Carl J. Hauser

Circulating Mitochondrial DAMPs Cause Inflammatory Responses to Injury

Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors. Similarly, cellular injury can release endogenous ‘damage’-associated molecular patterns (DAMPs) that activate innate immunity. Mitochondria are evolutionary endosymbionts that were derived from bacteria and so might bear bacterial molecular motifs. Here we show that injury releases mitochondrial DAMPs (MTDs) into the circulation with functionally important immune consequences. MTDs include formyl peptides and mitochondrial DNA. These activate human polymorphonuclear neutrophils (PMNs) through formyl peptide receptor-1 and Toll-like receptor (TLR) 9, respectively. MTDs promote PMN Ca2+ flux and phosphorylation of mitogen-activated protein (MAP) kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTDs can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.

The coagulopathy of trauma: a review of mechanisms.

BACKGROUND Bleeding is the most frequent cause of preventable death after severe injury. Coagulopathy associated with severe injury complicates the control of bleeding and is associated with increased morbidity and mortality in trauma patients. The causes and mechanisms are multiple and yet to be clearly defined. METHODS Articles addressing the causes and consequences of trauma-associated coagulopathy were identified and reviewed. Clinical situations in which the various mechanistic causes are important were sought along with quantitative estimates of their importance. RESULTS Coagulopathy associated with traumatic injury is the result of multiple independent but interacting mechanisms. Early coagulopathy is driven by shock and requires thrombin generation from tissue injury as an initiator. Initiation of coagulation occurs with activation of anticoagulant and fibrinolytic pathways. This Acute Coagulopathy of Trauma-Shock is altered by subsequent events and medical therapies, in particular acidemia, hypothermia, and dilution. There is significant interplay between all mechanisms. CONCLUSIONS There is limited understanding of the mechanisms by which tissue trauma, shock, and inflammation initiate trauma coagulopathy. Acute Coagulopathy of Trauma-Shock should be considered distinct from disseminated intravascular coagulation as described in other conditions. Rapid diagnosis and directed interventions are important areas for future research.

Blood transfusion, independent of shock severity, is associated with worse outcome in trauma.

BACKGROUND We have previously shown that blood transfusion in the first 24 hours is an independent predictor of mortality, intensive care unit (ICU) admission, and increased ICU length of stay in the acute trauma setting when controlling for Injury Severity Score, Glasgow Coma Scale score, and age. Indices of shock such as base deficit, serum lactate level, and admission hemodynamic status (systolic blood pressure, heart rate) and admission hematocrit were considered potential confounding variables in that study. The objectives of this study were to evaluate admission anemia and blood transfusion within the first 24 hours as independent predictors of mortality, ICU admission, ICU length of stay (LOS), and hospital LOS, with serum lactate level, base deficit, and shock index (heart rate/systolic blood pressure) as covariates. METHODS Prospective data were collected on 15,534 patients admitted to a Level I trauma center over a 3-year period (1998-2000) and stratified by age, gender, race, Glasgow Coma Scale score, and Injury Severity Score. Admission anemia and blood transfusion were assessed as independent predictors of mortality, ICU admission, ICU LOS, and hospital LOS by logistic regression analysis, with base deficit, serum lactate, and shock index as covariates. RESULTS Blood transfusion was a strong independent predictor of mortality (odds ratio [OR], 2.83; 95% confidence interval [CI], 1.82-4.40; p < 0.001), ICU admission (OR, 3.27; 95% CI, 2.69-3.99; p < 0.001), ICU LOS (p < 0.001), and hospital LOS (Coef, 4.37; 95% CI, 2.79-5.94; p < 0.001) when stratified by indices of shock (base deficit, serum lactate, shock index, and anemia). Patients who underwent blood transfusion were almost three times more likely to die and greater than three times more likely to be admitted to the ICU. Admission anemia (hematocrit < 36%) was an independent predictor of ICU admission (p = 0.008), ICU LOS (p = 0.012), and hospital LOS (p < 0.001). CONCLUSION Blood transfusion is confirmed as an independent predictor of mortality, ICU admission, ICU LOS, and hospital LOS in trauma after controlling for severity of shock by admission base deficit, lactate, shock index, and anemia. The use of other hemoglobin-based oxygen-carrying resuscitation fluids (such as human or bovine hemoglobin substitutes) in the acute postinjury period warrants further investigation.

External fixation or arteriogram in bleeding pelvic fracture: initial therapy guided by markers of arterial hemorrhage.

BACKGROUND Bleeding pelvic fractures (BPF) carry mortality as high as 60%, yet controversy remains over optimal initial management. Some base initial intervention on fracture pattern, with immediate external fixation (EX FIX) in amenable fractures aimed at controlling venous bleeding. Others feel ongoing hemodynamic instability indicates arterial bleeding, and prefer early angiography (ANGIO) before EX-FIX. Our aim was to evaluate markers of arterial bleeding in patients with BPF, thus identifying patients requiring early ANGIO regardless of fracture pattern. METHODS Patients with pelvis fracture were identified from a Level I trauma center registry over a 7-year period and records reviewed. From this group, two subsets were analyzed: those with initial hypotension related to pelvic fracture, and those without hypotension who underwent pelvic ANGIO. Data included hemodynamics, response to resuscitation, presence of contrast blush on CT, fracture treatment and outcome. Adequate response to initial resuscitation (R) was defined as a sustained (>2 hours) improvement of systolic blood pressure to >90 mm Hg systolic after the administration of < or = 2 units packed red blood cells. Those with repeated episodes of hypotension despite resuscitation were classified as non-responders (NR) RESULTS: From 1/94-1/01, 1171 patients were admitted with pelvic ring fracture. Thirty-five (0.3%) had hypotension attributable to pelvis fracture. 28 fell into the NR group, and 26 of these underwent ANGIO. Nineteen (73%) showed arterial bleeding while 3 resuscitation response patients underwent ANGIO with none demonstrating bleeding (p = 0.03). Sensitivity and specificity of inadequate response to initial resuscitation for predicting the presence of arterial bleeding on ANGIO were 100% and 30% respectively while negative and positive predictive value were 100% and 73%. In patients with fractures amenable to external fixation (n = 16), 44% had arterial bleeding on ANGIO, and all were in the NR group. An additional 17 patients without hypotension also underwent ANGIO. Contrast blush on admission CT was seen in 4, 3 of which had arterial bleeding seen on ANGIO (75%). Sensitivity and specificity for contrast blush in predicting bleeding on ANGIO were 60% and 92% with positive and negative predictive value being 75% and 85%. CONCLUSIONS In patients with hypotension and pelvic fracture, therapy selection based on initial response to resuscitation in BPF yields a 73% positive ANGIO rate in NR patients. Delay in ANGIO for EX FIX in patients with amenable fractures would have delayed embolization in the face of ongoing arterial bleeding in 44% of patients. In stable patients with pelvic fracture, contrast blush also indicates a high likelihood of arterial injury and ANGIO is indicated. Optimal therapy in the face of BPF requires early determination of the presence of arterial bleeding so that ANGIO can be rapidly obtained, and response to initial resuscitation as well as the presence of contrast blush aid in this decision.

Use of a transcutaneous PO2 regional perfusion index to quantify tissue perfusion in peripheral vascular disease.

In order to develop transcutaneous oxygen tension (PtcO2) measurements into a practical method for assessing peripheral vascular disease, the relationships between extremity and chest wall PtcO2 were examined in subjects with and without systemic atherosclerotic disease. The ratio of extremity to chest PtcO2, or transcutaneous regional perfusion index (RPI) assessed limb oxygenation more reliably than did direct PtcO2 measurement by obviating the effects of changes in systemic oxygen delivery upon local PtcO2. The authors find that transcutaneous oximetry can be used during treadmill exercise testing and that the RPI is unchanged by exercise in all normal subjects. PtcO2 and RPI were then measured during rest, position change, and exercise testing in patients with intermittent claudication. Whereas normal subjects maintain a constant thigh and calf RPI during exercise, patients with intermittent claudication consistently manifested large decreases in RPI in these areas when they were exercised until symptomatic. The authors find no overlap between the responses of normal subjects and patients with claudication; positive findings are, therefore, highly specific for exercise-induced limb ischemia. Since transcutaneous RPI exercise testing is easily performed and highly reproducible, it is well suited to clinical use in the diagnosis and documentation of intermittent claudication. Furthermore, since limb ischemia can be quantified, this method lends itsetf both to grading the severity of disease and to evaluating clinical progression of disease. It is suggested that such a quantitative approach to evaluation of intermittent claudication may allow refinement and extension of the indications for operative intervention in patients with intermittent claudication.

Purinergic Signaling: A Fundamental Mechanism in Neutrophil Activation

Neutrophil activation by infectious and inflammatory signals requires ATP release and its feedback through purinergic receptors. Feedback for Function Neutrophils migrate to sites of infection, where they kill pathogens by processes such as phagocytosis and the release of reactive oxygen species. However, activated neutrophils can also result in tissue damage and inflammatory diseases in the host; thus, a better understanding of the mechanisms that regulate neutrophil activation could help in the development of therapies that could curb their destructive side effects. Chen et al. found that neutrophils responded to a range of infectious and inflammatory signals by releasing adenosine triphosphate (ATP). In addition to its role as a cellular energy source, ATP and its metabolites function as intercellular signaling molecules by stimulating purinergic receptors. The authors found that stimulation of formyl peptide receptors (FPRs) on neutrophils triggered the release, through pannexin-1 hemichannels, of ATP that signaled in an autocrine fashion through P2Y2 receptors. Moreover, this autocrine signal was required for neutrophil activation. In addition, mice deficient in P2Y2 receptors were less capable of clearing bacteria than were their wild-type counterparts. Together, these data suggest that feedback signaling by ATP released by neutrophils contributes to their activation. Efficient activation of neutrophils is a key requirement for effective immune responses. We found that neutrophils released cellular adenosine triphosphate (ATP) in response to exogenous stimuli such as formylated bacterial peptides and inflammatory mediators that activated Fcγ, interleukin-8, C5a complement, and leukotriene B4 receptors. Stimulation of the formyl peptide receptor (FPR) led to ATP release through pannexin-1 (panx1) hemichannels, and FPRs colocalized with P2Y2 nucleotide receptors on the cell surface to form a purinergic signaling system that facilitated neutrophil activation. Disruption of this purinergic signaling system by inhibiting or silencing panx1 hemichannels or P2Y2 receptors blocked neutrophil activation and impaired innate host responses to bacterial infection. Thus, purinergic signaling is a fundamental mechanism required for neutrophil activation and immune defense.

Bone Marrow Failure Following Severe Injury in Humans

Background: Hematopoietic failure has been observed in experimental animals following shock and injury. In humans, bone marrow dysfunction has been observed in the red cell component and characterized by a persistent anemia, low reticulocyte counts, and the need for repeated transfusions despite adequate iron stores. While a quantitative defect in white blood cell count has not been noted, an alteration in white blood cell function manifesting as an increased susceptibility to infection is well established. Since the etiology of this anemia remains unknown and the bone marrow has been rarely studied following injury, we measured various parameters of hematopoiesis directly using bone marrow from trauma patients and tested the hypothesis that trauma results in profound bone marrow dysfunction, which could explain both the persistent anemia and the alteration in white blood cell function. Methods: Bone marrow aspirates and peripheral blood were obtained between day 1 and 7 following injury from 45 multiple trauma patients. Normal volunteers served as controls. Peripheral blood was assayed for hemoglobin concentration, reticulocyte count, erythropoietin levels, white blood cell count, and differential. Peripheral blood and bone marrow were cultured for hematopoietic progenitors (CFU-GM, BFU-E, and CFU-E colonies). Results: Bone marrow CFU-GM, BFU-E, and CFU-E colony formation was significantly reduced while peripheral blood CFU-GM, BFU-E, and CFU-E was increased in the trauma patients compared with normal volunteers. Bone marrow stroma failed to grow to confluence by day 14 in >90% of trauma patients. In contrast, bone marrow stroma from volunteers always reached confluence between days 10 and 14 in culture. The mean hemoglobin concentration and reticulocyte counts of the trauma patients were 8.6 ± 1.0 g/dL and 2.75 ± 0.7% respectively, while their plasma erythropoietin levels were 2 to 10 times greater than control values. Conclusions: Release of immature white blood cells into the circulation may also contribute to a failure to clear infection and an increased propensity to organ failure. Concomitantly, profound changes occur within the bone marrow, which include the increased release of erythroid and myeloid progenitors into the circulation, a decrease in progenitor cell growth within the bone marrow, and an impaired growth of the bone marrow stroma. Erythropoietin levels are preserved following trauma, implying that the persistent anemia of injury is related to the failure of the bone marrow to respond to erythropoietin.

The immune microenvironment of human fracture/soft-tissue hematomas and its relationship to systemic immunity.

The immune environment of human soft-tissue injury is unstudied. We studied fracture soft-tissue hematomas (FxSTH) in 56 patients with high-energy bony fractures. FxSTH serum and mononuclear cells (MNC) as well as fracture patient plasma and blood MNC were studied. Twenty healthy controls donated plasma and MNC. Soluble tumor necrosis factor (TNF)-alpha, interleukin (IL-1 beta, IL-2, 6, 8, 10, 12, and interferon-gamma were studied by enzyme linked immunosorbent assay. Cells were studied by flow cytometry after cell-membrane stains for CD-14, TNF-alpha (mTNF), and human leukocyte antigen-DR, or intracellular stains for TNF (icTNF) and IL-10. Thirty-six patients with Injury Severity Score < 15 were analyzed further to evaluate the effects of isolated fracture on systemic immunity. Cytokines were rarely detectable in control plasma. TNF-alpha, IL-1 beta, IL-2, and interferon-gamma were rarely found in FxSTH serum or fracture patient plasma. All FxSTH sera were rich in IL-6, peaking before 48 hours (12,538 +/- 4,153 vs. 3,494 +/- 909 pg/mL, p = 0.02, U test). In Injury Severity Score < 15, IL-6 was not detectable in most early fracture patient plasma, but rose after 48 hours (p = 0.028). FxSTH serum IL-8 peaked after 48 hours (440 +/- 289 vs. 4,542 +/- 1,219 pg/mL, p = 0.006) and circulating IL-8 appeared after 72 hours. IL-6 and IL-8 showed gradients from FxSTH serum to paired PtS (p < 0.05, Wilcoxon). IL-10 was abundant (884 +/- 229 pg/mL) in FxSTH serum < 24 hours old. FxSTH serum IL-12 peaked late (3,323 +/- 799 pg/mL, day 4-7) then fell (p < 0.001, analysis of variance). Only IL-12 was higher in fracture patient plasma (1,279 +/- 602 pg/mL) than FxSTH serum (591 +/- 327 pg/mL) during the first 48 hours (p = 0.032, U test). On flow cytometry, control monocytes expressed 201 +/- 31 mTNF sites/cell, but icTNF was absent. mTNF was up-regulated after injury more in FxSTH monocytes (3,202 +/- 870 sites/cell) than peripheral blood monocytes (584 +/- 186 sites/cell) (p < 0.05 vs. peripheral blood monocytes by Wilcoxon, p < 0.001 vs. control monocytes by U test). Intracellular IL-10 was abundant in all MNC, but varied widely after injury. Fracture and peripheral blood monocytes expressed far less human leukocyte antigen-DR than control monocytes. Fractures create an inflammatory local environment. Proximal mediators are cell-associated and relatively confined to the wound, but soluble IL-6, IL-8, and IL-10 are abundant and probably exported. Systemic MNC have complex responses to local injuries. These may reflect the combined impact of multiple soluble cytokines initially generated within the wound. FxSTH appear to be a potentially important source of immunomodulatory cytokines in trauma.

Hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock

There is evidence suggesting that the ischemic gut is a major source of factors that lead to neutrophil activation, and that neutrophil activation can be reduced by hypertonic saline resuscitation. Thus, we tested whether trauma-hemorrhagic shock-induced neutrophil activation can be reduced by hypertonic saline resuscitation, as well as whether hypertonic saline reduces the ability of mesenteric lymph from shocked animals to activate neutrophils. Male Sprague-Dawley rats subjected to trauma (laparotomy), plus 90 min of shock [mean arterial pressure (MAP) MAP = 30 mmHg] or sham shock were resuscitated with Ringers lactate or 7.5% hypertonic saline at an equivalent sodium load. Whole blood samples were collected before shock as well as at 1 and 2 h after the end of the shock period for neutrophil CD11b and CD18 expression measurements. In a second set of experiments, mesenteric lymph samples collected from rats subjected to trauma plus hemorrhagic shock (T/HS) or trauma plus sham-shock (T/SS) and resuscitated with Ringers lactate or hypertonic saline were tested for their ability to modulate PMN CD11b, CD18, or L-selectin expression, as well as prime PMN for an augmented respiratory burst. To avoid confounding results due to interspecies differences, while at the same time looking at potential human responses, both naive rat and human PMN were tested. Both CD11b and CD18 expression were increased in PMN harvested from rats subjected to T/HS and resuscitated with Ringers lactate solution, but not in T/HS rats resuscitated with hypertonic saline. These results indicate that PMN activation is increased to a greater extent in Ringers lactate-resuscitated than hypertonic saline-resuscitated animals. Likewise, mesenteric lymph from the T/HS rats resuscitated with Ringers lactate increased naive rat and human PMN CD11b and CD18 expression to a greater extent than did T/HS lymph from the hypertonic saline-treated rats. Additionally, T/HS lymph from the Ringers lactate-but not the hypertonic saline-treated rats induced PMN L-selectin shedding. Lastly, T/HS lymph from the Ringers lactate-treated rats induced the greatest PMN respiratory burst. These results indicate that resuscitation from T/HS with hypertonic saline is associated with less PMN activation than resuscitation with Ringers lactate, and that factors produced or released by the postischemic intestine and carried in the mesenteric lymph contribute to neutrophil activation after an episode of T/HS.

Mitochondrial DAMPs increase endothelial permeability through neutrophil dependent and independent pathways.

Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.