Published ahead of print on February 14, 2008, doi:10.1165/rcmb.2007-0315OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0315OC Neutrophil Elastase Is Needed for Neutrophil Emigration into Lungs in Ventilator-Induced Lung Injury1 Department of Critical Care Medicine, 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 3 Washington University School of Medicine, St. Louis, Missouri; and 4 Department of Environmental and Occupational Health, University of Pittsburgh, Graduate School Public Health, Pittsburgh, Pennsylvania Correspondence and requests for reprints should be addressed to A. Murat Kaynar, MD, Department of Critical Care Medicine, Scaife Hall 639, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261. E-mail: kaynarm{at}upmc.edu
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.
Key Words: neutrophil elastase • ventilator-induced lung injury • endothelial injury • emigration
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
Complete Blood Counts
Animal Preparation, Experimental Ventilation Protocols, and Physiological Measurements
Bronchoalveolar Lavage
Histopathological Examination of the Lungs
Measurement of Wet:Dry Lung Weights
Neutrophil Isolation, Chemotaxis, and Matrigel Invasion Assays
Transmigration of Neutrophils across Human Pulmonary Vein and Microvascular Endothelial Cell–Coated Membranes
Endothelial Cell Injury after Co-Incubation and Transmigration of Neutrophils across hPVEC-Coated Membranes
ICAM-1 Expression on hPVEC after Co-Incubation with Neutrophils
Data Analysis
Complete Blood Counts 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).
Total Cell and Neutrophil Content in BAL after Mechanical Ventilation 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 Lung Compliance after Mechanical Ventilation in WT and NE–/– Mice 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).
Lung Injury as Measured by Wet:Dry Lung Weights after Mechanical Ventilation 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).
Neutrophil Emigration from the Pulmonary Vasculature into the Alveolar Space after Mechanical Ventilation 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).
Chemotaxis of WT and NE–/– Neutrophils 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.
Migration of WT and NE–/– Neutrophils through Matrigel 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.
Penetration of WT and NE–/– Neutrophils across hPVEC- and hPMVEC-Coated Membranes
Ability of WT and NE–/– Neutrophils to Cause Endothelial Cell Injury upon Prolonged Contact and Transmigration
Capacity of NE–/– and WT Neutrophils to Cleave Endothelial Surface ICAM-1 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. Received in original form April 25, 2007 Accepted in final form January 25, 2008
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Abstract
CLINICAL RELEVANCE
–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. 
