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Interferon gamma induces dendritic cell apoptosis via both nitric oxide-dependent and independent mechanisms
SBAS MEETING
Abstracts From the 11th Annual Society of Black Academic Surgeons Meeting 1 Interferon gamma induces dendritic cell apoptosis via both nitric oxidedependent and independent mechanisms Stanford A, Chen Y, Hoffman R, Ford HR. From the Departments of Surgery, Children’s Hospital of Pittsburgh and University of Pittsburgh. Pittsburgh, Pennsylvania. Purpose: Dendritic cells (DCs) play a crucial role in amplifying the immune response by promoting antigen presentation, T-lymphocyte proliferation, proinflammatory cytokine and nitric oxide (NO) production. However, excess production of cytokines and NO may exert profound regulatory effects on DC function by inducing apoptosis. We have previously shown that the NO donor, s-nitroso-N-acetyl penicillamine (SNAP), induces DC apoptosis via a caspasedependent pathway. In this study we examined whether interferon-␥ (IFN-␥) can induce DC apoptosis, and the inherent mechanisms involved. Methods: DC2.4 cells, a bone marrow derived cell line (Dana Farber Cancer Institute), were incubated with IFN-␥ (100 u/mL) in the presence or absence of 1 mM NIO, a specific inhibitor of inducible NO synthase. Supernatant nitrite (NO⫺ 2 ) levels were measured using the Greiss reaction. Flow cytometry with Annexin V and propidium iodide staining were used to measure apoptosis and necrosis. Cell surface expression of Fas was measured using FITC-labeled antibody. Results: IFN-␥ induced DC2.4 apoptosis and necrosis (see Table). Addition of NIO suppressed NO⫺ 2 generation and cell death, and conversely increased live cells, although not to the level of control. There was a 4-fold increase in Fas expression in IFN-␥ treated cells, which was unaffected by addition of NIO. Media †IFN-␥ IFN-␥ ⫹NIO
% Live
% Death
NO2ⴚ (uM)
Fas
84.1 ⫾ 3.2 23.7 ⫾ 3.4 61.1 ⫾ 3.4
15.8 ⫾ 3.2 76.2 ⫾ 3.4 38.8 ⫾ 3.4
4.0 ⫾ 0.3 53.5 ⫾ 12 3.1 ⫾ 1.1
14.8 ⫾ 10 64.9 ⫾ 19 66.8 ⫾ 21
Conclusions: IFN-␥-induced DC2.4 apoptosis is partially dependent upon NO production but independent of Fas upregulation. These observations suggest that proinflammatory cytokine production in sepsis may mediate DC apoptosis via both NO-dependent, as well as NO-independent pathways. †p ⬍ .05, versus IFN-␥ ⫹ NIO % live cells, % cell death, and NO⫺ 2.
2 Venous smooth muscle cell responses to shear and oxidative stresses Jessie ML,* Al-Badawi H,* Bonheur J,* Kadowaki H,*† Crittenden M,¶ Birjiniuk V,¶ Khuri SF,¶ Patton GM,* ‡ Watkins MT.* § From the VA Boston Healthcare System and the Departments of *Surgery, †Biochemistry, ‡Medicine, and §Pathology, Boston University School of Medicine and the ¶Department of Surgery, Harvard Medical School. Boston, Massachusetts. Introduction: Intimal hyperplasia, the leading cause of early vein graft failure, is characterized by smooth muscle cell (SMC) proliferation and migration due to synthesis of a number of growth factors including basic fibroblast growth factor (bFGF). In transition from venous to arterial conditions, human saphenous veins are exposed to simultaneous alterations in mechanical (shear stress) and oxidative (arterial oxygen tension) stresses. The stimuli responsible for SMC synthesis of bFGF remain unclear. These studies were designed to: (1) 500
Determine whether human saphenous vein SMC respond to shear stress, and (2) Determine whether the combination of the mechanical and oxidative stresses provoke SMC to synthesize bFGF. Methods: Human SMC were cultured from segments of saphenous veins remaining after bypass surgery, and seeded onto microcarrier beads. To determine whether these SMC respond to shear stress, cells were exposed to various levels of shear by stepwise increases in the stirring rate within the microcarrier flask from 5 to 40 rpm. Medium was assayed for prostaglandin E2 (PGE) by ELISA. To assess the effect of combined shear and oxidative stress on SMC synthesis of bFGF, the cells were allowed to accommodate to venous conditions of low shear stress (10 rpm) and venous oxygen tension (3-4% O2) for 30 hours. Following venous accommodation, the cells were acutely exposed to a simultaneous increase in shear (30 rpm) and arterial oxygen tension (⬵20%) for 4 hours to simulate in vitro arterialization. Conditioned medium was assayed for bFGF using an ELISA. Measurements of bFGF and PGE were normalized to cellular protein. Results: Human venous SMC exposed to sequential increases in shear stress synthesized increasing amounts of PGE (p ⬍ 0.0001, ANOVA, n ⫽ 5). A significant and sustained increase in PGE synthesis was observed between 5 and 20 rpm (1.5 ⫾ 0.3 vs 8.0 ⫾ 1.0 pg/g protein, p ⬍ 0.01). bFGF levels increased significantly (p ⬍ 0.001) after 30 minutes of in vitro arterialization and remained elevated throughout the 4-hour period. Conclusions: Human saphenous vein SMC exhibit a classic shear response as indicated by the sequential increases in PGE synthesis during brief 30-minute intervals. SMC exhibit a significant increase in bFGF synthesis in response to the combination of shear and oxidative stresses. The SMC sensors for shear and/or oxidative stress may represent genetic or pharmacologic targets to inhibit bFGF synthesis, and thereby limit the development of intimal hyperplasia.
3 Differential end organ activation of map kinases in response to sepsis and hemorrhage Carter Y, Liu G, Mendez C. From the Department of Surgery, University of South Florida, Tampa, Florida. Background: It is known that TNF and other cytokines, play pivotal roles in the initiation and perpetuation of the inflammatory response seen in the sepsis syndrome. Certain kinases, particularly p44/42 and SAPK/JNK, are thought to be involved in the pathways which lead to the production of TNF and other cytokines; and although the in vitro response has been extensively studied, the in vivo pathway activation, has not been specifically defined. The purpose of this study was to explore the difference in MAP kinase activity in various organs, in response to LPS, and to a tolerizing sublethal hemorrhage. Methods: Rats were made tolerant by SLH (femoral artery cannulation with controlled bleeding, to maintain a mean arterial pressure of 30 mmHg for 15 minutes) or sham operation. One day later, they were exposed to LPS (40 mg/kg intraperitoneally). Lung, liver and splenic tissues were harvested for protein extraction 15, 30, 45 and 60 minutes after operation (day 1) or LPS (day 2), and western blots were performed to detect the phosphorylation of p44/42 and SAPK/JNK. Results: SLH and sham operation both led to the phosphorylation of p44/42 and SAPK/JNK MAP kinases in all tissues. Activity was expressed earliest at 15
CURRENT SURGERY • © 2001 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/01/$20.00
Abstracts From the 11th Annual Society of Black Academic Surgeons Meeting 1 Interferon gamma induces dendritic cell apoptosis via both nitric oxidedependent and independent mechanisms Stanford A, Chen Y, Hoffman R, Ford HR. From the Departments of Surgery, Children’s Hospital of Pittsburgh and University of Pittsburgh. Pittsburgh, Pennsylvania. Purpose: Dendritic cells (DCs) play a crucial role in amplifying the immune response by promoting antigen presentation, T-lymphocyte proliferation, proinflammatory cytokine and nitric oxide (NO) production. However, excess production of cytokines and NO may exert profound regulatory effects on DC function by inducing apoptosis. We have previously shown that the NO donor, s-nitroso-N-acetyl penicillamine (SNAP), induces DC apoptosis via a caspasedependent pathway. In this study we examined whether interferon-␥ (IFN-␥) can induce DC apoptosis, and the inherent mechanisms involved. Methods: DC2.4 cells, a bone marrow derived cell line (Dana Farber Cancer Institute), were incubated with IFN-␥ (100 u/mL) in the presence or absence of 1 mM NIO, a specific inhibitor of inducible NO synthase. Supernatant nitrite (NO⫺ 2 ) levels were measured using the Greiss reaction. Flow cytometry with Annexin V and propidium iodide staining were used to measure apoptosis and necrosis. Cell surface expression of Fas was measured using FITC-labeled antibody. Results: IFN-␥ induced DC2.4 apoptosis and necrosis (see Table). Addition of NIO suppressed NO⫺ 2 generation and cell death, and conversely increased live cells, although not to the level of control. There was a 4-fold increase in Fas expression in IFN-␥ treated cells, which was unaffected by addition of NIO. Media †IFN-␥ IFN-␥ ⫹NIO
% Live
% Death
NO2ⴚ (uM)
Fas
84.1 ⫾ 3.2 23.7 ⫾ 3.4 61.1 ⫾ 3.4
15.8 ⫾ 3.2 76.2 ⫾ 3.4 38.8 ⫾ 3.4
4.0 ⫾ 0.3 53.5 ⫾ 12 3.1 ⫾ 1.1
14.8 ⫾ 10 64.9 ⫾ 19 66.8 ⫾ 21
Conclusions: IFN-␥-induced DC2.4 apoptosis is partially dependent upon NO production but independent of Fas upregulation. These observations suggest that proinflammatory cytokine production in sepsis may mediate DC apoptosis via both NO-dependent, as well as NO-independent pathways. †p ⬍ .05, versus IFN-␥ ⫹ NIO % live cells, % cell death, and NO⫺ 2.
2 Venous smooth muscle cell responses to shear and oxidative stresses Jessie ML,* Al-Badawi H,* Bonheur J,* Kadowaki H,*† Crittenden M,¶ Birjiniuk V,¶ Khuri SF,¶ Patton GM,* ‡ Watkins MT.* § From the VA Boston Healthcare System and the Departments of *Surgery, †Biochemistry, ‡Medicine, and §Pathology, Boston University School of Medicine and the ¶Department of Surgery, Harvard Medical School. Boston, Massachusetts. Introduction: Intimal hyperplasia, the leading cause of early vein graft failure, is characterized by smooth muscle cell (SMC) proliferation and migration due to synthesis of a number of growth factors including basic fibroblast growth factor (bFGF). In transition from venous to arterial conditions, human saphenous veins are exposed to simultaneous alterations in mechanical (shear stress) and oxidative (arterial oxygen tension) stresses. The stimuli responsible for SMC synthesis of bFGF remain unclear. These studies were designed to: (1) 500
Determine whether human saphenous vein SMC respond to shear stress, and (2) Determine whether the combination of the mechanical and oxidative stresses provoke SMC to synthesize bFGF. Methods: Human SMC were cultured from segments of saphenous veins remaining after bypass surgery, and seeded onto microcarrier beads. To determine whether these SMC respond to shear stress, cells were exposed to various levels of shear by stepwise increases in the stirring rate within the microcarrier flask from 5 to 40 rpm. Medium was assayed for prostaglandin E2 (PGE) by ELISA. To assess the effect of combined shear and oxidative stress on SMC synthesis of bFGF, the cells were allowed to accommodate to venous conditions of low shear stress (10 rpm) and venous oxygen tension (3-4% O2) for 30 hours. Following venous accommodation, the cells were acutely exposed to a simultaneous increase in shear (30 rpm) and arterial oxygen tension (⬵20%) for 4 hours to simulate in vitro arterialization. Conditioned medium was assayed for bFGF using an ELISA. Measurements of bFGF and PGE were normalized to cellular protein. Results: Human venous SMC exposed to sequential increases in shear stress synthesized increasing amounts of PGE (p ⬍ 0.0001, ANOVA, n ⫽ 5). A significant and sustained increase in PGE synthesis was observed between 5 and 20 rpm (1.5 ⫾ 0.3 vs 8.0 ⫾ 1.0 pg/g protein, p ⬍ 0.01). bFGF levels increased significantly (p ⬍ 0.001) after 30 minutes of in vitro arterialization and remained elevated throughout the 4-hour period. Conclusions: Human saphenous vein SMC exhibit a classic shear response as indicated by the sequential increases in PGE synthesis during brief 30-minute intervals. SMC exhibit a significant increase in bFGF synthesis in response to the combination of shear and oxidative stresses. The SMC sensors for shear and/or oxidative stress may represent genetic or pharmacologic targets to inhibit bFGF synthesis, and thereby limit the development of intimal hyperplasia.
3 Differential end organ activation of map kinases in response to sepsis and hemorrhage Carter Y, Liu G, Mendez C. From the Department of Surgery, University of South Florida, Tampa, Florida. Background: It is known that TNF and other cytokines, play pivotal roles in the initiation and perpetuation of the inflammatory response seen in the sepsis syndrome. Certain kinases, particularly p44/42 and SAPK/JNK, are thought to be involved in the pathways which lead to the production of TNF and other cytokines; and although the in vitro response has been extensively studied, the in vivo pathway activation, has not been specifically defined. The purpose of this study was to explore the difference in MAP kinase activity in various organs, in response to LPS, and to a tolerizing sublethal hemorrhage. Methods: Rats were made tolerant by SLH (femoral artery cannulation with controlled bleeding, to maintain a mean arterial pressure of 30 mmHg for 15 minutes) or sham operation. One day later, they were exposed to LPS (40 mg/kg intraperitoneally). Lung, liver and splenic tissues were harvested for protein extraction 15, 30, 45 and 60 minutes after operation (day 1) or LPS (day 2), and western blots were performed to detect the phosphorylation of p44/42 and SAPK/JNK. Results: SLH and sham operation both led to the phosphorylation of p44/42 and SAPK/JNK MAP kinases in all tissues. Activity was expressed earliest at 15
CURRENT SURGERY • © 2001 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/01/$20.00