American Journal of Respiratory and Critical Care Medicine

Mechanical distortion of blood vessels is known to activate endothelial cells. Whether airway distension likewise activates the vascular endothelium within the airway wall is unknown. Using intravital microscopy in the rat trachea, we investigated if airway distention with the application of positive end-expiratory pressure (PEEP) caused leukocyte recruitment to the airway. Tracheal postcapillary venules were visualized and leukocyte kinetics monitored in anesthetized, mechanically ventilated rats (80 breaths/minute, 6 ml/kg VT, 1 cm H2O PEEP). Leukocyte rolling velocity (Vwbc) and the number of adherent cells were not altered with normal ventilation over the course of 2 hours. Ventilation with sustained PEEP (8 cm H2O) for 1 hour reduced Vwbc and increased adhesion, reaching a maximum at 1 hour of PEEP. Intermittent (2× and 5×) 8 cm H2O PEEP also induced a similar reduction in Vwbc, accompanied by an increase in adhesion. However, leukocyte recruitment after airway distension is localized to the airways because increased PEEP did not induce leukocyte recruitment in the mesenteric microcirculation or when PEEP was applied to the lung distal to the site of measurement. Pretreatment with endothelin receptor and selectin inhibitors blocked the effects of distension on leukocyte recruitment, suggesting their involvement in the proinflammatory response.

Recently there has been a growing interest in the effects of mechanical strain on cells comprising the lung parenchyma, airways, and pulmonary vasculature (1). Many studies have focused on the effects of increased Vt and deleterious levels of positive end-expiratory pressure (PEEP) (2). The extent of airway distension with the application of PEEP has been carefully documented (3). However, the effects of mechanical distortion of the vascular endothelium lining the walls of the airways are unknown. Stretch-induced activation of endothelial cell monolayers has demonstrated endothelial cell activation (4, 5), release of inflammatory mediators (6), changes in monolayer permeability (7), and increased adhesion molecule expression (8). Whether each of these deleterious effects would be observed in relevant in vivo models has not been substantiated. We have recently characterized a new application of intravital microscopy in the rat trachea that enables visualization of leukocyte kinetics in the airway circulation in vivo (9). Using this model, we can systematically investigate inflammatory cell velocity and adherence in the tracheal circulation after endothelial cell activation with mechanical distortion. Thus, the aim of this study was to determine the effect of airway distension with the application of PEEP on leukocyte recruitment in the airway microcirculation. Some of the results of these studies have been previously reported in abstract form (10, 11).

Animals

Male Sprague–Dawley rats (200–250 g) were obtained from Harlan (Indianapolis, IN) and housed in controlled pathogen-free animal facilities. They were maintained on a standard chow pellet diet with tap water ad libitum, using a 12-hour light/dark cycle. All procedures were approved by the Johns Hopkins Institution Animal Care and Use Committee.

Surgical Procedures

Rats were anesthetized by intraperitoneal injection with 75 mg/kg sodium pentobarbital (Sigma, St. Louis, MO) in saline. Each animal was intubated with an endotracheal tube (size 14 catheter; Johnson and Johnson, Arlington, TX) inserted past the level of the thyroid gland and mechanically ventilated (80 breaths/minute, 1.5–2 ml Vt), with a small rodent ventilator (Harvard 683; Harvard Instruments, Holliston, MA). Pulmonary inflation pressure was monitored with a pressure transducer attached to a side port of the tracheal cannula. The femoral artery and vein were cannulated with P10 luer catheters (Becton Dickinson, Sparks, MD) for the measurement of systemic blood pressure and the infusion of additional anesthetic (80 μg/minute), respectively. Systemic arterial blood pressure and pulmonary inflation pressure were monitored and recorded on a Grass recorder (Grass model 79D; Grass Instruments, Quincy, MA). Succinylcholine (4 mg/kg) was administered subcutaneously to prevent voluntary movements.

For intravital microscopy of the trachea, a midline incision was made in the ventral surface of the neck and the trachea was exposed and superfused with warmed (37°C) buffer. Care was taken not to disrupt the blood vessels supplying the trachea. Single postcapillary venules were selected in each rat, visualized for up to 2 hours and recorded on video for offline analysis. In separate experiments, postcapillary venules were visualized and recorded in the mesentery (12). For these studies, an incision was made in the abdomen, and the mesentery and small intestine were exteriorized. Leukocyte trafficking was quantified offline by measuring: (1) leukocyte rolling velocity, (2) the number of rolling cells, and (3) the number of adherent cells in a 100-μm length of vessel per time point. Red blood cell velocity was measured online using an optical Doppler velocitometer (Microcirculation Research Institute, Texas A&M University, TX), and mean red blood cell velocity was determined as red blood cell velocity/1.6 (13). Wall shear rate was calculated by the Newtonian definition: shear rate = 8,000 × (mean red blood cell velocity/diameter of vessel).

Ventilation Protocols
PEEP.

Figure 1

illustrates the various PEEP protocols performed. Normal ventilation was maintained at 80 breaths/minute, with 6 ml/kg Vt (14) and 1 cm H2O PEEP. Vt and ventilatory rate were kept constant throughout the experimental protocols in which PEEP was altered. PEEP was increased to 8 cm H2O either continuously (for 1 hour, sustained) or intermittently twice (8 cm H2O × 2) or five times (8 cm H2O × 5), every 10 minutes for a duration of 1 minute. An intermediate level of intermittent PEEP (4 cm H2O, five times; 4 cm H2O × 5) was also applied to evaluate the pressure threshold for leukocyte recruitment. Rolling and adhesion were measured at baseline, 5 minutes, and at 60 minutes after the final PEEP maneuver for the intermittent PEEP protocols, and every 20 minutes for the sustained PEEP protocol. The application of 8 cm H2O PEEP demonstrated, in preliminary studies using high-resolution computed tomography, an approximate 10% increase in average tracheal lumenal area (see Figure E1 in the online supplement).

Low intubation.

To determine whether changes in leukocyte kinetics were due to local distension of the trachea or whether distension of the lung parenchyma contributed to changes observed in the trachea, a separate set of experiments was performed in which the endotracheal tube was inserted deeper into the trachea, past the area of observation. A suture was tied firmly around the end of tube to ensure that no air leakage occurred during the experimental procedure. With this low intubation, control animals were ventilated with 1 cm H2O PEEP for 2 hours, whereas laboratory animals were ventilated using the 8 cm H2O × 5 protocol, and the leukocyte trafficking parameters were recorded.

Mesentery.

Additional experiments were performed to determine whether the alterations in leukocyte kinetics were specific to the trachea. In separate experiments, the microcirculation of the rat mesentery was examined for comparison with the trachea using the 8 cm H2O × 5 protocol.

Pharmacologic Inhibition

To address two potential mechanisms by which excessive airway distension mediates changes in leukocyte kinetics, we studied the effects of endothelin receptor blockade and selectin inhibition before the application of 8 cm H2O × 5 cm H2O PEEP in additional animals.

Endothelin receptor inhibition.

Both endothelin receptor (ETA and ETB) antagonists BQ123 and BQ788 in 0.1% dimethyl sulfoxide (Sigma) were added at a concentration of 0.1 μM to the Krebs buffer superfusion after a suitable vessel was located and baseline parameters were recorded. The drugs were superfused over the trachea for 20 minutes before the first PEEP maneuver (using the 8 cm H2O × 5 protocol) and during the 2-hour observation period. Control animals received dimethyl sulfoxide vehicle (0.01%) superfusion in parallel.

Selectin inhibition.

After a suitable tracheal postcapillary venule was located and baseline parameters were recorded, we delivered fucoidin (0.75 mg; Sigma), known to block both P- and L-selectin (15), through the femoral venous catheter over the course of 10 minutes (0.3 ml). A supplemental dose (0.75 mg) was delivered during the 8 cm H2O × 5 distension protocol. Leukocyte kinetics were studied before (0 time), 5 minutes, and 60 minutes after distension. Control rats were studied with the same fucoidin delivery but without airway distension.

Histology

Leukocyte accumulation in the trachea was assessed histologically in control rats and after the maximum PEEP protocol of 8 cm H2O × 5. At the end of the experiment, animals were killed by pentobarbital overdose and were perfused transcardially using phosphate-buffered saline with heparin for 15 minutes, followed by fixing the entire trachea with 4% paraformaldehyde at 4°C overnight. Tissues were extracted and frozen on dry ice in optimum cutting temperature compound and stored at −70°C. Cryostat-cut sections were thaw-mounted onto polylysine-coated slides, stained using diaminobenzidine (for peroxidase, 3 minutes), and counterstained with hemotoxylin (1.5 minutes). Tracheas from n = 3 animals per treatment and four areas of each trachea were sectioned serially. Two sections were chosen per area, with good morphology and staining, and the number of leukocytes were counted in the intercartillagenous region (0.75 mm2). Both the control and experimental groups were counted in the same way. The number of leukocytes counted are expressed as the total number of leukocytes per tissue for all eight sections evaluated.

Data Analysis

Data are reported as mean ± SEM of n animals per group. Statistical differences were calculated on original values using unpaired tests (histology, vessel diameters) or one-way analysis of variance followed by a Bonferroni/Dunn post hoc test for intergroup comparisons. A value of p less than 0.05 was accepted as significant.

Mechanical ventilation during control conditions (6 ml/kg, 80 breaths/minute, 1 cm H2O PEEP) for up to 2 hours did not alter leukocyte trafficking parameters (rolling velocity or adhesion) or blood pressure (n = 6) compared with time 0. Peak inspiratory pressure averaged 7.6 ± 0.4 cm H2O, and mean arterial blood pressure averaged 94 ± 7.5 mm Hg. The changes in peak inspiratory pressures during the increase in PEEP are shown in Table 1

TABLE 1. Changes in peak inspiratory pressure during the various positive end-expiratory pressure protocols





Peak Inspiratory Pressure (cm H2O)
Protocol
n
PEEP
Before PEEP
During PEEP
Control617.5 ± 0.37.5 ± 0.3
4 cm H2O × 5448.3 ± 0.512.5 ± 0.8
8 cm H2O × 2488.3 ± 0.921.0 ± 1.1*
Sustained 8 cm H2O489.0 ± 0.721.3 ± 0.5*
8 cm H2O × 5, trachea586.5 ± 0.618.2 ± 1.0*
8 cm H2O × 5, mesentery586.6 ± 0.418.6 ± 1.2*
8 cm H2O × 5, low intubation
3
8
7.3 ± 0.3
18.3 ± 2.0*

*p Value less than 0.05 versus baseline values.

Definition of abbreviation: PEEP = positive end-expiratory pressure.

Normal ventilation was maintained at 80 breaths/minute, with 6 ml/kg Vt and 1 cm H2O PEEP. Airway distension was induced by increasing PEEP to either 4 or 8 cm H2O either intermittently twice (8 cm H2O × 2) or five times (4 cm H2O × 5 or 8 cm H2O × 5) every 10 minutes for a duration of 1 minute; or continuously (for 1 hour, sustained) for up to 60 minutes.

Data are means ± SEM of n = 3 to 6 animals per experimental group.

. See the online supplement (Figures E2 and E3) for actual video recordings of postcapillary venules before and after the application of PEEP.

Effects of Continuous Distension on Leukocyte Rolling and Adhesion

Continuous application of 8 cm H2O PEEP for 1 hour (n = 4) resulted in a time-dependent reduction in rolling velocity (Figure 2A)

together with an increase in the number of adherent cells (Figure 2B). This was accompanied by a 3.1- ± 1.9-fold increase in the number of rolling cells. The increase in the number of rolling and adherent cells was transient, returning to baseline levels 60 minutes after the cessation of PEEP. An initial decrease in arterial blood pressure was observed immediately after the application of PEEP, which returned to control level within 30 minutes (Figure 4A). This was mirrored by a decrease in vessel shear rate that was not due to a change in vessel diameter. However, leukocyte rolling velocity remained reduced by 41.7 ± 12.3% at the final time point, even after blood pressure had stabilized.

Effects of Intermittent Distension on Leukocyte Rolling and Adhesion in the Trachea

To determine whether shorter periods of distension would similarly alter leukocytic–endothelial interactions, we examined intermittent short periods of distension with the application of PEEP. Animals were ventilated normally throughout the course of the experiment, apart from several 1-minute bouts of increased PEEP. The application of PEEP twice with 8 cm H2O PEEP evoked a significant reduction in rolling velocity, 5 minutes after the second PEEP maneuver (p < 0.05, n = 4). This was accompanied by a concomitant increase in leukocyte adhesion (Figure 3)

. An hour after the cessation of airway distension with PEEP, the rolling velocity remained significantly reduced (50 ± 7% reduction, p < 0.05), and adhesion increased compared with baseline. Similarly, five distensions resulted in a significant reduction in rolling velocity at the first time point measured after PEEP was removed (n = 6), together with a concurrent increase in adhesion (5.3 ± 2.3 adherent cells). Both persisted for the duration of the experiment (Figure 3). In this case, the number of rolling leukocytes trended toward an increase (59 ± 22.9%, p = 0.09). These PEEP maneuvers induced a sharp decrease in arterial blood pressure (32.6 ± 3.3% decrease), which was restored when the increased PEEP was removed (Figure 4B) . Furthermore, with the application of PEEP, vessel shear rate decreased simultaneously with arterial blood pressure but recovered each time PEEP was removed. Application of five intermediate bouts of PEEP (4 cm H2O × 5, n = 4; Figure 3) failed to significantly alter either leukocyte rolling velocities or the number of adherent cells counted. This suggests that a threshold level of end-expiratory pressure is required for significant enhancement of leukocytic–endothelial interactions and recruitment to occur in the airway microcirculation. There was no correlation between the maximum change in blood pressure and the number of adherent cells in the trachea when evaluating all PEEP protocols (r2 = 0.295; p > 0.05).

Confirmation of Venular Distension with Increased PEEP

To confirm that the application of PEEP resulted in a change in the dimensions of tracheal postcapillary venules, we measured venular diameter at end-expiration during control ventilation and during the application of the highest level of PEEP studied (8 cm H2O; n = 3 rats). Venular diameter increased from 29.0 ± 2.3 μm (1 cm H2O PEEP) to 32.5 ± μm (8 cm H2O PEEP; p = 0.04).

Effect of Intermittent Airway Distension on the Mesenteric Microcirculation

To determine whether the effects of increased PEEP on leukocyte recruitment were due to airway distension and not a result of decreased systemic arterial pressure and blood flow, we studied the effects of PEEP (8 cm H2O × 5) on leukocyte trafficking in the rat mesentery (n = 5). Higher levels of baseline adhesion and number of rolling cells were observed compared with the trachea, similar to those previously observed in the rat mesentery (16). However, when compared with baseline, there were no significant changes in leukocyte rolling velocity or adhesion at any time points measured after airway distension with PEEP (Figure 5)

.

Effect of Low Intubation and PEEP on Leukocyte Rolling and Adhesion in the Trachea

Additional experiments were performed to determine whether the effects of PEEP were due to the direct effects of tracheal distension or whether lung distension resulted in the release of circulating factors into the pulmonary circulation that then affected the tracheal venular endothelium. Table 2

TABLE 2. Changes in leukocyte kinetics after positive end-expiratory pressure and without tracheal distension



Control (Low Intubation)

8 cm H2O × 5 (Low Intubation)
Parameter
0 min
5 min
60 min
0 min
5 min
60 min
Velocity, μm/s38.8 ± 5.234.0 ± 4.336.4 ± 3.441.9 ± 4.536.5 ± 1.332.5 ± 0.4
Adhesion, no. of cells
2.0 ± 0.6
2.7 ± 0.7
2.3 ± 0.7
1.3 ± 0.7
2.7 ± 0.3
2.7 ± 0.9

Definition of abbreviation: PEEP = positive end-expiratory pressure.

Control ventilation was maintained at 80 breaths/minute, with 6 ml/kg VT and 1 cm H2O PEEP. Airway distension was induced by increasing PEEP to 8 cm H2O five times every 10 minutes for a duration of 1 minute. Time points of measurements are before (0), 5, and 60 minutes after the last distension with PEEP. The endotracheal tube was advanced beyond the site of venule observation and the tip of the tube was secured with a ligature.

Data are mean ± SEM of n = 3 animals per experimental group.

p > 0.05 versus 0 time values.

shows the results in control rats (n = 3) and rats exposed to PEEP (8 cm H2O × 5) when the endotracheal tube was positioned deeper into the trachea beyond the site of venule observation. No change in either leukocyte velocity or adhesion was observed after distension. Changes in systemic arterial pressure were similar to those recorded previously (Figure 4B).

Mechanism of Response

Addition of the ET receptor antagonists, BQ123, and BQ788 to the superfusion buffer before the first distension with PEEP did not alter basal leukocyte trafficking parameters (Table 3

TABLE 3. Changes in leukocyte kinetics after positive end-expiratory pressure and selectin or endothelin receptor a/b inhibition



Selectin Inhibition

ETA/ETB Inhibition
Parameter
Baseline
0 min
5 min
60 min
Baseline
0 min
5 min
60 min
Velocity, μm/s30.0 ± 3.926.9 ± 2.224.1 ± 3.531.2 ± 7.254.0 ± 5.153.6 ± 5.645.8 ± 8.451.5 ± 4.6
Adhesion, no. of cells
0
0
0
0
0.2 ± 0.2
1.2 ± 0.4
2.2 ± 0.9
1.6 ± 0.6

Definition of abbreviations: ETA/ETB = endothelin receptor A/B; PEEP = positive end-expiratory pressure.

Control ventilation was maintained at 80 breaths/minute, with 6 ml/kg VT and 1 cm H2O PEEP. Airway distension was induced by increasing PEEP to 8 cm H2O five times every 10 minutes for a duration of 1 minute. Time points of measurements were before pharmacologic inhibitors (baseline) and before (0), 5, and 60 minutes after the last distension with PEEP.

Data are mean ± SEM of n = 4 to 5 animals per experimental group.

p > 0.05 versus 0 time values.

; n = 5) or blood pressure (data not shown). However, addition of BQ123 and BQ788 inhibited the significant reduction in leukocyte rolling velocity immediately and 60 minutes after the removal of PEEP. Leukocyte velocity after distension was unchanged from baseline (before treatment) as well as time 0 (before the application of the distension protocol; p = 0.1408). In addition, the PEEP-induced enhancement of leukocyte adhesion seen previously was not observed. Adhesion after distension was unchanged from baseline (before treatment) and time 0 (p = 0.1017), suggesting that endothelin receptor binding was involved in the PEEP-mediated alterations in leukocytic–endothelial interactions. Superfusion with vehicle (dimethyl sulfoxide; n = 2 control rats; 1 PEEP) alone resulted in expected responses (i.e., no changes in control animals, usual decrease in velocity/increase in adherence with PEEP).

Pretreatment with the P- and L-selectin inhibitor fucoidin resulted in complete inhibition of distension-induced changes in leukocyte velocity and adhesion (Table 3, n = 4). Although average leukocyte velocity was lower in this series of experiments, distension had no effect on leukocyte recruitment or adhesion (p = 0.6465). Fucoidin alone had no effect on measured parameters in control rats (n = 2).

Identification of Leukocyte Subtypes in the Trachea

Figure 6

shows representative sections of rat trachea from control animals and rats exposed to intermittent PEEP (8 cm H2O × 5). Few extravasated leukocytes (Figure 6A) were observed in control rats. However, histologic sections from rats exposed to airway distension with PEEP showed substantially increased numbers of leukocytes, primarily neutrophils. Interestingly, as Figure 6B illustrates, neutrophils were observed on the luminal aspect of the airway as well as within the adventitial region. Quantitative assessment of the superficial adventitial region (0.75 mm2 area) in control rats (n = 3; eight sections each) showed a total of 28 ± 12 neutrophils. In rats exposed to airway distension (n = 3; 8 sections each), the number of neutrophils counted was significantly increased to 118 ± 20 neutrophils (p = 0.0096).

In the present study, we demonstrated that airway distension induced by the application of PEEP (8 cm H2O) affects the tracheal vascular endothelium resulting in enhanced leukocytic–endothelial interactions and leukocyte recruitment in the trachea. We predicted that applying PEEP would cause changes consistent with endothelial cell activation, and we confirmed an increase in venular diameter during the application of PEEP. Indeed, increasing PEEP to 8 cm H2O resulted in increased leukocyte rolling (reduced rolling velocities) and adhesion in the microvasculature of the upper airways, accompanied by local leukocyte (neutrophil) infiltration. The extent and intensity of airway distension needed to produce an effect on leukocyte recruitment was examined by increasing the number of perturbations as well as the magnitude of tracheal distension. PEEP was applied for 1 minute intermittently (every 10 minutes) either twice or five times. Our data suggest that a critical threshold of PEEP (between 4 and 8 cm H2O) is necessary to exert a significant response in leukocyte trafficking because a mild level of distension (4 cm H2O) was not sufficient to cause changes in leukocytic–endothelial interactions. From these results, it appears that more significant airway distension induced by the application of higher levels of PEEP will have a more profound effect on leukocyte trafficking in the airway circulation.

The selectivity of the effects of airway distension by the application of PEEP was assessed in two different series of experiments. The application of PEEP had no effect on leukocyte recruitment in the mesenteric circulation demonstrating that leukocytes generally were not activated (Figure 5). In addition, when applying PEEP to the lung and airways distal to end of the endotracheal tube when the tube had been advanced beyond the site of venule observation (Table 2), no change in leukocyte kinetics was observed in the upper trachea that had not been distended. We interpret this observation to suggest that lung distension did not activate leukocytes or elicit release of factors that circulated to and affected the tracheal venular endothelium.

Each time PEEP was applied, systemic blood pressure and vessel shear rates fell simultaneously, likely due to a decreased Q̇ in response to the reduced venous return (17). However, the reductions in blood pressure and red cell velocity were transient and recovered each time PEEP was removed. However, it is possible that the reduction in arterial blood pressure and shear rate in the tracheal vessels under observation could contribute to the increased leukocytic–endothelial interactions seen in the airway circulation. Alterations in flow rates may lead to the induction of specific signaling pathways (18), which upregulate the expression of adhesion molecules such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (19). A relevant approach to address this hemodynamic issue in our study would be to visualize the same tracheal vasculature, but reduce blood flow to a similar extent without the application of PEEP. It is clear that extracting volumes of blood from an animal would result in a reduced blood pressure and flow. However, this approach was not feasible in our study, as the circulating leukocyte counts needed to be constant for accurate measurement of leukocyte trafficking and as the changes in blood flow evoked by PEEP were rapid and transient. Thus, we addressed the issue by observing the effects of PEEP on a remote systemic vascular bed, the mesenteric microvasculature. No alterations in leukocyte rolling or adhesion were observed in the mesenteric microcirculation, despite similar reductions in systemic arterial pressure and mesenteric vessel shear rate. This implies that the leukocyte trafficking seen in the airways after airway distension was not due to the hemodynamic effects of PEEP but was a selective effect of airway distension. Furthermore, similar decreases in systemic blood pressure were observed when the lung and lower airways distal to the site of venule observation were distended with PEEP. However, no changes were seen in leukocyte kinetics. Thus, it appears that changes in blood pressure and shear rate are not responsible for enhanced leukocyte recruitment to the tracheal wall after airway distension with PEEP.

The cellular mechanism whereby endothelial distortion leads to enhanced leukocytic–endothelial interactions is not known. Endothelial cell stretch has been examined previously using innovative in vitro systems applying mechanical stretch of confluent endothelial cell monolayers. Cyclical stretch has been shown to induce the release of various inflammatory mediators (6), which can lead to leukocyte adhesion in vitro (20). It has been shown that cyclic stretch of endothelial cells in vitro results in the induction of chemokines such as interleukin-8 and monocyte chemoattractant protein-1, which are important in the recruitment of neutrophils and monocytes (21). Interleukin-8 is upregulated during cyclical stretch of airway epithelial cells (22), and could play a key role, together with adhesion molecules such as P-selectin, in the selective recruitment of leukocytes into the airways. Previous studies have also addressed the importance of endothelin-1 during endothelial stress (23) and mechanical stretch of endothelial cells (24, 25). Furthermore, exogenous application of endothelin-1 (either topically or systemically) induces leukocyte adhesion in the rat mesenteric microcirculation (26), which was found to be P-selectin–dependent (27). We studied the effects of endothelin receptor blockade and selectin inhibition in the distension-induced responses to begin to assess potential mechanisms of the observed response. We found that both endothelin receptor (ETA and ETB) blockade as well as use of the pan selectin inhibitor fucoidin, interrupted distension-induced leukocyte recruitment to the trachea. The precise molecular mechanism of response requires further experimentation but may involve the interaction of P-selectin and endothelin, both of which are stored within endothelial cell Weibel-Palade bodies (28).

Although this study focused specifically on leukocyte kinetics in the tracheal vasculature, there has been tremendous recent interest in the effects of mechanical strain on all cells of the lung (11). Many studies have focused on the effects of increased Vt and deleterious levels of PEEP (2). However, the effects of a comparable level of PEEP (7.5 cm H2O) on leukocyte kinetics in the pulmonary circulation have been shown to be largely transient and likely the effects of increased compression and leukocyte trapping (29).

In conclusion, our data demonstrate that airway distension induced by the application of PEEP can lead to increased inflammatory leukocyte trafficking in the airways, and a critical level of PEEP is necessary to evoke this response. Furthermore, this observation is selective for the airways, as leukocyte recruitment did not occur in the mesenteric microcirculation after identical ventilation procedures.

The authors thank Dr. Bruce Bochner and Dr. Stuart Mazzone for helpful discussion and critical review of the manuscript.

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Correspondence and requests for reprints should be addressed to Elizabeth M. Wagner, Ph.D., Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail:

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American Journal of Respiratory and Critical Care Medicine
168
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