The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region
Raquel Herrero, … , Xiaoyun Fu, Thomas R. Martin
Raquel Herrero, … , Xiaoyun Fu, Thomas R. Martin
Published February 1, 2011
Citation Information: J Clin Invest. 2011;121(3):1174-1190. https://doi.org/10.1172/JCI43004.
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The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region

Abstract

Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.

Authors

Raquel Herrero, Osamu Kajikawa, Gustavo Matute-Bello, Yi Wang, Naoki Hagimoto, Steve Mongovin, Venus Wong, David R. Park, Nathan Brot, Jay W. Heinecke, Henry Rosen, Richard B. Goodman, Xiaoyun Fu, Thomas R. Martin

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Figure 6

Effect of the stalk region at the N terminus on the biological activity of human sFasL in vivo.

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Effect of the stalk region at the N terminus on the biological activity ...
(A) Structure of the short and long variants of human sFasL produced in HEK cells. The short rh-sFasL contains only the TNF domain. Both proteins have a leading 15–amino acid linker peptide encoded by the expression vector. (B–H) Effects of long and short rh-sFasL in mouse lungs 16 hours after intratracheal instillation (25 ng/g body weight). (B) Representative lung tissue sections stained with hematoxylin and eosin, showing alveolar wall thickening, vascular congestion, alveolar hemorrhage, and neutrophilic infiltrates in mice treated with long rh-sFasL. h-sFasL, human sFasL. (C–F) Effect of long rh-sFasL on lung wet weight, concentration of IgM in BAL fluid and PMNs in BAL fluid, and MPO activity in lung homogenates. (G) Analysis of cell death in lung tissue sections (TUNEL, green dots) and the overlap of differential interference contrast (DIC) image and TUNEL fluorescence signal (DIC, TUNEL). (H) Measurement of TUNEL-positive cells in lung sections (mean ± SD). (I) Single section of mouse lung, dually stained with the TUNEL method, followed by staining with an antibody to cytokeratin, and then visualized by fluorescence microscopy using a red wavelength (cytokeratin) or a green wavelength (TUNEL) and light microscopy (DIC). The merged image shows cells that are dual labeled by the TUNEL method and the cytokeratin antibody (epithelial cells). (J) Caspase-3 activity in lung homogenates. Each dot represents an individual mouse. Horizontal bars represent medians. *P < 0.01; **P < 0.001. Original magnification, ×200 (B, G, and I); insets (×400).