ORIGINAL ARTICLE Reducing Leukocyte Trafficking Preserves Hepatic Function After Sepsis Toan Huynh, MD, Nhat Nguyen, MS, Steven Keller, MS, Cathy Moore, PhD, Min C. Shin, PhD, and Iain H. McKillop, PhD Introduction: Leukocyte trafficking may induce hepatic dysfunction in sepsis. Herein, we hypothesize that reduction in leukocyte adhesion and, hence, leukocyte-endothelial interaction by activated protein C (aPC) may preserve hepatic function after sepsis. Methods: Rats underwent sham or cecal ligation and puncture, followed by saline or aPC (1 mg/kg intravenously) infusion, twice daily for 4 days. Cytokine levels were determined by enzyme-linked immunosorbent assay. Liver function and injury were assessed by bile production and plasma aspartate transaminase, respectively. In parallel experiments, neutrophils were labeled with Rhodamine 6G, and trafficking determined by cell motion tracking using intravital microscopy. Leukocyte trafficking and traveling velocity were computed at baseline and at 10 minutes and 40 minutes after endothelin-1 infusion. Results: Sepsis induced 90% mortality and elevated levels of interleukin (IL)-2 (167 pg/mL  39 pg/mL vs. 68 pg/mL  2 pg/mL, p  0.05), IL-6 (5,806 pg/mL  3,389 pg/mL vs. 0 pg/mL  0 pg/mL, p  0.05), and IL-8 (492 pg/mL  22 pg/mL vs. 21 pg/mL  17 pg/mL, p  0.05). Aspartate transaminase levels increased (227 IU/L  14 IU/L vs. 51 IU/L  7 IU/L, p  0.05) in cecal ligation and puncture animals, whereas bile production decreased by fivefold compared with sham (436 g/kg/h  247 g/kg/h vs. 2,357 g/kg/h  147 g/kg/h, p  0.05). Hepatic leukocyte adhesion increased threefold in septic animals (42.7 WBC per image  7.3 WBC per image vs. 14.8 WBC per image  3.8 WBC per image, p  0.01), whereas leukocyte velocity decreased compared with sham (10.5 m/s  2.2 m/s vs. 22.3 m/s  2.4 m/s, p  0.01). By contrast, aPC treatment reduced mortality to 60%, attenuated inflammatory cytokines, reduced leukocyte trafficking, and preserved hepatic function. Conclusions: Our data demonstrate that sepsis may, in part, induce hepatic dysfunction by augmenting leukocyte trafficking into hepatic sinusoids. Treatment with aPC attenuated leukocyte trafficking and, in doing so, preserved hepatic function and improved survival. Collectively, these data suggest an important role for protein C-dependent leukocyte-endothelial interaction in sepsis. Key Words: Sepsis, Leukocytes, Cell trafficking, Liver, Microcirculation. (J Trauma. 2010;69: 360 –367) C urrent guidelines in the management of sepsis, including goal-direct resuscitation, early broad-spectrum antimicro- bial therapy, and low tidal volume ventilation, have led to improved mortalities. 1 However, when sepsis deteriorates to septic shock with multiple-organ dysfunction syndrome, sur- vival remains poor at 40%. 2 A multitude of anti-inflammatory 3 and anticytokine trials 4,5 report limited efficacy in the treat- ment of sepsis. Failure of these unidirectional and targeted modalities results from an incomplete understanding of the pathophysiology of sepsis. Indeed, emerging evidence suggests a complex interplay among the host systemic inflammatory response syndrome (SIRS), leukocyte-endothelial cell interac- tion, and the microcirculation in the pathogenesis of sepsis and multiple organ failure (MOF). 6 Previous studies have demonstrated that the inflamma- tory and procoagulant host responses are closely inter- twined 7–11 during systemic stress. After sepsis, inflammatory cytokines, such as tumor necrosis factor- , interleukin (IL)-1, and IL-6, stimulate coagulation and inhibit fibrinolysis. Simulta- neously, the procoagulant thrombin initiates several inflam- matory pathways leading to activation of the endothelial system. This activation results in microvascular thrombosis, diffuse endovascular injury, and, ultimately, end-organ dys- function. 7–11 Hepatic dysfunction, an early hallmark of MOF, results, in part, from microcirculatory disturbances causing a mismatch of blood supply to metabolic demand. The altered microcirculation, in conjunction with leukocyte-endothelial interaction, creates a heterogeneous pattern of perfusion, further compromises substrate supply, and leads to hepa- tocellular injury. 12,13 As such, leukocyte-endothelial inter- action during systemic stress 14 may potentiate regional microcirculatory dysfunction leading, ultimately, to organ failure. 15–17 Activated protein C (aPC) plays an important role in modulating coagulation and inflammation in sepsis. Previous studies substantiated a link among coagulation, inflammation, and apoptosis, mediated by aPC. 18 In the context of accel- erated systemic inflammation, septic patients demonstrate reduced levels of aPC. 19 Compelling evidence has also supported the efficacy of aPC in the treatment of septic patients. 14,20 More recently, we reported that aPC treatment improves hepatic function and ameliorates endothelin (ET)-1 Submitted for publication September 24, 2009. Accepted for publication May 5, 2010. Copyright © 2010 by Lippincott Williams & Wilkins From the F.H. “Sammy” Ross Jr. Trauma Center (T.H., S.K., C.M.) and Depart- ment of Surgery (T.H., M.C.S., I.H.M.), Carolinas Medical Center, Charlotte, North Carolina; and Department of Computer Science (N.N.), University of North Carolina at Charlotte, Charlotte, North Carolina. Supported, in part, by a grant from the Education and Research Foundation, Carolinas Health Care System (to T.H.). Presented at the 68th Annual Meeting of the American Association for the Surgery of Trauma, October 1–3, 2009, Pittsburgh, Pennsylvania. Address for reprints: Toan Huynh, MD, FACS, FCCM, The F.H. “Sammy” Ross Jr. Trauma Center, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28232-2861; email: toan.huynh@carolinashealthcare.org. DOI: 10.1097/TA.0b013e3181e65133 360 The Journal of TRAUMA ® Injury, Infection, and Critical Care • Volume 69, Number 2, August 2010