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Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes
Abstract:
Fas ligand (FasL) is a type II membrane protein. Binding of FasL to its receptor, Fas, induces apoptosis. Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form. Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris. The purified rFasL was found to be glycosylated and to exist as a trimer. The rFasL was effective in inducing apoptosis in a Fas-expressing T cell or a fibroblast cell line. The ID50 of rFasL for mouse Fas-expressing T cells was about 0.5 ng/ml. The killing process with rFasL was quick. That is, >80% Fas-expressing mouse cells were killed within 1 h by a saturation concentration of human rFasL. Intravenous administration of 500 microg of human rFasL had a lethal effect in mice. When the mice were pretreated with Propionibacterium acnes, the subsequent injection of 30 microg of human rFasL induced hepatic failure and killed the mice within 24 h. These results indicated that the soluble human FasL is active in inducing apoptosis in vitro and in vivo, and its deleterious effect may be strengthened in patients who are suffering from bacterial infection.
Insights
Recombinant soluble Fas ligand (FasL) effectively induces apoptosis in vitro and in vivo. This soluble FasL has lethal effects in mice, which are amplified by bacterial infection, suggesting potential risks for infected patients.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Fas ligand (FasL) is a type II membrane protein that induces apoptosis upon binding its receptor, Fas.
- Matrix metalloproteinases cleave membrane-bound FasL to generate its active soluble form.
Purpose of the Study:
- To produce and characterize recombinant human soluble FasL (rFasL).
- To evaluate the apoptotic activity and in vivo effects of rFasL.
Main Methods:
- Production of human soluble rFasL in Pichia pastoris.
- Purification and characterization of rFasL (glycosylation, trimerization).
- In vitro apoptosis assays using Fas-expressing cell lines.
- In vivo studies involving intravenous administration of rFasL in mice, with and without Propionibacterium acnes pretreatment.
Main Results:
- Purified rFasL was glycosylated and existed as a trimer.
- rFasL effectively induced apoptosis in Fas-expressing T cells and fibroblasts with an ID50 of approximately 0.5 ng/ml for mouse T cells.
- >80% of Fas-expressing mouse cells were killed within 1 hour by saturation concentrations of rFasL.
- Intravenous administration of 500 µg rFasL was lethal to mice.
- Pretreatment with Propionibacterium acnes enhanced the lethality of a lower dose (30 µg) of rFasL, causing hepatic failure and death within 24 hours.
Conclusions:
- Soluble human FasL is biologically active in inducing apoptosis both in vitro and in vivo.
- The deleterious effects of soluble FasL can be potentiated in the context of bacterial infection, indicating potential clinical implications.