Biologically Active Fas Antigen and Its Cognate Ligand Are Expressed on Plasma Membrane-Derived Extracellular Vesicles

Abstract

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.

Apheresis platelet concentrates contain platelet‐derived and endothelial cell‐derived microparticles

“…As EMP carry cytoplasmic components and cell surface antigens of the donor, the effect of transfused EMP on patients might be perturbing: EMP surface expression of cell adhesion molecules, such as E‐selectin and ICAM‐1, is associated with increased binding and activation of both monocytes [31] and leucocytes [32]. In addition, EMP decrease endothelial nitric oxide release in vitro [33,34] and increase arterial stiffness, stimulate activation and apoptosis of cells [35] and may also serve as potential carriers of immunogenic and pathogenic compounds. As EMP carry HLA antigens in high density [20], transfer of EMP into the recipient might counteract the aim of reducing transfused HLA load by leucodepletion.…”

Serum cytochrome c indicates in vivo apoptosis and can serve as a prognostic marker during cancer therapy

“…A number of proteins, such as HIV‐Tat, thioredoxin, interleukin 1β and basic fibroblast growth factor, which lack a signal peptide, are exported by alternative, not well‐defined pathways 26. For example, death ligands, such as CD95L and TRAIL, are stored in microvesicles that are released on demand, upon activation or apoptosis 27, 28. However, pharmacologic inhibitor experiments (data not shown) argue against a related release mechanism by cytochrome c.…”

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

“…Whether these vesicles are derived from the plasma membrane of APC or internal sources remains an important unanswered question. Albanese et al [37] described release of vesicles expressing Fas and Fas ligand (FasL) from the plasma membrane of cancer cells. Such "exfoliation" was postulated as a means for remote killing of Fas-expressing target cells.…”