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Mechanical ventilation, often required to maintain normal gas exchange in critically ill patients, may itself cause lung injury. Lung-protective ventilatory strategies with low tidal volume have been a major success in the management of acute respiratory distress syndrome (ARDS). Volutrauma causes mechanical injury and induces an acute inflammatory response. Our objective was to determine whether neutrophil elastase (NE), a potent proteolytic enzyme in neutrophils, would contribute to ventilator-induced lung injury. NE-deficient (NE−/−) and wild-type mice were mechanically ventilated at set tidal volumes (10, 20, and 30 ml/kg) with 0 cm H2O of positive end-expiratory pressure for 3 hours. Lung physiology and markers of lung injury were measured. Neutrophils from wild-type and NE−/− mice were also used for in vitro studies of neutrophil migration, intercellular adhesion molecule (ICAM)-1 cleavage, and endothelial cell injury. Surprisingly, in the absence of NE, mice were not protected, but developed worse ventilator-induced lung injury despite having lower numbers of neutrophils in alveolar spaces. The possible explanation for this finding is that NE cleaves ICAM-1, allowing neutrophils to egress from the endothelium. In the absence of NE, impaired neutrophil egression and prolonged contact between neutrophils and endothelial cells leads to tissue injury and increased permeability. NE is required for neutrophil egression from the vasculature into the alveolar space, and interfering with this process leads to neutrophil-related endothelial cell injury.
This study suggests a role for neutrophil elastase in neutrophil emigration out of vasculature during mechanical ventilation. The lack of the enzyme caused prolonged interaction between neutrophils and endothelium leading to endothelial injury.
Mechanical ventilation provides effective gas exchange in patients with respiratory failure. Since its inception, questions about the possibility of mechanical ventilation causing lung injury have been raised, giving rise to the term “ventilator-induced lung injury” (VILI) (1). A growing body of literature documented harmful effects of alveolar overdistention during mechanical ventilation (2). In animal models, increasing tidal volumes cause an acute inflammatory response with neutrophil infiltration and disruption of the endothelial and epithelial barriers (1, 3).
Pre-existing lung injury may further exacerbate inflammation and VILI (4). As most patients requiring mechanical ventilation have a primary pulmonary insult, distinguishing mechanical ventilator-induced injury from an underlying disease process has been difficult. Moreover, with heterogeneous injury to the lung, the relatively spared, disease-free lung tissue, may be particularly sensitive to overdistention (5). Based upon these concerns, strategies using low tidal volume ventilation have been adopted, and resulted in improvement in mortality in acute respiratory distress syndrome (ARDS) (6).
How pure mechanical ventilation leads to neutrophil sequestration into the lung tissue with subsequent injury in VILI is not well understood. Raised alveolar and transpulmonary pressures may produce temporary neutrophil entrapment due to compression of alveolar capillaries (7). Neutrophil recruitment can be sustained by induction of local inflammatory mediators (3) such as CXC chemokines (KC and MIP-2) that have been shown in murine models of VILI to cause neutrophil and microvascular endothelial cell stiffening, activation, migration, and endothelial cell discontinuity with resultant edema (7–9).
In the systemic circulation, neutrophils egress from vasculature through post-capillary venules, whereas this event occurs in alveolar capillaries in the lung (10). Within the pulmonary capillaries, activated neutrophils become stiff and trapped and then tightly adhere to capillary endothelium. These neutrophils migrate directly through pulmonary capillaries through a process that is only partially β2-integrin–dependent (11). Neutrophils subsequently detach from high-affinity binding sites, alter their cytoskeleton, and open tight junctions between endothelial cells, allowing them to egress from the vasculature (12). The role of proteinases in neutrophil migration and lung injury, particularly neutrophil elastase (NE), has been a major focus of investigation for years. Because of its destructive potential, NE has been thought to cause or amplify acute and chronic lung injury including ARDS (13). With respect to neutrophil migration, most cell-based studies failed to support the hypothesis that NE promotes neutrophil migration through proteolytic degradation of extracellular matrix components (14), but NE might participate through other mechanisms. We and others have previously generated NE-null mutant mice (NE−/−) to study the functional properties of NE. Our main finding was that NE was required for neutrophil antimicrobial activity. Otherwise, neutrophil development and function was unaltered (15, 16). NE−/− mice have not been used to directly assess neutrophil migration through endothelial barriers.
The purpose of this study was to investigate the role of NE in the earliest phases of VILI. In contrast to our initial hypothesis that mice lacking NE would be protected from VILI, we found that lack of NE promoted lung injury due to prolonged endothelial–neutrophil interaction in the absence of NE. Further studies demonstrated the role for NE in mediating transvascular egress via cleavage of intercellular adhesion molecule (ICAM)-1.
Additional methodological details are provided in the online supplement.
Mice deficient in NE (NE−/−) were generated in our laboratory by gene targeting (15) and backcrossed 10 generations into the C57BL/6J background. Of note, deletion of NE did not alter expression of other proteinases and did not alter neutrophil development.
Complete blood and leukocyte differential counts were determined in NE−/− and wild-type (WT) mice using a Baker Instruments 9000 Hematologic Series cell counter (BioImmuno Chem, Allentown, PA) after cytocentrifugation and staining with Diff Quick Stain (Harleco, Gibbstown, NY).
Mice were ventilated using a small animal ventilator (flexiVent, SCIREQ, Montreal, Quebec, Canada) via tracheostomy, and a polyethylene catheter was introduced into right carotid artery for monitoring arterial pressures and infusion of fluids. We created VILI by applying various tidal volumes—10, 20, or 30 ml/kg—during a 3-hour experimental period with 0 cm H2O of positive end-expiratory pressure (PEEP). Quasi-static pressure–volume (P–V) curves were generated every 30 minutes. Static compliance was calculated from the slope of each curve.
Total bronchoalveolar lavage (BAL) cell and differential leukocyte counts were performed at the end of experiments using PBS in 0.8 ml volume with five replicates.
Lung tissues fixed with zinc fixative were sectioned and stained with Gr1 antibody for neutrophils. Total lung field, alveolar, and intravascular neutrophils were quantified. The intra-alveolar/intravascular ratio has previously been used to measure the extravasability of neutrophils into lung tissue (17).
At the end of experiments, wet weight of the lungs were measured and then incubated in a 72°C oven for 24 hours. Wet:dry ratio of the left lung was measured as a surrogate marker of lung injury.
Neutrophils from NE−/− mice and WT littermates were collected after intraperitoneal injection of thioglycollate with greater than 90% purity. Neutrophils were used either in micro-chemotaxis or Matrigel invasion chambers to assess their chemotactic ability toward formyl-Met-Leu-Phe (fMLP), leukotriene B4 (LTB4), zymosan-activated serum (ZAS), or culture media as a negative control.
To assess the ability of neutrophils to migrate across pulmonary endothelium, we seeded Transwell inserts with human pulmonary vein endothelial cells (hPVEC) or human pulmonary microvascular endothelial cells (hPMVEC), and they were grown to confluence. Neutrophils (1 × 106) were allowed to migrate toward fMLP (10−5 M) or ZAS.
To assess whether neutrophils in prolonged contact with endothelial cells would cause cell injury upon transmigration, we exposed 51Cr-labeled hPVEC grown to confluence on Transwell inserts to activated neutrophils. Neutrophils were allowed to co-incubate with endothelial cells for 18 hours. In additional experiments, neutrophils were allowed to migrate to the lower chamber in response to fMLP (10−5 M). Release of 51Cr was used as marker for endothelial cell injury.
To assess whether the increased NE−/− neutrophil adhesion to endothelial cells is mediated by their impaired ability to cleave ICAM-1, we co-incubated PMA-activated WT and NE−/− neutrophils with TNF-α–activated endothelial cells (ratio 10:1) at 37°C for 2 hours and cells were imaged using an inverted microscope. The cells were then either lysed and subjected to Western blot analysis for ICAM-1, or cells were fixed and stained for cell surface ICAM-1 expression by immunofluorescence.
Data are expressed as means ± SD. Pairwise comparisons were made by Student's t tests. A P value of < 0.05 was considered significant. The values of the P–V loop areas were compared by use of paired two-tailed t tests.
Previous reports have linked mutations in the NE gene to the syndrome of cyclic neutropenia in humans (18). To assess potential effects of NE deficiency on circulating neutrophils, we measured WBC and neutrophil counts over a period of 22 days, the described cycle length for the recurrence of neutropenia. We found no difference in WBC and neutrophil counts obtained on Days 1, 3, 8, 10, 12, 15, 17, and 22 between WT and NE−/− mice (Table 1).
To study the role of NE in the migration of inflammatory cells into the alveolar spaces, we measured the number of total cells and neutrophils in the BAL in WT and NE−/− mice. We found that the numbers of total cells in BAL increased with increasing tidal volumes but were similar in WT and NE−/− mice for similar ventilation groups (see online supplement). The difference between the two genotypes was not statistically significant. Neutrophils represent a small but important subset of inflammatory cells. Total neutrophil count in BAL also increased proportionally to Tv in the WT mice, but this increase in the alveolar neutrophil counts was significantly blunted in NE −/− mice (P < 0.05) (Figure 1).
Static compliance, which was used as a marker of lung injury severity, was measured every 30 minutes throughout the 3-hour experimental period after recruitment maneuvers to normalize physiologic properties. There was no difference in baseline lung compliance between the two genotypes, nor were there differences between the groups at 10 ml/kg Tv (WT: 0.068 and NE−/−: 0.079 ml/cm H2O). As expected, with increasing tidal volumes, lung functions worsened over time. In the 20 and 30 ml/kg Tv groups, compliance decreased over the course of the experimental period in both WT and NE−/− mice as compared with 10 ml/kg group (P < 0.05) (Figure 2). Unexpectedly, the static lung compliance of NE−/− mice was significantly worse as compared with sex- and age-matched control WT mice under similar ventilation conditions (P < 0.05).
Wet:dry lung weight ratios of WT and NE−/− mice (n = 5) after 3-hour ventilation at varying tidal volumes were measured (Figure 3). In the 20 and 30 ml/kg Tv groups, both genotypes had significantly higher wet:dry ratios as compared with 10 ml/kg groups in their respective genotypes. At 20 ml/kg and 30 ml/kg Tv, NE−/− mice had significantly higher extravascular lung water, consistent with increased lung stiffness above and suggestive of worse lung injury and edema in NE−/− than WT control mice (P < 0.05).
We examined Gr1 immunostained lung sections under light microscopy to determine the ratio of neutrophils located in the airspace as opposed to those located in the intravascular space (Figure 4A). In WT mice following mechanical ventilation with 20 and 30 ml/kg Tv, the ratio of intra-alveolar/intravascular neutrophils was significantly higher in WT than NE−/− mice in both Tv groups (P < 0.05), suggesting impaired ability of NE−/− neutrophils to egress from the vasculature into the alveolar space. Representative images at 20 ml/kg tidal volume are shown (Figure 4B).
To determine the basis for the defect in neutrophil recruitment in NE−/− mice, we first assessed neutrophil chemotaxis. Neutrophils from WT and NE−/− mice were placed into a micro-chemotaxis (Boyden) chamber and the cells were incubated in the presence of agents known to be chemotactic for neutrophils: LTB4, ZAS, and fMLP. WT and NE−/− neutrophils displayed equal movement in response to the various chemotactic stimuli (Figure 5A). Hence, impaired transvascular migration in NE−/− mice was not due to impaired motility or processing of chemokines.
Previous studies have used NE inhibitors to assess the role of NE in neutrophil basement membrane penetration in vitro. Here, we applied neutrophils from WT and NE−/− mice, obtained from the peritoneum after thioglycollate injection, to a Matrigel invasion assay. We found no difference in the numbers of WT and NE−/− neutrophils that penetrated the Matrigel basement membrane matrix in response to neutrophil chemokines (Figure 5B). This is consistent with most previous studies that failed to inhibit neutrophil movement with NE inhibitors.
The ability of neutrophils to cross pulmonary vein and microvascular endothelium in vitro was determined using a Transwell assay. In response to fMLP, WT neutrophils penetrated the endothelial barriers much more efficiently than NE−/− neutrophils (hPVEC: 143.26 ± 10 and 16.8 ± 15.6 neutrophils/hpf for WT and NE−/−, respectively [P < 0.05], and hPMVEC: 55.7 ± 16.2 and 27.4 ± 5.3 neutrophils/hpf for WT and NE−/−, respectively [P < 0.05]). In contrast, penetration of the endothelial microvasculature was similar in WT and NE−/− neutrophils in response to ZAS (Figure 5C).
The ability of neutrophils to cross pulmonary endothelium and cause cellular injury in vitro was determined using a radioactive tracer (Na51CrO4). Neutrophils co-incubated with endothelial cells loaded with radioactive 51Cr in a 96-well tissue chamber plate caused more injury in WT than in NE−/− mice, as indicated by release of 51Cr into the conditioned media, suggesting that NE-containing neutrophils, when in contact with endothelial cells, cause more injury than in its absence (Figure 6A). However, when WT and NE−/− neutrophils are allowed to migrate through the endothelial barriers in response to fMLP, neutrophils from NE−/− mice penetrated the endothelial barrier less effectively than neutrophils derived from WT mice and caused increased cellular injury as compared with WT (Figure 6B). The findings of increased endothelial cell injury upon prolonged exposure to NE−/− neutrophils may explain the worsening lung injury (increased lung edema and lower static compliance) associated with the NE−/− mice.
We hypothesized that the enhanced migration of WT as compared with NE−/− neutrophils through endothelial cell barriers was due to NE-mediated cleavage of ICAM-1, releasing the neutrophil from the endothelial cell. To test this, we incubated activated WT and NE−/− neutrophils with activated endothelial cells for 2 hours and assessed endothelial surface ICAM-1 expression using both Western blot analysis and immunofluorescence with an antibody raised against the extracellular domain of ICAM-1 (Figure 7). Despite greater numbers of adherent NE−/− neutrophils in contact with endothelial cells (Figure 7A), there was much less cleavage of ICAM-1 observed both by Western blot (Figure 7B) and immunofluorescence (Figure 7C) by NE−/− neutrophils as compared with WT neutrophils. Impaired NE-mediated cleavage of ICAM-1 would explain the prolonged neutrophil–endothelial cell contact leading to endothelial cell injury and increased permeability observed in NE−/− mice.
This study demonstrates that after the onset of mechanical ventilation, mice developed decreased lung compliance and accumulated lung water in proportion to tidal volumes and time on the ventilator. In the absence of NE, mice developed worse parameters of lung injury than controls, particularly at higher tidal volumes and increased ventilator times. NE−/− mice had a greater proportion of neutrophils “stuck” in the endothelial compartment as opposed to WT, where more neutrophils did egress into alveolar spaces. Previous studies in cell culture revealed that presence or absence of NE did not influence the ability of neutrophils to respond to chemotactic signals or traverse extracellular matrix barriers (14). However, in this study, in the presence of intact endothelium, WT neutrophils migrated through the endothelial barrier in response to chemotactic signals much more efficiently than NE−/− neutrophils. This was likely related to NE-mediated cleavage of the firm attachment of the neutrophil to ICAM-1 allowing neutrophils to egress through the endothelium. Prolonged contact of activated neutrophils with endothelial cells results in cell injury, as demonstrated by chromium release assays. In fact, there was greater cell injury when neutrophils contained NE than in its absence. However, when allowed to traverse the endothelial barrier in response to chemotactic stimuli, there were fewer WT as compared with NE−/− neutrophils in contact with endothelial cells and less endothelial cell injury. Proteases and reactive oxygen species in addition to NE could injure the endothelium, which may explain our findings (19–21). These results suggest a role for NE in facilitating egression of neutrophils from the vasculature into alveolar spaces during mechanical ventilation in mice, and that continued contact of neutrophils with endothelial cells could lead to lung injury.
Neutrophil elastase is a 28-kD serine proteinase, characterized by conserved His, Asp, Ser residues that form a charge relay system resulting in a powerful nucleophile able to cleave peptide bonds of the substrate (22). Neutrophil serine proteinases are only expressed during the myelomonocytic stage of leukocyte development, and are stored for later use as active enzymes within the neutrophil's primary granules. Upon activation, neutrophils release approximately 2% of their NE content into the extracellular space, where they can reach high local concentrations overwhelming inhibitors transiently (23). In addition, about 12% of NE content is mobilized to the cell membrane, where it is catalytically active and resistant to inhibitors. Cell surface NE is strategically positioned for cell–cell and cell–matrix interactions and localized extracellular proteolysis (23).
NE has a broad range of susceptible extracellular matrix substrates including elastin, interstitial collagens (I-III), and basement membrane proteins (24). Other relevant substrates include coagulation factors, fibronectin, plasminogen, immunoglobulins, and C5a (25, 26). Hence, NE has been thought to play a role in a variety of destructive inflammatory diseases including ARDS (27). NE has also been postulated to “clear paths” through extracellular and promote neutrophil trafficking, but as discussed above few studies support this concept.
We and others previously generated NE−/− mice, which helped establish a role for NE in host defense. Specifically, NE is required for intracellular neutrophil killing Gram-negative bacteria via degradation of outer membrane proteins (Omps) (15, 28, 29). Neutrophil function was otherwise normal; however, the role of NE in neutrophil migration assays within the context of VILI was not previously reported. Application of NE−/− mice to a model of cigarette smoke–induced emphysema demonstrated that NE contributed to monocyte, but not neutrophil, recruitment into the lung and was in part responsible for airspace enlargement (30). In response to bleomycin, there were no differences in neutrophil inflammation, but NE−/− mice were also protected from fibrosis secondary to NE-mediated activation of TGF-β (31).
In this study, to determine the role of NE in VILI, we applied NE−/− mice to mechanical ventilation. Beginning with classic studies of mechanical ventilation in dog models, and extending to rodents, investigators describe increased numbers of intra-alveolar and intravascular leukocytes and macrophages after VILI using high tidal volumes (1, 32). High tidal volumes in animal models of VILI have been criticized for not reflecting the actual practice at bedside, but these Tv (20–30 ml/kg) are comparable to the excessive overdistention expected in some parts of the lungs in ARDS due to its heterogeneous nature (5). We used a volume-based lung injury model along with 0 cm H2O of PEEP to induce VILI. Surprisingly, in the absence of NE, mice developed worse injury than when this potentially destructive enzyme was present.
NE's beneficial role in VILI could relate to its requirement for neutrophils to egress from the pulmonary vasculature. In the absence of NE, prolonged endothelial cell–neutrophil contact led to endothelial cell injury and increased permeability, similar to a previous report on neutrophil–epithelial cell interaction (33). The role of NE in neutrophil trafficking remains of great interest and controversial. As discussed, most cell culture studies failed inhibit NE migration using NE inhibitors (14, 34). However, there are published studies showing inhibition of NE resulting in diminished neutrophil chemotaxis (35) and matrix (Matrigel) invasion (36). This study shows that upon the addition of an endothelial cell barrier, NE is required for transvascular egression. The effect of NE on neutrophil trafficking in vivo varies depending upon the model. In some studies of acute lung injury, NE inhibitors (endogenous, small molecular weight compounds, or antibodies) or lack of NE ameliorate neutrophil accumulation in vivo (35, 37, 38). Yet, application of NE−/− mice to other animal models such as bleomycin and emphysema (above), as well as lipopolysaccharide (16), did not demonstrate a role for NE in neutrophil accumulation in the lung.
We performed further experiments to determine the mechanism by which NE promotes transvascular migration in the context of an activated pulmonary endothelium. First, we observed that NE-deficient neutrophils as compared with WT neutrophils had impaired migration through endothelial monolayers in response to fMLP, but not zymosan. Since fMLP, but not zymosan, induces endothelial ICAM-1 and displays NE-mediated neutrophil migration, this suggested a role for NE in β2-integrin–mediated migration. Previously, NE has been shown to release neutrophils from endothelial cells noncatalytically by interfering with CD11b/CD18–ICAM-1 interactions (39). Yet, NE was also shown to catalytically cleave ICAM-1 on monocytic cell lines (39, 40). Our data suggest a role for NE in catalytically disrupting ICAM-1–CD11b/CD18 bonds releasing neutrophils from endothelial cells, allowing them to follow the chemotactic gradient.
Despite the fact that NE itself is an injurious proteinase, the NE-deficient neutrophil contains a variety of toxic oxidants and other proteinases that can harm endothelial cells upon prolonged contact. Hence, while WT neutrophils are more harmful to endothelial cells than NE−/− neutrophils, it is in the absence of NE that they have prolonged contact and hence cause cell injury.
This study suggests that retention of neutrophils within pulmonary vasculature has an acute injurious effect on the lung in mice. Whether the detrimental effects of NE-containing neutrophils within the alveolar space ultimately have more severe consequences on lung injury over time is not known. However, it is interesting to note in the context of ARDS that a clinical trial using small-molecular-weight NE inhibitor resulted in a negative trend in long-term mortality, with no superiority in ventilator-free days or 28-day all-cause mortality over placebo (41). Whether this is a function of NE's antimicrobial activity or its role in neutrophil egress, the mechanisms invoked here or others, is not known (15). Given NE's physiological functions to release neutrophils from the vasculature and kill bacteria within the neutrophil, perhaps an ideal inhibitor would be one that did not penetrate the neutrophil and introduced to (and limited to) the alveolar space. These findings also reinforce the importance of limiting mechanical ventilation volumes with consequent reduction in inflammation contributing to volutrauma.
This work was supported by grants FAER-RTG (A.M.K.), National Institutes of Health HL65697 (B.R.P.), and NIH HL054853-11A1 (S.D.S.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2007-0315OC on February 14, 2008
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.