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 expression of FasL on cellular as well as islet transplants results in accelerated rejection by neutrophils. The mechanism of the neutrophilic response to FasL expression 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 neutrophil migration by creating a non-cleavable mutant of FasL.


METHODS
Three mutants of FasL with serial deletions in the putative proteolytic cleavage site of human FasL were made using inverse polymerase chain reaction. The relative fractions of sFasL and membrane-bound FasL were assessed by Western blot and immunoprecipitation, as well as by cytotoxicity assay using Fas-expressing target cells. The fully non-cleavable mutant was transduced 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 FasL resulted in a fully non-cleavable mutant of FasL (ncFasL). Expression of ncFasL in tumor 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 expressing ncFasL also induced neutrophil infiltration.


CONCLUSIONS
Membrane-bound Fas ligand is fully capable of inducing a neutrophilic response to transplants, suggesting an activation by Fas ligand of neutrophil chemotactic factors.
Kang
S M
SM
Department of Surgery, Hormone Research Institute, and University of California, San Francisco 94143, USA.
Braat
D
D
Schneider
D B
DB
O'Rourke
R W
RW
Lin
Z
Z
Ascher
N L
NL
Dichek
D A
DA
Baekkeskov
S
S
Stock
P G
PG
D000818:Animals; D019556:COS Cells; D053222:Fas Ligand Protein; D006801:Humans; D007515:Islets of Langerhans / Q000473:pathology*; D016381:Islets of Langerhans Transplantation; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology* / Q000627:therapeutic use; D051379:Mice; D009154:Mutation; D009504:Neutrophils / Q000502:physiology*; D013329:Structure-Activity Relationship
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins