Departments of 1Physiology and 2Pathology and 3Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; and 4Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Submitted 21 August 2003 ; accepted in final form 8 November 2003
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ABSTRACT |
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vascular permeability; acute respiratory distress syndrome; bronchoalveolar lavage; pulmonary edema
Although increases in BALF albumin and total protein are used to track progression of lung injury, there is recent interest in using the appearance in plasma of lung specific proteins as markers of lung injury. The smallest of these proteins and, presumably, the most sensitive to injury is Clara cell secretory protein (CCSP), a 16-kDa protein in the epithelial lining fluids of the lung (16). Clara cells are abundant in the epithelium of small conducting airways down to the level of the terminal bronchioles, and CCSP represents 2-3% of the total protein concentration in human BALF (27). Because CCSP is produced in high concentrations only by airway epithelium, CCSP concentration in BALF has been used to assess damage to the respiratory epithelium. The appearance of CCSP in plasma has been used to indicate a loss of air-blood barrier integrity in clinical disease states as well as animal models of lung injury (16). Elevated serum levels of CCSP have been measured in firefighters after smoke inhalation (4) and also in patients with sarcoidosis (18). Broeckaert et al. (5) demonstrated the sensitivity of increased serum levels of CCSP as a marker of increased vascular permeability in studies of cyclists exposed to ozone. There was essentially no safety margin for ozone exposure that did not produce detectable increases in serum CCSP. The appearance of increased amounts of CCSP in the blood of rats and mice has also been used as a sensitive indicator of acute lung injury after exposure to lipopolysaccharide (2), ozone (1, 5), high-volume ventilation (21), and arachis oil and -naphthylthiourea (17). However, the pattern of CCSP fluxes from lung to blood in relation to ventilation times and peak inflation pressures (PIP), as well as larger protein markers of permeability and lung edema, has not been systematically studied.
In the present study, we measured accumulation of three proteins of known hydrodynamic radii in response to ventilation for 2 h with a progressive increase in PIP and as a function of time during ventilation with a PIP observed to produce lung injury. We assessed vascular permeability by measuring changes in CCSP concentration in plasma as well as albumin and IgG in BALF. Because of the differences in molecular radii of CCSP (1.9 nm), albumin (3.6 nm), and IgG (5.6 nm), larger-diameter aqueous pathways would be required for exchange of IgG than albumin and for exchange of albumin than CCSP between blood and alveolar fluid. The clearance of each protein was calculated as an indicator of transvascular flux in each group. We also used least-squares regression analysis after graded injuries with different PIP values and time periods to determine relations between plasma CCSP concentrations, BALF levels of albumin, IgG, and total protein, and lung wet-to-dry weight ratios. Transmission electron microscopy was used to assess structural changes in each group. We observed increases in protein transport related to molecular radii and ultrastructural changes in proportion to PIP levels and longer ventilation times. Increases in plasma CCSP concentration occurred at the lowest PIP and shortest ventilation time, whereas increases in IgG fluxes occurred at the highest PIP and longest ventilation time.
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METHODS |
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Experimental protocol. Mice were ventilated with PIP of 15 cmH2O (n = 3), 35 cmH2O (n = 3), 45 cmH2O (n = 3), and 55 cmH2O (n = 7) with 2.5 cmH2O positive end-expiratory pressure (PEEP) for 2 h. Constant pH and PCO2 values were maintained at the tidal volume required to produce these PIP values by adjustment of the respiratory rates between groups (Table 1). A control group (n = 6) was ventilated only immediately after tracheal intubation for a very brief time during blood sampling. After the ventilation period, blood was sampled by cardiac puncture for gas analysis and measurement of CCSP, IgG, and albumin. Separate groups were ventilated with 55 cmH2O PIP for 30 min (n = 3) and 1 h (n = 3) to establish a time course for injury.
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BAL. After collection of blood samples, the right lung and the lower left lung lobe were lavaged three times with 0.5 ml of phosphate-buffered saline.
Albumin and IgG concentrations in BALF and serum. Albumin and IgG concentrations were measured in BALF and serum using an ELISA kit (Bethyl, Montgomery, TX).
Total protein concentration. Total protein in BALF was assayed using the modified method of Bradford with bovine serum albumin as a standard.
CCSP. CCSP was assayed in BALF, plasma, and lung homogenate. After lavage, the right and left lower lobes were homogenized in 1 ml of RIPA buffer and centrifuged at 4°C to remove debris. CCSP was measured in BALF, plasma, and lung supernatant by an ELISA based on an anti-CCSP antibody previously described (28, 29).
Protein clearances. Changes in protein clearance from plasma to BALF produced by ventilation were calculated for albumin and IgG as follows
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Changes in protein CCSP clearance from airway fluid to plasma produced by ventilation were calculated as follows
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RNase protection assay. Total RNA was isolated from lung homogenate, and an RNase protection assay was performed as previously described (19). S1 nuclease protection probes for mouse CCSP have also been described previously (34). The S1 5' primer has been described elsewhere (30), and the 3' primer was 5'-CTGGAGGCAGCTGGCCCTTG-3'. After amplification, the product was purified using a Majic polymerase purification kit (Promega), and dried gels were read on a PhosphorImager (DuPont, Boston, MA).
Lung wet-to-dry weight ratios. The upper half of the left lung was weighed wet and desiccated at 80°C for 1 wk before the dry weight was measured for the wet-to-dry lung weight ratio.
Electron microscopy. Right upper lung lobes from each experimental group were fixed in 3% glutaraldehyde in cacodylate buffer by immersion. Each lobe was sectioned into five 1-mm3 blocks for light and transmission electron microscopy. The tissue was then rinsed in cacodylate buffer, postfixed for 1 h with 1% osmium tetroxide, dehydrated using a graded alcohol series, and embedded in PolyBed 812 resin (Polysciences, Warrington, PA). Thick sections (1 µm) were cut from each block with glass knives and stained with 1% toluidine blue for light microscopy. Two thin sections (80 nm) were cut from each block with a diamond knife and then stained with uranyl acetate and Reynold's lead citrate. Sections were examined and photographed using a Philips CM 100 transmission electron microscope (FEI, Hillsboro, OR).
Statistics. Values are means ± SE unless otherwise stated. Analysis of the data was performed by one-way ANOVA followed by Student-Newman-Keuls test. A significant difference was determined where P < 0.05.
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RESULTS |
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The relation of BALF protein concentration to edema formation is shown in Fig. 2. BALF albumin, total protein, and IgG concentrations significantly correlated with lung wet-to-dry weight ratios. These correlations suggest that convective coupling of protein transport to an increased fluid filtration across the alveolar-capillary barrier was a mechanism for protein movement into the airway fluid after mechanical injury to the lung.
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Effect of PIP on CCSP in BALF, lung homogenate, and plasma. BALF CCSP concentrations were significantly decreased after ventilation for 2 h with 15 cmH2O PIP, even though concentrations of BALF CCSP at 45 and 55 cmH2O PIP were not significantly different from control values (Fig. 3A). Lung homogenate CCSP content (Fig. 3C) normalized to 1 mg of protein was not significantly different from control after ventilation with 15 and 55 cmH2O PIP. Unfortunately, samples for the measurement of BALF CCSP at 35 cmH2O PIP and lung homogenate samples for the 35 and 55 cmH2O PIP groups were lost. Plasma CCSP concentrations (Fig. 3B) increased in a PIP-dependent manner, with significant increases in plasma CCSP levels after ventilation with 35 cmH2O PIP, the lowest PIP for any of the proteins. There was also a significant correlation of plasma CCSP with PIP (Fig. 3D) for individual data points (r2 = 0.69). In addition, no significant differences were observed for CCSP mRNA in lung homogenate between PIP groups using a nuclease protection assay (data not shown).
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Time course of BALF protein concentrations and edema formation. Figure 4 shows the effects of ventilation with 55 cmH2O PIP for 0.5, 1, and 2 h on BALF albumin, IgG, and total protein concentrations and lung wet-to-dry weight ratios. All variables increased significantly as a function of time during high-PIP ventilation. BALF albumin and total protein concentrations increased significantly compared with control after only 0.5 h of ventilation, whereas BALF IgG concentrations and wet-to-dry lung weight ratios increased significantly only after 2 h of 55 cmH2O PIP.
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Time course of plasma and BALF CCSP during ventilation. Figure 5 shows the time course of CCSP in plasma and BALF in mice after 0, 0.5, 1, and 2 h of ventilation with 55 cmH2O PIP. A significant increase in plasma CCSP levels was observed at 0.5 h, and these levels remained constant for up to 2 h and did not increase progressively with time. BALF CCSP concentrations did not change significantly relative to control over the 2-h ventilation period.
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Correlations of plasma CCSP with BALF proteins and lung edema. When the plasma CCSP measurements from individual experiments were tested as a dependent variable against other indexes of lung injury (Fig. 6), significant correlations were observed with BALF albumin, total protein, and lung wet-to-dry weight ratios, but not with BALF IgG concentrations. The highest correlation was obtained between plasma CCSP and lung wet-to-dry weight ratio (r2 = 0.57), which suggests a graded increase in CCSP with greater degrees of edema and injury.
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Time and PIP dependence of protein clearances. Changes in protein clearance from baseline unventilated control levels as a function time and PIP are shown in Fig. 7. Clearances are shown as percentage of maximum mean values to compare all proteins on the same scale. Maximal clearances calculated were 2.08 ± 0.36, 0.053 ± 0.012, and 0.105 ± 0.008 µl/h for albumin, IgG, and CCSP, respectively. Figure 7A shows protein clearances as a function of PIP after 2 h of ventilation. CCSP increased progressively even at 15 cmH2O PIP to a maximum at 45 cmH2O PIP, whereas clearance was maximal at 55 cmH2O PIP for albumin and IgG. BALF CCSP concentration in mice ventilated at 35 cmH2O PIP was assumed to equal that in mice ventilated at 45 cmH2O PIP for these calculations, because BALF CCSP concentrations did not change markedly between PIP groups. Figure 7B shows the time course of the changes in clearance for the three proteins across the alveolar-capillary barrier in the lung during ventilation with 55 cmH2O PIP. CCSP was cleared at a maximal rate at 0.5 h and then subsequently declined, albumin clearance was near maximal at all time periods, and IgG clearance increased progressively to a maximum at 2 h.
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Ultrastructure of lung injury. Figure 8 shows transmission electron micrographs of lungs from mice ventilated at the different PIP levels and different time periods. In general, the Clara cells did not show obvious damage at any of the pressures or time periods investigated. Vessels 15-30 µm diameter were the structures affected first and the structures exhibiting the most severe injury. Capillaries were affected later and less severely, but in all cases the pattern of injury was heterogeneous, with apparently normal areas, even in the most severely injured group. Injuries consisted of endothelium and epithelium that were lifted to form blebs and basement membrane that was disorganized or pulled apart. Some artifactual spaces were present because of loss of mitochondria and surfactant granules from type II cells during fixation, but these could be separated from blebs by their positions. Obvious intercellular gaps at endothelial cell junctions were rarely observed. Occasional small perivascular edema cuffs were observed after 2 h of ventilation with 15 cmH2O PIP, but no blebs were found; however, the number and size of edema cuffs increased proportionally as PIP was increased. Figure 8A shows a small (
15-µm-diameter) vessel with an edema cuff from the group ventilated at 15 cmH2O PIP. Figure 8B shows a small bronchiole lined with Clara cells adjacent to two alveoli from the group ventilated for 2 h at 35 cmH2O PIP. Note the CCSP-filled inclusions in the Clara cells and the proximity of the Clara cells to alveolar capillaries. Blebs were not apparent in small vessels or capillaries in this group. Figure 8C shows minimal widening of the basement membrane and early bleb formation in a
15-µm-diameter vessel from the group ventilated for 2 h at 45 cmH2O PIP. Occasional bleb formation in small vessels was observed in this group. Figure 8D shows extensive bleb formation and widening of the basement membrane around nearly the total circumference of a small lung vessel from the group ventilated for 2 h at 55 cmH2O PIP. Figure 8E from the same group shows separation of endothelium and epithelium from the basement membrane and apparent separation of basement membrane layers in a higher magnification of a small vessel bleb. Figure 8F shows extensive blebbing and widening of the basement membrane in a capillary from this group. Figure 8G shows the effect of 30 min of ventilation with 55 cmH2O PIP on a small (
15-µm-diameter) vessel. Some regions of blebbing are seen in this vessel. Figure 8H shows endothelial blebbing in a small (
15-µm-diameter) vessel after 1 h of ventilation with 55 cmH2O PIP with relatively little injury to an adjacent capillary.
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DISCUSSION |
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Early studies by Taylor and Gaar (31) reported equivalent pore radii of 0.6-1.0 nm for alveolar epithelium and 4.0-5.8 nm for the capillary endothelial barrier. Later studies of lung epithelium indicated the presence of infrequent larger pores of 3.4-nm radius for passage of macromolecules (3). Although active vesicular transport of protein across epithelium may contribute to transport (20), the size and concentration dependence of endogenous proteins in BALF and the size-selective clearance of solutes from the alveolar spaces reported in most studies suggest that paracellular pathways are the major transport route for proteins entering and leaving the alveolar compartment (16). Because the molecular radius of CCSP is 1.9 nm (19), restriction of this small molecule by the epithelium would be easily lost at an early phase of lung injury. Egan (11) demonstrated a progressive increase in epithelial equivalent pore radius with lung overinflation. An equivalent pore radius of 2.0 nm occurred at 82% of vital capacity, and unrestrictive leaks occurred at 100% vital capacity. Thus CCSP would leak very rapidly as the lung was distended to total lung capacity. This was confirmed by the significant increases in plasma CCSP at the lowest PIP. The sensitivity of CCSP transport to injury was demonstrated by Broeckaert et al. (5), who observed that plasma CCSP increased in mice and humans breathing ozone levels below the permissible air quality standards, which did not cause respiratory distress. In patients with lung failure, plasma CCSP also was strongly inversely correlated to the arterial PO2-to-inspired O2 fraction ratio but less correlated to the histological indexes of severe lung injury, suggesting leak of CCSP before inflammatory processes sufficient to produce significant tissue disruption (9). In the present study, CCSP transport also occurred before microscopic evidence of injury. Because most macromolecules <40 kDa are believed to cross the air-blood barrier mainly by passive diffusion through paracellular pathways (7), the concentration gradient of CCSP between the epithelium lining fluid and blood of
10,000 times provides a large driving force for diffusion (16). Paracellular epithelial pores sufficiently large to allow passage of CCSP (1.6-nm radius) would not allow passage of albumin or IgG, whereas pathways that permitted passage of albumin (3.6-nm radius) would not allow passage of significant amounts of IgG (5.6-nm radius). Thus significant transport of IgG occurred only at the highest PIP (55 cmH2O).
The bidirectional flux of proteins across the air-blood barrier, even in normal lungs, is indicated by the presence of trace amounts of serum albumin and IgG in BALF and of CCSP in plasma (12, 14). Because the concentration of CCSP in plasma reflects a balance between the rate of synthesis and/or CCSP leak from the air spaces and the rate of clearance from the circulation, it can be affected by glomerular filtration rate (9, 15). It is possible that high PIP ventilation might contribute to the increase in plasma CCSP levels by a reduced renal clearance if systemic blood pressure and cardiac output were significantly decreased by an increased intrathoracic pressure. However, a progressive increase in plasma CCSP concentrations with time would be expected with renal failure, and this was not observed over the time course of these experiments, even in mice ventilated with 55 cmH2O PIP.
We can exclude an increased secretion of CCSP into the air space at high PIP ventilation as a probable cause of the increase in plasma CCSP, because BALF CCSP concentrations were significantly lower in mice ventilated with 15 cmH2O PIP than in control mice, and BALF CCSP concentrations returned to control values in groups ventilated using higher PIP values. Because neither CCSP protein nor mRNA levels in lung homogenate changed as PIP increased, it is unlikely that the increase in plasma CCSP concentration is the consequence of increased synthesis of CCSP by the Clara cell. Also, there was no apparent ultrastructural injury to Clara cells in the present study. Lesur et al. (21) reported a decrease in BALF CCSP in rats during high-tidal-volume ventilation proportional to tidal volume that was suggestive of Clara cell injury. The use of 2.5 cmH2O PEEP in our experiments may have minimized the small airway injury and prevented a decrease in BALF CCSP (24).
Increases in plasma CCSP preceded any ultrastructural indication of vessel injury, because no ultrastructural evidence of endothelial and epithelial injury was observed at 15 and 35 cmH2O PIP. Injury became apparent as occasional bleb formation after 2 h of ventilation at 45 cmH2O PIP, and this was accompanied by significant increases in BALF albumin. The ultrastructural evidence of injury was greater after 0.5 h of ventilation at 55 cmH2O PIP than after 2 h at 45 cmH2O PIP, and BALF albumin was significantly increased by about the same amount in both groups. Increases in BALF IgG occurred only after 2 h of ventilation at 55 cmH2O PIP, which was associated with extensive endothelial blebbing of small (15- to 30-µm-diameter) vessels and separation of endothelium and epithelium with widening of basement membranes in the alveolar capillaries. Muscedere et al. (24) observed significant histological evidence of bronchiolar injury in rats ventilated without PEEP, but we did not observe significant small airway injury to the epithelium in our mice ventilated with 2.5 cmH2O PEEP. Although the extent of ultrastructural changes was related to PIP and time of ventilation, the exact pathway for movement of the proteins across the air-blood barrier could not be deduced. No obvious endothelial gaps were observed in the samples examined. The small plasma membrane breaks reported by Fu et al. (13) at high airway pressures were also not observed in our study. However, separation of endothelium from the basement membrane suggests that the cell junctions may be stronger than the focal adhesions binding endothelial cells to the basement membranes.
In summary, VILI in mice produced by high-PIP ventilation increased plasma CCSP levels, BALF albumin, IgG, and total protein, and lung edema formation in a time- and pressure-dependent manner. The smallest protein, CCSP, increased in plasma at the lowest PIP and earliest sampling period; the largest protein, IgG, increased significantly only at the highest PIP and the longest ventilation time of 2 h. The surface area of separated endothelium and epithelium in small vessels and capillaries was related to ventilation time and PIP levels and protein clearances, but plasma CCSP levels increased before any ultrastructural evidence of vascular injury. Therefore, plasma CCSP levels would be useful for early detection of injury and quantification of mild-to-moderate injury, BALF albumin concentration increases would be useful for detection of progressive development of injury, and BALF IgG would increase only during severe injury when obvious histological evidence of tissue destruction is present.
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported by National Heart, Lung, and Blood Institute Grant P01 HL-66299 and the Lung Biology Center.
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FOOTNOTES |
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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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REFERENCES |
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