Severe gunshot injuries in a porcine model: impact on central markers of innate immunity.

BACKGROUND: the mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll-like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. METHODS: nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor-α (TNF-α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. RESULTS: trauma and treatment caused a significant decline in the LPS-stimulated concentrations of TNF-α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF-α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. CONCLUSION: the results suggest that trauma-induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.

Early time course of altered leukocyte response to lipopolysaccharide and peptidoglycan in porcine gunshot injury.

BACKGROUND: Penetrating injuries are frequently combined with polybacterial soiling. Clearance of the microorganisms depends on the ability to activate immune responses, but post-traumatic hyporeactivity of immune cells is almost universal. The aim of this study was to map the early time course of this altered leukocyte reactivity, and to compare the reactions to subsequent Gram-positive or Gram-negative challenges. METHODS: Twelve juvenile pigs sustained two standardized rounds, one through the right femur and one through the left upper abdomen. First aid treatment and acute surgery were started immediately. Blood samples were drawn before trauma and after 10, 30, 60, and 90 min, and thereafter stimulated in ex vivo whole blood for 3 h with lipopolysaccharide (LPS, 10 ng/ml), peptidoglycan (PepG, 1 microg/ml), or an equivalent amount of normal saline. The leukocyte response was evaluated by measurement of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, IL-8, and IL-10 in the supernatant. RESULTS: In the post-traumatic in vivo serum, the concentration of TNF-alpha increased steadily (significant after 60 min). A reduced ex vivo reaction to LPS was evident after 10 min, and was statistically significant after 30 min. The lowest levels were reached after 90 min. The ex vivo synthesis of TNF-alpha after stimulation with PepG remained unaltered. A similar development was seen for IL-6. IL-1 beta levels did not change, while IL-8 increased significantly only after 60 and 90 min. CONCLUSIONS: Trauma almost instantaneously reprogrammed circulating leukocytes. As measured with TNF-alpha, a profound hyporeactivity to LPS, but not to PepG, was induced. In addition, no global down-regulation of leukocyte function was found after stimulation with LPS.

Response of circulating immune cells to major gunshot injury, haemorrhage, and acute surgery.

PURPOSE: The purpose of this study was to use an established porcine model to investigate the effects on immune function of severe gunshot injury. METHODS: Twelve pigs sustained two standardised rounds, one through right femur and one through left upper abdomen. First aid treatment and acute surgery was started immediately. Blood samples were drawn before shooting and after 75 min. Circulating neutrophils were isolated and reactive oxygen species (ROS) measured. Serum levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-10 were determined at 0, 75 min, as well as 2h after incubation with 1 microg/ml endotoxin in an ex vivo whole blood model. RESULTS: TNF-alpha, IL-1beta, and IL-6 significantly increased at 75 min. ROS in circulating granulocytes tended to increase (NS). Incubation with endotoxin led to a more than 100-fold increase of TNF-alpha pre-trauma, compared to a three-fold increase post-trauma (p<0.0001 between groups). A similar pattern was obtained for IL-1beta, and IL-6. IL-10 was below detection in all samples. The granulocytes maintained their ability to react to the protein kinase C activator phorbol myristate acetate (PMA) after trauma. CONCLUSION: Severe gunshot injury and peritraumatic stress rapidly activate circulating immune cells, but reduce their capacity to react to a subsequent challenge to endotoxin.