1 Department of Anaesthesiology and Critical Care Medicine, University Hospital, Hugstetterstraße 55, 79106 Freiburg, Germany. 2 Department of Anaesthesiology, University of Florida, PO Box 100254, Gainesville, FL 326100254, USA
* Corresponding author. E-mail: edgar.kirchner{at}uni-freiburg.de
Accepted for publication December 13, 2004.
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Abstract |
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Methods. Isolated perfused rabbit lungs were randomly allocated to one of three groups: zero end-expiratory pressure (ZEEP) to induce repeated alveolar collapse (n=6), high PEEP to induce cyclic alveolar overdistension (n=6) and repeated PEEP adjustments based on intratidal compliancevolume curve analysis by the slice method to minimize repeated alveolar collapse and overdistension (n=9). All lungs were ventilated with a tidal volume of 6 ml kg1 bodyweight for 120 min. Thereafter, activation of transcription factors NF-B, AP-1 and CREB in lung tissue was analysed by electrophoretic mobility shift assay.
Results. High PEEP was associated with the highest activation of NF-B and AP-1 and repeated PEEP adjustments with the lowest activation when compared with the other two study groups (P<0.001). In contrast, activation of CREB did not differ between groups. Activated NF-
B and AP-1 protein complexes consisted mainly of the transactivators p50/p65 and c-Fos/Jun, respectively.
Conclusions. In isolated perfused rabbit lungs, repeated adjustments of PEEP based on the continuously analysed intratidal compliancevolume curve were associated with less activation of early steps of inflammatory signalling cascades than ventilation with ZEEP or high PEEP.
Keywords:
biochemistry, transcription factors, activator protein-1
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biochemistry, transcription factors, cAMP responsive element binding protein
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biochemistry, transcription factors, nuclear factor-B
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lung, model
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model, rabbit
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ventilation, positive end-expiratory pressure
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Introduction |
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Use of PEEP is part of routine ventilatory management. Optimum PEEP may be defined as one which prevents repeated end-expiratory alveolar collapse without causing cyclic alveolar overdistension. Such repeated alveolar collapse is detectable in the isolated perfused rabbit lung by continuous analysis of the intratidal compliancevolume curve using the slice method.11 The slice method serves to determine the dynamic compliance (Cslice) within the VT.12 We recently demonstrated in the isolated perfused lung that repeated adjustments of PEEP based on the intratidal course of Cslice result in full end-expiratory alveolar recruitment and low risk of cyclic alveolar overdistension.13 However, the impact of this approach on pulmonary transcription factor activation remains to be defined. Accordingly, the aim of this study was to investigate the effect of this ventilatory management on early steps of pro-inflammatory signalling cascades in the lung. We hypothesized that intrapulmonary activation of the immunomodulatory transcription factors NF-B, AP-1 and CREB, which induce the expression of various chemokines and cytokines, would be reduced by the new ventilatory approach.
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Materials and methods |
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Measurement of pulmonary mechanics
A detailed description of measurements and analysis of respiratory mechanics is given elsewhere.11 13 Briefly, airway pressure was measured with a transducer (1210A, ICSensors, Milpitas, CA, USA) at the site of a heated pneumotachograph (Fleisch No. 0, Metabo, Epalinges, Switzerland) connected to the endotracheal tube via a three-way stopcock (Discofix-3, B. Braun, Melsungen, Germany). The signals acquired were digitized and data were processed online using a slightly modified slice method11 12 which automatically considers intrinsic PEEP.14 The slice method divides every VT into six equal portions (slices). One value of compliance and resistance is determined for each slice by multiple linear regression analysis.12 Volume-dependent compliance (Cslice) within VT and pressurevolume loops were continuously monitored.
Experimental protocol
After initial preparation, 30 lungs were randomly assigned to one of three groups (see below) by drawing lots. After a subsequent 45 min stabilization period, during which ventilation was maintained constant in all groups (VT=6 ml kg1, 10 ventilations per minute), and standardization of volume history (sustained inflation with 20 cm H2O for 30 s), lungs were included in the study if the following criteria were met: homogeneous white appearance with no signs of haemostasis, oedema,or atelectasis, pulmonary artery and ventilation pressures in the normal range, no air leak (continuous online monitoring of pressurevolume loops) and no pH instability. By these criteria, 21 of the 30 initially randomized lungs were included in the following three groups.
Two hours after the start of the respective ventilatory strategy, lungs were snap frozen, minced in liquid nitrogen and stored at 70°C.
Total protein extracts of lung tissue and electrophoretic mobility shift assay (EMSA)
Frozen lung samples were homogenized in high-salt detergent buffer [HEPES (pH 7.9) 20 mM, NaCl 350 mM), glycerol 20% (vol vol1), Nonidet P-40 1% (vol vol1), MgCl2 1 mM, EDTA 0.5 mM, EGTA 0.1 mM, dithiothreitol 1 mM, aprotinin 10 µg ml1, Leupeptin 25 µM, NaF 10 mM, phenylmethylsulphonylfluoride 0.1% and phosphatase inhibitor cocktail 10 µl ml1 (Calbiochem, La Jolla, CA, USA). Preparation of whole-cell extracts and EMSA for NF-B, AP-1 and CREB were performed as previously described.15 16 Equal amounts of total cellular protein (1025 µg) were used for incubation with [
-32P]-labelled NF-
B, AP-1, and CREB oligonucleotides. To demonstrate specific binding of oligonucleotides, the binding reactions were co-incubated with a 50-fold molar excess of either unlabelled specific or non-specific competitor. DNA binding proteins were identified by supershift experiments. Reaction mixtures were co-incubated with antibodies against the respective transcription factor (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA). After gel electrophoresis, gels were dried and specific signals were quantified using a phosphorimager system (FLA 2000, Raytest, Straubenhardt, Germany), analysed with AIDA software (version 3.10, raytest, Straubenhardt, Germany) and subsequently exposed to X-ray films with an intensifying screen at 70°C.
Data analysis
EMSA, PEEP and Pplat data were analysed by one-way ANOVA or two-way ANOVA for repeated measurements, where appropriate, followed by the Tukey test (P<0.05) using the SigmaStat software package (version 2.03, SPSS, Chicago, IL, USA). Data are presented as medians (IQR) or means (SD).
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Results |
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Discussion |
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A relationship between intratidal pulmonary mechanics and the pulmonary inflammatory response has previously been demonstrated during constant-flow ventilation.17 18 In contrast with our study, ventilation was adjusted on the basis of the inspiratory pressuretime curve (stress index).17 The algorithm of the slice method used in the present study analyses both inspiratory and expiratory changes of the compliancevolume relationship during non-constant flow conditions.12
In vitro, cyclic mechanical stress apparently causes an inflammatory response predominantly in human lung macrophages but also in alveolar type II like epithelial cells and bronchial epithelial cells.8 10 A similar inflammatory response can be induced in isolated lungs during artificial ventilation: Whereas previous investigators concentrated on pro-inflammatory mediator expression,2 17 we focused on the early steps of the pro-inflammatory signalling cascades. Activation of NF-B and AP-1 represents a crucial early event during activation of inflammatory signalling pathways in pulmonary cells.19 20 Our finding of activation of these transcription factors by ventilation with high PEEP is consistent with previous studies showing activation by cyclic stretch in pulmonary cells810 and lung parenchyma.21 In accordance with findings in bronchial epithelial cells treated with cyclic stretch,10 we detected CREB activation in whole-lung tissue. It is not clear why CREB activation in isolated lungs did not differ between study groups. In vitro, CREB activation caused by cyclic mechanical stress seems to follow a different course than activation of NF-
B and AP-1.10 22 Therefore we cannot exclude the possibility that ventilation with different PEEP settings might have caused a difference in CREB activation before or after the 2 h observation point of our study. Pulmonary activation of NF-
B during ventilation with large tidal volumes has previously been reported in isolated lungs,3 but neither activation of NF-
B during ventilation with small tidal volumes nor activation of CREB and AP-1 during mechanical ventilation in general have been reported.AP-1 is comprised of homodimeric and heterodimeric complexes of members of the Jun and Fos families and constitutes an important regulator of cell proliferation and related events. Moreover, there is increasing evidence that activation of AP-1 plays a pivotal role in the pro-inflammatory signalling pathways of acute lung injury.19 21 23 AP-1 activation leads to IL-8 transcription9 which, in turn, is responsible for the chemoattraction of neutrophils to the lung, a critical step in the development of VILI.24
Limitations
There are several limitations to this study. Data were obtained in the isolated perfused rabbit lung. For obvious reasons, this model does not per se reflect clinical reality. Nevertheless, it represents an accepted model in the study of VILI-related problems.13 18 2527 It remains to be determined whether a reduction of repeated end-expiratory alveolar collapse and cyclic alveolar overdistension are the main mechanisms by which R-PEEP resulted in less activation of the transcription factors than ZEEP and H-PEEP. The degree of correlation between early intrapulmonary activation of the transcription factors on the one hand and indices of lung injury (e.g. oxygenation, histology) and other inflammatory responses (e.g. broncho-alveolar release of cytokines, PMN infiltration) on the other remains to be defined. Clearly, the observed activation of transcription factors cannot be equated with cytokine expression. However, ventilation with high PEEP induced the NF-B heterodimer p50/p65 and the AP-1 heterodimer c-Fos/Jun which are strong transactivators of gene transcription.5 28 The activation of NF-
B and AP-1 during ventilation with ZEEP observed in our model is in accordance with the induction of pro-inflammatory cytokines observed in other recent experimental studies.4 29 30 Nevertheless, direct translation of the results into clinical practice is unwarranted.
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Conclusions |
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Acknowledgments |
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During the course of this work Gerd Hermle died unexpectedly. We have lost a skilled scientist, a dedicated physician and a close friend.
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References |
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2 Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS. Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997; 99: 94452
3 Held HD, Boettcher S, Hamann L, Uhlig S. Ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappaB and is blocked by steroids. Am J Respir Crit Care Med 2001; 163: 71116
4 Caruso P, Meireles SI, Reis LF, Mauad T, Martins MA, Deheinzelin D. Low tidal volume ventilation induces proinflammatory and profibrogenic response in lungs of rats. Intensive Care Med 2003; 29: 180811[CrossRef][ISI][Medline]
5 Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997; 9: 2406[CrossRef][ISI][Medline]
6 Barnes PJ, Adcock IM. Transcription factors and asthma. Eur Respir J 1998; 12: 22134
7 Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999; 18: 685366[CrossRef][ISI][Medline]
8 Pugin J, Dunn I, Jolliet P, et al. Activation of human macrophages by mechanical ventilation in vitro. Am J Physiol 1998; 275: L104050[ISI][Medline]
9 Li L-F, Ouyang B, Choukroun G, et al. Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-kappaB-inducing kinases. Am J Physiol Lung Cell Mol Physiol 2003; 285: L46475
10 Oudin S, Pugin J. Role of MAP kinase activation in interleukin-8 production by human BEAS-2B bronchial epithelial cells submitted to cyclic stretch. Am J Respir Cell Mol Biol 2002; 27: 10714
11 Mols G, Hermle G, Schubert J, et al. Volume-dependent compliance and ventilation-perfusion mismatch in surfactant-depleted isolated rabbit lungs. Crit Care Med 2001; 29: 14451[ISI][Medline]
12 Guttmann J, Eberhard L, Fabry B, et al. Determination of volume-dependent respiratory system mechanics in mechanically ventilated patients using the new SLICE method. Technol Health Care 1994; 2: 17591
13 Hermle G, Mols G, Zugel A, et al. Intratidal compliancevolume curve as an alternative basis to adjust positive end-expiratory pressure: a study in isolated perfused rabbit lungs. Crit Care Med 2002; 30: 158997[CrossRef][ISI][Medline]
14 Eberhard L, Guttmann J, Wolff G, et al. Intrinsic PEEP monitored in the ventilated ARDS patient with a mathematical method. J Appl Physiol 1992; 73: 47985[ISI][Medline]
15 Mueller JM, Pahl HL. Assaying NF-kappa B and AP-1 DNA-binding and transcriptional activity. Methods Mol Biol 2000; 99: 20516[Medline]
16 Pahl HL, Krauss B, Schulze-Osthoff K, et al. The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-kappaB. J Exp Med 1996; 183: 182940
17 Ranieri VM, Zhang H, Mascia L, et al. Pressuretime curve predicts minimally injurious ventilatory strategy in an isolated rat lung model. Anesthesiology 2000; 93: 13208[CrossRef][ISI][Medline]
18 Gama de Abreu M, Heintz M, Heller A, Szechenyi R, Albrecht DM, Koch T. One-lung ventilation with high tidal volumes and zero positive end-expiratory pressure is injurious in the isolated rabbit lung model. Anesth Analg 2003; 96: 2208
19 Rahman I, MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 1998; 53: 60112
20 Guo RF, Lentsch AB, Sarma JV, et al. Activator protein-1 activation in acute lung injury. Am J Pathol 2002; 161: 27582
21 Kumar A, Lnu S, Malya R, et al. Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma. FASEB J 2003; 17: 180011
22 Du W, Mills I, Sumpio BE. Cyclic strain causes heterogeneous induction of transcription factors, AP-1, CRE binding protein and NF-kB, in endothelial cells: species and vascular bed diversity. J Biomech 1995; 28: 148591[CrossRef][ISI][Medline]
23 Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 2000; 16: 53454
24 Imai Y, Kawano T, Miyasaka K, Takata M, Imai T, Okuyama K. Inflammatory chemical mediators during conventional ventilation and during high frequency oscillatory ventilation. Am J Respir Crit Care Med 1994; 150: 15504[Abstract]
25 Broccard AF, Hotchkiss JR, Kuwayama N, et al. Consequences of vascular flow on lung injury induced by mechanical ventilation. Am J Respir Crit Care Med 1998; 157: 193542[ISI][Medline]
26 Broccard AF, Vannay C, Feihl F, Schaller MD. Impact of low pulmonary vascular pressure on ventilator-induced lung injury. Crit Care Med 2002; 30: 218390[CrossRef][ISI][Medline]
27 Mols G, Hermle G, Fries G, et al. Different strategies to keep the lung open: a study in isolated perfused rabbit lungs. Crit Care Med 2002; 30: 1598604[CrossRef][ISI][Medline]
28 Schmitz ML, Baeuerle PA. The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B. EMBO J 1991; 10: 380517[Abstract]
29 Naik AS, Kallapur SG, Bachurski CJ, et al. Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung. Am J Respir Crit Care Med 2001; 164: 4948
30 Bregeon F, Roch A, Delpierre S, et al. Conventional mechanical ventilation of healthy lungs induced pro-inflammatory cytokine gene transcription. Respir Physiol Neurobiol 2002; 132: 191203[CrossRef][ISI]