Circulating histones are mediators of trauma-associated lung injury

Abstract

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.

Figure 1.

Extensive cell death in trauma patients elevates circulating histones to levels that damage endothelial cells in vivo. (A) Circulating nucleosome levels at admission in 250 trauma patients (Table E1) were detected by ELISA. The means ± SD in healthy donors (Normal, n = 20) and patients grouped by injury severity score (ISS) (0–3, n = 38; 4–24, n = 111; >25, n = 101; total, 250 patients) are presented. *Analysis of variance (ANOVA) test shows significant increases in patients with higher ISS compared with normal and low-ISS groups (0–3). (B) The dynamic change in circulating nucleosomes and histones in patients with severe trauma. Maximal values at a time point were set up as 100% and relative percentages are shown (means ± SD). (C) Box plot of total circulating histones in healthy donors and 52 patients with severe nonthoracic blunt trauma; median test, P < 0.01. (D) EA.hy926 cells and human pulmonary microvascular endothelial cells (HPMECs) were treated for 1 hour with serum from trauma patients on admission and the cell survival rate was measured (see Methods). Survival rates (means ± SD) relative to that of healthy donor cells treated with serum (Normal, designated 100%) are presented. *ANOVA test shows a significant decrease compared with normal (P < 0.05). (E) EA.hy926 cells were incubated for 1 hour with serum-free medium or 80% human serum from three healthy donors supplemented with various concentrations of calf thymus histones. The normalized percentage of surviving cells (means ± SD) from three independent experiments are shown. *ANOVA test shows significant reduction compared with untreated cells (P < 0.05). Histone effect became significant at a concentration as low as 10 μg/ml in serum-free medium, and 20 μg/ml in serum. (F) EA.hy926 cells were treated with serum from healthy donors (Normal) or from patients with trauma, sepsis, or pancreatitis in the absence or presence of anti-histone scFv (ahscFv) or control scFv (cscFv) (200 μg/ml). Sera from patients containing histones at more than 50 μg/ml were selected and each group had at least three patients. *ANOVA test shows significant reduction in cell survival rates compared with normal (P < 0.05). ^#ANOVA test shows significant increase in cell survival rates in the presence of ahscFv compared with that without ahscFv or with cscFv (P < 0.05).

Figure 2.

Histone-induced toxicity in mice. (A) Hematoxylin and eosin (H&E)-stained sections: (a) lung from an untreated mouse and (b) lung, (c) liver, and (d) kidney from a mouse 4 hours after infusion with histones (60 mg/kg). Obvious pathological changes were found in the lungs, such as edema (black arrows), microvascular congestion (red arrow), and hemorrhage (blue arrow) (b). Less obvious changes were found in (c) the liver and (d) kidneys. (B) Survival curves of mice injected with calf thymus (Cth) histones (75 mg/kg; a lethal dose) preincubated without (none of seven survived) or with anti-histone scFv (ahscFv, 10 mg/kg) (seven of seven survived) and control scFv (cscFv, 10 mg/ml) (none of seven survived); log-rank test, P < 0.01. (C) Top: An example of histone H3 detected by Western blot in the blood of mice after trauma. Bottom: The mean ± SD of circulating histone H3 of 10 mice. *Analysis of variance (ANOVA) test, P < 0.01. (D) H&E-stained sections: (a) lung from an untreated mouse; (b) lung, (c) liver, and (d) kidney, respectively, from a mouse 4 hours after major trauma. Similar but less severe pathological changes than those in A were observed, with arrows indicating edema (black), congestion (red), and hemorrhage (blue). Scale bar: 50 μm. (E) Liver function, as measured by aspartate transaminase (AST) and alanine transaminase (ALT), and renal function, as measured by urea and creatinine, in the blood of untreated mice, mice infused with histone at 50 mg/kg (nonlethal dose) or 75 mg/kg (lethal dose), and mice after severe trauma (10 per group). Blood was taken 4 hours after treatment or just before death in the group infused with a lethal dose of histones. Mean from untreated group was designated as 100% and the relative percentages are presented. *ANOVA test shows a significant increase compared with the untreated group (P < 0.05).

Figure 3.

Histone-induced endothelial damage and permeability increase. (A) Box plot shows the medians of circulating soluble thrombomodulin (sTM) levels in 20 healthy donors (Normal) and 52 patients with severe nonthoracic trauma. *Median test, P = 0.01. (B) Mean ± SD of circulating sTM in mice infused with saline (Control), calf thymus histones (Histones, 50 mg/ml; a nonlethal dose) or histones plus anti-histone scFv (10 mg/kg) (Hist+ahscFv) (10 mice per group). *Analysis of variance (ANOVA) test, P < 0.05 compared with the Control and Hist+ahscFv groups. (C) Mean ± SD of circulating sTM in mice treated with anesthetics alone (Control), mice subjected to trauma (Trauma), and mice infused with ahscFv (10 mg/kg) 10 minutes prior to trauma (Trauma+ahscFv) (10 mice per group). *ANOVA test, P < 0.05 compared with Control and Trauma+ahscFv groups of the same time point. (D) In vitro permeability assay, using Transwells plated with EA.hy926 cells and human pulmonary microvascular endothelial cells (HPMECs; see Methods). Fully confluent monolayers of cells on the membrane of Transwells were treated with histones (50 μg/ml) or 2 units of thrombin preincubated without or with ahscFv (200 μg/ml) or control scFv (cscFv). Fold changes (means ± SD) compared with controls (treated with buffer alone) from five independent experiments are shown. *ANOVA test, P < 0.05 compared with control. ^#Student t test shows significant reduction compared with histones alone, P < 0.05. (E) Means ± SD of right lung wet-to-dry weight ratios (Methods) of mice 4 hours after infusion with calf thymus histones (60 mg/kg) or trauma models (10 mice per group). *ANOVA test, P < 0.05 compared with control (saline infusion or anesthetics alone). ^#Student t test shows significant reduction compared with the treatment group, P < 0.05.

Figure 4.

Histone-induced coagulation activation in vivo. (A) Box plot shows the medians of thrombin–anti-thrombin (TAT) levels in 20 healthy donors (Normal) and 52 patients with severe nonthoracic trauma. *Median test, P = 0.001. (B) Mean ± SD of TAT in mice infused with saline (Control), calf thymus histones (Histones, 50 mg/ml), or histones plus anti-histone scFv (ahscFv, 10 mg/kg) (Hist+ahscFv) (10 mice per group). *Analysis of variance (ANOVA) test, P < 0.05 compared with Control and Hist+ahscFv groups. (C) Mean ± SD of TAT in mice treated with anesthetics alone (Control), mice subjected to trauma (Trauma), and mice infused with ahscFv (10 mg/kg) 10 minutes before trauma (Trauma+ahscFv) (10 mice per group). *ANOVA test, P < 0.05 compared with Control and Trauma+ahscFv groups. (D) Tissue sections from (a and c) untreated mice and (b and d) mice infused with a 75-mg/kg concentration of calf thymus histones. (b) Hematoxylin and eosin (H&E)–stained lung section with microvascular thrombi (arrows). (d) H&E-stained kidney section with thrombi (arrow). (E) Immunohistochemical staining of lung sections from (a) an untreated mouse and (b–d) mice after infusion of histones (75 mg/kg). (a and d) Stained with anti-fibrin; (b) stained with secondary antibody only; (c) probed with anti-fibrin preincubated with fibrin (Sigma) as controls. Arrow in d indicates thrombosis in an anti-fibrin–stained lung section. Scale bars: 50 μm.

Figure 5.

Membrane binding and calcium influx determine histone toxicity in endothelial cells. (A) Confocal images of EA.hy926 cells 10 minutes after incubation with fluorescein isothiocyanate (FITC)–labeled histones (10 μg/ml) alone (left) or FITC-labeled histones preincubated with anti-histone scFv (ahscFv; 100 μg/ml) (right). Arrows indicate the membrane associated with FITC-labeled histones. Scale bar: 20 μm. (B) Immunohistochemical staining of histone H3 in tissues from a mouse 4 hours after infusion with histones at 60 mg/kg (left) and an untreated mouse (right). Red arrows indicate endothelial nuclei and blue arrows point to a continuous line between endothelial nuclei stained with anti-histones to indicate association of histones with the plasma membranes of endothelial cells. Scale bar: 20 μm. (C) Representative whole-cell currents recorded from EA.hy926 cells when histones (20 μg/ml) were applied to the extracellular bathing solution. Currents generated by application of histones were reversible by washing after a short exposure (30–60 s). (D) Example of elevation of intracellular Ca²⁺ recorded with a Hitachi F-7000 fluorescence spectrometer when EA.hy926 cells were exposed to various histone concentrations. (E) Example of intracellular Ca²⁺ elevation triggered by histones (20 μg/ml) that was nearly abolished by removal of Ca²⁺ from extracellular medium. (F) Survival rates (mean ± SD) of cells incubated for 1 hour with medium containing 0–3 mM Ca²⁺ in the presence or absence of histones (20 μg/ml), from three independent experiments. *Significant reduction in cell survival rate compared with that without Ca²⁺ (analysis of variance test, P < 0.05). ^#Student t test shows significantly higher survival rates than when treated with histones (P < 0.05).

Figure 6.

Histone-triggered cytokine release. (A) Circulating IL-6 levels in both the mouse trauma model (Trauma) and the histone infusion model with or without the coinfusion of anti-histone scFv (ahscFv, 10 mg/kg). With 10 mice per group, the means ± SD of IL-6 are shown. *Analysis of variance (ANOVA) test shows a significant increase compared with that before treatment (Before), P < 0.01. ^#Student t test shows significant reduction compared with that without ahscFv coinfusion, P < 0.05. (B) Left: Direct flow cytometric analysis of human leukocytes from healthy donors. R1 = neutrophils; R2 = monocytes; R3 = lymphocytes. Right: Flow cytometric analysis of the same sample but fluorescently stained with rat IgG–phycoerythrin (PE) (black), rat anti-human IL-6–PE (blue), anti-CDs–fluorescein isothiocyanate (FITC) (purple) (R1, CD15 for neutrophils; R2, CD14 for monocytes; and R3, CD3 for lymphocytes) to demonstrate the storage of presynthesized IL-6 in peripheral leukocytes from healthy donors. (C) IL-6 release from freshly isolated leukocytes incubated with various histone concentrations for 2 hours. A typical dose–response curve is shown. (D) Isolated leukocytes were incubated with histones (50 μg/ml) in the absence (blue) or presence of ahscFv (200 μg/ml) (red) and control scFv (cscFv) (green). The supernatants were collected at various time points for determination of IL-6 concentration. A time course of means ± SD is shown from three repeats. *ANOVA test shows significant increase compared with time zero (P < 0.05). ^#ANOVA test shows significant reduction compared with the same time point but without ahscFv (P < 0.05). (E) Extracellular Ca²⁺ effects on IL-6 release from leukocytes treated with histones (50 μg/ml) for 2 hours. A typical dose–response curve is shown.

Figure 7.

Effects of histones on neutrophils. (A) Anti-myeloperoxidase (MPO)–stained lung sections. Left: A mouse with trauma shows MPO-positive cell infiltration (arrows). Right: A mouse infused with calf thymus histones (60 mg/kg) shows MPO inside alveoli (arrows), a possible neutrophil breakdown product. Scale bar: 50 μm. (B) Increased release of MPO from isolated human neutrophil after histone treatment (Methods). The means ± SD of extracellular MPO from three independent experiments are shown. *Analysis of variance test shows a significant increase compared with untreated (UT), P < 0.05. (C) Neutrophil extracellular trap (NET) formation after treatment with histone at 50 mg/ml (Methods). Left: A typical NET-like structure stained with propidium iodide (red). Arrows indicate that DNA fibers are outside the cell nucleus. Scale bar: 10 μm. Right: Most cells develop NET-like structures after 3 hours of histone treatment. Scale bar: 50 μm. (D) Anti–citrullinated (cit)-histone–stained lung section from a mouse infused with histones (60 mg/kg). Arrows indicate cit-histone–positive neutrophils congested inside alveolar walls. Scale bar: 20 μm.