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Modulation of CD177 Expression in Neutrophils
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Conclusions: Here we show that the loss of liver iNKT cells appears to be due to transmigration (exodus) of cells out of the liver and this is markedly effect by PD-1 expression. Whether this is a direct/indirect result of PD-1 ligation and/or signal remains to be determined.
45.8. Modulation of CD177 Expression in Neutrophils. B. Chung, M. De, A. De, P. Bankey; University of Rochester Medical Center, Rochester, NY Introduction: We recently demonstrated that the mRNA and expression of CD177, a membrane bound neutrophil antigen is significantly elevated in trauma patients. However, the mediators of CD177 expression are currently unknown. Therefore, we tested the in vitro effects of lipopolysaccharide (LPS) and dexamethasone on CD177 in neutrophils (PMNs). Methods: A prospective study was performed to measure expression of CD177 in healthy subjects (n¼6). Peripheral blood draws were performed and PMNs isolated by Ficoll-Hypaque density centrifugation and dextran sedimentation. PMNs were cultured in the presence of dexamethasone (1ug/ml) or LPS (100U/ml) for six hours. Flow cytometry was performed to measure CD177 expression and CD66b, a marker that is upregulated in activated PMNs. Results: CD177 is significantly upregulated when cultured in the presence of LPS (mean MFI 47.3+-25.1, p-value 0.03), but not significantly increased when cultured with dexamethasone (mean MFI 33.1+-15.5, p-value 0.06) when compared with control culture conditions (mean MFI 31.6+-15.5). There were no significant increases in the percentage of CD177 expressing PMNs with LPS (mean 56.6%+-16.2, p-value 0.12), and dexamethasone (mean 54.9%+-15.7, p-value 0.28) compared to control conditions (mean 53.0%+-17.1). Conclusions: Our results demonstrate that LPS significantly elevates CD177 expression. Recent studies have suggested that PMNs may be important for host defense through inTABLE
Control LPS Dexamethasone
CD66b (mean MFI 6 SEM)
CD66b (% Positive Cells 6 SEM)
CD177 (mean MFI6 SEM)
CD177 (% Positive Cells 6 SEM)
140.9 6 27.3 210.0 6 54.5 192.2 6 41.5
97.6 6 0.8 97.9 6 0.5 98.1 6 0.5
31.6 6 15.5 47.3 6 25.1 31.6 6 15.4
53.0 6 17.1 56.58 6 16.2 54.91 6 15.7
teractions with platelets. CD177 binds to PECAM-1, a platelet endothelial cell adhesion molecule, and may facilitate this PMN-platelet interaction. LPS is a major element of gram-negative sepsis, and modulation of CD177 by LPS may be an important factor for host defense and a potential therapeutic target unrecognized in trauma patients.
45.9. Alcohol Administration Suppresses LKS Cell Proliferation During the Granulopoietic Response to E. Coli Bacteremia. X. Shi, M. D. Basson, P. Zhang; Department of Surgery, Michigan State University College of Human Medicine, Lansing, MI Introduction: Excessive alcohol consumption injures the immune system, predisposing the host to the development of severe infections. Indeed, alcoholic patients with systemic bacterial infection frequently present with granulocytopenia which is an indicator for increased mortality. How alcohol impairs the granulopoietic response remains unclear. Methods: To determine whether alcohol affects the increase in primitive hematopoietic precursor cell commitment to granulocyte lineage development in response to bacteremia, acute alcohol intoxication was induced in male Balb/c mice maintained on a Lieber-DeCarli low fat liquid alcohol diet for 5 weeks by intraperitoneal injection (i.p.) of alcohol (20% alcohol in saline at a dose of 5 g alcohol/kg). Control animals on the Lieber-DeCarli low fat liquid control
331
diet received i.p. injection of saline. Thirty minutes after the i.p. injection, E. coli (E11775, ATCC, 1 x 106 or 5 x 107 CFUs/mouse) or saline was given to mice via penile vein injection. Intravenous BrdU (1 mg/ mouse) was administered 24 hours before the termination of each experiment. Results: The number of lineage (lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)+ (LKS) cells (primitive hematopoietic precursors enriched with hematopoietic stem cells) increased significantly in the bone marrow 24 hours following E. coli bacteremia in control mice. Bacteremia resulted in a significant increase in BrdU incorporation into LKS cells in control mice, reflecting a marked activation of proliferation by these primitive hematopoietic precursors. Alcohol administration impaired this LKS cell response to bacteremia. in control mice, enhancement of Sca-1 expression was also observed in granulocyte lineage committed cells following E. coli challenge. the number of Sca-1+ cells in cells bearing granulocyte lineage marker Gr1 was significantly increased in control mice with bacteremia. Alcohol treatment suppressed this increase in Sca-1 expression in marrow Gr1+ cells. in addition, alcohol treatment blocked the increase in the number circulating PMNs 48 hours after intravenous challenge with 5 x 107 E. coli and impaired bacterial clearance from the circulation as well as vital organ tissues. Conclusions: These data demonstrate that acute alcohol intoxication superimposed upon chronic alcohol intake impairs the proliferative expansion of marrow LKS cells and restricts the granulopoietic response to bacteremia at the initial stage of increase in primitive hematopoietic precursor cell commitment to granulocyte lineage development. Our observations highlight a potential target at the upstream hematopoietic precursor level for intervention to restore immune function in alcoholic patients with serious bacterial infection.
45.10. Polymicrobial Sepsis is Associated With Decreased Hepatic Oxidative Phosphorylation and an Altered Metabolic Profile. S. Whelan,1 E. H. Carchman,1 B. S. Zuckerbraun1,2; 1University of Pittsburgh, Piitsburgh, PA; 2 VA Pittsburgh Healthcare System, Pittsburgh, PA Introduction: Organ failure in sepsis accounts for significant mortality world wide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus the organ. Adaptive responses in metabolism are critical to the recovery of the cell. the purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration in the liver. Methods: C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or a operation without CLP. Mice were euthanized from 0-24 hours after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity was measured. Global biochemical profiles of 329 metabolites were performed at the 8 hour time point (n¼8) analyzed by GC/MS and LC/MS/MS platforms by Metabolon. the influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. Results: CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measure at 12 hours. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every TCA Cycle intermediate and derivative measured (including citrate, fumarate, malate, and succinylcarnitine), suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. Conclusions: Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration. This occurs prior to significant changes in hemodynamics. the metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.
45.8. Modulation of CD177 Expression in Neutrophils. B. Chung, M. De, A. De, P. Bankey; University of Rochester Medical Center, Rochester, NY Introduction: We recently demonstrated that the mRNA and expression of CD177, a membrane bound neutrophil antigen is significantly elevated in trauma patients. However, the mediators of CD177 expression are currently unknown. Therefore, we tested the in vitro effects of lipopolysaccharide (LPS) and dexamethasone on CD177 in neutrophils (PMNs). Methods: A prospective study was performed to measure expression of CD177 in healthy subjects (n¼6). Peripheral blood draws were performed and PMNs isolated by Ficoll-Hypaque density centrifugation and dextran sedimentation. PMNs were cultured in the presence of dexamethasone (1ug/ml) or LPS (100U/ml) for six hours. Flow cytometry was performed to measure CD177 expression and CD66b, a marker that is upregulated in activated PMNs. Results: CD177 is significantly upregulated when cultured in the presence of LPS (mean MFI 47.3+-25.1, p-value 0.03), but not significantly increased when cultured with dexamethasone (mean MFI 33.1+-15.5, p-value 0.06) when compared with control culture conditions (mean MFI 31.6+-15.5). There were no significant increases in the percentage of CD177 expressing PMNs with LPS (mean 56.6%+-16.2, p-value 0.12), and dexamethasone (mean 54.9%+-15.7, p-value 0.28) compared to control conditions (mean 53.0%+-17.1). Conclusions: Our results demonstrate that LPS significantly elevates CD177 expression. Recent studies have suggested that PMNs may be important for host defense through inTABLE
Control LPS Dexamethasone
CD66b (mean MFI 6 SEM)
CD66b (% Positive Cells 6 SEM)
CD177 (mean MFI6 SEM)
CD177 (% Positive Cells 6 SEM)
140.9 6 27.3 210.0 6 54.5 192.2 6 41.5
97.6 6 0.8 97.9 6 0.5 98.1 6 0.5
31.6 6 15.5 47.3 6 25.1 31.6 6 15.4
53.0 6 17.1 56.58 6 16.2 54.91 6 15.7
teractions with platelets. CD177 binds to PECAM-1, a platelet endothelial cell adhesion molecule, and may facilitate this PMN-platelet interaction. LPS is a major element of gram-negative sepsis, and modulation of CD177 by LPS may be an important factor for host defense and a potential therapeutic target unrecognized in trauma patients.
45.9. Alcohol Administration Suppresses LKS Cell Proliferation During the Granulopoietic Response to E. Coli Bacteremia. X. Shi, M. D. Basson, P. Zhang; Department of Surgery, Michigan State University College of Human Medicine, Lansing, MI Introduction: Excessive alcohol consumption injures the immune system, predisposing the host to the development of severe infections. Indeed, alcoholic patients with systemic bacterial infection frequently present with granulocytopenia which is an indicator for increased mortality. How alcohol impairs the granulopoietic response remains unclear. Methods: To determine whether alcohol affects the increase in primitive hematopoietic precursor cell commitment to granulocyte lineage development in response to bacteremia, acute alcohol intoxication was induced in male Balb/c mice maintained on a Lieber-DeCarli low fat liquid alcohol diet for 5 weeks by intraperitoneal injection (i.p.) of alcohol (20% alcohol in saline at a dose of 5 g alcohol/kg). Control animals on the Lieber-DeCarli low fat liquid control
331
diet received i.p. injection of saline. Thirty minutes after the i.p. injection, E. coli (E11775, ATCC, 1 x 106 or 5 x 107 CFUs/mouse) or saline was given to mice via penile vein injection. Intravenous BrdU (1 mg/ mouse) was administered 24 hours before the termination of each experiment. Results: The number of lineage (lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)+ (LKS) cells (primitive hematopoietic precursors enriched with hematopoietic stem cells) increased significantly in the bone marrow 24 hours following E. coli bacteremia in control mice. Bacteremia resulted in a significant increase in BrdU incorporation into LKS cells in control mice, reflecting a marked activation of proliferation by these primitive hematopoietic precursors. Alcohol administration impaired this LKS cell response to bacteremia. in control mice, enhancement of Sca-1 expression was also observed in granulocyte lineage committed cells following E. coli challenge. the number of Sca-1+ cells in cells bearing granulocyte lineage marker Gr1 was significantly increased in control mice with bacteremia. Alcohol treatment suppressed this increase in Sca-1 expression in marrow Gr1+ cells. in addition, alcohol treatment blocked the increase in the number circulating PMNs 48 hours after intravenous challenge with 5 x 107 E. coli and impaired bacterial clearance from the circulation as well as vital organ tissues. Conclusions: These data demonstrate that acute alcohol intoxication superimposed upon chronic alcohol intake impairs the proliferative expansion of marrow LKS cells and restricts the granulopoietic response to bacteremia at the initial stage of increase in primitive hematopoietic precursor cell commitment to granulocyte lineage development. Our observations highlight a potential target at the upstream hematopoietic precursor level for intervention to restore immune function in alcoholic patients with serious bacterial infection.
45.10. Polymicrobial Sepsis is Associated With Decreased Hepatic Oxidative Phosphorylation and an Altered Metabolic Profile. S. Whelan,1 E. H. Carchman,1 B. S. Zuckerbraun1,2; 1University of Pittsburgh, Piitsburgh, PA; 2 VA Pittsburgh Healthcare System, Pittsburgh, PA Introduction: Organ failure in sepsis accounts for significant mortality world wide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus the organ. Adaptive responses in metabolism are critical to the recovery of the cell. the purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration in the liver. Methods: C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or a operation without CLP. Mice were euthanized from 0-24 hours after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity was measured. Global biochemical profiles of 329 metabolites were performed at the 8 hour time point (n¼8) analyzed by GC/MS and LC/MS/MS platforms by Metabolon. the influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. Results: CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measure at 12 hours. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every TCA Cycle intermediate and derivative measured (including citrate, fumarate, malate, and succinylcarnitine), suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. Conclusions: Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration. This occurs prior to significant changes in hemodynamics. the metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.