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

Background Fas ligand (FasL) mediates apoptosis of susceptible Fas-expressi ng lymphocytes, and may contribute to the maintenance of peripheral toleran ce. In transplantation models, however, artificial expression of Fast on ce llular as well as islet transplants results in accelerated rejection by neu trophils. The mechanism of the neutrophilic response to Fast expression is unknown, Fast, like other members of the tumor necrosis factor family, is c leaved to a soluble form by metalloproteases, We tested the hypothesis that soluble Fast (sFasL) was responsible for neutrophil migration by creating a non-cleavable mutant of Fast. Methods. Three mutants of Fast with serial deletions in the putative proteo lytic cleavage site of human Fast were made using inverse polymerase chain reaction. The relative fractions of sFasL and membrane-bound Fast were asse ssed by-Western blot and immunoprecipitation, as well as by cytotoxicity as say using Fas-expressing target cells. The fully non-cleavable mutant was t ransduced into murine islets as well as myoblasts and tumor cell lines, and tested in a murine transplantation model. Results. Serial deletions in the putative metalloprotease site of Fast resu lted in a fully non-cleavable mutant of Fast (ncFasL). Expression of ncFasL in tumor lines induced higher levels of apoptosis in Fas bearing targets t han wild-type Fast. Transplantation of ncFasL-expressing islets under the k idney capsule of allogenic mice resulted in accelerated rejection identical to that seen with wild-type Fas ligand-expressing islets, Myoblasts and tu mor cell lines expressing ncFasL also induced neutrophil infiltration. Conclusions. Membrane-bound Fas ligand is fully capable of inducing a neutr ophilic response to transplants, suggesting an activation by Fas ligand of neutrophil chemotactic factors.