Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity

Abstract

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-α undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-α) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

A novel mechanism for HIV1-mediated bystander CD4+ T-cell death: neighboring dying cells drive the capacity of HIV1 to kill noncycling primary CD4+ T cells

“…The 39-40 kDa band corresponds to the described MW of the unprocessed, microvesicle-bound form of CD95L. 52,56 (d) Purified unstimulated primary CD4 þ T cells (10 6 /ml) were incubated with viruses (500 ng p24/ml) purified from living HeLa cells transfected with NL4-3 or with NL4-3/Denv, and with either supernatants from the Neuro-2a FasL cell line (Neuro-2a FasL), which contain microvesicle-bound, unprocessed CD95L, 57 51,[53][54][55] Western blot analysis of the purified supernatants of living or dying (serum deprived) control HeLa cells, using a CD95L-specific antibody, showed the presence of a 39-40 kDa CD95L in the latter (as in the positive control Jurkat cell cytoplasmic lysates) and no detectable CD95L in the former (Figure 5c). To further investigate the potential role of CD95L, we incubated CD4 þ T cells with supernatants from the Neuro-2a FasL cell line, which contain microvesicle-bound, unprocessed CD95L released by these cells.…”

“…The microvesicle-bound, unprocessed form of CD95L has a higher aggregation degree, and hence death signaling efficiency, than its soluble 26 kDa form. 54 However, a similar aggregation degree can be achieved by crosslinking soluble recombinant (sr) FLAG-tagged CD95L, using FLAG-specific antibodies. 54 We incubated CD4 þ T cells with either FLAG-specific antibody-crosslinked soluble recombinant FLAG-tagged CD95L (srCD95L), noncrosslinked srCD95L, or the FLAGspecific antibody alone, in the presence of either NL4-3 or NL4-3/Denv viruses purified from living transfected HeLa cells.…”

“…54 However, a similar aggregation degree can be achieved by crosslinking soluble recombinant (sr) FLAG-tagged CD95L, using FLAG-specific antibodies. 54 We incubated CD4 þ T cells with either FLAG-specific antibody-crosslinked soluble recombinant FLAG-tagged CD95L (srCD95L), noncrosslinked srCD95L, or the FLAGspecific antibody alone, in the presence of either NL4-3 or NL4-3/Denv viruses purified from living transfected HeLa cells. Crosslinked srCD95L synergized in a dose-dependent manner with NL4-3, but not with NL4-3/Denv in inducing CD4 þ T-cell killing (Figure 5d), while neither nonaggregated srCD95L nor the anti-FLAG antibody affected CD4 þ T-cell survival (data not shown).…”

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…sFasL can either cause apoptosis at a distant site 6,7 or alternatively dampen the apoptotic response by blocking Fas receptors. 8,9 The fate of the remaining membrane-anchored N-terminal portion of FasL, which comprises a transmembrane region and the proline-rich FasL intracellular domain (FasL ICD), has not been studied so far.…”

Combination of Human Fas (CD95/Apo-1) Ligand with Adriamycin Significantly Enhances the Efficacy of Antitumor Response

“…Several studies have shown that FasL expression is implicated in HCC and is enhanced in regions with infiltrating inflammatory cells on the margins of the cancerous tissue (18)(19)(20). In vitro studies have revealed that the capacity of naturally processed sFasL to induce apoptosis is less potent than that of cell surface FasL, possibly due to decreased capability to cross-link Fas (21,22). On the other hand, in vivo studies have shown that injection of sFasL can induce tumor cell apoptosis and hepatocyte apoptosis, which cause liver failure (23,24).…”