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Role of signal transducer and activator of transcription pathways (STAT4 and STAT6) in pancreatitis associated lung injury
198
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
tivating drug. Levels of phosphorylated ERK1/2 and the NE marker Chromogranin A (CgA) were determined by Western Blot. Cellular growth was measured by MTT cell-proliferation assay. Results: At baseline, PC-1 2 cells had very little phosporylated ERK1/2, similar to other NE tumors. Treatment of PC-12 cells with increasing dosages of ZM336372 resulted in increased phosphorylated ERK1/2. Importantly, ZM336372 inhibited pheochromocytoma cellular proliferation. Furthermore, Raf-1 pathway activation by ZM336372 wasassociated with suppression of NE marker, CgA, by the tumor cells. Conclusions: These data suggest that Raf-1/MEK/ERK1/2 pathway activation may be a novel strategy to treat pheochromocytoma and other catecholamine-producing tumors. In pheochromocytoma cells, ZM336372 blocks cellular proliferation, and suppresses NE vasoactive peptide production. Thus, ZM336372 may be used for both therapeutic and palliative treatment for patients with pheochromocytomas.
GI II-Inflammation and Injury 98. ROLE OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION PATHWAYS (STAT4 AND STAT6) IN PANCREATITIS ASSOCIATED LUNG INJURY. Simovic MO, Ballard BR, Gray KD, Stain SC; Meharry Medical College Background: The STAT pathways are integral to the inflammatory response, and these proteins provide a direct link between the cytokine receptors and cytokine-induced gene transcription. We examined the roles of STAT4 and STAT6 in lung injury following caerulein-induced severe acute pancreatitis. We hypothesize that a modified organ expression of cytokines and chemokines that occurs in transgenic mice may affect the remote response to severe acute pancreatitis. Methods: Acute pancreatitis [13-hourly i.p. injections of caerulein (50g/kg b.w., 0.2 mL) or the same volume of saline] was induced in wild-type (BALB/cJ) and transgenic (STAT4 ⫺/⫺ or STAT 6 ⫺/⫺) mice of the same background, 7-8 weeks old. The lung tissue was collected at 1, 6, 12, 24 hours after the completion of caerulein administration. Standard histological staining (H&E) was performed, and blindly scored by a pathologist for evidence of lung injury (pulmonary edema, accumulations of neutrophils and mononuclear cells, thickness of alveolar-capillary membrane, perivascular infiltrate and hemorrhage). Pulmonary leukocyte sequestration was assessed by myeloperoxidase (MPO) activity at time of maximal enzyme activity (6 hours, n ⫽ 5) as determined in wild-type mice with pancreatitis. Results: Caerulein treated wild-type mice exhibited increased lung injury score at 1 through 12 hours, as compared to saline controls. As compared to wild-type, STAT6 - deficient mice had increased lung injury from 1 to 6 hours, with full recovery by 12 hours. An opposite pattern was observed in STAT4 - deficient mice with mild injury seen at 1 and 6 hours, and maximal injury at 12 hours. MPO activity was significantly increased at 6 hours in caerulein-treated wild-type mice compared to saline-treated controls. Caerulein treated STAT6 and STAT4 mice had increased MPO activity as compared to their saline controls. Both caerulein-treated STAT4- and STAT6-deficient mice had significantly increased MPO activity in comparison with wild-type mice with pancreatitis at 6 hours. The lung injury in STAT4 mice was reduced in the first 6 hours, but increased by 12 hours. Conclusion: We found the maximal lung injury after cerulean induced pancreatitis occurred at different time points in STAT4 and STAT6 - deficient mice. These temporal differences may suggest alternative roles in the systemic inflammatory response associated with pancreatitis. 99. IL-1 SHORT INTERFERING RNA (SIRNA) AMELIORATES LUNG INFLAMMATORY RESPONSE FOLLOWING LIVER INJURY. S. C. Glasgow 1, S. Ramachandran 1, T. S. Blackwell 2, T. Mohanakumar 1, W. C. Chapman 1; 1Wash-
ington University, St. Louis, MO, 2Vanderbilt University, Nashville, TN. INTRODUCTION: Hepatic cryoablation produces systemic and pulmonary inflammatory responses, which are notable for acutely increased levels of IL-1 and activation of NF-B-dependent pathways. We sought to reduce this lung inflammation by inhibiting IL-1 signaling in a model of hepatic ischemia-reperfusion (I/R) injury, using both transgenic mice pretreated with siRNA and homozygous knockouts. METHODS: Cultured murine alveolar macrophages were used to select an optimized IL-1-specific siRNA sequence, which then was encapsulated in liposomes and administered intraperitoneally to HLL mice (5’ HIV-LTR-Luciferase; 5’ HIV-LTR is an NF-B promoter). HLL and IL-1 receptor 1 knockout mice (IL1R1KO) underwent 35% hepatic volume cryoablation. NF-B activity in HLL mice was assessed by ex vivo bioluminescence, serum cytokines and lung chemokines assayed by ELISA, and pulmonary neutrophil content measured using a myeloperoxidase (MPO) assay. Data were analyzed by ANOVA and expressed as mean ⫾ SEM. RESULTS: IL-1 siRNA pretreatment effectively reduced circulating IL-1 levels at 4 hours post-hepatic injury (203 ⫾ 39 pg/mL vs. 500 ⫾ 68 for controls and 445 ⫾ 23 for IL1R1KO; p ⬍ 0.05). IL-6, a downstream pro-inflammatory cytokine, also was suppressed in IL-1 signaling pathway-deficient mice (IL-1 siRNA: 103 ⫾ 29 pg/mL; controls: 978 ⫾ 313; IL1R1KO: 43 ⫾ 13; p ⬍ 0.05). NF-B activation in the non-injured liver remnant of HLL mice pretreated with IL-1 siRNA was less than observed in controls (63754 ⫾ 10250 vs. 177138 ⫾ 21040 for controls; p ⬍ 0.05). Likewise, pulmonary NF-B activity in this group was significantly diminished (graph). Similarly, lung MPO content at 24 hours was unchanged from baseline in IL-1 siRNA treated animals (0.22 ⫾ 0.02 ⌬OD/min/mg protein vs. 0.29 ⫾ 0.02; p ⬍ 0.05). CONCLUSIONS: Liver injury-induced lung inflammation is mediated predominantly by IL-1. Knockdown of IL-1 expression prior to hepatic cryoablation led to significant reductions in both pro-inflammatory cytokine production and hepatic and pulmonary NF-B activation. This translated to reduced acute lung injury as demonstrated by neutrophilic influx. Similar interventions may have a role in diminishing graft dysfunction resulting from I/R injury.
100. DEVELOPMENT OF CERULEIN-INDUCED ACUTE PANCREATITIS IS INDEPENDENT OF CROSSTOLERANCE AFTER HEAT SHOCK IN MICE. Granger SR, Firpo MA, Kime M, Yang DT, Mulvihill SJ, Benjamin IJ, Glasgow RE; University of Utah
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
tivating drug. Levels of phosphorylated ERK1/2 and the NE marker Chromogranin A (CgA) were determined by Western Blot. Cellular growth was measured by MTT cell-proliferation assay. Results: At baseline, PC-1 2 cells had very little phosporylated ERK1/2, similar to other NE tumors. Treatment of PC-12 cells with increasing dosages of ZM336372 resulted in increased phosphorylated ERK1/2. Importantly, ZM336372 inhibited pheochromocytoma cellular proliferation. Furthermore, Raf-1 pathway activation by ZM336372 wasassociated with suppression of NE marker, CgA, by the tumor cells. Conclusions: These data suggest that Raf-1/MEK/ERK1/2 pathway activation may be a novel strategy to treat pheochromocytoma and other catecholamine-producing tumors. In pheochromocytoma cells, ZM336372 blocks cellular proliferation, and suppresses NE vasoactive peptide production. Thus, ZM336372 may be used for both therapeutic and palliative treatment for patients with pheochromocytomas.
GI II-Inflammation and Injury 98. ROLE OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION PATHWAYS (STAT4 AND STAT6) IN PANCREATITIS ASSOCIATED LUNG INJURY. Simovic MO, Ballard BR, Gray KD, Stain SC; Meharry Medical College Background: The STAT pathways are integral to the inflammatory response, and these proteins provide a direct link between the cytokine receptors and cytokine-induced gene transcription. We examined the roles of STAT4 and STAT6 in lung injury following caerulein-induced severe acute pancreatitis. We hypothesize that a modified organ expression of cytokines and chemokines that occurs in transgenic mice may affect the remote response to severe acute pancreatitis. Methods: Acute pancreatitis [13-hourly i.p. injections of caerulein (50g/kg b.w., 0.2 mL) or the same volume of saline] was induced in wild-type (BALB/cJ) and transgenic (STAT4 ⫺/⫺ or STAT 6 ⫺/⫺) mice of the same background, 7-8 weeks old. The lung tissue was collected at 1, 6, 12, 24 hours after the completion of caerulein administration. Standard histological staining (H&E) was performed, and blindly scored by a pathologist for evidence of lung injury (pulmonary edema, accumulations of neutrophils and mononuclear cells, thickness of alveolar-capillary membrane, perivascular infiltrate and hemorrhage). Pulmonary leukocyte sequestration was assessed by myeloperoxidase (MPO) activity at time of maximal enzyme activity (6 hours, n ⫽ 5) as determined in wild-type mice with pancreatitis. Results: Caerulein treated wild-type mice exhibited increased lung injury score at 1 through 12 hours, as compared to saline controls. As compared to wild-type, STAT6 - deficient mice had increased lung injury from 1 to 6 hours, with full recovery by 12 hours. An opposite pattern was observed in STAT4 - deficient mice with mild injury seen at 1 and 6 hours, and maximal injury at 12 hours. MPO activity was significantly increased at 6 hours in caerulein-treated wild-type mice compared to saline-treated controls. Caerulein treated STAT6 and STAT4 mice had increased MPO activity as compared to their saline controls. Both caerulein-treated STAT4- and STAT6-deficient mice had significantly increased MPO activity in comparison with wild-type mice with pancreatitis at 6 hours. The lung injury in STAT4 mice was reduced in the first 6 hours, but increased by 12 hours. Conclusion: We found the maximal lung injury after cerulean induced pancreatitis occurred at different time points in STAT4 and STAT6 - deficient mice. These temporal differences may suggest alternative roles in the systemic inflammatory response associated with pancreatitis. 99. IL-1 SHORT INTERFERING RNA (SIRNA) AMELIORATES LUNG INFLAMMATORY RESPONSE FOLLOWING LIVER INJURY. S. C. Glasgow 1, S. Ramachandran 1, T. S. Blackwell 2, T. Mohanakumar 1, W. C. Chapman 1; 1Wash-
ington University, St. Louis, MO, 2Vanderbilt University, Nashville, TN. INTRODUCTION: Hepatic cryoablation produces systemic and pulmonary inflammatory responses, which are notable for acutely increased levels of IL-1 and activation of NF-B-dependent pathways. We sought to reduce this lung inflammation by inhibiting IL-1 signaling in a model of hepatic ischemia-reperfusion (I/R) injury, using both transgenic mice pretreated with siRNA and homozygous knockouts. METHODS: Cultured murine alveolar macrophages were used to select an optimized IL-1-specific siRNA sequence, which then was encapsulated in liposomes and administered intraperitoneally to HLL mice (5’ HIV-LTR-Luciferase; 5’ HIV-LTR is an NF-B promoter). HLL and IL-1 receptor 1 knockout mice (IL1R1KO) underwent 35% hepatic volume cryoablation. NF-B activity in HLL mice was assessed by ex vivo bioluminescence, serum cytokines and lung chemokines assayed by ELISA, and pulmonary neutrophil content measured using a myeloperoxidase (MPO) assay. Data were analyzed by ANOVA and expressed as mean ⫾ SEM. RESULTS: IL-1 siRNA pretreatment effectively reduced circulating IL-1 levels at 4 hours post-hepatic injury (203 ⫾ 39 pg/mL vs. 500 ⫾ 68 for controls and 445 ⫾ 23 for IL1R1KO; p ⬍ 0.05). IL-6, a downstream pro-inflammatory cytokine, also was suppressed in IL-1 signaling pathway-deficient mice (IL-1 siRNA: 103 ⫾ 29 pg/mL; controls: 978 ⫾ 313; IL1R1KO: 43 ⫾ 13; p ⬍ 0.05). NF-B activation in the non-injured liver remnant of HLL mice pretreated with IL-1 siRNA was less than observed in controls (63754 ⫾ 10250 vs. 177138 ⫾ 21040 for controls; p ⬍ 0.05). Likewise, pulmonary NF-B activity in this group was significantly diminished (graph). Similarly, lung MPO content at 24 hours was unchanged from baseline in IL-1 siRNA treated animals (0.22 ⫾ 0.02 ⌬OD/min/mg protein vs. 0.29 ⫾ 0.02; p ⬍ 0.05). CONCLUSIONS: Liver injury-induced lung inflammation is mediated predominantly by IL-1. Knockdown of IL-1 expression prior to hepatic cryoablation led to significant reductions in both pro-inflammatory cytokine production and hepatic and pulmonary NF-B activation. This translated to reduced acute lung injury as demonstrated by neutrophilic influx. Similar interventions may have a role in diminishing graft dysfunction resulting from I/R injury.
100. DEVELOPMENT OF CERULEIN-INDUCED ACUTE PANCREATITIS IS INDEPENDENT OF CROSSTOLERANCE AFTER HEAT SHOCK IN MICE. Granger SR, Firpo MA, Kime M, Yang DT, Mulvihill SJ, Benjamin IJ, Glasgow RE; University of Utah