Granzyme B is dispensable for immunologic tolerance to self in a murine model of systemic lupus erythematosus.
OBJECTIVE
Proteolytic autoantigen cleavage by the serine protease granzyme B has been implicated in the development of systemic autoimmune disease; however, there has been no conclusive demonstration of a pathogenic role for granzyme B in autoimmunity. In this study, we evaluated the role of granzyme B in a murine model of autoimmunity.


METHODS
To identify potential novel granzyme B substrates, complementary DNAs encoding nuclear factor 45 (NF45) and NF90 were used to generate (35)S-methionine-labeled proteins by coupled in vitro transcription/translation. Radiolabeled proteins were then incubated with purified recombinant granzyme B or caspases, and the cleavage products were analyzed by autoradiography. We also immunized granzyme B-deficient and granzyme B-intact mice with the mineral oil pristane. Production of autoantibodies directed against granzyme B substrates in response to pristane was evaluated by Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay.


RESULTS
The double-stranded RNA-binding protein NF90 was identified as a novel substrate for caspases and granzyme B, both in vitro and in vivo. NF90 is uniquely cleaved by granzyme B in vitro; however, pristane immunization still induced anti-NF90 antibodies in granzyme B-deficient mice. Pristane-treated granzyme B-deficient mice also produced antibodies directed against the U1-70-kd antigen, a previously identified granzyme B substrate. Last, antibodies directed against U1-70 kd arose spontaneously in granzyme B-deficient mice.


CONCLUSIONS
These results demonstrate that granzyme B is not required for the production of autoantibodies directed against antigens that are granzyme B substrates in vitro. The data also suggest a protective role for this proapoptotic protease in systemic autoimmunity.
Graham
Kareem L
KL
Stanford University School of Medicine, Stanford, California, USA.
Thibault
Donna L
DL
Steinman
Jonathan B
JB
Okeke
Lance
L
Kao
Peter N
PN
Utz
Paul J
PJ
D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D015551:Autoimmunity / Q000276:immunology*; D005260:Female; D053804:Granzymes; D007108:Immune Tolerance / Q000276:immunology*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D051379:Mice; D023421:Models, Animal; D012697:Serine Endopeptidases / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases