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Clara cell secretory protein and phospholipase A₂ activity modulate acute ventilator-induced lung injury in mice

¹Physiology and ²Pediatrics, ³Center for Lung Biology, University of South Alabama, Mobile, Alabama; and ⁴Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 12 October 2004 ; accepted in final form 19 November 2004

	ABSTRACT

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Lung vascular permeability is acutely increased by high-pressure and high-volume ventilation. To determine the roles of mechanically activated cytosolic PLA₂ (cPLA₂) and Clara cell secretory protein (CCSP), a modulator of cPLA₂ activity, we compared lung injury with and without a PLA₂ inhibitor in wild-type mice and CCSP-null mice (CCSP^–/–) ventilated with high and low peak inflation pressures (PIP) for 2- or 4-h periods. After ventilation with high PIP, we observed significant increases in the bronchoalveolar lavage albumin concentrations, lung wet-to-dry weight ratios, and lung myeloperoxidase in both genotypes compared with unventilated controls and low-PIP ventilated mice. All injury variables except myeloperoxidase were significantly greater in the CCSP^–/– mice relative to wild-type mice. Inhibition of cPLA₂ in wild-type and CCSP^–/– mice ventilated at high PIP for 4 h significantly reduced bronchoalveolar lavage albumin and total protein and lung wet-to-dry weight ratios compared with vehicle-treated mice of the same genotype. Membrane phospho-cPLA₂ and cPLA₂ activities were significantly elevated in lung homogenates of high-PIP ventilated mice of both genotypes but were significantly higher in the CCSP^–/– mice relative to the wild-type mice. Inhibition of cPLA₂ significantly attenuated both the phospho-cPLA₂ increase and increased cPLA₂ activity due to high-PIP ventilation. We propose that mechanical activation of the cPLA₂ pathway contributes to acute high PIP-induced lung injury and that CCSP may reduce this injury through inhibition of the cPLA₂ pathway and reduction of proinflammatory products produced by this pathway.

capillary permeability; arachidonyl trifluoromethyl ketone; mechanical stress; knockout mice

A pathway that rapidly responds to mechanical stress but has not previously been linked to VILI is the phospholipase A₂ (PLA₂) pathway. Activated PLA₂ can have myriad proinflammatory effects by catalyzing the hydrolysis of phospholipids to form arachidonic acid and lysophospholipids (2), which are further metabolized to prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, or platelet-activating factor. Many of these products can increase vascular permeability, recruit neutrophils, and injure cells (26). PLA₂ activity was increased in cultured endothelial cells subjected to mechanical stress via a p42 MAPK pathway, so overdistention of the lung would be expected to activate PLA₂ (14, 30).

The Clara cell secretory protein (CCSP) is an endogenous modulator of lung inflammation that has not previously been investigated with respect to the etiology of mechanical injury. Because CCSP has an inhibitory effect on PLA₂ activity, it is likely that this inhibition is a mechanism for its inflammation-suppressing action (5). This protein represents 2–3% of the total protein content of human bronchoalveolar lavage (BAL) fluid (12). Mice deficient in CCSP had increased lung injury and inflammatory responses after exposure to oxidant stress or virus challenge (11, 17, 23), and lavage CCSP was found in higher levels in patients who survived acute respiratory distress syndrome than in those who did not (6). Because CCSP inhibits both PLA₂ and transglutaminase, which amplifies the activity of PLA₂, the injurious effects of the multiple downstream mediators would be reduced (2, 25).

In the present study, we investigated the contributions of PLA₂ and CCSP to the lung permeability response to VILI using an inhibitor of PLA₂ in wild-type and CCSP knockout mice. Lavage albumin concentration and wet-to-dry lung weight (W/D) ratios were used to assess vascular permeability and pulmonary edema formation, respectively, and lung myeloperoxidase and lavage cell counts were used to track the inflammatory response after ventilation with high and low peak inflation pressures (PIP). CCSP-deficient (CCSP^–/–) mice were more susceptible to high-PIP ventilation injury than genetically matched controls, and inhibition of cytosolic PLA₂ (cPLA₂) attenuated injury in both wild-type and CCSP^–/– mice. Injury was also correlated with an increased phosphorylation state and enzymatic activity of membrane-localized cPLA₂.

	METHODS

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Animal Preparation

All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of South Alabama. Male C57BL/6 strain mice, which were CCSP^–/–, and strain-matched wild-type control mice were obtained after weaning from breeding colonies at the University of Pittsburgh and maintained as specific pathogen-free in-house colonies at the University of South Alabama animal facility until used for experiments.

Experiments were conducted on 50 mice, 24 wild-type and 26 CCSP^–/– mice, which were divided into 10 groups. Mice ranging in weight from 27.8 to 33.2 g (mean of 29.0 ± 0.5 g) were anesthetized with pentobarbital (65 mg/kg). A cannula was inserted through a tracheostomy into the trachea, and mice were ventilated using a rodent ventilator (Harvard, model 683, South Natick, MA) as previously described (40). Airway pressure was measured using a Cobe pressure transducer (Lakewood, CO), and an electrocardiogram was monitored using a polygraph (Grass, model 70, Quincy, MA).

Experimental Protocols

All protocols and analyses were similar for each group except for the ventilation periods of either 2 or 4 h. Inhibition of cPLA₂ was obtained by intraperitoneal injection of 20 mg/kg arachidonyl trifuoromethyl ketone (ATK; Cayman Chemical, Ann Arbor, MI) in the groups indicated 1 h before the experiments to allow equilibration of the drug.

Two-hour ventilation protocols.   Wild-type mice were divided into three experimental groups: 1) a high-PIP group (n = 6), 2) a low-PIP group (n = 3), and 3) a control group (n = 5). Groups of CCSP^–/– mice consisted of 1) a high-PIP group (n = 6) and 2) a control group (n = 5).

Four-hour ventilation protocols.   Wild-type mice were divided into 1) a high-PIP vehicle-treated group (n = 5), 2) a high-PIP group treated with ATK (n = 5), and 3) a low-PIP group (n = 5). Groups of CCSP^–/– mice consisted of 1) a high-PIP vehicle-treated group (n = 5) and 2) a high-PIP group treated with ATK (n = 5).

The high-PIP wild-type and CCSP^–/– groups were ventilated with 50 cmH₂O of PIP and 2.5 cmH₂O positive end-expiratory pressure at 17 breaths/min for either 2 or 4 h. The low-PIP groups of wild-type mice were ventilated at a PIP of 15 cmH₂O for 2 or 4 h at 120 breaths/min with a tidal volume of 0.29 ml. A low-PIP ventilation group was not done for the CCSP^–/– mice because sufficient mice were not available. The control groups of wild-type and CCSP^–/– mice were intubated and ventilated for a brief period with a PIP of 15 cmH₂O at 120 breaths/min to allow blood sampling. The low ventilatory rate in the high-PIP groups was necessary to maintain pH and blood gases comparable to other groups. A delivered tidal volume of 1 ml was calculated from the airway pressures and lung compliances for the high-PIP groups (35). Tidal volume was adjusted as necessary to maintain PIP constant throughout the experiment. Blood was sampled by cardiac puncture for gas analysis after ventilation.

BAL

The right lung was lavaged four times with 0.5 ml of phosphate buffered saline. Total cell counts were measured using a hematocytometer, and albumin concentrations were measured with an ELIZA (Bethyl, Montgomery, TX). Total protein was measured using a modified Bradford assay. Differential cell counts were performed using Wright’s stain.

Myeloperioxidase Activity

After BAL collection, the right middle lung lobe was removed and homogenized in 0.5% hexadecyl trimethylammonium bromide in phosphate buffered saline. Samples were centrifuged, and supernatant was reacted with tetramethylbenzidine (1.6 mM) and 0.1 mM H₂O₂ for 6 min. Myeloperioxidase (MPO) activity was expressed as a change in absorbency at 450 nm.

W/D Ratios

The left lung was weighed for wet weight and desiccated at 80°C for 1 wk to obtain the dry weight for the W/D ratio. The W/D ratios in the 4-h groups were corrected for the dry weight of alveolar total protein by assuming the same protein leak in both lungs such that

where W and D are wet and dry weights, respectively, of the left lung and V_BAL and C_TP are volume and total protein concentrations, respectively, of the BAL fluid.

Immunoblotting

The right lower lung lobe was frozen in liquid nitrogen. The lungs were homogenized in buffer and centrifuged at 100,000 g for 1 h. The cytosolic supernatant was separated, and the membrane pellet was resuspended in buffer. Fractions were mixed with Laemmli buffer, and equal amounts of proteins (50 µg) were subjected to 7.5% SDS-PAGE. Protein was transferred to nitrocellulose membranes and probed with anti-cPLA₂ antibody and anti-phospho cPLA₂ (Ser505) antibody (Cell Signaling, Beverly, MA). Bound antibody was detected by a chemiluminescent detection system kit (Cell Signaling), quantified by using a computer scanner, and density calculated using Sigmagel software.

PLA₂ Activity

Activity of calcium-dependent cPLA₂ was determined in lung homogenates using a cPLA₂ assay kit obtained from Cayman Chemical (Ann Arbor, MI). Reaction of cPLA₂ with arachidonyl thio-phosphorylcholine was indicated using Ellman’s reagent, and the color was measured using a plate reader at 414 nm.

Statistics

All values are expressed as means ± SE. Comparison between groups was performed using a two-way ANOVA following Fishers least significant difference analysis. Significant differences were determined where P < 0.05.

	RESULTS

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Tidal Volume

Tidal volume per gram of body weight required to produce an initial PIP of 50 cmH₂O was not significantly different between the two genotypes (wild type: 0.090 ± 0.002 ml/g; CCSP^–/–: 0.092 ± 0.002 ml/g), indicating similar lung mechanics between groups. Over a 2-h period of high-PIP ventilation at a fixed tidal volume, PIP decreased by 3.7 ± 0.2 cmH₂O in wild-type mice and by 3.8 ± 0.3 cmH₂O in CCSP^–/– mice, suggesting a small increase in compliance during prolonged overdistention. Terminal arterial blood gases are summarized in Table 1. Because only one sample could be analyzed after the experimental period, it was only possible to correct ventilation parameters for the next experiment and not during an experiment. These blood gases suggest a moderate amount of hyperventilation in all mechanically ventilated groups. The high PO₂ values obtained after lung injury are surprising but may relate to supplemental oxygen administered during experiments, where indicated, and possibly to gas exchange during the open-chest cardiac-puncture procedure.

Two-hour ventilation studies.   Albumin concentration in lavage was used as an indicator of increased vascular permeability after lung injury induced by ventilation (Fig. 1). No significant difference was observed between the lavage albumin concentrations of wild-type and CCSP^–/– control mice. High-PIP ventilation induced a significant 4.2-fold increase in lavage albumin concentration in wild-type mice and a significant 6.7-fold increase in CCSP^–/– mice. The lavage albumin concentration was significantly higher in CCSP^–/– mice ventilated with high-PIP compared with wild-type mice.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Relationship of lavage albumin concentrations in wild-type and Clara cell secretory protein-null (CCSP–/–) mice in the low-peak inflation pressure (PIP) group ventilated with a PIP of 15 cmH2O for 2 h, unventilated control groups, and high-PIP groups ventilated with a PIP of 50 cmH2O for 2 h. *P < 0.05 vs. control and low-PIP groups. #P < 0.05 vs. wild-type high-PIP group.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Relationship of lavage albumin (A) and total protein (B) concentrations in wild-type and CCSP–/– mice in the low-PIP group ventilated with a PIP of 15 cmH2O for 4 h and high-PIP groups ventilated with a PIP of 50 cmH2O for 4 h with and without pretreatment with the PLA2 inhibitor arachidonyl trifluoromethyl ketone (ATK; 40 mg/kg). *P < 0.05 vs. 4-h low-PIP group. #P < 0.05 vs. vehicle-treated high-PIP group of same genotype. P < 0.05 vs. untreated high-PIP wild-type group.

W/D Ratios

Two-hour ventilation studies.   W/D ratio was used as an indicator of edema formation induced by ventilation (Fig. 3). No significant difference was observed in the W/D ratios from wild-type control and CCSP^–/– control mice. High-PIP ventilation for 2 h induced significant increases in the W/D ratio to 18% in wild-type mice and 39% in CCSP^–/– mice compared with control values. W/D ratios in CCSP^–/– mice were significantly greater by 16% than those of wild-type mice.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Relationship of wet-to-dry lung weight ratios in wild-type and CCSP–/– mice in unventilated control groups and high-PIP groups ventilated with a PIP of 50 cmH2O for 2 h. *P < 0.05 vs. unventilated control group. #P < 0.05 vs. wild-type high-PIP group.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Relationship of lung wet-to-dry weight ratios in wild-type and CCSP–/– mice in the low-PIP group ventilated with a PIP of 15 cmH2O for 4 h and high-PIP groups ventilated with a PIP of 50 cmH2O for 4 h with and without pretreatment with ATK (40 mg/kg). *P < 0.05 vs. 4-h low-PIP group. #P < 0.05 vs. vehicle-treated high-PIP group of same genotype. P < 0.05 vs. high-PIP wild-type group treated with ATK. P < 0.05 high-PIP CCSP–/– vs. high-PIP wild type.

Total cell count and MPO activity for groups ventilated for 2 and 4 h are summarized in Table 2. There were no differences in lavage total cell count between wild-type and CCSP^–/– control mice. The cell counts in all 2- and 4-h high-PIP ventilation groups were significantly reduced relative to unventilated controls, but the 2- and 4-h low-PIP groups were not different from controls. There were no statistical differences in lavage total cell counts between the genotypes after ventilation with high PIP for 2 or 4 h or in the leukocyte percentages. Differential counts indicated 68.0 ± 0.6% of macrophages and 32.0 ± 0.6% of lymphocytes in wild-type mice and 67.3 ± 0.8% of macrophages and 32.7 ± 0.8% of lymphocytes in CCSP^–/– mice after 2 h of high-PIP ventilation. Numerous red cells were present in the lavage of the high-PIP groups but were not present in the low-PIP controls after both 2 and 4 h of high-PIP ventilation.

Level of Membrane Localized Phospho-cPLA₂

The levels of total cPLA₂ expression were not significantly different between the control groups of wild-type and CCSP^–/– mice, and 2 h of high-PIP ventilation did not significantly change the total cPLA₂ levels in either group (Fig. 5A) or the ratio of the membrane fraction of cPLA₂ to total cPLA₂ in either phenotype. Protein bands shown are from the same gel exposed for 15 s for total cPLA₂ levels and 60 s for phospho-cPLA₂ levels to obtain densities in an appropriate range for quantitation. In contrast, the mean (n = 4) phospho-cPLA₂ in the membrane fraction (Fig. 5B) and the ratio of phospho-cPLA₂ to total cPLA₂ were significantly increased in wild-type (2.1-fold) and CCSP^–/– mice (2.7-fold) after 2 h of high-PIP ventilation when normalized to wild-type control levels. In addition, the membrane phospho-cPLA₂ after high-PIP ventilation was significantly higher by 36% in lungs in the CCSP^–/– group compared with the wild-type high-PIP group and was increased by 6.1-fold relative to the CCSP^–/– control value.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Relationship of cytosolic PLA2 (cPLA2) and phospho-cPLA2 expression in membrane and cytosol fractions in wild-type and CCSP–/– mice in unventilated control groups and high-PIP groups ventilated with a PIP of 50 cmH2O for 2 h. A: representative immunoblot showing cPLA2 and phopho-cPLA2 bands from 1 gel. B: phospho-cPLA2 in the membrane fraction of lung homogenates normalized for that of wild-type control mice. Immunoblots from 3 independent experiments were averaged to obtain mean density values. *P < 0.05 vs. control. #P < 0.05 vs. wild-type high-PIP group.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Relationship of phospho-cPLA2 expression in membrane fraction in wild-type mice low-PIP and high-PIP groups ventilated for 4 h. A: representative immunoblot showing phopho-cPLA2 bands from lung membrane fractions of lung homogenates from low-PIP and high-PIP groups from the same gel. B: mean values of phospho-cPLA2 in the membrane fraction of lung homogenates from low-PIP and high-PIP groups normalized for that of wild-type control mice. Immunoblots from 3 independent experiments were averaged to obtain mean density values. *P < 0.05 vs. low-PIP group.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Relationship of calcium-dependent cytoplasmic PLA2 activity in wild-type and CCSP–/– mice ventilated for 4 h at low PIP and high PIP with and without treatment with ATK (40 mg/kg). *P < 0.05 vs. 4-h low-PIP group. #P < 0.05 vs. vehicle-treated high-PIP group of same genotype.

	DISCUSSION

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The time course for lung injury and inflammation during VILI has previously been studied in both mice and rats. Yoshikawa et al (39) previously observed time- and pressure-dependent increases in lung transcapillary protein fluxes and edema in intact mice. A size-selective transcapillary exchange of CCSP, albumin, and IgG increased as the PIP and ventilation periods were increased. The present study supports these observations since lavage albumin, total protein, and W/D ratios were greater in the high-PIP groups compared with low-PIP groups and in the 4-h compared with 2-h high-PIP groups for both wild-type and CCSP^–/– mice. Because there were no differences in normalized tidal volumes required to produce a PIP of 50 cmH₂O, it is unlikely that differences in the baseline mechanics of the lungs accounted for the differences between genotypes. In previous studies, no differences in production of surfactant mRNA or surfactant were observed between the lungs of CCSP^–/– and wild-type mice (15, 34).

The relationship of the vascular permeability lesion to the inflammatory response after VILI has generated considerable controversy because both perfused and unperfused isolated lung preparations readily produce inflammatory cytokines such as TNF-, whereas ventilated intact animals have much less of an inflammatory response (7, 36). In intact mice ventilated at high PIP, the increase in lung vascular permeability preceded the appearance of inflammatory cytokines in BAL by over 1 h, and there were no differences in the BAL albumin and W/D ratios between TNF--null mice and wild-type mice (39). The rapid initial permeability response suggests that rapidly formed mediators or rapid signal events, such as response elements of the PLA₂ pathway, may help initiate this acute response. The greater albumin and W/D ratio responses observed in the CCSP^–/– compared with CCSP^+/+ mice suggest that CCSP has a role in partially reducing both the increase in lung permeability and inflammatory responses after mechanical damage in intact animals, as has previously been demonstrated for infectious and chemical lung insults (11, 17, 23).

The ability of CCSP to inhibit PLA₂ activity is the best studied chemical action of CCSP (5, 22). In cultured-cell experiments, CCSP was shown to inhibit secretory PLA₂ by binding calcium (2), a cofactor of secretory PLA₂ activation, or phosphatydilcholine, a substrate of PLA₂. CCSP also lowered the cPLA₂ activity in fibroblasts stimulated by interleukin-1 (21). However, C57BL/6 mice have an inbred deficiency in secretory PLA₂ but not cPLA₂, which can be activated by increased intracellular Ca²⁺ or phosphorylation by activated MAPK (ERK, JNK, p38) (20, 41). Both increased Ca²⁺ and MAPK activation occur in stretched endothelial cells and in endothelial and epithelial cells in intact lungs during distention (3, 28). Likewise, PLA₂ activation has been reported after mechanical stress in endothelial cells in culture (30) and in intact blood-perfused lungs (19). On activation, cPLA₂ translocates to the perinuclear and intercellular junctional membranes (9, 20), but maximal activation requires phosphorylation of cPLA₂ (29).

The evidence for a causative link between CCSP and PLA₂ activity and VILI is as follows. First, BAL albumin and W/D ratios were significantly higher after 2 and 4 h of high-PIP ventilation in CCSP^–/– mice compared with wild-type mice, which is indicative of a greater vascular permeability increase in CCSP^–/– mice. Second, high-PIP ventilation in the CCSP^–/– mice resulted in significantly higher phosphorylation of cPLA₂ after 2 h, and the cPLA₂ activity was also marginally greater in the CCSP^–/– mice (45%; P < 0.056). Third, inhibition of cPLA₂ activity with ATK significantly reduced indexes of injury in both CCSP^–/– and wild-type mice after 4 h of high-PIP ventilation. The reduction in BAL albumin concentration of 92 mg/ml for CCSP^–/– mice vs. 67 mg/ml in wild-type mice and the greater reduction in cPLA₂ activity in CCSP^–/– mice of 50 vs. 28% in wild-type mice suggest that the cPLA₂ activity increase may have contributed more to the injury in CCSP^–/– mice than that of wild-type mice. These effects strongly suggest that CCSP modulates mechanical lung injury primarily via its effect on PLA₂ activity. However, ATK pretreatment reduced lavage albumin and edema by only about one-third in high-PIP ventilation groups, so additional factors besides cPLA₂ products would be expected to contribute to the mechanical injury. In addition, the significantly higher edema formation, which persisted in CCSP^–/– mice after ATK treatment relative to wild-type mice, also suggests that CCSP may have additional modulating effects in addition to inhibition of cPLA₂ activity.

Inhibition of cPLA₂ could attenuate VILI by reducing numerous downstream products. cPLA₂ is a proinflammatory enzyme that hydrolyzes membrane phospholipids to free arachidonic acid, lysophosphatidic acid, and platelet-activating factor (20, 26). Arachidonic acid is further metabolized by cyclo-oxygenase, lipoxygenase, and P450 pathways to inflammatory mediators such as prostaglandins, thromboxanes, leukotrienes, and epoxyeicosatrienoic acids (41). Candidate mediators for the increased lung microvascular permeability are direct actions of platelet-activating factor (10) or lysophosphatidic acid (13), prostaglandin E₂ induction of proteases (33), leukotriene B₄ attraction of neutrophils (38), or P450 metabolites (1). Recently, Alvarez et al. (1) showed a significant permeability increase in isolated rat lungs in response to infusion of the epoxyeicosatrienoic acid P450 derivatives, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid. Because CCSP inhibited arachidonic acid release in an adenocarcinoma cell line (31) and ablation of the cPLA₂ gene in mice inhibited lung injury due to sepsis, zymosan infusion, or acid aspiration (27), one might speculate that the presence of CCSP would alter the inflammatory response to high-PIP ventilation by altering cPLA₂ activity.

Neutrophil accumulation in the lung is one of the pathological features of VILI, and neutrophil accumulation increases progressively with time during high-PIP ventilation (16, 18). High-PIP ventilation increased MPO activity in a time- and pressure-dependent manner in the present study. MPO increased significantly by 75% in both genotypes after 2 h of ventilation compared with unventilated controls and by 35-fold after 4 h of high-PIP ventilation. MPO after 4 h of low-PIP ventilation was also 65% greater than in unventilated controls. Although mean MPO in the CCSP^–/– mice was 17% higher than that of the CCSP^+/+ mice after 2 h of high-PIP ventilation and 35% greater after 4 h, the difference was not significant between the two genotypes. Consequently, a difference in neutrophil accumulation is unlikely to explain the augmented permeability response in the CCSP^–/– mice. However, simple neutrophil numbers may not accurately represent the complex interactions of neutrophils with endothelial cells because neutrophil activation was also increased by mechanical ventilation (24). Activation of neutrophils could be increased by several arachidonic acid derivatives. However, previous studies in isolated perfused rat lungs ventilated with high PIP suggest a permeability response that occurs much more rapidly than that attributed to neutrophil recruitment (39, 41).

Paradoxically, the lavage total cell counts actually decreased significantly, suggesting that the MPO measurements represented marginating or interstitial neutrophils. The decrease in total cell count in lavage may represent the movement of activated macrophages into the interstitial spaces as previously described (16). Quinn et al (32) ventilated rats for 2 h at high tidal volumes (20 ml/kg) and observed significant increases in lung W/D ratios, but lavage macrophage inflammatory protein-2 and neutrophil counts did not change. Lung MPO also was not elevated after 2 h of high-PIP ventilation in their study but increased significantly after an additional 6-h wait. In a previous study using our ventilated mouse preparation, lavage cell counts also were not increased significantly after only 2 h of high-PIP ventilation in the CCSP^+/+ mice (40). The increased MPO suggests that neutrophils may adhere to lung vessels within 2 h of high-PIP ventilation without massive immigration into the alveolar spaces.

	GRANTS

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This study was supported by National Heart, Lung, and Blood Institute Grant P01 HL-66299 and the Center for Lung Biology.

	ACKNOWLEDGMENTS

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The authors thank Anna Penton for technical assistance and Kelli Roberson for secretarial assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. C. Parker, Dept. of Physiology, MSB 3074, Univ. of South Alabama, Mobile, AL 36688 (E-mail: jparker{at}usouthal.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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