Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury. Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause–effect relationship was studied using cells and mouse models. Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

S100P, an EF-hand calcium-binding protein, has been reported to be associated with the progression of many types of cancers. Transfection of an expression vector for S100P into a benign, nonmetastatic rat mammary cell line causes a 4- to 6-fold increase in its level in all four transformant cell clones. When the resultant transformant cell lines are introduced in turn into the mammary fat pads of syngeneic Furth-Wistar rats, there is a significant 3-fold increase in local muscle invasion and a significant induction of metastasis in 64% to 75% of tumor-bearing animals. In a group of 303 breast cancer patients followed for up to 20 years, antibodies to S100P immunocytochemically stain 161 primary tumors. Survival of patients with S100P-positive carcinomas is significantly worse by about 7-fold than for those with negatively stained carcinomas. There is also a significant association between the class level of immunocytochemical staining of the carcinoma cells and decreased patient survival. Positive staining for S100P is significantly associated with that for two other metastasis-inducing proteins, S100A4 and osteopontin. Patients with tumors that stained positively for both S100P and S100A4 have a significantly reduced survival of 1.1% over patients with either S100 protein alone. Multivariate regression analysis identifies S100P, S100A4, and osteopontin as the most significant independent indicators of death in this group of patients. These results suggest that stratification of patients into groups according to expression of multiple metastasis-inducing proteins may lead to a more accurate prediction of patient survival. (Cancer Res 2006; 66(2): 1199-207)

C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP–histone complexes in serum from patients with both elevated CRP and histones. CRP is able to compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans.

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K_d = 0.5 μm; IIB, K_d = 8 μm; IIC, K_d = 1.0 μm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Objectives Extracellular histones are rapidly cleared by the liver and rarely detectable in the circulation unless there is extensive cell death, as in severe trauma and sepsis. This study investigated whether circulating histones are elevated in experimental acute pancreatitis models and correlate to disease severity. Methods Acute pancreatitis was induced in mice by: (1) 4 or (2) 12 intraperitoneal injections of cerulein (50 μg/kg) at 1 hour apart; (3) retrograde infusion of 3.5% sodium taurocholate into the biliopancreatic duct. Mice were sacrificed at various time points to collect blood and tissues. Severity of pancreatitis was assessed by biochemical markers and histopathology. Circulating histones were determined by Western blotting. Results Four cerulein injections induced edematous pancreatitis, whereas 12 cerulein injections and ductal taurocholate infusion caused necrotizing pancreatitis. Circulating histones were barely detectable in the blood of animals with edematous pancreatitis but significantly increased in necrotizing pancreatitis. The levels of circulating histones were strongly correlated to histopathological scores of necrosis of the pancreas. Conclusions Circulating histones increased significantly in necrotizing pancreatitis due to extensive pancreatic acinar cell death. Levels of circulating histones may have translational potential as a biomarker of disease severity.

With the widespread use of yeast two-hybrid systems, many heterodimeric forms of S100 proteins have been found, although their biological significance is unknown. In the present study, S100A1 was found to interact with another S100 protein, S100P, by using the yeast two-hybrid system. The binding parameters of the interaction were obtained using an optical biosensor and show that S100P has a slightly higher affinity for S100A1 (K_d=10–20 nM) when compared with that for self-association (K_d=40–120 nM). The physical interaction of S100A1 and S100P was also demonstrated in living mammalian cells using a fluorescence resonance energy transfer technique. Preincubation of recombinant S100P with S100A1, before the biosensor assay, reduced by up to 50% the binding of S100P to a recombinant C-terminal fragment of non-muscle myosin A, one of its target molecules. Site-specific mutations of S100P and S100A1, combined with homology modelling of an S100P/S100A1 heterodimer using known S100P and S100A1 structures, allowed the hydrophobic interactions at the dimeric interface of the heterodimer to be defined and provide an explanation for the heterodimerization of S100P and S100A1 at the molecular level. These results have revealed the similarities and the differences between the S100P homodimer and the S100A1/S100P heterodimer.