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Wolthuis, Esther K.*†‡; Vlaar, Alexander P. J.*‡; Choi, Goda*‡§; Roelofs, Joris J. T. H.; Haitsma, Jack J.∥**; van der Poll, Tom§††‡‡; Juffermans, Nicole P.*‡; Zweers, Machteld M.; Schultz, Marcus J.*‡§§

doi: 10.1097/SHK.0b013e31817d42dd
Basic Science Aspects

Ventilator-induced lung injury is mediated, at least in part, by TNF-α. We determined the effect of a recombinant human soluble TNF receptor fusion protein (etanercept) on mechanical ventilation (MV)-induced changes in a murine ventilator-induced lung injury model. After pretreatment with etanercept or placebo, C57Bl/6 mice were anesthetized and randomized to MV with either low tidal volumes (VT, ∼7.5 mL/kg) or high VT (∼15 mL/kg) for 5 h. Instrumented but spontaneously breathing mice served as controls. End points were lung wet-to-dry ratios, lung histopathology scores, protein levels, neutrophil cell counts and thrombin-antithrombin complex levels in bronchoalveolar lavage fluid (BALF), and cytokine levels in lung homogenates. The number of caspase 3-positive cells was used as a measure for apoptosis. Etanercept treatment attenuated MV-induced changes, in particular, in MV with high VT. Compared with placebo, etanercept reduced the number of neutrophils in BALF and thrombin-antithrombin complex levels in BALF and cytokine levels in lung homogenates. Lung wet-to-dry ratios, histopathology scores, and local protein levels in BALF, however, were not influenced by etanercept treatment. The number of caspase 3-positive cells was significantly higher in etanercept-treated animals. Inhibition of TNF by etanercept attenuates, in part, MV-induced changes.

Departments of *Intensive Care Medicine and Anesthesiology; Laboratory for Experimental Intensive Care and Anesthesiology; Departments of §Internal Medicine and Pathology, Academic Medical Center at the University of Amsterdam, Amsterdam; Department of Anesthesiology, Erasmus Medical Center, Rotterdam, The Netherlands; **Interdepartmental Division of Critical Care Medicine, University of Toronto, Keenan Research Center, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Canada; ††Center of Infection and Immunity and ‡‡Center for Experimental and Molecular Medicine Academic Medical Center at the University of Amsterdam; and §§HERMES Critical Care Group, Amsterdam, The Netherlands.

Received 27 Dec 2007; first review completed 14 Jan 2008; accepted in final form 17 Apr 2008

Address reprint requests to Esther K. Wolthuis, Department of Intensive Care Medicine, Academic Medical Center, University of Amsterdam, C3-423, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail:

This study has been performed at the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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Mechanical ventilation (MV) may aggravate or even initiate lung injury, a phenomenon frequently referred to as "ventilator-induced lung injury" (VILI) in experimental models of MV or "ventilator-associated lung injury" (VALI) in mechanically ventilated patients (1, 2). Low tidal volume (VT) ventilation reduces mortality in critically ill patients with acute lung injury (3). In addition, low tidal volumes should also be considered inpatients without lung injury because retrospective clinical studies suggest that the use of large VT favors the development of lung injury (4).

VILI/VALI occurs in the absence of gross structural lung damage. The biotrauma hypothesis proposes that biophysical forces are responsible for alteration of normal cellular physiology in the lungs, leading to a proinflammatory milieu (5), disturbances in alveolar fibrin turnover (6, 7), changes in pulmonary repair and remodeling, and mechanisms involving programmed cell death. Mechanical ventilation injures the pulmonary epithelium through either apoptotic or nonapoptotic cell death. In vitro mechanical strain induces proapoptotic changes in human lung epithelial cells (8, 9). Furthermore, in vivo animal studies demonstrate impairment of apoptosis pathways to limit pulmonary inflammation, lung injury, and to protect against multiple organ failure and death (10, 11). Therefore, it has been proposed that intra-alveolar apoptosis is a potentially harmful process that can be targeted in the treatment of VILI or VALI (12).

Ventilator-induced lung injury possesses a significant TNF-α-dependent component (13), including the optional capacity to induce apoptosis. In addition, neutrophil recruitment is substantially attenuated in TNF receptor knockout mice and mice treated with intratracheally administered anti-TNF antibodies (13). Etanercept is a humanized dimeric fusion protein consisting of the extracellular ligand-binding portion of the 75-kd TNF receptor linked to the Fc portion of immunoglobulin G1. It can bind to two TNF molecules blocking their interaction with cell surface TNF receptors, thereby interfering with biological activity of TNF. TNF inactivation by etanercept is a thousand times stronger than TNF inactivation by p75 monomeric TNF receptor (14). Etanercept has been proven to inhibit the activity of TNF in several animal model systems of inflammatory and autoimmune diseases (15-18). It has been tested in numerous clinical trials, and it has been approved for rheumatic disorders.

Present strategies aiming at minimizing VALI in the critically ill patients consist of using low tidal volumes (VT) (19). However, additional strategies to attenuate pulmonary inflammation may be useful to further reduce VALI. The aim of the present investigation was to determine the effect of etanercept on VILI using low and high VT.

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The Animal Care and Use Committee of the Academic Medical Center approved all experiments. Animal procedures were carried out in compliance with Institutional Standards for Human Care and Use of Laboratory Animals.

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Seventy-two female C57Bl/6 mice (6 - 8 weeks old, 17 - 20 g; Charles River, Maastricht, The Netherlands) were maintained at the animal care facility of the Academic Medical Center according to institutional guidelines. Control animals (n=6) served as nonventilated controls. The other animals were all mechanically ventilated for 5 h with two different MV strategies and 2 different pretreatments (either placebo or etanercept). Thus, six groups of animals were formed (n = 72). In each group, half of the mice were used for bronchoalveolar lavage (BAL, right lung) and wet-to-dry ratio (W/D; left lung). The other half was used for blood sampling from the carotid artery and blood gas analysis; the lungs of these mice were homogenized (right lung) and used for histopathology (left lung).

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Anesthesia protocol

Mice received an intraperitoneal bolus of 1 mL 0.9% saline 1 h before start of anesthesia and initiation of MV. A tracheostomy (Y-tube connector; outer diameter, 1.0 mm; inner diameter, 0.6 mm; VBM Medizintechnik GmbH, Sulz, Germany) was inserted under general anesthesia with 126 mg/kg ketamine, 0.2 mg/kg medetomidine, and 0.5 mg/kg atropine (i.p.). Body temperature was kept constant at 36.5 to 37.5°C with the use of rectal temperature monitoring and a warming device. Maintenance anesthesia consisted of 36 mg/kg ketamine, 0.04 mg/kg medetomidine, and 0.075 mg/kg atropine. Maintenance mix was administered via an intraperitoneal catheter (PE 10 tubing; BD, Breda, the Netherlands) every hour. To correct for hypovolemia, sodium bicarbonate was administered via the intraperitoneal catheter every 30 min.

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Mechanical ventilation

Mice were placed in a supine position and connected to a human ventilator (Servo 900 C; Siemens, Solna, Sweden). Pressure-controlled MV was initiated with either an inspiratory pressure of 10 cm H2O (resulting in VT ∼7.5 mL/kg; low VT) or an inspiratory pressure of 18 cm H2O (resulting in VT ∼15 mL/kg; high VT). The respiratory rate was set at 120 breaths per minute with low VT and 70 breaths per minute with high VT, respectively. Positive end-expiratory pressure was set at 2 cm H2O in both MV strategies. The inspiration-to-expiration ratio was kept at 1:1 throughout the experiment. A sigh (sustained inflation with 30 cm H2O) for five breaths was performed every 30 min to recruit atelectatic lung tissue. Fio2 was set at 0.5. Control mice, receiving half the dose of induction anesthesia, were instrumented, but not ventilated.

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Hemodynamic and ventilatory monitoring

A subset of mice were used for monitoring of noninvasive blood pressure and pulse every hour during the 5-h period of MV using a murine blood pressure/pulse monitor (Visitech Systems, Apex, NC). Tidal volume was monitored hourly with aspecially designed mouse Fleisch tube connected to the body plethysmograph. The flow signal was integrated from a differential pressure transducer, and data were recorded and digitized online using a 16-channel data acquisition program (ATCODAS; Dataq Instruments, Inc., Akron, Ohio) and stored on a computer for postacquisition off-line analysis. A minimum of five consecutive breaths were selected for analysis of the digitized VT signals. After 5 h of MV, arterial blood from the carotid artery was taken for blood gas analysis.

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Etanercept treatment

To neutralize TNF, 100 μg etanercept (Enbrel Wyeth Pharmaceuticals BV, Hoofddorp, The Netherlands) was administered 15 and 1 h (i.p.) before start of MV. The dose of 100 μg had been shown to neutralize TNF (20-23). Etanercept was reconstituted at a concentration of 25 mg/mL in sterile water as suggested by the manufacturer and was further diluted in phosphate-buffered saline to a final concentration of 500 μg/mL. Control mice received the same volume of intraperitoneal sterile saline.

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Bronchoalveolar lavage

Bronchoalveolar lavage was performed by instilling three times 0.5-mL aliquots of saline by a 22-gauge Abbocath-T catheter (Abbott, Sligo, Ireland) into the trachea. Approximately 1.2 mL of lavage fluid was retrieved per mouse, and cell counts were determined using a hemacytometer (Beckman Coulter, Fullerton, Calif). Subsequently, differential counts were done on Giemsa-stained cytospin preparations. Cell-free supernatants were stored at −20°C.

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W/D ratio

Lungs were weighed and subsequently dried for 3 days in a 65°C stove. The W/D ratio represents tissue edema.

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Lung homogenates

Lungs were homogenized in four volumes of sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, Okla). Lung homogenates were diluted 1:1 inlysis buffer (150 mM NaCl; 15 mM Tris; 1 mM MgCl H2O; 1 mM CaCl2; 1% Triton X-100; 100 μg/mL pepstatin A, leupeptin, and aprotinin, pH 7.4) and incubated at 4°C for 30 min. Cell-free supernatants were obtained by centrifugation at 1,500g for 15 min and stored at −20°C.

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Lungs were fixed in 4% formalin and embedded in paraffin. Four-micrometer sections were stained with hematoxylin-eosin and analyzed by a pathologist who was blinded for group identity. To score lung injury, we used a modified VILI histology scoring system as previously described (24). In short, four pathologic parameters were scored on a scale of 0 to 4: (1) alveolar congestion, (2) hemorrhage, (3) leukocyte infiltration, and (4) thickness of alveolar wall/hyaline membranes. A score of 0 represents normal lungs; 1, mild, less than 25% lung involvement; 2, moderate, 25% to 50% lung involvement; 3, severe, 50% to 75% lung involvement and 4, very severe, greater than 75% lung involvement. The total histological score was expressed as the sum of the score for all parameters.

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BAL fluid (BALF) total protein levels were determined using a Bradford protein assay kit (OZ Biosciences, Marseille, France) according to manufacturers' instructions. Thrombin-antithrombin complex was measured with a mouse-specific enzyme-linked immunosorbent assay (25). Cytokine and chemokine levels in lung homogenates were measured by enzyme-linked immunosorbent assay according to the manufacturer's instructions. TNF, IL-6, macrophage inflammatory protein (MIP) 2, and keratinocyte chemoattractant (KC) assays were all obtained from R and D Systems (Abingdon, UK).

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Caspase 3 immunohistochemistry

Activated caspase 3, a distal enzyme in the caspase cascade, can be detected in cell and tissues using antibodies specific for (cleaved) activated form of caspase 3. In short, deparaffinized slides were boiled in citrate buffer (pH 6.0). After blocking of nonspecific binding and endogenous peroxidase activity, slides were incubated with rabbit antihuman active caspase 3 polyclonal antibody (Cell Signaling, Beverly, Mass), followed by biotinylated swine antirabbit antibody (Dako, Glostrup, Denmark) (26). At a magnification of 400×, we counted the number of caspase 3-positive cells in 10 randomly chosen fields. The total number of caspase 3-positive cells was the summation of caspase 3-positive cells in 10 fields.

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Statistical analysis

All data are expressed as means (SEM) or median (interquartile range [IQR]), depending on normal distribution of data or not. To detect differences, the Dunnett method or Mann-Whitney U test in conjunction with two-way ANOVA was used. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 12.0.2 (SPSS, Chicago, Ill).

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Physiological measurements

Body temperature was strictly controlled between 36.5 and 37.5°C in all experiments to achieve normal homeostasis during prolonged procedures. Systolic blood pressure was well maintained during the 5-h MV period in both MV groups in both placebo and treatment mice. Blood gas parameters were also maintained within physiological range and were not different among groups (data not shown).

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Lung injury with low and high tidal volume

Wet-to-dry ratio, histopathology, and total BALF protein levels were all affected by MV (Fig. 1). Wet-to-dry ratios were higher in high-VT mice as compared with low-VT mice (P < 0.001) and control mice (P < 0.001). The pulmonary histopathology scores were similar in high VT and low-VT mice (3.5 [1.0] in high-VT mice and 3.0 [0.8] in low-VT mice) but higher as compared with control mice (0.0 [0.0]; P < 0.05). Total BALF protein levels were significantly higher in high VT as compared with low VT (P < 0.001) and control mice (P < 0.001).

The total number of cells in the BALF was not influenced by either MV strategy. However, the number of neutrophils was significantly higher in high VT as compared with low VT (P < 0.001) and control mice (P < 0.001; Fig. 1).

Thrombin-antithrombin complex levels in BALF were significantly higher in high VT as compared with low-VT mice (P < 0.001) and nonventilated controls (P < 0.001; Fig. 2).

Pulmonary levels of TNF, IL-6, MIP-2, and KC were all influenced by MV (Fig. 3). Levels of TNF and IL-6 were higher in both ventilated groups as compared with control mice (P < 0.001 and 0.001), with higher levels in high-VT mice (P = 0.014 and P < 0.001). Similarly, pulmonary levels of MIP-2 and KC were higher in both ventilated groups as compared with control mice (P < 0.001 and 0.001), with higher levels in high-VT mice (P < 0.001 and 001).

The mean number of caspase 3-positive cells was higher in high-VT mice as compared with low-VT mice in controls, although the difference did not reach statistical significance (P = 0.055; Fig. 4).

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Effect of etanercept on VILI

Treatment with etanercept did not influence the W/D ratios (Fig. 1) nor the pulmonary histopathology score. Treatment with etanercept also did not influence the protein level in BALF (Fig. 1).

Etanercept attenuated neutrophil influx (Fig. 1). In high VT and low-VT mice, the number of neutrophils was significantly lower after administration of etanercept (P = 0.017 vs. placebo treatment in high VT and low-VT mice).

Etanercept also attenuated activation of coagulation with MV (Fig. 2). Thrombin-antithrombin complex levels were lower in high-VT mice after etanercept treatment as compared with placebo-treated animals (P = 0.03). In lower VT mice, etanercept did not influence activation of coagulation.

Pulmonary levels of IL-6, MIP-2, and KC were all significantly lower in both ventilated groups after etanercept treatment (P = 0.001 and 0.028 for IL-6, P = 0.037 and 0.009 for MIP-2, and P = 0.002 for KC vs. placebo treatment in high VT and low-VT mice; Fig. 3).

Etanercept treatment induced an increase in the number of caspase 3-positive cells in control and low-VT mice (P = 0.032 and 0.01 vs. placebo treatment in low-VT mice and controls), but not in high-VT mice (Fig. 4).

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In the present study, evaluating the effect of etanercept on VILI, both low and high VT, induces lung injury. Whereas treatment with etanercept does not influence the W/D, histopathology, or total protein level in BALF, neutrophil influx, pulmonary coagulation, and pulmonary levels of IL-6, MIP-2, and KC are affected by etanercept treatment. This effect is found in both MV groups, although to a lesser extent in low-VT mice. Finally, etanercept treatment has a proapoptotic effect in the lungs in low-VT mice and in nonventilated animals.

Wet-to-dry ratio, histopathology, and local protein levels in BALF were not influenced by pretreatment with etanercept. These findings are in line with results from a previous study (13). Indeed, double-receptor TNF (TNFR1 and TNFR2) knockout mice had a similar protein level in BALF with high or low VT MV as compared with wild-type animals, indicating a comparable degree of lung injury and pulmonary edema.

Wilson et al. (13) demonstrated pulmonary neutrophil recruitment in response to ventilation with high VT to be dependent on a TNF-mediated component. Indeed, intratracheal administration of anti-TNF antibodies attenuated alveolar neutrophil recruitment. In addition, in TNF receptor knockout animals, neutrophil recruitment was lower. Similarly, Imai et al. (27) showed pretreatment with intratracheal administration of anti-TNF antibodies to attenuate VILI in a saline-lavaged rabbit model. Our data are in agreement with these studies, demonstrating a decline in the number of neutrophils after treatment with etanercept, with less coagulation activation and a decline in the pulmonary cytokine and chemokine levels.

Clinical studies have provided convincing evidence that use of high VT leads to an increase in pulmonary inflammation. Indeed, Ranieri et al. (28) demonstrated conventional ventilation with high VT (∼11 mL/kg) to increase local TNF levels in patients with established acute lung injury. Protective ventilation with lower VT (∼8 mL/kg) attenuated neutrophil influx into the lungs. Here, we show etanercept to be capable of preventing, in part, neutrophil influx, pulmonary coagulopathy, and inflammation caused by high VT. We also show that even with lower VT, etanercept treatment attenuates pulmonary inflammation. Etanercept has been tested in numerous clinical trials, and it has been approved for treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis (29). This new drug is now being evaluated for treating many other clinical disorders in which up-regulation of TNF seems to play a pathophysiological role. Our data suggest etanercept may be of value in patients at risk for VALI.

We showed etanercept to have proapoptotic properties in the lungs. In other inflammatory conditions, etanercept may limit the inflammatory response through induction of apoptosis. Indeed, in patients with Crohn disease, induction of apoptosis of activated inflammatory cells has been proposed as a mechanism of the protective effect of blocking TNF activity (30). In addition, in mice lacking TNF receptor 1, apoptosis of ileal crypt epithelial cells was increased in a model of cecal ligation and puncture (31). It remains to be established whether etanercept limits VILI via apoptosis.

Our model has some limitations. First, lung-protective MV with low VT may promote atelectasis. Low VT used in the present study is equal to that of spontaneously breathing C57Bl/6 mice (6 - 9 mL/kg) (32). In our model, we used deep inflation every 30 min to prevent atelectasis. Allen et al. (33) demonstrated that periodic recruitment with relatively deep inflations during ventilation with low VT can improve oxygenation, ventilation, and lung mechanical function with no evidence of lung injury by 2 h in mechanically ventilated mice. They suggest a threshold tolerance of deep inflation frequency, beyond which the intrinsic reparative properties of the lung epithelium are overwhelmed. Some pulmonary inflammation seen in low-VT mice may have been caused by atelectrauma. Alternatively, even MV using a VT of 7.5 mL/kg may still cause overdistension of alveoli. Second, the VT used in high-VT mice is quite large. Although there is still underuse of low VT ventilation, VT's have declined gradually during the past 10 years. However, VT's of as large as 15 mL/kg are reported to be used in human clinical studies (34, 35). Therefore, our comparison may still reveal relevant information on lung injury caused by MV. Third, different amounts of anesthetics were administered to ventilated and control mice. Most anesthetics have an effect on innate immunity. Ketamine has an immunosuppressive effect on inflammatory cytokine production (36). Therefore, differences in inflammatory response between ventilated and control mice may not just result from differences in ketamine administration.

In conclusion, we found that etanercept attenuates neutrophil influx, procoagulant, and inflammatory changes in VILI. Blockade of TNF signaling may have therapeutic potential to reduce pulmonary inflammation in patients at risk for VALI. Further investigations are necessary to prove this hypothesis.

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Artificial respiration; tidal volume; adult respiratory distress syndrome; pulmonary inflammation; ventilator-induced lung injury

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