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Class I MHC mediates programmed cell death in a Fas-independent manner
ELSEVIER
Class I MHC Mediates Programmed Fas-Independent Manner E.S. Woodle,
D.M. Smith, J.A. Bluestone,
Cell Death in a
D.R. Green, and E.W. Skowronski
P
rogrammed cell death has recently been shown to play a central role in several basic immunologic processes, including T and B cell negative selection, downregulation of immune responses, and immune privilege.‘,’ Although Fas/ Fas ligand interactions have been implicated mechanistically in immune privilege,* other death-signaling pathways complementary to Fas may provide a means for achieving or enhancing immune privilege. We have previously shown that 5H7, a MAb specific for a monomorphic determinant expressed by the alpha 3 domain of human class I MHC molecules, induces apoptosis in lymphoid cells with a potency equivalent to that of anti-Fas MAb. Studies were conducted to examine the nature of the class I MHC death signal. METHODS
Apoptosis was quantitated using several techniques including ethidium bromideiacridine orange staining, viable cell recovery, FACS analysis following propidium iodide staining, and a TUNELbased FACS assay.
MHC-induced apoptosis was not affected. Finally, 5H7 and anti-Fas mAb demonstrated additive effects in inducing apoptosis. DISCUSSION
Of the various signaling pathways for induction of apoptosis, the Fas pathway has been the most extensively characterized. Previous reports have indicated that the Fas signaling pathway does not require gene expression, and that ceramide production may be involved in Fas-mediated death signaling.” Interleukin-1 converting enzyme activity has also been proposed to play a central role in Fasmediated death signaling.4 The present studies indicate that the Class I MHC-mediated death signal is distinct from the Fas pathway. The observation that Fas- and Class I MHCderived death signals provide additive effects raises the intriguing possibility that these two signals may act in concert during basic immunologic processes such as T cell killing and the veto phenomenon. CONCLUSIONS
RESULTS
Like Fas, 5H7-induced programmed cell death did not require the induction of gene expression as inhibitors of protein (cycloheximide) and RNA synthesis (actinomycin D) did not influence 5H7-induced apoptosis. However, two biochemical processes involved in the Fas signaling pathway (ceramide production via sphingomyelinase activity, and activation of ICE (interleukin-1 converting enzyme) were not involved in 5H7-mediated apoptosis. The sphingomyelinase/ceramide pathway was evaluated using LCL lines generated from patients with Niemann-Pick disease (a congenital disorder characterized by a deficiency of acidic sphingomyelinase). Niemann-Pick (but not control) LCL lines were resistant to Fas-induced apoptosis (although they expressed Fas antigen), whereas 5H7 induced marked apoptosis. Phorbol ester (which antagonizes the ceramide pathway) did not antagonize 5H7-induced apoptosis, thus supporting the lack of a role for ceramide in Class I MHC-induced apoptosis. Specific inhibition of ICE activity via ICE inhibitors (Y-VAD and Z-VAD) completely abrogated Fas-mediated apoptosis in LCL lines, whereas Class I
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
(1) Like Fas, induction of gene expression is not required for transduction of the Class I MHC death signal; (2) unlike Fas, Class I MHC-mediated death signaling does not involve either ceramide production or activation of ICE activity; and (3) simultaneous signaling via Fas and Class I MHC augments apoptosis induction. REFERENCES 1. Thompson CB: Science 267:1456, 1995 2. Griffith TS, Brunner T, Fletcher SM, et al: Science 270:1189, 1995 3. Tepper 4. Enari 1996
CC, Jayadev M. Talanian
S, Liu B, et al: PNAS 92:8443, RV, Wong
WW, et al: Nature
1995 380:72X
From the Section of Transplantation, Department of Surgery, University of Chicago; La Jolla Institute for Allergy and Immunology.
0041-1345/97/$17.00 PII SO041 -1345(96)00429-O
1101
Transplantation
Proceedings, 29, 1101 (1997)
Class I MHC Mediates Programmed Fas-Independent Manner E.S. Woodle,
D.M. Smith, J.A. Bluestone,
Cell Death in a
D.R. Green, and E.W. Skowronski
P
rogrammed cell death has recently been shown to play a central role in several basic immunologic processes, including T and B cell negative selection, downregulation of immune responses, and immune privilege.‘,’ Although Fas/ Fas ligand interactions have been implicated mechanistically in immune privilege,* other death-signaling pathways complementary to Fas may provide a means for achieving or enhancing immune privilege. We have previously shown that 5H7, a MAb specific for a monomorphic determinant expressed by the alpha 3 domain of human class I MHC molecules, induces apoptosis in lymphoid cells with a potency equivalent to that of anti-Fas MAb. Studies were conducted to examine the nature of the class I MHC death signal. METHODS
Apoptosis was quantitated using several techniques including ethidium bromideiacridine orange staining, viable cell recovery, FACS analysis following propidium iodide staining, and a TUNELbased FACS assay.
MHC-induced apoptosis was not affected. Finally, 5H7 and anti-Fas mAb demonstrated additive effects in inducing apoptosis. DISCUSSION
Of the various signaling pathways for induction of apoptosis, the Fas pathway has been the most extensively characterized. Previous reports have indicated that the Fas signaling pathway does not require gene expression, and that ceramide production may be involved in Fas-mediated death signaling.” Interleukin-1 converting enzyme activity has also been proposed to play a central role in Fasmediated death signaling.4 The present studies indicate that the Class I MHC-mediated death signal is distinct from the Fas pathway. The observation that Fas- and Class I MHCderived death signals provide additive effects raises the intriguing possibility that these two signals may act in concert during basic immunologic processes such as T cell killing and the veto phenomenon. CONCLUSIONS
RESULTS
Like Fas, 5H7-induced programmed cell death did not require the induction of gene expression as inhibitors of protein (cycloheximide) and RNA synthesis (actinomycin D) did not influence 5H7-induced apoptosis. However, two biochemical processes involved in the Fas signaling pathway (ceramide production via sphingomyelinase activity, and activation of ICE (interleukin-1 converting enzyme) were not involved in 5H7-mediated apoptosis. The sphingomyelinase/ceramide pathway was evaluated using LCL lines generated from patients with Niemann-Pick disease (a congenital disorder characterized by a deficiency of acidic sphingomyelinase). Niemann-Pick (but not control) LCL lines were resistant to Fas-induced apoptosis (although they expressed Fas antigen), whereas 5H7 induced marked apoptosis. Phorbol ester (which antagonizes the ceramide pathway) did not antagonize 5H7-induced apoptosis, thus supporting the lack of a role for ceramide in Class I MHC-induced apoptosis. Specific inhibition of ICE activity via ICE inhibitors (Y-VAD and Z-VAD) completely abrogated Fas-mediated apoptosis in LCL lines, whereas Class I
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
(1) Like Fas, induction of gene expression is not required for transduction of the Class I MHC death signal; (2) unlike Fas, Class I MHC-mediated death signaling does not involve either ceramide production or activation of ICE activity; and (3) simultaneous signaling via Fas and Class I MHC augments apoptosis induction. REFERENCES 1. Thompson CB: Science 267:1456, 1995 2. Griffith TS, Brunner T, Fletcher SM, et al: Science 270:1189, 1995 3. Tepper 4. Enari 1996
CC, Jayadev M. Talanian
S, Liu B, et al: PNAS 92:8443, RV, Wong
WW, et al: Nature
1995 380:72X
From the Section of Transplantation, Department of Surgery, University of Chicago; La Jolla Institute for Allergy and Immunology.
0041-1345/97/$17.00 PII SO041 -1345(96)00429-O
1101
Transplantation
Proceedings, 29, 1101 (1997)