Mechanical ventilation may cause lung injury through the excitation of an inflammatory response and the release of mediators, such as cytokines. The authors tested the hypothesis that intratracheal lipopolysaccharide amplifies the cytokine response to mechanical ventilation.
Rat lungs were intratracheally instilled with lipopolysaccharide followed by ex vivo mechanical ventilation for 2 h with low tidal volume of 7 ml/kg with 3 cm H2O positive end-expiratory pressure (PEEP), high tidal volume of 40 ml/kg with zero PEEP, medium tidal volume of 15 ml/kg with 3 cm H2O PEEP, or medium tidal volume and zero PEEP.
In the absence of lipopolysaccharide, lung lavage concentrations of tumor necrosis factor and interleukin 1 beta but not macrophage inflammatory protein 2 were significantly higher in lungs ventilated at high tidal volume/zero PEEP than at low tidal volume. There was a marked increase in lavage tumor necrosis factor and macrophage inflammatory protein 2 concentrations in lungs ventilated at low tidal volume after exposure to intratracheal lipopolysaccharide at doses of 100 ng/ml or greater. However, in lungs ventilated at high tidal volume, this response to lipopolysaccharide was markedly reduced. In addition, the number of alveolar macrophages recovered in the lavage was significantly lower in lungs ventilated at high tidal volume.
Ventilation strategy can modify lung cytokine responses to lipopolysaccharide, likely through an effect on the alveolar macrophage population.
MECHANICAL ventilation is often essential to preserve life in the critically ill, but it may exacerbate lung injury. Animal studies have implicated overdistension of the alveoli and the repetitive opening and closing of small airways as injurious features of mechanical ventilation.1,2Clinical studies have indicated that in patients with acute respiratory distress syndrome, mortality is lower when ventilation is performed using lower tidal volumes (VT).3However, it is not precisely known why such a ventilation strategy leads to an improved outcome, particularly because most deaths from acute respiratory distress syndrome are attributable to multiple organ failure rather than the lung condition per se .4
One possibility is that mechanical ventilation initiates or augments an inflammatory response, with release of mediators such as tumor necrosis factor-α (TNF-α), which influence distal organ function.5In support of this hypothesis was the finding by Tremblay et al. 6that ventilation of isolated rat lungs with high VTs and zero end-expiratory pressure (ZEEP) led to far higher lung lavage concentrations of several cytokines, including TNF-α, interleukin 1β (IL-1β), and macrophage inflammatory protein 2 (MIP-2), than ventilation at low VT.
Using similar isolated, nonperfused lungs of rats ventilated for 2 h with 7 ml/kg VTand 3 cm H2O positive end-expiratory pressure (PEEP) or 42 ml/kg VTand ZEEP, Ricard et al. 7reported that IL-1β and MIP-2 concentrations were higher in lung lavage fluids of lungs ventilated with the high VTgroup, whereas there was no difference for TNF-α, as compared with the lungs that were maintained for 2 h in a statically inflated state at 7 cm H2O airway pressure. In an in vivo model of small rats, Verbrugge et al. 8found no pulmonary TNF-α release after mechanical ventilation with a strategy of 32 cm H2O peak inspiratory pressure and 6 cm H2O PEEP. Many factors may have effects on cytokine production. These include differences in the lung size to body weight ratio,9the degree of external stress experienced by the rats before studies, and differences in species and the immunoassays used. Another possibility is that the lungs may have been “primed” by an external cytokine inducer, such as lipopolysaccharide,7which renders the lung more sensitive to the production of large quantities of cytokines when exposed to the additional stress of mechanical ventilation.
In this study, we sought to address the specific issue of whether exposure to lipopolysaccharide, delivered intratracheally, augments the cytokine response of high VTventilation. We used a well-established isolated rat lung model similar to that used in earlier studies for ventilator-induced lung injury. Unexpectedly, we found that the cytokine response of the lung to lipopolysaccharide is attenuated by ventilation using high VTor ZEEP or both. We report here that an alteration in alveolar macrophage activity or location or both, induced by the mechanical ventilation, may underlie this observation.
Materials and Methods
The protocol was approved by the animal care committee of the University of Toronto. Pathogen-free male Sprague-Dawley rats weighing 350–400 g (Charles River, St. Constant, Quebec, Canada) were used. Animals were anesthetized with intraperitoneal injection of 30 mg ketamine (Ketaset®; Wyeth-Ayerst Canada Inc., Guelph, Ontario, Canada) and 6 mg xylazine (Rompun®; Bayer, Toronto, Ontario, Canada). A tracheostomy was performed, and the trachea was cannulated with a sterile 14-gauge cannula (Angiocath; Becton Dickson, Sandy, UT). The abdomen was opened, and the animal was exsanguinated by division of the great abdominal vessels. The thoracic cavity was then opened, and the heart and lungs were exposed. The right and left auricles were excised, and the pulmonary circulation was flushed using 30 ml sterile normal saline (Baxter, Toronto, Ontario, Canada) injected into the main pulmonary artery at a rate of 15 ml/min via a 22-gauge needle. During this period of pulmonary artery flushing, the lungs were ventilated using a volume-cycling rodent ventilator (Harvard model 55-3438; Harvard Apparatus, South Natick, MA) at 7 ml/kg/kg VTwith 3 cm H2O PEEP at 12 breaths/min. After this flushing procedure, the lungs appeared white. The heart and lungs were then excised en bloc .
The lungs were suspended in a humidified box thermostatically maintained at 37°C, and mechanical ventilation was commenced. To reach similar distribution of lipopolysaccharide in all cases, all lungs were ventilated for 15 min using 7 ml/kg VTand 3 cm H2O PEEP at a rate of 40 breaths/min. After this period, lungs were allocated, according to the randomization, to one of four ventilation strategies: (1) low VTof 7 ml/kg with 3 cm H2O PEEP (VT7), (2) high VTof 40 ml/kg with ZEEP (VT40), (3) medium VTof 15 ml/kg with 3 cm H2O PEEP (VT15 PEEP), or (4) medium VTand ZEEP (VT15 ZEEP).
The study was designed so that five doses of lipopolysaccharide (0, 1, 10, 100, 1,000 ng) were tested for the VT7 and VT40 ventilation strategies. For each of the intermediate ventilation strategies (VT15 PEEP and VT15 ZEEP), only lipopolysaccharide doses of 0 and 100 ng were used. Thus, there were 14 groups, each with five animals. Briefly, the isolated lungs were recruited three times by inflation with 6 ml air and randomly allocated to receive different doses of intratracheal injection of lipopolysaccharide (Escherichia coli serotype 0111:B4; Sigma, Oakville, Ontario, Canada) and different ventilation strategies. The lipopolysaccharide was given in blinded fashion in 0.5 ml sterile normal saline injected intratracheally in a 1-ml syringe, followed by three further inflations with 6 ml air. Ventilation was then continued for a further 2 h at a rate of 40 breaths/min with room air. At the end of this period, lung lavage was performed with 5 × 10 ml cold normal saline, and the effluent pooled. The lavage fluid was centrifuged at 12,000g at 4°C for 10 min (Eppendorf, Hamburg, Germany). Aliquots of the supernatant were frozen at −70°C for subsequent analysis.
Quantification and Differentiation of Cells in Bronchoalveolar Alveolar Lavage
After centrifugation of the lung lavage, the cell pellet was resuspended in 0.5 ml cold saline. Cell numbers and viability were assessed by exclusion of trypan blue using a hemacytometer at 100× magnification. Identification of the cells in the lavage was studied by cytospin (Cytospin 2; Shandon, Astmoor, United Kingdom) followed by eosin and methylene blue staining (Harleco, EM Science, Gibbstown, NJ). Cell slides were then analyzed.
Assays of Cytokines
Lung lavage assays of TNF-α, IL1-β, and MIP-2 were performed using commercially available enzyme-linked immunosorbent assay kits (Biosource International, Inc., Camarillo, CA).
ED-1 Staining for Alveolar Macrophages
To clarify the location of alveolar macrophages after mechanical ventilation, a second series of experiments was conducted using excised isolated lungs in which the pulmonary circulation had been flushed. The lungs were randomly allocated to receive no ventilation, ventilation at VT7, or VT40 for 30, 60, or 120 min (n = 3 for each condition). Without preceding lavage, lungs were then inflated with 4% formaldehyde in phosphate-buffered saline. Staining of sections was performed using a specific mouse anti-rat macrophage marker ED-1 (Serotec, Inc., Raleigh, NC) applied at 1:250, followed by biotinylated horse anti-mouse (1:200) and then Vectastain® Elite ABC kit (Vector Laboratories Inc., Burlingame, CA). Counterstaining was performed with Mayer’s hematoxylin. These lung sections were reviewed by a pathologist (B. M.) who was blinded to the study group.
Results are expressed as mean ± SEM unless otherwise indicated. Data were analyzed using one-way analysis of variance followed by the Tukey-Kramer test. P values less than 0.05 were considered statistically significant.
Peak airway pressures are shown in table 1. Peak airway pressures increased progressively, concomitant with the increase in VT. In the VT15 groups, peak airway pressure was significantly greater in the absence of PEEP (VT15 ZEEP) than in its presence (VT15 PEEP) at 60 and 120 min. This probably reflects the greater tendency for airways to collapse on expiration in the absence of PEEP, which then require higher pressure to reopen on inspiration.10
Lavage Cytokine Concentrations in the Absence of Lipopolysaccharide
Figure 1shows lung lavage cytokine concentrations from lungs randomly allocated to the four ventilation strategies that received vehicle normal saline intratracheally but no lipopolysaccharide. Lung TNF-α and IL-1β concentrations were significantly greater in the VT40 group than in the VT7 group (16-fold and 4-fold higher, respectively). These results are consistent with those previously reported from this laboratory.6There was no difference in MIP-2 concentrations among the groups.
Lavage Cytokine Concentrations after Intratracheal Lipopolysaccharide
In lungs ventilated at low VTwith PEEP, much higher TNF-α and MIP-2 concentrations are seen after intratracheal instillation of lipopolysaccharide at concentrations of 100 ng or greater (fig. 2). In the VT7 group, ventilation after 1,000 ng intratracheal lipopolysaccharide was associated with lavage TNF-α concentrations around 140 times greater than those seen after saline vehicle alone. By contrast, in the VT40 group, there was no significant increase in lavage TNF-α concentrations with increasing concentrations of lipopolysaccharide (fig. 2).
Although in the VT40 group the concentrations of lavage MIP-2 after 1,000 ng lipopolysaccharide were significantly greater than that after no lipopolysaccharide, the magnitude of this difference was far less than in the VT7 groups (approximately 4-fold increase as opposed to a 26-fold increase). There was no significant effect of increasing concentrations of lipopolysaccharide on lavage IL1-β concentrations irrespective of the ventilation strategy (fig. 2).
At intermediate ventilation strategies of VT15 ml/kg with and without PEEP (VT15 PEEP and VT15 ZEEP), there was tendency to a lower cytokine production in response to an intratracheal lipopolysaccharide dose of 100 ng for TNF-α and MIP-2 but not for IL-1β (fig. 3).
Cells Recovered from the Lavage Fluid
In all instances, the identity of the lavaged cells (excluding erythrocytes), as assessed in cytospin preparations, was greater than 99.5% mononuclear cells (data not shown). The proportion of cells that were viable, as judged by exclusion of trypan blue, tended to be higher after low VTthan high VTventilation, but these differences did not reach statistical significance.
At all concentrations of intratracheal lipopolysaccharide, the number of mononuclear cells seen in the lavage fluid was significantly greater in the lungs that had been ventilated at VT7 compared with those ventilated at VT40 (fig. 4). Cell counts were also decreased in the intermediate ventilation strategies of VT15 with and without PEEP.
Compared with the ventilation strategy, the concentration of intratracheal lipopolysaccharide has a far less marked effect on the number of cells recovered in the lavage fluid (figs. 4).
Immunostaining of Alveolar Macrophages
Lungs that had not undergone lavage after mechanical ventilation were immunostained with ED-1 for analysis of the distribution of macrophage in the lung sections. In normal lung, the distribution of macrophages was approximately 50% in intraalveolar space and 50% in interstitial space including intraparenchymal and septal locations (fig. 5). Lungs that had been ventilated for more than 60 min with VT40 had markedly fewer macrophages in the alveolar space but much more in interstitial space than those ventilated with VT7, a less injurious strategy (fig. 5).
Figure 6shows the representative lung slides after ventilation using two different strategies for 120 min and stained with the anti-rat macrophage ED-1 antibody. In the lung ventilated with VT7, intraalveolar macrophages are evident, whereas no macrophages are seen within the alveolar space in lung sections after ventilation at VT40.
The main findings of the study are that the ventilation strategy used has great effects on lung cytokine response to lipopolysaccharide, which was associated with changes in the distribution of macrophages in the lung.
The model we used of an ex vivo nonperfused lung has advantages and disadvantages as a model system. The main advantages are that it allows one to directly assess the impact of mechanical factors (i.e. , due to mechanical ventilation) on mediator release without the complications inherent in an in vivo model in which large VTs impact hemodynamics, which could alter mediator release, lead to activation of circulating inflammatory cells, or both. In addition, because there is no chest wall, at end-expiration with zero PEEP, there is complete collapse of the lung, magnifying atelectrauma11(damage due to collapse and repetitive reopening of collapsed lung regions). These factors likely account for the lack of a cytokine response noted in in vivo studies of animals with normal lungs.8,12A number of the advantages described above are disadvantageous in terms of direct application of the findings to the clinical setting. In vivo , the interaction between the lung and the pulmonary circulation is likely to be important in the response to mechanical ventilation.13In addition, the degree of stress imposed by overdistension and atelectrauma in this model is much greater than that observed in vivo . Finally, the lungs are ischemic for more than 2 h, and this may add to the injury being studied. Nonetheless, based on human studies, it seems that many of the findings observed in this model have been observed in the clinical setting.3,14,15
In the absence of lipopolysaccharide, ventilation with a VTof 40 ml/kg and ZEEP led to significantly higher concentrations of TNF-α and IL-1β in lung lavage than ventilation with a VTof 7 ml/kg. In this respect, our results are qualitatively similar to those obtained in this laboratory by Tremblay et al. 6but differ from those of Ricard et al. 7Unlike both groups, we did not find a significant difference in lavage MIP-2 concentrations between the different ventilation strategies in the absence of lipopolysaccharide, although there was a trend toward greater concentrations with the more injurious ventilation. A possible explanation for the discrepancy in MIP-2 observations may lie in the experimental conditions distinct in this study from the previous. In the current study, we ventilated lungs in which the pulmonary circulation had been flushed, thereby removing or reducing the influence of immune cells such as neutrophils that may themselves produce MIP-2 or influence its production by other cells.16
Tremblay et al. 6found that large VTventilation led to release of high concentrations of various inflammatory cytokines, including TNF-α, IL-1β, and MIP-2 in isolated rat lung. In the current study, we tested the hypothesis that exposure of the lungs to lipopolysaccharide before ventilation might augment the release of inflammatory cytokines in response to mechanical ventilation. In vitro studies suggested that this might be the case. Pugin et al. 17exposed primary human alveolar macrophages to cyclic mechanical stretch in the presence and absence of lipopolysaccharide. Stretch alone (without lipopolysaccharide) led to significantly greater production of interleukin 8, the human homolog of rodent MIP-2, compared with static controls, but no increase in TNF-α or IL-6. Exposure of the resting macrophages to lipopolysaccharide led to significant release of all three cytokines, but their release was considerably greater in the presence of cyclic stretch. Similarly, Mourgeon et al. 18showed that mechanical stretch of fetal rat lung cells increased MIP-2 secretion in response to lipopolysaccharide.
In our study, lipopolysaccharide was delivered at low dose (≤ 1 μg) into the lungs by the intratracheal route immediately before ventilation. In the earlier studies using isolated lungs, the effect of lipopolysaccharide on cytokine production was studied by administering intravenous lipopolysaccharide approximately 1 h before removal of the lungs.6,7Because the cytokine response of lipopolysaccharide is largely confined to the compartment into which the lipopolysaccharide is delivered,19we reasoned that intratracheal lipopolysaccharide would exert a much greater influence on the cytokine response to ventilation. Our results showed an effect opposite to what we had hypothesized and indicate that high VTventilation can markedly reduce the release of inflammatory cytokines in response to intratracheal lipopolysaccharide. This was most striking with respect to TNF-α production. These results suggest that the cell response to lipopolysaccharide stimulation may vary depending on ventilatory strategies. Given that the pulmonary vasculature had been flushed to minimize neutrophil involvement in cytokine production after mechanical ventilation, we focused on the role of alveolar macrophages because they are an important cell type to produce lung cytokines and can transmigrate and relocate across intraalveolar spaces.
In a previous study using immunohistochemistry staining technique, we demonstrated that pulmonary epithelium is an important source of cytokine production in response to mechanical ventilation.20In that study, we did not examine the contribution of epithelium to the quantitative cytokine response. In the current study, we extend this observation by suggesting that pulmonary macrophages may also play a role in the cytokine response using this model. It is not clear from the current study whether the response is solely dependent on any one cell type or whether there is cross-talk between pulmonary macrophages and the pulmonary epithelium in eliciting release of cytokines. In addition, it is unknown whether this mechanism is also involved in the ventilator-induced decompartmentalization of TNF-α in the absence of PEEP as previously reported by other investigators.21–24
We focused the effect of different ventilatory strategies on alveolar macrophage population and found that far fewer macrophages were recovered in the lung lavage after high VTthan low VTventilation. Therefore, our results suggest that ventilation with high VTexerts an effect on the alveolar macrophage such that they are less easy to recover in lung lavage and have a reduced capacity to respond to lipopolysaccharide exposure. Immunohistochemistry staining using the specific marker ED-1 confirmed the reduction in the number of alveolar macrophages after high VTmechanical ventilation. It was not possible to judge whether the integration of alveolar macrophages was altered by our assays; however, it seemed that the alveolar macrophages were relocalized from alveolar space to interstitial space including intraparenchymal and septal sites.
These observations are consistent with those reported by Imanaka et al. ,25who studied the effect of different ventilation strategies on alveolar macrophages using an in vivo rat model. They found that fewer macrophages (150 × 103vs. 1,000 × 103) were recovered in those animals who were ventilated for 40 min with a peak airway pressure of 45 cm H2O than those ventilated with 7 cm H2O. However, the authors did not discuss why fewer alveolar macrophages were recovered after high peak airway pressure ventilation.
Broadly speaking, there are several possible explanations for the reduced recovery of alveolar macrophages seen in our study. An increased adherence of the alveolar macrophages to alveolar epithelium may make them less easily recovered by lavage. In support of this hypothesis was the finding of increased expression of the adhesion molecules CD11b and CD54 in the relatively few alveolar macrophages recovered after high peak airway pressure ventilation in the work of Imanaka et al. 25However, we did not observe the presence of alveolar macrophages within the alveoli of lungs that were ventilated with high VT.
Death of the alveolar macrophages induced by high VTmay have resulted in the low population. This might take the form of apoptosis or necrosis. However, apoptosis of alveolar macrophages would not in itself necessarily lead to their absence in the lung lavage. Also, in this ex vivo model, we have not found greater evidence of apoptosis by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining after high VTcompared with low VTventilation. Furthermore, the proportion of viable alveolar macrophages was not different between the low and high VTgroups. Necrosis of the alveolar macrophages, with total loss of cellular architecture, may be a potential explanation for the reduced numbers seen after high VTventilation seen in our study and that of others.25However, it would be surprising if ventilation was responsible for such a dramatic degree of damage to the alveolar macrophages within a short period of 2 h.
Relocation of alveolar macrophages outside the alveolar compartment is the most likely explanation. We were able to show that the number of interstitial macrophages was greater than that in alveolar compartment after high VT. It is probable that the decreased TNF-α secretion in response to intratracheal lipopolysaccharide seen with high VTventilation was due to a reduction of alveolar macrophage population.
In recent years, considerable evidence has accumulated suggesting that mechanical ventilation, particularly of injured lungs, causes the release of inflammatory mediators, which may then pass into the circulation.6,14,26This provides a possible explanation of how mechanical ventilation causes or exacerbates distal organ dysfunction.5Alveolar macrophages are central in the early response of the lung to air-space pathogens. A reduction in this population with high VTventilation, suggested by our results and others,25may increase susceptibility to infection. In addition, the macrophage may have other important roles in acute respiratory distress syndrome, regulating the inflammatory process by clearing apoptotic neutrophils.27In a study of patients with acute respiratory distress syndrome, Steinberg et al. 28found that increased numbers of alveolar macrophages correlated with survival, suggesting that these cells have a beneficial role in the condition, although this may simply be a reflection of less severe underlying lung injury. We believe that further studies are needed to substantiate our findings, which suggest that the macrophage population and the early innate immune response of the lung can be profoundly affected by mechanical ventilation.