collagen production, t‐test P < 0.05 compared to MCAA treatment (b). N = 6 ELISA wells in each treatment group.