BACKGROUND Transfusion of stored blood is associated with increased complications. Microparticles (MPs) are small vesicles released from RBCs that can induce cellular dysfunction, but the role of RBC-derived MPs in resuscitation from hemorrhagic shock is unknown. In the current study, we examined the effects of RBC-derived MPs on the host response to hemorrhage and resuscitation. STUDY DESIGN MPs were isolated from murine packed RBC units, quantified using flow cytometry, and injected into healthy mice. Separate groups of mice underwent hemorrhage and resuscitation with and without packed RBC–derived MPs. Lungs were harvested for histology and neutrophil accumulation and assessed by myeloperoxidase content. Human neutrophils were treated with human RBC-derived MPs and CD11b expression, superoxide production, and phagocytic activity were determined. RESULTS Stored murine packed RBC units contained increased numbers of RBC-derived MPs compared with fresh units. Hemorrhaged mice resuscitated with MPs demonstrated substantially increased pulmonary neutrophil accumulation and altered lung histology compared with mice resuscitated without MPs. Intravenous injection of MPs into normal mice resulted in neutrophil priming, evidenced by increased neutrophil CD11b expression. Human neutrophils treated with RBC-derived MPs demonstrated increased CD11b expression, increased superoxide production, and enanced phagocytic ability compared with untreated neutrophils. CONCLUSIONS Stored packed RBC units contain increased numbers of RBC-derived MPs. These MPs appear to contribute to neutrophil priming and activation. The presence of MPs in stored units can be associated with adverse effects, including lung injury, after transfusion.

Background and Aim The purpose of the present study was to determine the effects of IL-37 on liver cells and on liver inflammation induced by hepatic ischemia/reperfusion (I/R). Materials and methods Mice were subjected to I/R. Some mice received recombinant IL-37 (IL-37) at the time of reperfusion. Serum levels of alanine amino transferase, liver myeloperoxidase content were assessed. Serum and liver TNFα, MIP-2 and KC were also assessed. Hepatic reactive oxygen species (ROS) levels were assessed. For in vitro experiments, isolated hepatocytes and Kupffer cells were treated with IL-37 and inflammatory stimulants. Cytokine and chemokine production by these cells were assessed. Primary hepatocytes were induced cell injury and treated with IL-37 concurrently. Hepatocyte cytotoxicity and Bcl-2 expression were determined. Isolated neutrophils were treated with TNFα and IL-37 and neurtrophil activation and respiratory burst were assessed. Results IL-37 reduced hepatocyte injury and neutrophil accumulation in the liver after I/R. These effects were accompanied by reduced serum levels of TNFα and MIP-2 and hepatic ROS levels.IL-37 significantly reduced MIP-2 and KC productions from LPS-stimulated hepatocytes and Kupffer cells. IL-37 significantly reduced cell death and increased Bcl-2 expression in hepatocytes. IL-37 significantly suppressed TNFα induced-neutrophil activation. Conclusions IL-37 is protective against hepatic I/R injury. These effects are related to the ability of IL-37 to reduce proinflammatory cytokine and chemokine production by hepatocytes and Kupffer cells as well as having a direct protective effect on hepatocytes. In addition, IL-37 contributes to reduce liver injury through suppression of neutrophil activity.

BACKGROUND Resuscitation with blood products instead of crystalloid in the treatment of hemorrhagic shock has been associated with improved outcomes in trauma patients requiring massive transfusions and transfusion of fresh products results in reduced morbidity and mortality compared with aged blood. Processes to eliminate harmful components of aged blood are under investigation. We hypothesized that washing blood would reduce levels of proinflammatory mediators in stored units, and resuscitation with washed units would attenuate the proinflammatory response in mice after hemorrhagic shock. METHODS Mice underwent pressure-controlled hemorrhage and resuscitation with fresh packed red blood cells (pRBCs) or 15-day-old washed or unwashed pRBCs. Cytokine concentrations in donor samples and recipient serum were measured. In addition, cytokine concentrations were measured in 15-day-old units that underwent three interval washes versus one poststorage wash. RESULTS Blood stored for 15 days demonstrated increased levels of interleukin lα, keratinocyte chemoattractant, macrophage inflammatory protein 1α, and macrophage inflammatory protein 2 compared with fresh units. Washing 15-day-old pRBCs reduced concentrations of these cytokines. Cytokine levels in stored units that underwent multiple washes versus a single wash were not different. Mice resuscitated with 15-day-old unwashed pRBCs had increased levels of serum cytokines compared with mice resuscitated with fresh and 15-day-old washed pRBCs. CONCLUSION Aged pRBC units have elevated levels of proinflammatory cytokines compared with fresh units, and washing aged units after storage reduces cytokine concentrations. Resuscitation with washed units blunts the proinflammatory response in mice after hemorrhage. Washing aged pRBCs may improve the safety profile of aged units and may result in improved outcomes in subjects after hemorrhagic shock and resuscitation.

Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. MDC signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer’s solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by ELISA. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines KC, MIP-2, and MIP-1α as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. MDC neutralization may provide a therapeutic strategy to mitigate this inflammatory response.

Background Severe hemorrhagic shock and resuscitation initiates a dysfunctional systemic inflammatory response leading to end-organ injury. Clinical evidence supports the transfusion of high ratios of plasma and packed red blood cells (pRBCs) in the treatment of hemorrhagic shock. The effects of resuscitation with different ratios of fresh blood products on inflammation and organ injury have not yet been characterized. Materials and Methods Mice underwent femoral artery cannulation and pressure-controlled hemorrhage for 60 minutes, then resuscitation with fresh plasma and pRBCs collected from donor mice. Plasma alone, pRBCs alone, and ratios of 2:1, 1:1, and 1:2 plasma:pRBCs were used for resuscitation strategies. Mice were sacrificed to determine biochemical and hematologic parameters, serum cytokine concentrations, tissue myeloperoxidase levels, and vascular permeability. Results As compared to other resuscitation strategies, mice resuscitated with pRBCs alone exhibited increased hemoglobin levels, while other hematologic and biochemical parameters were not significantly different among groups. Compared to 1:1, mice resuscitated with varying ratios of plasma:pRBCs exhibited increased cytokine concentrations of KC, MIP-lα, and MIP-2, and increased intestinal and lung myeloperoxidase levels. Mice resuscitated with 1:1 had decreased vascular permeability in the intestine and lung as compared to other groups. Conclusions Resuscitation with a 1:1 ratio of fresh plasma:pRBCs results in decreased systemic inflammation and attenuated organ injury. These findings support the potential advantage of transfusing blood products in physiologic ratios to improve the treatment of severe hemorrhagic shock.

Hemorrhagic shock is a primary injury amongst combat casualties. Aeromedical evacuation (AE) of casualties exposes patients to a hypobaric, hypoxic environment. The effect of this environment on the host response to hemorrhagic shock is unknown. In the present study, we sought to determine the effect of simulated AE on systemic inflammation and organ injury using a murine model of hemorrhagic shock. Mice underwent femoral artery cannulation and were hemorrhaged for 60 minutes. Mice were then resuscitated with a 1:1 ratio of plasma:packed red blood cells. At 1 or 24 hours after resuscitation, mice were exposed to a 5-hour simulated AE or remained at ground level (control). Serum was analyzed for cytokine concentrations and organs were assessed for neutrophil accumulation and vascular permeability. Mice in the simulated AE groups demonstrated reduced arterial oxygen saturation compared to ground controls. Serum cytokine concentrations, neutrophil recruitment, and vascular permeability in the lung, ileum, and colon in the simulated AE groups were not different from the ground controls. Our results demonstrate that mice exposed to simulated AE following hemorrhagic shock do not exhibit worsened systemic inflammation or organ injury compared to controls. The data suggest that AE has no adverse effect on isolated hemorrhagic shock.

No abstract