Salicylate enhances endotoxin-induced pro- and anti-inflammatory cytokine expression

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

CRITICAL CARE PARALLEL SESSION 18. Gut-Derived Leukotriene B4 Following Hemorrhagic Shock is a Pivotal Mediator of Neutrophil Priming. E. L. Sarin, M.D., E. E. Moore, M.D., F. R. Sheppard, M.D., J. L. Johnson, M.D., A. Banerjee, Ph.D., C. C. Silliman, M.D., Ph.D. University of Colorado. Introduction: The lipid fraction of mesenteric lymph collected after hemorrhagic shock (HS) and reperfusion primes the neutrophil oxidative burst in vitro. Recently, we have identified the culprit agent to be a neutral lipid consistent with Leukotriene B4 (LTB4). We hypothesized that plasma LTB4 increases after HS and that diversion of mesenteric lymph (DML) abrogates this effect. Furthermore, we posited that administration of an LTB4 antagonist blocks the priming effect of mesenteric lymph on neutrophils (PMNs). Methods: Adult male Sprague-Dawley rats (325–375 g) underwent laparotomy with cannulation of the mesenteric lymphatics for lymph diversion (DML) or laparotomy without mesenteric cannulation (Sham). Additionally, DML rats had lymph collected both pre- and post-shock. All rats underwent cannulation of the femoral artery and vein. HS to a MAP of 30 mmHg for 45 minutes was achieved via venous exsanguination. Animals were resuscitated with shed blood and with 2x the shed blood volume of NS over two hours. After resuscitation, plasma was collected for LTB4 measurement by ELISA. Lymph collected in the resuscitation phase was added to isolated PMNs that were then measured for superoxide release in the presence and absence of the LTB4 receptor antagonist CP105,696. Results: LTB4 concentration in the lymph collected from the DML group measured 281.25 ⫾ 53.81 ng/ml pre-shock, increasing to 692.13 ⫾ 176.91 ng/ml post-shock (p ⬍ 0.05). LTB4 plasma concentrations after resuscitation differed based on lymph diversion. LTB4 concentration for DML animals measured 2745 ⫾ 214.77 pg/ml vs. Sham animals, 4012 ⫾ 628.93 pg/ml (p ⬍ 0.05). PMN pre-treatment with the LTB4 receptor antagonist completely abrogated superoxide release in response to lymph exposure compared with the untreated control, 1.39 ⫾ 0.23 vs. 4.22 ⫾ 0.68 nmol O 2-/3.75 x 10 5 cells/mL/min respectively (p ⬍ 0.05). Conclusion: These data suggest that splanchnic ischemia secondary to HS increases concentrations of the known priming agent LTB4 in mesenteric lymph and the systemic circulation. Furthermore, selective blockade of LTB4 markedly attenuates the effect of mesenteric lymph on PMN priming, indicating a potential therapeutic location for disruption of the dysfunctional PMN priming/activation sequence. 19. Whole Body Hyperthermia Preserves Endothelial Function and Restores Mesenteric Blood Flow After Resuscitated Hemorrhage. G. Singh, M.B.B.S., W. Flynn, Jr., M.D., J. Ostberg, Ph.D., R. van Schie, Ph.D., G. Miller. State University of New York at Buffalo, Department of Surgery, Buffalo, NY. Endothelial dysfunction has been implicated in regional blood flow deficits after resuscitated shock. Whole body hyperthermia generates heat shock proteins (HSP) that attenuate protein denaturation and preserve cellular function. This study was performed to determine the effect of whole body hyperthermia on endothelial function and mesenteric blood flow after resuscitated hemorrhage. Sprague Dawley rats were hemorrhaged to 30% of blood volume and resuscitated with shed blood and an equal of volume of Ringer’s Lactate (Standard Resuscitation, SR). Intravital microscopy was used to determine first order arteriolar (A1) diameters and blood flow (QA1) in the neurovascularly intact ileum at baseline and 90 minutes post resuscitation (T90). Animals treated with SR and whole body hyperthermia (SR-H) were placed into a ventilated heating chamber following hemorrhage and resuscitation to achieve a temperature of 39.5– 40 oC for 6 hours. Identical microvascular studies were performed 18 hours later. Endothelial function was quantitated by measuring changes in vessel diameter to topical application of the

endothelial dependent dilator acetylcholine (Ach, 10 -5 M). A1 diameter is reported in microns and QA1 as nanoliters per second (⫹/SEM). Tissue was taken from the ileum for HSP72 determination via Western analysis. This study demonstrated that whole body hyper-

TABLE—ABSTRACT 19

Baseline T90

SR SR-H SR SR-H

Ach

QA1

HSP72

85 ⫾ 5 90 ⫾ 5 48 ⫾ 8 88 ⫾ 5*

161 ⫾ 15 226 ⫾ 17 31 ⫾ 7 157 ⫾ 20*

.124 ⫾ .1 .124 ⫾ .1 .241 ⫾ .1 .924 ⫾ .2*

* P ⫽ ⬍0.05 vs. T90 SR for ACH, QA1 and HSP72 by Student’s t test.

thermia applied after resuscitated hemorrhage results in an upregulation of HSP expression that is associated with preserved endothelial function and restoration of mesenteric blood flow. These results suggest that whole body hyperthermia may serve as an adjunct to standard resuscitation in restoring blood flow and attenuating subsequent organ dysfunction. 20. Selective and Non-Selective Cox Inhibitors Up-Regulate Heat Shock Protein-70 Expression and Protect Human Monocytes and Macrophages from the Cytotoxic Effect of Hydrogen Peroxide. S. M. Kipnis, M.D., B. Haimovich, Ph.D. Robert Wood Johnson Medical School, New Brunswick, NJ. Heat shock protein 70 (HSP-70) is a molecular chaperone that protects cells against stress stimuli. The expression of HSP-70 is normally up regulated in response to stress. However, in patients with severe sepsis, HSP-70 expression is suppressed. We hypothesized that inhibitors of cyclooxygenase (COX) could up regulate the expression of HSP-70 in human macrophages and monocytes, which in turn will improve the resistance of these cells to stress. Human monocytes and macrophages were untreated or treated with aspirin, ibuprofen or NS-398, a selective COX-2 inhibitor, for 1.5 to 24 hrs. A parallel group was exposed to a 30-min heat shock at 42 oC. Western blotting and real-time polymerase chain reaction quantified, respectively, time dependent changes in HSP-70 protein expression and mRNA production. To evaluate whether elevated HSP-70 expression protects the cells from stress, cell viability was determined after exposure of cells to cytotoxic concentrations of hydrogen peroxide. Changes in cell viability were monitored using propidium iodide and fluorescent microscopy. Treatment of the monocytes or macrophage cultures with either aspirin, ibuprofen or NS-398 caused a statistically significant increase (p ⬍ 0.05; n ⫽ 4) of about 2.5 fold in HSP-70 mRNA expression that peaked at 1.5 hrs. Heat shock caused a 25-fold increase in HSP-70 mRNA expression at 1.5 hrs. The COX inhibitors also triggered a statistically significant increase (p ⬍ 0.05; n ⫽ 8) of about 2-fold in HSP-70 expression within 1.5 hrs of treatment, while the heat shock triggered a 3–5 fold increase in both cell populations. The protein levels remained elevated for 24 hrs in all treated groups. Significantly, pretreatment of the cells with either the COX inhibitors or the heat shock protected the cells from the cytotoxic effect of hydrogen peroxide. These findings suggest that both selective and non-selective COX inhibitors may be as effective as a febrile response in augmenting the body’s resistance to stress. 21. Salicylate Enhances Endotoxin-Induced Pro- and AntiInflammatory Cytokine Expression. S. D. West, M.D., K. S. Helmer, M.D., L. K. Chang, B.S., Y. Cui, M.D., D. W. Mercer, M.D. Ut Houston Medical School.

246

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

Introduction: Salicylate is frequently given as an antiinflammatory agent, and its effects are primarily due to cyclooxygenase inhibition and a resultant decrease in the synthesis of prostaglandins. However, salicylate also has effects beyond COX inhibition that may be pro-inflammatory. We have shown that salicylate enhances the expression of the pro-inflammatory enzyme iNOS in the gastric mucosa during endotoxemia. For this reason, this study examined the effects of salicylate on other pro-inflammatory and antiinflammatory mediators in endotoxic rats. We hypothesized that salicylate would alter cytokine expression during endotoxemia. Methods: Rats were given intraperitoneal saline or salicylate (100 mg/kg). One hour later, rats were given intraperitoneal saline or LPS (20 mg/kg). Five hours later, serum was collected for determination of the pro-inflammatory cytokines IL-1␣, IL-6 and IL-12 and the anti-inflammatory cytokine IL-10 by ELISA. Data are reported in mg/ml as means ⫾ SEM (n ⫽ 5; ANOVA). Results: LPS significantly increased serum IL-1␣, IL-6, IL-12 and IL-10 when compared to controls. Salicylate enhanced LPS-induced changes in IL-1␣, IL-6, IL-12 and IL-10 protein. In the absence of LPS, salicylate had no effect on these cytokines. Conclusion: The data indicate that salicylate augments the effects of LPS on both pro- and antiinflammatory cytokines. Whether this effect is due to inhibition of COX-1 or COX-2 or another mechanism remains to be determined. However, while salicylate may be anti-inflammatory in some tissues, caution should be exercised in its administration to septic patients given its potential for increasing expression of pro-inflammatory mediators that could have deleterious effects.

TABLE—ABSTRACT 21

Saline/Sal Saline/LPS Salicylate/Sal Salicylate/LPS

IL-1␣

IL-6

IL-12

IL-10

0.1 ⫾ 0.09 0.8 ⫾ 0.2* .01 ⫾ .002 2.8 ⫾ 0.5**

1.9 ⫾ 1.6 46.6 ⫾ 6.1* 0.5 ⫾ 0.2 82.8 ⫾ 5.9**

2.3 ⫾ 0.1 11.9 ⫾ 1.4* 1.6 ⫾ 0.2 17.3 ⫾ 1.3**

0.03 ⫾ 0.01 1.2 ⫾ 0.2* 0.02 ⫾ 0.01 2.4 ⫾ 0.3**

* P ⬍ 0.05 vs. saline counterpart. ** P ⬍ 0.05 vs. Saline/LPS.

22. Induction of BLP Tolerance Protects TLR4 Deficient Mice Against Gram Negative Sepsis Via Enhanced Bacterial Clearance, in a Process Independent of the Neutrophil. G. C. O’Brien, M.D., J. H. Wang, M.D., B. J. Manning, M.D., M. Doyle, M.D., P. Redmond, M.D. Cork University Hospital Introduction: Toll like receptors (TLR’s) are highly conserved pathogen pattern recognition receptors. As a result, TLR4 deficient mice are highly susceptible to Gram Negative (G-ve) sepsis. We assessed whether pretreatment of TLR4 deficient mice with BLP, a TLR2 ligand, could induce tolerance to a subsequent G-ve Salmonella Typhimurium (S.Typhi) challenge. Methods: In three separate experiments C3H/HeN, C3H/HeJ and BLP pre-treated C3H/HeJ mice (each n ⫽ 10) were exposed to 0.25 x 10 6 CFU S.Typhi. Firstly, mortality was observed. Secondly, animals were divided and harvested at 24 and 48 hours. Blood, lungs, liver and spleen were harvested and homogenized in 2mls sterile PBS. Following serial dilution, samples were incubated on trypticase soy agar for 24 hrs at 37 oC for determination of bacterial CFU. Thirdly, all animals were depleted of neutrophils by pretreatment with cyclophosphamide (250mg/kg I.P.) and bacterial CFU were determined in a similar manner. Results: Pre-treatment with BLP results in a significant survival benefit in C3H/HeJ mice (p ⬍ 0.0001) following challenge with 0.25 x 10 6 CFU S.Typhi. This phenomenon is partially explained by the differences seen with bacterial clearance rates. BLP pre-treatment results in significantly increased rates of bacterial clearance from the bloodstream and significant attenuation of bac-

terial colonization of the lung, liver and spleen. Neutrophil depletion did not reduce the beneficial differences observed in bacterial clearance rates. Conclusion: BLP, a ligand for TLR2, induces tolerance

TABLE—ABSTRACT 22 HeN

HeJ

Blood 68 (⫾58) 11 ⫻ 10 6 Lungs 13,720 (⫾3,161) 8.7 ⫻ 10 6 Liver 17,280 (⫾6,255) 74 ⫻ 10 6 Spleen 20,400 (⫾2,960) 30 ⫻ 10 6

HeJ&BLP

1,000 (⫾360) (⫾19 ⫻ 10 6) (⫾7.9 ⫻ 10 6) 46,080 (⫾35,000) (⫾78 ⫻ 10 6) 88,360 (⫾51,878) 3,200 (⫾1,020) (⫾50 ⫻ 10 6)

All results shown as Mean (CFU/ml) ⫾ Standard Deviation. in C3H/HeJ mice to a G-ve S.Typhi challenge. This phenomenon appears to be mediated by greatly enhanced bacterial clearance from both the bloodstream and the solid organs in a process independent of the neutrophil. 23. Carbon Monoxide Paradoxically Protects Against Hypoxia. B. S. Zuckerbraun, M.D., T. R. Billiar, M.D. University of Pittsburgh. Background: Tissue hypoxia is fundamental to the development of cellular injury caused by multiple processes, including hemorrhagic shock. Others and we have previously demonstrated that endogenous production of carbon monoxide (CO) by heme oxygenase or exogenous administration of CO can protect against organ injury induced by hemorrhage. The purpose of these experiments was to test the hypotheses that CO abrogates the development of tissue hypoxia in vivo and diminishes effects of hypoxia in vitro. Methods: Male C57/BL6 mice were hemorrhaged to a mean arterial pressure of 25 mmHg. Control animals underwent the same procedure but were not hemorrhaged (sham). Inhalational CO exposure (250 ppm) was initiated concurrently with hemorrhage Mice were administered EF5 (10 ␮l/g; ip), which is an agent that irreversibly binds to intracellular proteins under hypoxic conditions and can then be detected by immunohistochemistry. Mice were sacrificed after 75 minutes of shock and livers were harvested. For in vitro experiments, primary mouse hepatocytes were exposed to normoxia (21% O 2) or hypoxia 1% O 2) with and without CO (250 ppm). Cells were harvested and assayed for viability, ATP content, or VEGF production. Results: Untreated hemorrhaged mice had a 17-fold increase in EF5 staining compared to shams. Hepatic hypoxia was most pronounced around the central veins. CO-treated hemorrhaged mice had a 79⫾13% reduction in EF5 staining compared to untreated hemorrhaged mice (P ⬍ 0.05). Additionally, CO-treated sham mice demonstrated no significant liver hypoxia. In vitro, CO significantly protected against hypoxia-induced cell death (P ⬍ 0.05). Hepatocytes exposed to hypoxia demonstrated a 54 ⫾ 4% reduction in intracellular ATP levels after 6 hours, whereas CO treatment of hypoxic cells resulted in only a 9 ⫾ 3% reduction in ATP (P ⬍ 0.01). Likewise, CO significantly inhibited VEGF elaboration by hypoxic hepatocytes. Conclusions: Inhaled low dose CO can paradoxically abrogate liver hypoxia during hemorrhage. Additionally, CO diminishes the consequences of hypoxia in vitro. CO may exert these effects by decreasing cellular metabolic activity and oxygen requirements, though further investigation is required. CO may prove to be a useful clinical adjunct in the treatment of hemorrhagic shock. 24. Alterations in Hepatic Microcirculation, Mitochondrial Redox State and Tissue Oxygenation after Trauma and Sepsis. S. A. Keller, M.S., M. Paxian, M.D., J. H. Ashburn, M.S., M. G. Clemens, Ph.D., T. T. Huynh, M.D. Carolinas Healthcare System, Carolinas Medical Center.