Department of Anaesthetics and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom
Submitted 10 August 2004 ; accepted in final form 13 October 2004
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ABSTRACT |
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tumor necrosis factor receptor knockout mice; anti-tumor necrosis factor antibody; chemokine; ventilator-induced lung injury
In the presence of underlying lung injury, a number of animal studies of VILI have reported upregulation of TNF within the lung (2, 7, 25, 31, 45), and increased TNF levels have been observed in bronchoalveolar lavage fluid from ARDS patients receiving conventional nonprotective ventilation (37, 44). We previously found in rabbits that lung injury induced by mechanical ventilation following saline lung lavage was attenuated by anti-TNF antibody (25). However, with such a "two-hit" approach, there are inherent difficulties in distinguishing the component of inflammation due to the preexisting lung injury from that due to ventilation per se (14). Our saline-lavaged rabbit study could not precisely determine to what extent anti-TNF treatment attenuated the injury induced by the lavage procedure and the injury induced by ventilation. In addition, two-hit models may sometimes be associated with complicated, potentially model-specific interactions between preinjury and ventilation, making the interpretation of results difficult (15, 54).
In the absence of predisposing lung injury, there has been considerable debate in the literature as to whether high-stretch ventilation alone can initiate TNF-mediated pulmonary inflammation (14, 50). Studies of such "one-hit" animal VILI models, which should provide a clearer answer regarding the effect of mechanical ventilation per se, have in fact produced conflicting and inconclusive results regarding upregulation of TNF within the lung (11, 20, 27, 38, 47, 48, 51). We have recently addressed this controversy in a mouse model of VILI by demonstrating that TNF protein upregulation is highly transient in response to mechanical lung stretch in vivo and therefore often difficult to detect during the course of experiments (55). However, demonstrating the presence of TNF during VILI does not necessarily indicate a significant biological role, and the in vivo physiological impact of TNF signaling in mediating stretch-induced pulmonary inflammation has yet to be determined.
In this study, we therefore investigated the role of TNF in the inflammatory response to high-stretch mechanical ventilation in the absence of underlying injury in mice. The involvement of TNF signaling was assessed using genetically modified mice lacking TNF receptors and wild-type (WT) mice treated with anti-TNF antibody. Mice were ventilated by high-stretch ventilation until lung injury started to become apparent, and then ventilation was continued using a noninjurious strategy. This protocol allowed the animal to survive long enough after the onset of injury to develop detectable intra-alveolar neutrophil infiltration in response to a highly standardized, short-duration mechanical stretch. The results demonstrated that pulmonary inflammation induced by high-stretch ventilation without underlying injury has a substantial TNF-mediated component.
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METHODS |
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Experiments were carried out under the guidelines of the Animals (Scientific Procedures) Act 1986, United Kingdom, using WT male C57BL6 mice (Charles River) aged 913 wk (2228 g) or age-matched male TNF receptor double knockout (DKO) mice (p55/p75/) (32) backcrossed onto their WT C57BL6 strain for five generations (generous gift from Amgen, Thousand Oaks, CA). To investigate the effects of high-stretch ventilation on pulmonary inflammation, we used a modification of our previous mouse model of VILI (10, 55). Mice were anesthetized by intraperitoneal injection of 2.5 ml/kg of Hypnorm (0.8 mg/kg of fentanyl and 25 mg/kg of fluanisone) and 2.5 ml/kg of midazolam (12.5mg/kg) and acutely instrumented as described in detail previously (55). In brief, animals were ventilated via endotracheal tube by a custom-made, flow-regulated mouse ventilator-pulmonary function testing system using O2 supplemented with 04% CO2 as required to avoid hypocapnia. Cannulae were placed in the left carotid artery for monitoring blood pressure (BP), blood gas analysis, and fluid infusion (0.9% NaCl containing 10 U/ml of heparin at 0.4 ml/h) and in the right jugular vein for antibody administration. During surgery and stabilization, animals were ventilated with a tidal volume (VT) of 910 ml/kg, a positive end-expiratory pressure (PEEP) of 2.5 cmH2O, and a respiratory rate (RR) of 120 breaths/min using O2 supplemented with 2% CO2. Inspiratory:expiratory ratio was kept at 1:2 throughout the experiment. After instrumentation, sustained inflation of 35 cmH2O for 5 s was given two times to standardize volume history of the lungs.
Chronic Inhibition of TNF Signaling
After baseline physiological measurements, WT and TNF receptor DKO mice were randomly allocated to receive either low- or high-stretch ventilation protocols. 1) Low stretch group: mice continued to be ventilated with the baseline low-stretch ventilation used during surgery (described above) for a further 240 min using O2 with 2% CO2. Sustained inflation of 35 cmH2O for 5 s was performed every 30 min throughout the experiments to prevent atelectasis. 2) High-stretch group: mice were ventilated with the same settings as the low-stretch group except for a short period of high-stretch ventilation (Fig. 1). Mice were first ventilated with a VT of 4344 ml/kg, zero PEEP, and RR of 90 breaths/min using O2 supplemented with 4% CO2. Initial peak inspiratory pressure (PIP) attained with these settings was 48.8 ± 0.8 (means ± SD) cmH2O. At the start of high stretch, animals received an intra-arterial bolus of 200 µl of saline. High-stretch ventilation was terminated when PIP started to increase by 510% (average duration 67 ± 18 min). We have previously shown that high-stretch ventilation of this level, if maintained longer (>2 h), produces progressive VILI in C57BL6 mice with a substantial pulmonary edema, decrease in lung compliance, and lung pathology characterized by airway epithelial damage and hyaline membrane formation (55). After the high-stretch period, animals were returned to baseline low-stretch ventilation (VT of 910 ml/kg) for a further 180 min to allow the animals to survive long enough to develop intra-alveolar neutrophil infiltration, since continuing the high-stretch ventilation leads to very rapidly progressing pulmonary edema and death in rodents (13, 16, 55). During this period, animals were ventilated using O2 with 0% CO2, since impaired gas exchange induced by the high-stretch ventilation meant that use of additional CO2 was not required. The level of mechanical lung injury induced by high stretch was standardized by ensuring that on return to low stretch, PIP was 4050% increased compared with pre-high-stretch values. Animals that showed <30% (4/22 mice) or >70% (3/22 mice) increases in the post-high-stretch PIP values were excluded from the study.
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WT mice were ventilated with high-stretch ventilation and then received anti-TNF antibody either intratracheally or intravenously. 1) Intratracheal antibody group: mice were ventilated using the same high-stretch protocol described above. Immediately after ventilation was returned from the high-stretch to low-stretch settings, 50 µg of anti-TNF- antibody (hamster anti-mouse TNF-
monoclonal antibody, TN3-19.12; BD Pharmingen, Oxford, UK) or isotype control antibody (hamster IgG, BD Pharmingen) were administered via the endotracheal tube in a total volume of 100 µl. Antibody administrations were carried out only after the high-stretch period to ensure a consistent degree of stretch-induced mechanical lung injury, since intratracheal administration of any fluid before high stretch will have somewhat unpredictable effects on lung mechanics, making it more difficult to standardize lung injury by monitoring PIP. Pilot studies indicated that intratracheal fluid administration produced severe hypoxemia (presumably due to airway obstruction and resultant ventilation/perfusion mismatch) after the return to low-stretch ventilation if the animals were ventilated with 2.5 cmH2O of PEEP. To counteract this, low-stretch ventilation after the high-stretch period was carried out with 5 cmH2O of PEEP. 2) Intravenous antibody group: WT mice were ventilated using the same high-stretch protocol described above. Immediately after ventilation was returned to the low-stretch settings, 50 µg of anti-TNF-
antibody or isotype control were administered in a volume of 200 µl via the right jugular vein over 10 min.
As in the chronic TNF inhibition experiments, animals that showed <30% (1/20 mice) or >70% (2/20 mice) increases in PIP immediately after high stretch (before the administration of antibody) were excluded from the study. The TN3-19.12 antibody has been shown to inhibit TNF-mediated cell cytotoxic activity >95% at a dose of 510 ng/pg of mouse TNF (40). Assuming 2 ml of circulating blood volume for a 25-g mouse, the dose of the antibody used in this study (50 µg) should neutralize 2,5005,000 pg/ml of mouse TNF activity in blood.
Physiological Measurements
Airway pressure, airway flow, and mean BP were monitored continuously, and blood gas analyses and determination of respiratory system compliance using the end-inflation occlusion technique (18) were carried out at intervals throughout the experiments. No animals died during any of the protocols used.
Lung Lavage
At the end of the protocols, mice were euthanized by anesthetic overdose and subjected to lung lavage as described previously (55). Lavage fluids recovered were centrifuged (5 min, 1,500 rpm, 4°C) and supernatants were stored at 80°C. Cell pellets were used for cell counting by hemocytometer, and differential cell analysis by Diff-Quik staining of samples was prepared by Cytospin (Shandon, Runcorn, UK).
Protein and Chemokine Measurements
Protein concentration in lavage fluid was determined by the Bradford method (6) using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK) with BSA (Sigma-Aldrich, Gillingham, UK) as a standard. Macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC) protein levels were measured using commercially available sandwich ELISA kits (R&D Systems, Abingdon, UK).
Data Analysis
Data are expressed as means ± SD. Statistical analysis was carried out by t-tests or ANOVA with Scheffé's tests. A P value of <0.05 was considered significant.
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RESULTS |
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Physiological measurements. In the low-stretch group, PIP was not affected by ventilation in either WT or DKO mice, remaining at baseline levels throughout (data not shown). In the high-stretch group, the period of high-stretch ventilation led to an increase in PIP in both WT and DKO animals (Fig. 1), and when this increased by 510% of the starting value, the ventilator settings were returned to the baseline low-stretch ventilation. The total length of experiments in the high-stretch group varied depending on the time required to reach this predetermined level of mechanical lung injury and was not different between the WT and DKO mice (255 ± 15 min for WT vs. 237 ± 18 min for DKO). On return to low-stretch ventilation, PIP was significantly increased compared with pre-high-stretch values (P < 0.01), with decreases in respiratory system compliance of 46 ± 4% for WT (n = 8) and 52 ± 11% for DKO (n = 7). There were no differences in these parameters between DKO and WT animals, demonstrating that a standardized level of mechanical lung injury was successfully achieved in all animals in the high-stretch group.
Mean BP was well maintained during both the low- and high-stretch protocols in both WT and DKO animals (Table 1) within the range expected for C57BL6 mice anesthetized with Hypnorm and midazolam (57), consistent with our previous observations in similar high VT-induced VILI models in mice (10, 55). Blood gas parameters were also maintained within a physiological range and were not different between WT and DKO mice throughout the experiment. The high-stretch group displayed relative hypoxemia on return to baseline low-stretch ventilation.
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Physiological measurements. The high-stretch protocol produced the expected changes in PIP and blood gases, which were not different between animals receiving intratracheal anti-TNF or isotype control antibodies, confirming a similar degree of mechanical lung injury in all animals (Table 2). Total length of ventilation was not different between the animals receiving either antibody (264 ± 20 min for isotype vs. 266 ± 15 min for anti-TNF).
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The high-stretch protocol produced the anticipated changes in physiological parameters, as in the animals receiving intratracheal antibodies (data not shown). Intravenous administration of anti-TNF antibody had no effect on lavage PMN percentage or number recovered compared with animals receiving isotype control (Fig. 6).
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DISCUSSION |
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To investigate the physiological role of TNF in mediating a high stretch-induced pulmonary inflammation, we modified our previously described mouse model of high-VT VILI (10, 55). In this model, high lung stretch in conjunction with potential collapse and reopening of small airways due to zero PEEP induces a highly reproducible lung injury. Mice were ventilated with high VT for a short period of time until physiological signs of mechanical lung injury started to become apparent (PIP 510% increased) and then returned to the baseline low-stretch ventilation. We have previously demonstrated in mice that at this early point of high stretch-induced lung injury, expression of bioactive TNF (which is transient and becomes undetectable in the later stages) was present in the alveolar space (55), and neutrophil sequestration within the lung vasculature was commencing (10). The protocol enabled the animals to survive long enough after this starting point of lung injury to develop intra-alveolar neutrophil infiltration, a clinically relevant, clearly detectable, and easily quantifiable sign of pulmonary inflammation (8, 9) and a crucial step in the progression of inflammatory injury during VILI (28, 37). The protocol also allowed us to create a highly standardized stretch-induced injury, necessary for proper evaluation of the differences in inflammatory response due to inhibition of TNF signaling. The high-stretch ventilation provoked a decrease in respiratory system compliance, increased PIP, decreased oxygenation, and increased protein levels in lavage fluid. The degree of injury as assessed by these parameters was identical between WT and DKO animals (see Fig. 1 and Table 1), confirming that a standardized degree of mechanical lung injury was achieved in all animals.
We found that the high-stretch protocol induced lung inflammation in both WT and DKO animals, but the degree of intra-alveolar neutrophil infiltration was significantly attenuated by 4045% in TNF receptor DKO mice. To confirm that the attenuated inflammation was due specifically to the inhibition of TNF signaling, rather than other molecules that potentially signal via TNF receptors, WT animals ventilated using the high-stretch protocol were treated with anti-TNF antibody. Neutrophil recruitment was dramatically reduced (
70% reduction) by intratracheal anti-TNF administration. Thus a substantial component (4070%) of high stretch-induced neutrophil infiltration is mediated by TNF signaling. In our study, it is difficult to directly compare the degree of attenuation between chronic receptor depletion and acute antibody inhibition because intratracheal administration of antibody itself somewhat worsened pulmonary inflammation, presumably due to airway obstruction by fluid, producing some increase in neutrophil recovery in animals given isotype control antibody compared with untreated high-stretch animals. However, lesser attenuation in TNF receptor DKO mice may reflect chronic compensatory upregulation of alternative cytokine or inflammatory pathways to TNF signaling, e.g., upregulation of IL-1 signaling or sensitization of MAPK and activation of NF-
B (46). Together, these data provide the first direct demonstration that TNF plays a significant physiological role in the lung inflammatory response to high-stretch ventilation in the absence of underlying injury.
Previous studies have demonstrated the involvement of the neutrophil attractants MIP-2 and KC in high stretch-induced lung inflammation (5, 36). Because expression of these CXC chemokines is partly TNF dependent in other models of pulmonary inflammation (4, 12, 56), we postulated that the observed effects of TNF may be mediated through regulating chemokine expression. However, we found that the chemokine response to high-stretch ventilation was similar between DKO and WT animals and between animals treated with anti-TNF or isotype control antibodies. Increased production of MIP-2 in response to high stretch has been demonstrated in vivo (5), in isolated lungs (38), and in cultured pulmonary cells (35, 52) in the absence of TNF upregulation. This indicates that such CXC chemokines can be increased directly in response to mechanical lung stretch regardless of prior TNF release, e.g., via activation of c-Jun NH2-terminal kinase (30) or NF-B (5). Our data therefore suggest that TNF and CXC chemokines may play at least partly independent roles in mediating high stretch-induced pulmonary neutrophil recruitment, although the data taken at a single time point may not reflect overall kinetics of chemokine expression across the course of the experiment (55). It is not clear precisely what mechanisms may be involved in modulation of neutrophil recruitment by TNF, but these could include the stimulation of adhesion molecule expression on endothelial cells via NF-
B activation (19) or the actions of neutrophil attractants other than MIP-2 and KC.
In contrast to intratracheal administration, intravenous administration of the same anti-TNF antibody at the same saturating dose had no impact on alveolar neutrophil recruitment. Previous in vivo studies using preinjured lungs (7, 23) and studies using isolated perfused lungs (24, 53) have suggested that injurious ventilation promotes the release of soluble mediators, including TNF into the circulation. If such decompartmentalization of TNF occurs in vivo in response to high-stretch ventilation in healthy lungs, and if this plays a role in neutrophil recruitment and migration, then intravenous anti-TNF administration would have attenuated recruitment in the current study. It is still possible that intravenous antibody could work at higher doses or with different timing, as with any negative experiments using antibodies. However, our results strongly suggest that TNF signaling in response to pure mechanical stretch is largely localized within the alveolar space. The mechanism by which this localized TNF signaling transmits to the circulation, ultimately leading to substantial neutrophil sequestration, adhesion, and migration, remains to be further investigated, although enhancement of calcium influx via arachidonate has been postulated to mediate TNF signaling between the alveolar epithelium and endothelial cells (29). Our results do not exclude the possibility that decompartmentalization of TNF may play some role during mechanical ventilation of preinjured lungs, mediating systemic propagation of lung inflammation leading to multiple system organ failure (42).
The current study was designed to investigate the impact of TNF signaling in VILI by evaluating an in vivo "impulse" response of pulmonary inflammation to a highly standardized, short-duration mechanical lung injury. At the end of the protocol, few differences were observed in physiological indexes of lung injury (PIP, blood gas, protein levels in lavage fluid etc.) between WT and DKO mice or between the animals with/without anti-TNF treatment. At first glance, this observation may be interpreted to mean that TNF signaling has little clinical relevance in the development of VILI. However, this is because in acute one-hit animal VILI models, these physiological parameters are predominantly a reflection of rapidly developing, mechanically induced pulmonary edema (13, 16). We intentionally controlled the degree of initial mechanical lung injury in all animals, and once a standardized injury was produced, no further deterioration of the physiological parameters occurred with 3 h of additional low-stretch ventilation, consistent with previous reports on the reversibility of stretch-induced pulmonary edema (21). Despite the similarity in physiological indexes of injury, we found a marked difference in intra-alveolar neutrophil infiltration, an inflammatory index of lung injury, between animals with/without TNF inhibition. This implies that mechanical lung injury induced by high stretch results in a more substantial pulmonary inflammatory response in the presence of TNF signaling. The ongoing neutrophil recruitment did not lead to further deterioration of lung physiological function within the time frame of our acute model, but previous studies have demonstrated the crucial importance of neutrophil-mediated inflammation in the progression of VILI in more prolonged models (5, 28, 39). Thus it is reasonable to assume that attenuation of neutrophil recruitment due to inhibition of TNF signaling would eventually lead to improved physiological outcome of VILI in clinically relevant conditions.
To reproducibly create high stretch-induced pulmonary inflammation and VILI in mice within a relatively short experimental period, we used a VT (4344 ml/kg) much greater than would be used in the clinical setting in humans. However, the VT employed is similar to those used in previous in vivo and ex vivo animal studies in the literature (13, 27, 38, 47) that have substantially contributed to our understanding of the mechanisms of VILI. It has also been suggested that such very high VT in healthy lungs may produce a similar degree of alveolar stretch to that observed regionally in heterogeneously injured lungs of ARDS patients receiving much lower VT (42, 47). Moreover, Soutiere and Mitzner (43) have recently demonstrated that intact mouse lungs can be temporarily inflated to pressures >60 cmH2O, relating to a VT of 6070 ml/kg, without reaching a traditionally defined total lung capacity (i.e., plateau of the pressure volume curve) or producing apparent morphological damage. Thus attempts to directly compare the absolute values of "injurious" VT between mice and other species, particularly humans, may be misleading due to the much larger compliance of the mouse respiratory system at high lung volumes. However, principles derived should give important insight into the pathophysiology of VILI in humans.
In conclusion, we have demonstrated directly for the first time that pulmonary neutrophil recruitment in response to high-stretch ventilation in the absence of underlying injury involves a significant TNF-mediated component. Present strategies aimed at minimizing VILI in the intensive care unit consist of avoiding high VT and pressures while maintaining the lung in an open state (1). However, the optimal ventilator settings are often controversial (17, 34), and because most patients with ARDS have highly heterogeneous lung injury, overstretching and/or repetitive collapse of the alveoli are inevitable in some regions of their lungs (22, 33, 42). Alternative therapies are therefore required, and strategies to attenuate pulmonary inflammation may help to reduce VILI and improve the outcome of acute lung injury. We found in the current study that local administration of anti-TNF antibodies directly into the alveolar space attenuated high stretch-induced pulmonary inflammation, with the intravenous route apparently ineffective. Our results are consistent with the hypothesis that local, as opposed to systemic, blockade of TNF signaling may have therapeutic potential to reduce pulmonary inflammation in ventilated patients.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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