A non-cleavable mutant of Fas ligand does not prevent neutrophilic destruction of islet transplants

Background. Fas ligand (FasL) mediates apoptosis of susceptible Fas-expressing lymphocytes, and may contribute to the maintenance of peripheral tolerance. In transplantation models, however, artificial expres- sion of FasL on cellular as well as islet transplants results in accelerated rejection by neutrophils. The mechanism of the neutrophilic response to FasL ex- pression is unknown. FasL, like other members of the tumor necrosis factor family, is cleaved to a soluble form by metalloproteases. We tested the hypothesis that soluble FasL (sFasL) was responsible for neutro- phil migration by creating a non-cleavable mutant of FasL. Methods. Three mutants of FasL with serial dele- tions in the putative proteolytic cleavage site of hu- man FasL were made using inverse polymerase chain reaction. The relative fractions of sFasL and mem- brane-bound FasL were assessed by Western blot and immunoprecipitation, as well as by cytotoxicity assay using Fas-expressing target cells. The fully non-cleav- able mutant was transduced into murine islets as well as myoblasts and tumor cell lines, and tested in a mu- rine transplantation model. Results. Serial deletions in the putative metallopro- tease site of FasL resulted in a fully non-cleavable mutant of FasL (ncFasL). Expression of ncFasL in tu- mor lines induced higher levels of apoptosis in Fas bearing targets than wild-type FasL. Transplantation of ncFasL-expressing islets under the kidney capsule of allogenic mice resulted in accelerated rejection identical to that seen with wild-type Fas ligand-expressing islets. Myoblasts and tumor cell lines express- ing ncFasL also induced neutrophil infiltration. Conclusions. Membrane-bound Fas ligand is fully ca- pable of inducing a neutrophilic response to trans- plants, suggesting an activation by Fas ligand of neu- trophil chemotactic factors.