Lymphocyte modulation with FTY720 improves hemorrhagic shock survival in swine

Abstract

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3(+) lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock.

Figure 1. Experimental design.

Liver injury was initiated at t = 0. Uncontrolled hemorrhage occurred until 1 hour, at which time the abdomen was packed and temporarily closed (pre-hospital phase). The animal was observed until 2 hours when hospital care was initiated. The liver was repaired and the abdomen definitively closed. The animal was then observed for a total of 72 hours. Blood transfusion was administered as indicated. Necropsy was performed when the animal expired or at 72 hours following euthanasia. The uncontrolled hemorrhage and pre-hospital phases were considered the hemorrhage period, while the hospital care phase was considered the reperfusion period. In the experimental group, FTY720 (0.3 mg/kg diluted in 250 mL of NS)) was administered 15 minutes following liver injury. Vehicle controls were treated with 250 mL of NS.

Figure 2. Hemodynamic profiles during hemorrhage period for FTY720 and vehicle control (in this and subsequent figures control is used for vehicle control) groups.

a) Heart rate. b) Mean arterial pressure. c) Cardiac output. There were no statistical differences between the control and experimental hemodynamic profiles.

Figure 3. Kaplan-Meier survival curve for experimental groups versus control with log-rank test for statistical comparison.

a) Overall survival. b) Reperfusion period survival. Dashed red line indicates end of hemorrhage and beginning of reperfusion period.

Figure 4. Peripheral leukocyte counts during hemorrhage and reperfusion periods.

Dashed line indicates end of hemorrhage and beginning of reperfusion period. Leukocytes were significantly decreased during the reperfusion period in the FTY720 group (p = 0.03). Data is depicted as mean ± SEM.

Figure 5. Central lymphocyte counts at time of necropsy.

a) Representative immunohistochemistry pictures (20× magnification) of anti-CD3 staining of normal, control, and FTY720 mesenteric lymph node and spleen tissue. b) Quantification of anti-CD3 reactivity by ACIS. * indicates p<0.05 compared to normal, unmanipulated tissue; † indicates p<0.05 compared to control. Data is depicted as mean ± SEM.

Figure 6. Peripheral neutrophil counts during hemorrhage and reperfusion periods.

Dashed line indicates end of hemorrhage and beginning of reperfusion period. Data is depicted as mean ± SEM.

Figure 7. Lung tissue neutrophil infiltration at time of necropsy.

a) Representative histology pictures (400× magnification) of normal, control and PATG tissue. b) Quantification of anti-MPO reactivity. * indicates p<0.05 compared to normal, unmanipulated tissue. † indicates p<0.05 compared to control group. Data is depicted as mean ± SEM.

Figure 8. Inflammatory gene transcript expression in liver tissue of FTY720 relative to the control group.

Mean value of relative fold expression is depicted on a logarithmic scale. * indicates p<0.05 compared to control group.