Sm. Kang et al., A non-cleavable mutant of Fas ligand does not prevent neutrophilic destruction of islet transplants, TRANSPLANT, 69(9), 2000, pp. 1813-1817
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.