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Pretreatment of HL60 cells with Deoxyspergualin enhances trail-induced apoptosis independent of caspase 3 activation
Pretreatment of HL60 Cells With Deoxyspergualin Enhances Trail-Induced Apoptosis Independent of Caspase 3 Activation J. Wu, J. He, S. Sooudi, X.L. Jiang, P. Ray, F. Thomas, and J.M. Thomas
D
EOXYSPERGUALIN (DSG) has a number of unique effects on the immune system.1 Our group recently demonstrated that DSG is a potent inducer of immune tolerance when used for only 14 days in combination with a T cell reducing agent. The mechanism of action of this tolerogenic effect has not been known until recently when our group demonstrated that this agent blocks the early pro-inflammatory cytokine storm seen after transplantation. In addition, studies of sessile dendritic cells showed that DSG caused arrest of DC maturation, which is felt to be a critical factor in modulating the immunogeneic to tolerogeneic orientation of the DC. Our group recently demonstrated that this DC maturation arrest was related to the ability of DSG to block Rel- translocation, thus blocking NFK.2 Ultimately, to block antigen presentation by a primed DC, the DC must undergo apoptosis however. Little is known concerning DC apoptosis but TRAILinduced APO has been implicated.3,4 In this study, we utilize the HL60 pro-monocytic cell line as a surrogate for the DC. APO of HL60 cells was induced by addition of TRAIL (APO-2) ligand to HL60 cell cultures with and without Deoxyspergualin at a pharmacological tissue level of 60 gm/dl. The robust APO (measured by Annexin V staining and flow cytometry) seen with TRAIL was enhanced a mean of 45.6 ⫾ 8% when DSG was added to the culture. DSG alone did not enhance APO of resting HL60 cells (P ⬎ .10) and HL60 cells activated by TNF␣ (P ⬎ .10). Caspase 3 was constitutively active in HL60 cells did but not mediate DSG amplification of TRAIL-induced APO. Western Blot studies demonstrated that Deoxyspergualin amplification of TRAIL-induced apoptosis was not associated with caspase 3 activation. Steady-state protein levels of
0041-1345/01/$–see front matter PII S0041-1345(00)02789-5
hsp70 were also decreased by DSG in a dose-dependent manner. In summary, DSG has been shown to block DC maturation, known to be a crucial factor in the orientation of the DC towards a tolerogenic state. In addition, this study suggests that DSG enhances TRAIL/APO2 induced apoptosis in cells of the human monocytic/DC lineage. This enhancement was independent of caspase 3 but associated with downregulation of the hsp70 and Rel-/p50 protein levels. These findings support the concept that DSG promotes tolerogenicity by both blocking DC maturation and by enhancing subsequent DC apoptosis via a TRAILmediated APO mechanism. REFERENCES 1. Thomas FT, Tepper MA, Thomas JM, et al: Ann NY Acad Sci 685:175, 1993 2. Thomas JM, Eckhoff DE, Contreras JL, et al: Transplantation 69:2497, 2000 3. Wang J, Zheng L, Lobito A, et al: Cell 98:47, 1999 4. Kaplan MJ, Ray D, Mo RR, et al: TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4⫹ T cell killing of antigenpresenting macrophages. J Immunol 164:2897, 2000 From the Division of Immunobiology, Department of Surgery at The University of Alabama at Birmingham, Birmingham, Alabama. Supported by NIH Grant #DK-57958-02 and a CRADA Grant from Novartis. Address reprint requests to Francis T. Thomas, MD, Department of Surgery, Suite 557, Boshell Diabetes Research and Education Building, 1808 Seventh Avenue, South, Birmingham, AL 35294-0012.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
278
Transplantation Proceedings, 33, 278 (2001)
D
EOXYSPERGUALIN (DSG) has a number of unique effects on the immune system.1 Our group recently demonstrated that DSG is a potent inducer of immune tolerance when used for only 14 days in combination with a T cell reducing agent. The mechanism of action of this tolerogenic effect has not been known until recently when our group demonstrated that this agent blocks the early pro-inflammatory cytokine storm seen after transplantation. In addition, studies of sessile dendritic cells showed that DSG caused arrest of DC maturation, which is felt to be a critical factor in modulating the immunogeneic to tolerogeneic orientation of the DC. Our group recently demonstrated that this DC maturation arrest was related to the ability of DSG to block Rel- translocation, thus blocking NFK.2 Ultimately, to block antigen presentation by a primed DC, the DC must undergo apoptosis however. Little is known concerning DC apoptosis but TRAILinduced APO has been implicated.3,4 In this study, we utilize the HL60 pro-monocytic cell line as a surrogate for the DC. APO of HL60 cells was induced by addition of TRAIL (APO-2) ligand to HL60 cell cultures with and without Deoxyspergualin at a pharmacological tissue level of 60 gm/dl. The robust APO (measured by Annexin V staining and flow cytometry) seen with TRAIL was enhanced a mean of 45.6 ⫾ 8% when DSG was added to the culture. DSG alone did not enhance APO of resting HL60 cells (P ⬎ .10) and HL60 cells activated by TNF␣ (P ⬎ .10). Caspase 3 was constitutively active in HL60 cells did but not mediate DSG amplification of TRAIL-induced APO. Western Blot studies demonstrated that Deoxyspergualin amplification of TRAIL-induced apoptosis was not associated with caspase 3 activation. Steady-state protein levels of
0041-1345/01/$–see front matter PII S0041-1345(00)02789-5
hsp70 were also decreased by DSG in a dose-dependent manner. In summary, DSG has been shown to block DC maturation, known to be a crucial factor in the orientation of the DC towards a tolerogenic state. In addition, this study suggests that DSG enhances TRAIL/APO2 induced apoptosis in cells of the human monocytic/DC lineage. This enhancement was independent of caspase 3 but associated with downregulation of the hsp70 and Rel-/p50 protein levels. These findings support the concept that DSG promotes tolerogenicity by both blocking DC maturation and by enhancing subsequent DC apoptosis via a TRAILmediated APO mechanism. REFERENCES 1. Thomas FT, Tepper MA, Thomas JM, et al: Ann NY Acad Sci 685:175, 1993 2. Thomas JM, Eckhoff DE, Contreras JL, et al: Transplantation 69:2497, 2000 3. Wang J, Zheng L, Lobito A, et al: Cell 98:47, 1999 4. Kaplan MJ, Ray D, Mo RR, et al: TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4⫹ T cell killing of antigenpresenting macrophages. J Immunol 164:2897, 2000 From the Division of Immunobiology, Department of Surgery at The University of Alabama at Birmingham, Birmingham, Alabama. Supported by NIH Grant #DK-57958-02 and a CRADA Grant from Novartis. Address reprint requests to Francis T. Thomas, MD, Department of Surgery, Suite 557, Boshell Diabetes Research and Education Building, 1808 Seventh Avenue, South, Birmingham, AL 35294-0012.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
278
Transplantation Proceedings, 33, 278 (2001)