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Hypertonic Saline Inhibits Leukotriene B4 And Arachidonic Acid Priming Of The Neutrophil Oxidase
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
Conclusions: We have shown that mechanical strain rapidly induces a proliferative, morphological, and functional response in abdominal wall fibroblasts which is dependent upon the continued presence of the strain signal and quickly lost when the force is removed. The loss of wound edge tension that occurs during laparotomy wound separation and hernia formation may contribute to impaired wound healing through loss of a key stimulatory mechanical signal.
TRAUMA & CRITICAL CARE 4: TRAUMA, BURNS, & BASIC SCIENCE 44.1. Hypertonic Saline Inhibits Leukotriene B4 And Arachidonic Acid Priming Of The Neutrophil Oxidase. L. Lee,1 M. R. Kelher,2 E. E. Moore,3 C. C. Silliman,2 J. Harr,3 D. Benson,3 A. Banerjee1; 1University of Colorado - Denver, Aurora, CO; 2Bonfils Blood Center, Denver, CO; 3Denver Health Medical Center, Denver, CO Introduction: Previous work shows that arachidonic acid (AA, and its leukotriene derivatives e.g.: LTB4) is an inflammatory mediator in post-shock mesenteric lymph (PSML), and in stored packed RBCs. AA appears to act as an agonist at LTB4 G-protein coupled receptors (GPCRs). These mediators prime neutrophils (PMNs) for an increased production of superoxide, resulting in ALI. Hypertonic Saline (HTS) have also been shown to have immunomodulatory effects such as attenuation of PMN respiratory burst via signaling GPCRs, thereby potentially attenuating ALI. We hypothesize that HTS inhibits priming of the PMN oxidase by these eicosanoid mediators. Methods: After PMNs were isolated from healthy donors, incubation was done in either isotonic buffer (control) or HTS (180mmol/L) for 5 minutes at 37 C. The PMNs were then primed for 5 minutes with AA [5mM] or LTB4 [10mM] and the oxidase was activated with 200ng/ml of phorbol 12-myristate 13-acetate (PMA), a non-GPCR activator, and superoxide anion generation was measured via reduction of cytochrome c. Results: Both 5 mM AA and 10 mM LTB4 significantly primed the PMA activated respiratory burst (p<0.05, N¼4) (Fig.). HTS abrogated both AA and LTB4 priming of the respiratory burst. Conclusions: These data indicate that HTS reduces the cytotoxicity of PMNs stimulated by these eicosanoid mediators in PSML and stored blood products, and further support the immunomodulatory effects of HTS. This may translate into directed therapy for AA/LTB4 stimulated PMN cytotoxicity in ALI and MOF.
44.2. Mammalian DNA Danger Signaling Induced Complement Factor B Requires HMGB1. J. P. Pribis,1 D. J. Kaczorowski,2 M. J. Scott,1 T. R. Billiar1; 1University of Pittsburgh, Pittsburgh, PA; 2University of Pennsylvania School of Medicine, Philadelphia, PA Introduction: Recent evidence suggests that DNA released by injured tissues is a major driver of the systemic inflammatory response following
trauma. Similarly, complement activation contributes to injury induced inflammation. We have previously linked these two pathways by showing that mammalian DNA can upregulate and release complement factor B (CfB) mRNA and protein, respectively. This was found to be dependent on p38, but independent of TLR9, JNK, ERK 1/2, and NF-kB signaling. However, the underlying mechanisms for endogenous DNA induced production of CfB remains poorly understood. We hypothesize that HMGB1, a danger signal and DNA binder may bind to exogenous DNA within macrophages allowing presentation to a DNA recognition element to stimulate extra-hepatic CfB upregulation. Methods: Murine macrophage-derived RAW 264.7 cells were stably transfected with control siRNA or pan-HMGB1 siRNA plasmids. The siRNA produced by this plasmid knocks down expression of HMGB1 as well as HMGB2 and HMGB3. HMGB1 knockdown was verified by western blot, and was almost completely knocked down in the cells used for this experiment. Control or HMGB1 knockdown cells were treated with fragmented calf thymus DNA and/or lipofectamine. Six hours after treatment total RNA was collected from cells, reverse transcribed and 1mg cDNA was used for qRTPCR detection of CfB. Results were normalized to b-actin expression. All results given are mean6SEM, n¼3. Groups comparisons were made using analysis of variance with Bonferroni post hoc test, a¼0.05. Results: HMGB1 siRNA effectively diminished HMGB1 protein expression in RAW cells while HMGB1 expression remained unchanged in cells transfected with control siRNA. Calf thymus DNA alone upregulated CfB message in control siRNA cells by 6.861.6 fold from baseline, compared to an increase of only 1.6860.22 fold in HMGB1 siRNA cells (p<0.05 control vs. HMGB1). Adding lipofectamine with DNA further increased CfB message levels in control siRNA cells to 162617.5 fold from baseline, but resulted in only a 10.161.26 fold increase in CfB message in HMGB1 siRNA cells (p<0.05 control vs. HMGB1). Lipofectamine only did not result in an increase in CfB message in either control siRNA or HMGB1 siRNA cells. Conclusions: Extra-hepatic complement production and release is a known contributor to tissue damage in sepsis and trauma. Cell free DNA, HMGB1 and complement have all been shown to play a major role in initiating and propagating inflammation following trauma. These results demonstrate a role for HMGB1 in mammalian DNA upregulation of complement factor B. Determining the mechanism of action of danger signals such as exogenous DNA released following trauma may help produce improved treatment for surgical and trauma patients.
44.3. Older Stored Red Blood Cells Impair Platelet Function. E. Gonzalez,1,2 E. Moore,1,2 W. Max,1,2 A. Banerjee,1 C. C. Silliman1,3; 1University of Colorado School of Medicine, Denver, CO; 2Denver Health Medical Center, Denver, CO; 3 Bonfils Blood Center, Denver, CO Introduction: Age of transfused red blood cells (RBCs) has been associated with adverse clinical outcomes. It has been reported that age of RBCs transfused correlates with inflammatory injury and immunomodulation. To determine whether duration of RBC storage is associated with effects on coagulation, we evaluated the effect of aged RBCs on plasma and platelet components by using thrombelastography (TEG) and it’s modified platelet specific assay - platelet mapping. Methods: Leukocyte reduced RBCs were obtained from the local blood bank on the day of collection and allowed to age for 42 days. At day 1, 14, 21, and 42, RBCs units were added to ABO-matched plasma (frozen within 24 hours from collection FP24) and apheresis platelets (PLTs). PLTs were used within 6 hrs. Of collection, and FP24 within 1 hr. Of thawing. RBCs, FFP and PLTs were combined in volumes in order to yield an Hct of 40% and a PLT count of aprox. 300 3 106, and immediately assessed by TEG, and TEG-platelet mapping - using adenosine diphosphate (ADP) or arachidonic acid (AA) to stimulate platelet aggregation. Platelet function is measured as% inhibition of aggregation. Statistical analyses were performed by ANOVA. Results: A statistically significant difference was noted in platelet aggregation by AA stimulation; increased inhibition of platelet aggregation was observed in the presence of RBCs stored 21 days vs. RBCs stored <21 days (34.6%67.4vs. 7.9%63.8, p<0.05). No significant difference was noted between any
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
Conclusions: We have shown that mechanical strain rapidly induces a proliferative, morphological, and functional response in abdominal wall fibroblasts which is dependent upon the continued presence of the strain signal and quickly lost when the force is removed. The loss of wound edge tension that occurs during laparotomy wound separation and hernia formation may contribute to impaired wound healing through loss of a key stimulatory mechanical signal.
TRAUMA & CRITICAL CARE 4: TRAUMA, BURNS, & BASIC SCIENCE 44.1. Hypertonic Saline Inhibits Leukotriene B4 And Arachidonic Acid Priming Of The Neutrophil Oxidase. L. Lee,1 M. R. Kelher,2 E. E. Moore,3 C. C. Silliman,2 J. Harr,3 D. Benson,3 A. Banerjee1; 1University of Colorado - Denver, Aurora, CO; 2Bonfils Blood Center, Denver, CO; 3Denver Health Medical Center, Denver, CO Introduction: Previous work shows that arachidonic acid (AA, and its leukotriene derivatives e.g.: LTB4) is an inflammatory mediator in post-shock mesenteric lymph (PSML), and in stored packed RBCs. AA appears to act as an agonist at LTB4 G-protein coupled receptors (GPCRs). These mediators prime neutrophils (PMNs) for an increased production of superoxide, resulting in ALI. Hypertonic Saline (HTS) have also been shown to have immunomodulatory effects such as attenuation of PMN respiratory burst via signaling GPCRs, thereby potentially attenuating ALI. We hypothesize that HTS inhibits priming of the PMN oxidase by these eicosanoid mediators. Methods: After PMNs were isolated from healthy donors, incubation was done in either isotonic buffer (control) or HTS (180mmol/L) for 5 minutes at 37 C. The PMNs were then primed for 5 minutes with AA [5mM] or LTB4 [10mM] and the oxidase was activated with 200ng/ml of phorbol 12-myristate 13-acetate (PMA), a non-GPCR activator, and superoxide anion generation was measured via reduction of cytochrome c. Results: Both 5 mM AA and 10 mM LTB4 significantly primed the PMA activated respiratory burst (p<0.05, N¼4) (Fig.). HTS abrogated both AA and LTB4 priming of the respiratory burst. Conclusions: These data indicate that HTS reduces the cytotoxicity of PMNs stimulated by these eicosanoid mediators in PSML and stored blood products, and further support the immunomodulatory effects of HTS. This may translate into directed therapy for AA/LTB4 stimulated PMN cytotoxicity in ALI and MOF.
44.2. Mammalian DNA Danger Signaling Induced Complement Factor B Requires HMGB1. J. P. Pribis,1 D. J. Kaczorowski,2 M. J. Scott,1 T. R. Billiar1; 1University of Pittsburgh, Pittsburgh, PA; 2University of Pennsylvania School of Medicine, Philadelphia, PA Introduction: Recent evidence suggests that DNA released by injured tissues is a major driver of the systemic inflammatory response following
trauma. Similarly, complement activation contributes to injury induced inflammation. We have previously linked these two pathways by showing that mammalian DNA can upregulate and release complement factor B (CfB) mRNA and protein, respectively. This was found to be dependent on p38, but independent of TLR9, JNK, ERK 1/2, and NF-kB signaling. However, the underlying mechanisms for endogenous DNA induced production of CfB remains poorly understood. We hypothesize that HMGB1, a danger signal and DNA binder may bind to exogenous DNA within macrophages allowing presentation to a DNA recognition element to stimulate extra-hepatic CfB upregulation. Methods: Murine macrophage-derived RAW 264.7 cells were stably transfected with control siRNA or pan-HMGB1 siRNA plasmids. The siRNA produced by this plasmid knocks down expression of HMGB1 as well as HMGB2 and HMGB3. HMGB1 knockdown was verified by western blot, and was almost completely knocked down in the cells used for this experiment. Control or HMGB1 knockdown cells were treated with fragmented calf thymus DNA and/or lipofectamine. Six hours after treatment total RNA was collected from cells, reverse transcribed and 1mg cDNA was used for qRTPCR detection of CfB. Results were normalized to b-actin expression. All results given are mean6SEM, n¼3. Groups comparisons were made using analysis of variance with Bonferroni post hoc test, a¼0.05. Results: HMGB1 siRNA effectively diminished HMGB1 protein expression in RAW cells while HMGB1 expression remained unchanged in cells transfected with control siRNA. Calf thymus DNA alone upregulated CfB message in control siRNA cells by 6.861.6 fold from baseline, compared to an increase of only 1.6860.22 fold in HMGB1 siRNA cells (p<0.05 control vs. HMGB1). Adding lipofectamine with DNA further increased CfB message levels in control siRNA cells to 162617.5 fold from baseline, but resulted in only a 10.161.26 fold increase in CfB message in HMGB1 siRNA cells (p<0.05 control vs. HMGB1). Lipofectamine only did not result in an increase in CfB message in either control siRNA or HMGB1 siRNA cells. Conclusions: Extra-hepatic complement production and release is a known contributor to tissue damage in sepsis and trauma. Cell free DNA, HMGB1 and complement have all been shown to play a major role in initiating and propagating inflammation following trauma. These results demonstrate a role for HMGB1 in mammalian DNA upregulation of complement factor B. Determining the mechanism of action of danger signals such as exogenous DNA released following trauma may help produce improved treatment for surgical and trauma patients.
44.3. Older Stored Red Blood Cells Impair Platelet Function. E. Gonzalez,1,2 E. Moore,1,2 W. Max,1,2 A. Banerjee,1 C. C. Silliman1,3; 1University of Colorado School of Medicine, Denver, CO; 2Denver Health Medical Center, Denver, CO; 3 Bonfils Blood Center, Denver, CO Introduction: Age of transfused red blood cells (RBCs) has been associated with adverse clinical outcomes. It has been reported that age of RBCs transfused correlates with inflammatory injury and immunomodulation. To determine whether duration of RBC storage is associated with effects on coagulation, we evaluated the effect of aged RBCs on plasma and platelet components by using thrombelastography (TEG) and it’s modified platelet specific assay - platelet mapping. Methods: Leukocyte reduced RBCs were obtained from the local blood bank on the day of collection and allowed to age for 42 days. At day 1, 14, 21, and 42, RBCs units were added to ABO-matched plasma (frozen within 24 hours from collection FP24) and apheresis platelets (PLTs). PLTs were used within 6 hrs. Of collection, and FP24 within 1 hr. Of thawing. RBCs, FFP and PLTs were combined in volumes in order to yield an Hct of 40% and a PLT count of aprox. 300 3 106, and immediately assessed by TEG, and TEG-platelet mapping - using adenosine diphosphate (ADP) or arachidonic acid (AA) to stimulate platelet aggregation. Platelet function is measured as% inhibition of aggregation. Statistical analyses were performed by ANOVA. Results: A statistically significant difference was noted in platelet aggregation by AA stimulation; increased inhibition of platelet aggregation was observed in the presence of RBCs stored 21 days vs. RBCs stored <21 days (34.6%67.4vs. 7.9%63.8, p<0.05). No significant difference was noted between any