Acute respiratory distress syndrome (ARDS) is characterized by severe acute hypoxemia caused by increased lung edema, atelectasis, intrapulmonary shunting, and hypoxemia.1 Although overall mortality seems to be decreasing because of recent improvements in supportive care, there are presently no proven, effective pharmacological therapies to treat ARDS or to prevent its associated complications.2 Exaggerated cytokine release from mononuclear cells has been observed in ARDS and ARDS is associated with endotoxemia.3
High mobility group box-1 protein (HMGB1) was originally as a DNA-binding nonhistone chromosomal protein implicated in diverse cellular functions such as stabilization of nucleosomal structure and regulation of transcriptional factors.4 On stimulation with lipopolysaccharides (LPS), HMGB1 is secreted from monocytes/macrophages and fosters inflammatory responses. HMGB1 is passively released from necrotic cells and mediates inflammation and immune responses.5 Recently, HMGB1 has been implicated as a putative danger signal involved in the pathogenesis of a variety of inflammatory conditions including septic shock, acute coronary syndrome, and disseminated intravascular coagulation.6 However, the regulation of HMGB1 levels in ARDS remained to be elucidated.
Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) plays a crucial role in the initiation and progression of various diseases including septic shock, and it has been measured as a biomarker of oxidative DNA damage.7,8 However, little is known about HMGB1 and 8-OHdG regulation in patients with ARDS.
Polymyxin B-immobilized fiber (PMX-F) treatment is safe and effective in septic shock patients. We reported previously that PMX-F treatment is effective in reducing various inflammatory mediators.9,10 Tsushima et al.11 reported that PMX-F treatment is effective against ARDS induced by direct or indirect pulmonary injury. We hypothesized that blood HMGB1 and urinary 8-OHdG are abnormally secreted in patients with ARDS and that PMX-F treatment would be effective in ameliorating these changes.
Patients and Methods
Our study group comprised 20 patients with ARDS (12 men and 8 women; mean age, 62 ± 8 years) who underwent PMX-F treatment and 20 age-matched healthy volunteers (12 men and 8 women; mean age, 60 ± 6 years). This study was approved by the local ethics committee for human research and was performed in the intensive care units of our hospitals. Informed consent was obtained from each patient and/or each patient's family. We excluded individuals with malignancy, ischemic heart disease, alcohol abuse, chronic respiratory failure including interstitial pneumonitis and tuberculosis, collagen disease, or liver disease. ARDS was defined according to the criteria of the American European Consensus Conference:12 acute onset of lung injury, diffuse bilateral infiltrates seen on chest radiography: a PaO2/FiO2 (PF) ratio of <200 torr, pulmonary artery occlusion pressure of <18 mm Hg, and no clinical evidence of left atrial hypertension. To rule out congestive heart failure, ultrasound cardiography was performed. For selection of patients, the condition underlying ARDS was pneumonia or another infection (Table 1). All patients were given several kinds of broad spectrum antibiotics on admission to other hospitals or clinics but were not given steroids before PMX-F treatment. On enrollment in the study, the same antibiotics were continued. The Acute Physiology and Chronic Health Evaluation (APACHE II) score,13 Sequential Organ Failure Assessment score,14 and lung injury score15 were determined at the time PMX-F treatment was started.
PMX-F hemoperfusion was as described previously.16,17 PMX-F treatment was performed in patients twice, with a 24-hour interval. Access to the circulation for direct hemoperfusion with PMX-F was obtained by means of a double-lumen catheter (Arrow International, Inc., Reading, PA) inserted into the femoral vein according to the Seldinger method. Direct hemoperfusion was carried out for 2 hours at a flow rate of 100 ml/min. Nafamostat mesilate (Torii Pharmaceutical, Torii, Japan) was used as anticoagulant. Nafamostat mesilate is a serine protease inhibitor that exerts its anticoagulatory effects primarily by inhibiting thrombin. The half-life of nafamostat mesilate is 8 minutes, and its anticoagulatory effects were observed only in the extracorporeal circuit. Blood pressure, heart rate, the PF ratio, white blood cell count, C-reactive protein (CRP), blood endotoxin, blood HMGB1, and urinary 8-OHdG levels were measured before and after the first and second PMX-F treatments.
Blood endotoxin levels were determined by the Endospecy test (Seikagaku Corp., Tokyo, Japan) according to the perchloric acid method1,16 described previously. The upper limit of normal is 9.8 pg/ml. The urinary 8-OHdG levels were estimated at 11:00 A.M. Twenty-four-hour urine samples were obtained starting at 11:00 A.M. and ending at 11:00 A.M. the next day. Urine samples were obtained from all patients (there were no anuric patients). The urinary 8-OHdG levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit that uses a highly sensitive monoclonal antibody, as described previously (8-OHdG Check, Nikken Foods, Fukuroi, Shizuoka, Japan).18,19 Blood HMGB1 levels were measured by ELISA kit obtained from Shino-Test Corp. (Kanagawa, Japan) as previously described.20 Validated inter-assay and intra-assay coefficients of variation were <10% and the detection limit of the ELISA system was 0.3 ng/ml.21
Normally distributed values are presented as mean ± SD. Differences were analyzed by Student's t test. Differences in values obtained before and after PMX-F treatment were assessed by Wilcoxon's signed-rank test. To analyze the relations between endotoxin, HNGB1, and urinary 8-OHdG levels, linear regression was performed. p values <0.05 were accepted as statistically significant.
Twenty-eight-day survival of patients with ARDS after PMX-F treatment was 80%. Clinical and laboratory data of the 20 patients with ARDS are shown in Table 1. Blood endotoxin levels (20.6 ± 8.2 pg/ml), blood HMGB1 levels (26.5 ± 12.5 ng/ml), and urinary 8-OHdG levels (38.5 ± 19.2 ng/mg.Cr) were significantly higher in patients with ARDS than in healthy volunteers (2.2 ± 0.6 pg/ml, p < 0.001; 0.68 ± 0.22 ng/ml, p < 0.001; 4.6 ± 2.8 ng/mg.Cr, and p < 0.001, respectively). Clinical and laboratory markers before and after PMX-F treatment are shown in Table 2. Systolic and diastolic blood pressures and the PF ratio increased significantly after the first (p < 0.05) and second (p < 0.01) PMX-F treatments. White blood cell and CRP levels decreased significantly after the first (p < 0.05) and second (p < 0.01) treatments. Heart rate changed little after PMX-F treatment. Blood endotoxin levels decreased significantly after the first (10.8 ± 4.0 pg/ml, p < 0.01) and second (4.0 ± 1.2 pg/ml, p < 0.001) PMX-F treatments. Blood HMGB1 levels decreased significantly after the first (12.0 ± 6.0 ng/ml, p < 0.01) and second (2.8 ± 0.6 ng/ml, p < 0.001) treatments. Urinary 8-OHdG levels also decreased significantly after the first (19.0 ± 7.8 ng/mg.Cr, p < 0.01) and second (6.8 ± 2.2 ng/mg.Cr, p < 0.001) treatments. Relations between blood endotoxin, blood HMGB1, and urinary 8-OHdG levels in patients with ARDS before and after the first and second PMX-F treatments are shown in Table 3. Blood endotoxin levels correlated significantly with blood HMGB1 levels before (p < 0.01) and after the first (p < 0.01) and second (p < 0.01) PMX-F treatments. Blood endotoxin levels correlated significantly with urinary 8-OHdG levels before (p < 0.001) and after the first (p < 0.001) and second (p < 0.001) treatments. Blood HMGB1 levels correlated significantly with urinary 8-OHdG levels before (p < 0.001) and after the first (p < 0.01) and second (p < 0.01) PMX-F treatments.
In this study, blood endotoxin, HMGB1, and urinary 8-OHdG levels in patients with ARDS were significantly higher than those in healthy controls and PMX-F treatment markedly improved oxygenation and blood pressure, blood endotoxin, blood HMGB1, and urinary 8-OHdG levels. Uriu et al.22 reported that improvement of hyperdynamic circulation was directly related to endotoxin removal and that endotoxin plays an important role in the development of septic shock. Tsushima et al.11 reported that in patients with ARDS, PMX-F treatment improved circulatory disturbance and oxygenation despite the underlying disease and that mortality improved remarkably compared with that before the induction of PMX-F treatment.
HMGB1 mediates the induction of delayed endotoxin lethality and acute lung injury.23 The pathophysiology of ARDS involves resident lung cells, including epithelial cells, as well as neutrophils, monocytes/macrophages, and platelets.24 HMGB1 is released by activated macrophages as a late-phase mediator during prolonged inflammation.25 Vascular smooth muscle cells positive for HMGB1 were shown to express CRP and matrix metalloproteinase (MMP)-9.25 MMPs play an important role in pathogenic pulmonary processes, and MMP-9 is essential for remodeling of the basement membrane in various pulmonary inflammatory diseases including ARDS.26 HMGB1 binds LPS in a concentration-dependent manner, and this binding was shown to be inhibited by polymyxin B.27 A mixture of HMGB1 and LPS vs. HMGB1 or LPS alone, results in a higher increase in tumor necrosis factor-alpha production in peripheral blood monocytes.27 Thus, we propose that HMGB1 plays an important role in ARDS with endotoxemia. Recently, HMGB1 levels were shown to be higher in bronchoalveolar lavage (BAL) fluid obtained from the site of infection in patients with pneumonia than in lavage fluid from healthy control subjects, suggesting that HMGB1 release may occur predominantly at the site of infection.28 However, little is known about HMGB1 levels in BAL fluid from patients with ARDS. Measuring HMGB1 has been quite challenging because no ELISA was available until recently.20,21 Earlier studies used blotting methods for measuring HMGB1.29 Gaini et al.30 reported that median HMGB1 levels measured by ELISA were 1.54 ng/ml in non-infected patients, 2.41 ng/ml in infected patients without sepsis, 2.24 ng/ml in patients with sepsis, and 2.18 ng/ml in patients with severe sepsis. In this study, blood HMGB1 levels in patients with ARDS were extremely high (mean: 26.5 ng/ml) based on the same ELISA system. Our data may be due, in part, to disease severity or to the time of blood sampling (because HMGB1 is a “late-onset” cytokine). Recently, Sakamoto et al.31 reported that HMGB1 levels improved significantly after successful PMX-F treatment and that the circulation dynamics of patients with septic shock can be improved by reducing HMGB1 levels.
Oxidant-mediated tissue injury is probably important in the pathogenesis of ARDS.32 Oxidants are generated as a result of the inflammatory response by phagocytic cells such as mononuclear cells.33 Levels of reactive species correlate with both the disease outcome and the severity of the injury to the alveolar epithelium in acute lung injury.33 The most frequently detected and studied oxidized nucleoside in nuclear and mitochondrial DNA lesions is 8-OHdG. On DNA repair, 8-OHdG is excreted into the urine.34 Recently, investigators have reported the importance of urinary 8-OHdG in both adult and pediatric medicine.35,36 In this study, urinary 8-OHdG levels were significantly higher in patients with ARDS than in healthy volunteers. Oxidative stress is induced by administration of LPS. We first showed that urinary 8-OHdG levels in patients with ARDS correlated with blood endotoxin levels. Li et al.37 reported that the degrees of inflammation and oxidative stress were positively related to LPS. It may be important to study correlation between urinary 8-OHdG and other known oxidative stress markers including plasma malondialdehyde, plasma F2 isoprostanes, and plasma thiobarbituric acid reactant substances in patients with ARDS. Some investigators reported that trace elements and vitamins that support antioxidant functions are safe and may be associated with a reduction in mortality in critically ill patients.38 Nathens et al.39 reported that the early antioxidant supplementation with alpha-tocopherol and ascorbic acid reduces the incidence of organ failure in critically ill patients including patients with ARDS. In contrast, oxidative stress is increased in critically ill patients according to antioxidant vitamin intake, independent of disease severity.40 In this study, PMX-F treatment significantly inhibited urinary 8-OHdG levels in patients with ARDS.
In this study, we could not compare between patients with ARDS with PMX-F treatment and those who did not receive PMX-F treatment. Patients with ARDS were severe, therefore, randomized study was ethically difficult. However, it would be needed to compare patients with ARDS treated with PMX-F treatment or not in future. Sakamoto et al.41 reported the effectiveness of continuous hemodiafiltration (CHDF) after PMX-F treatment of septic shock patients with endotoxemia. Nakada et al.42 reported that cytokine-oriented critical care using CHDF may be an effective strategy for the treatment of septic shock. It may be needed to compare patients with ARDS with endotoxemia treated with PMX-F and those treated with CHDF or those treated with both combinations.
In summary, we showed that blood HMGB1 and urinary 8-OHdG levels are increased in patients with ARDS and that PMX-F treatment is effective in decreasing these levels.
The authors thank Mr. Hisataka Shoji, Mr. Yoshihiro Nakamura, and Mr. Misao Hachiya, Toray Medical Co., Ltd., Tokyo, Japan, for their helpful suggestions. The authors also thank Mr. Yoshinobu Takahashi, Shinmatsudo Central General Hospital, Chiba, Japan, for his technical assistance.
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