Although acute lung injury (ALI) is associated with inflammation and surfactant dysfunction, the precise sequence of these changes remains poorly described. We used oleic acid to study the pathogenesis of ALI in spontaneously breathing anesthetized rats. We found that lung pathology can occur far more rapidly than previously appreciated. Lung neutrophils were increased approximately threefold within 5 min, and surfactant composition was dramatically altered within 15 min. Alveolar cholesterol increased by ∼200%, and even though disaturated phospholipids increased by ∼30% over 4 h, the disaturated phospholipid-to-total phospholipid ratio fell. Although the alveolocapillary barrier was profoundly disrupted after just 15 min, with marked elevations in lung fluid (99mTc-labeled diethylenetriamine pentaacetic acid) and 125I-labeled albumin flux, the lung rapidly began to regain its sieving properties. Despite the restoration in lung permeability, the animals remained hypoxic even though minute ventilation was increased approximately twofold and static compliance progressively deteriorated. This study highlights that ALI can set in motion a sequence of events continuing the respiratory failure irrespective of the alveolar surfactant pool size and the status of the alveolocapillary barrier.
- static lung compliance
- alveolocapillary permeability
- respiratory pathophysiology
- alveolar protein
the healthy lung, as the interface between the gaseous environment and the internal milieu, functions primarily to oxygenate blood and clear carbon dioxide. To achieve this, a large surface area interfaces with a complex microcirculatory system across a 0.1- to 0.2-μm barrier. The alveolocapillary barrier is normally remarkably effective at partitioning the plasma from the air spaces. However, given the extraordinary dimensions of the lung, it is hardly surprising that a variety of insults can increase alveolocapillary permeability and result in edema, atelectasis, and hypoxemia, a condition known as acute lung injury (ALI).
ALI is associated with the dysfunction of at least three interrelated physiological systems: the inflammatory response, lung fluid balance, and surfactant. Although numerous animal models have provided important insights into the pathogenesis of ALI, the physiological systems have largely been studied in isolation. Because of deficiencies in our knowledge, therapeutic strategies in ALI patients, including attempts to block the inflammatory cascade and the use of exogenous surfactant replacement therapy to improve gas exchange and survival, have been poorly targeted and largely ineffective. Moreover, although strategies to recruit atelectatic alveoli remain central to ALI patient management, ventilatory support continues to be driven by technological imperatives rather than by physiological understanding. Consequently, severe ALI, termed acute respiratory distress syndrome, continues to be associated with an unacceptably high mortality.
The lung incorporates three extracellular fluid compartments, the vascular fluid, the interstitial fluid, and the epithelial lining fluid (ELF), that are separated by the endothelium and epithelium. We used a triple radiolabeling technique to monitor relative changes in alveolar and interstitial lung fluid, protein flux, and vascular volume in one of the most commonly used rat models, oleic acid (OA)-induced lung injury. In addition, we studied the interrelationships among alveolar cytology, surfactant composition, and cardiorespiratory physiology to better understand the sequence of events in the pathogenesis and pathophysiology of ALI.
Induction of Lung Injury
Male Porton rats (200–330 g) were anesthetized with intraperitoneal methohexital sodium (60 mg/kg; Eli Lilly, Sydney, Australia) and pentobarbital sodium (40 mg/kg; Boehringer Ingelheim, Sydney, Australia) (12). The thorax and abdomen were shaved, and residual hair was removed (Veet hair removal cream, Reckitt and Colman Pharmaceuticals). A caudal vein and artery at the base of the tail were catheterized (12). Lines were fixed in place (Loctite 406 cyanoacrylate ester, Loctite Australia, Sydney, Australia), and heparinized saline (0.15 M, 2 U/ml) was infused (1 ml/h) to maintain the patency of the arterial line. The rats were placed in the prone position and infused via the caudal vein with OA (0.1 ml/kg) over ∼1 min. Additional methohexital sodium (40 mg/kg) and pentobarbital sodium (25 mg/kg) were administered every ∼45 min, providing a constant depth of anesthesia as monitored by the animal's cardiorespiratory and reflex responses. Body temperature was continuously monitored via a rectal probe and maintained at 37°C with a thermostatically controlled plate (12). Each OA-treated animal was matched with a saline control.
t), breathing frequency, and sigh frequency (defined as periodic deep breaths ∼2.5-fold greater than resting Vt) were monitored with purpose-built impedance pneumographs incorporating adhesive electrodes (25-mm, 3M red dot Ag-AgCl, 3M Health Care, London, ON) placed on either side of the chest (12). Arterial blood gases were analyzed with an ABL 5 (Radiometer, Copenhagen, Denmark) blood gas analyzer, and hematocrit was determined with heparinized capillary tubes (Chase Instrument, Norcross, GA).
Preparation and Infusion of Radiolabels
Preparation of radiolabeled red blood cells.
Approximately 1.4 ml of blood were drawn from a donor rat into a syringe containing heparin (5,000 U/ml, 75 μl) and acid citrate-dextrose (12.25 g glucose, 11 g sodium citrate, and 4 g citric acid/500 ml, 150 μl). The blood was centrifuged at ∼3,500g at room temperature (20°C) for 5 min. The plasma was discarded, and the cells were resuspended in 40 μl of phosphate-buffered saline (PBS; 145.3 mM sodium chloride, 7.5 mM disodium hydrogen phosphate, and 2.5 mM sodium dihydrogen phosphate) and 40 μl of acid citrate-dextrose. Sodium chromate (51Cr, 120 μCi) was added, and the cells were incubated at room temperature for 1 h. The labeled cells [51Cr-red blood cells (RBCs)] were pelleted as described above, washed three times in 0.7 ml of PBS, and finally resuspended in PBS to 1 ml.
Preparation of radiolabeled albumin and diethylenetriamine pentaacetic acid.
Human serum albumin (Alb) labeled with 125I (125I-Alb; ICN Biomedicals Australasia, Sydney, Australia) and 99mTc-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA; 1 and 20 μCi, respectively, per 100 g of recipient rat) were diluted in PBS to ∼200 μl.99mTc-DTPA was prepared from SnCl2, and DTPA was a gift from the Department of Nuclear Medicine (Flinders Medical Centre, Adelaide, Australia).
The radiolabeled preparations were infused over ∼5 s via the caudal vein exactly 10 min before the rats were give an overdose of pentobarbital sodium (120 mg/kg iv).
Compartmentalization of Radiolabels
Blood was sampled by puncturing the left ventricle, and the plasma was separated by centrifuging at ∼3,500 g at room temperature for 5 min. At the end of the experiment, the lungs were ventilated with air via a tracheal catheter at 60 cycles/min and a Vt of 10 ml/kg, with 2 cmH2O end-expiratory pressure to prevent the lungs from collapsing, and removed without perfusion. This procedure took <2 min. The right upper lobe was resected for the wet-to-dry weight ratio and radiolabel analysis. A kidney and ∼100-mg sections of adductor magnus muscle, duodenum, diaphragm, and liver were also resected for the same purpose, a procedure taking <1 min. The remaining lung was degassed at 0.5 atm for 60 s and lavaged at 2°C with three separate 32 ml/kg volumes of 0.15 M saline, each volume instilled and withdrawn three times. A 4-ml aliquot was used for radiolabel analysis.
Radiolabels were counted with a Cobra 5003 gamma counter (125I, 15–75 keV; 99mTc, 90–190 keV;51Cr, 240–400 keV; Auto-gamma 5000 series, Packard Instruments, Downers Grove, IL). Because 99mTc interferes with the counting of 125I and 51Cr, the latter two labels were recounted ∼3 days later after the 99mTc had decayed (half-life = ∼6 h). Compartmentalization of the radiolabels in either the lavage aliquot or tissue section is expressed as the percent volume by expressing the counts per minute (cpm) per gram of tissue relative to the counts per minute per milliliter of blood or plasma
Preparation of Alveolar Subfractions
The remaining lavage fluid was centrifuged at 150 g(average) for 5 min at 2°C to afford a cellular pellet. The supernatant was centrifuged at 1,000 g (average) for 25 min at 2°C to afford a pellet rich in tubular myelin [alveolar fraction-1 (Alv-1)] and a second supernatant poor in tubular myelin [alveolar fraction-2 (Alv-2)] (40).
Preparation of Lamellar Body and Microsomal Fractions
The lungs were homogenized, and a total lamellar body fraction was prepared by the method of Duck-Chong (18a) as modified by Power et al. (39). Microsomes were collected at a density between 0.8 and 1.0 M and a total lamellar body fraction between 0.4 and 0.5 M. The total lamellar body fraction was suspended in an equal volume of ice-cold water, and classic-appearing lamellar bodies [lamellar body fraction-A (LB-A)] were pelleted at 2°C by centrifuging at 2,000 g (average) for 30 min. The resulting supernatant was centrifuged at 150,000 g (average) for 1 h to sediment a vesicular fraction [lamellar body fraction-B (LB-B)] (39).
Cell numbers were estimated with a hemacytometer (Improved Neubauer B.S. 748, Weber Scientific International, Teddington, UK), and a differential cell count was performed with staining with Diff-Quik and Papanicolaou stains.
The lipids were extracted with the method of Bligh and Dyer (6a) and total phospholipid (PL) content was determined by measuring the amount of inorganic phosphorus with the method of Bartlett (2a). Disaturated phospholipids (DSPs) were separated with the method of Mason and associates (30a), and the PL content was determined (39,40).
PL classes were separated by HPLC (μPorasil Silica, Waters Millipore, Bedford, MA) and quantified with a mass detector (model 750/14, Applied Chromatography System, Cheshire, UK) coupled to a Delta chromatography data system program (Digital Solutions, Margate, Australia) (16). Sphingosine (Sigma, St. Louis, MO) was included as an internal standard. Neutral lipids were separated by HPLC with a Waters 18C Novapak column, and free cholesterol (Chol) was quantified by its absorption at 210 nm (11, 16).
Surfactant proteins A and B.
To free surfactant protein (SP) A and SP-B from any associated plasma or surfactant components, aliquots were first treated with EDTA, SDS, and Triton X-100 as previously described (15). SP-B was determined with a human-based ELISA inhibition assay (17) similar to the one that Doyle et al. (15) described for rat SP-A. The antibody reacts strongly with rat SP-B (50). All samples were assayed in duplicate at four serial dilutions. Standards, assayed in quadruplicate, were included in each ELISA plate at eight serial dilutions ranging from 7.8 to 1,000 ng/ml (r > 0.99).
Total protein was determined with a modification of the Lowry method (15).
In a separate cohort of matching rats, the heart and lungs were removed without perfusion and placed in a humidified chamber at 37°C, a procedure taking <3 min. The lungs were connected to a syringe and a pressure transducer (Sorenson Trans Pac), and relaxation pressures were recorded during inflation and then deflation in 1-ml steps between 0 and 30 cmH2O (11).
Labeled white blood cells (99mTc-WBCs) were prepared with SnF2 by the Department of Nuclear Medicine (Flinders Medical Centre). Briefly, ∼10 ml of blood were drawn from a donor rat into a syringe containing 0.5 ml of heparinized saline (1,000 U/ml). The blood was incubated for 1 h with 1.5 ml of a99mTc-SnF2 colloid. The blood was centrifuged at ∼3,000 g, and the plasma was discarded. The labeled cells were washed with three times with 1 ml in PBS and resuspended in PBS to 7.1 ml.
In a separate cohort of rats, ∼0.5 ml of 99mTc-WBCs was infused over ∼15 min via the caudal vein, and after 3 h, lung injury was induced by OA infusion as described in Induction of Lung Injury. After 5 min, the lungs were resected and counted, and the leukocyte volume was determined as above.
Control animals were treated in an identical manner except for the induction of lung injury.
Results are expressed as means ± SE. Data were analyzed with nonparametric methods: the Wilcoxon matched-pairs signed rank test or the Mann-Whitney U-test was used for all comparisons. For comparisons with n > 20 preparations, significance with the Mann-Whitney U-test was determined from the normal deviate Z with reference to normal frequency distribution tables. The correlation between variables was determined with the Spearman rank order correlation (r S) test. To allow for variations with anesthesia during the experiment, all findings are discussed relative to the matching control values.
Although there was a transient decrease in Vt at 45 min in both the control and the OA-infused rats, the relative Vt (Fig.1 A) and breathing frequency (Fig. 1 B) were increased throughout the study (allP < 0.01) such that minute ventilation (V˙e) was elevated approximately twofold (i.e., 200% increase relative to the original V˙e) at all time points (all P < 0.001; Fig. 1 C). Although sighing was initially abolished, its frequency began to increase after 45 min, and by 4 h, it was 1.6-fold that of the control value (P < 0.01; Fig. 1 D). OA infusion did not alter HR or mean BP, which were ∼350 beats/min and ∼100 mmHg, respectively.
Although arterial Pco 2, pH, and HCO3 − remained normal throughout the course of the study, arterial Po 2(PaO2) was decreased from ∼80 to ∼55 mmHg by 15 min (all P < 0.001; Fig.2).
Lung fluid volume.
The wet-to-dry weight ratio of the resected lobe was directly related to that of the remaining lung (r S = 0.61;P < 0.001; n = 39 preparations). Similarly, the percentage of 99mTc-DTPA was directly related to the wet-to-dry weight ratio of both the lobe (r S = 0.79; P < 0.001) and the remaining lung (r S = 0.81;P < 0.001).
OA infusion increased the wet-to-dry weight ratio of the lobe approximately twofold by 15 min, 45 min, and 4 h (allP < 0.001; Table 1). Similarly, the percentage of99mTc-DTPA in the lobe was increased ∼2.1-, ∼2.0-, and ∼1.6-fold, respectively, at these times (all P < 0.001). In contrast, the percentage of 99mTc-DTPA in the lavage fluid was increased ∼5.7-, ∼6.1-, and ∼4.1-fold, respectively (all P < 0.001).
Although the percentage of 125I-Alb in the resected lobe was increased ∼1.7- and ∼1.3-fold at 15 and 45 min, respectively (both P < 0.01; Table 1), it was normal by 4 h. On the other hand, the percentage of 125I-Alb in the lavage fluid was increased ∼7.4-, ∼5.4-, and ∼2.5-fold at 15 min, 45 min, and 4 h, respectively (all P < 0.001).
Total alveolar protein.
Total protein in the lavage fluid was elevated ∼5.1-, ∼7.1-, and ∼8.0-fold at 15 min, 45 min, and 4 h, respectively (allP < 0.001).
Although the percentage of 51Cr-RBCs in the resected lobe was normal at 15 min, it was decreased ∼0.5-fold (i.e., 50% relative to the original RBC volume) by 45 min and 4 h (bothP < 0.01; Table 1). Blood hematocrit did not change. Although the absolute percentage of 51Cr-RBCs in the alveoli was extremely small, it was increased ∼21.8-, ∼1.9-, and ∼1.8-fold at 15 min, 45 min, and 4 h, respectively (allP < 0.01).
The number of alveolar macrophages harvested after OA infusion was decreased ∼8.8- and ∼15.4-fold at 15 and 45 min, respectively (both P < 0.001; Fig.3). However, by 4 h, the cell number had returned to ∼0.4-fold of the normal value (P < 0.01).
Leukocyte recruitment as reflected by the percentage of99mTc-WBCs in the resected lung was increased from ∼0.16 to ∼0.42% within 5 min (P < 0.001;n = 5 preparations). Although neutrophils were not prevalent in the lavage fluid at 15 or 45 min, by 4 h they were the predominant cell type (Fig. 3).
The amount of surfactant was normalized to grams of dry lung weight.
Despite ∼1.6-fold (P < 0.01) and ∼1.4-fold (P < 0.05) increases in DSPs at 15 and 45 min, respectively, the DSP-to-PL ratio was not significantly altered (Table3). Although SP-B did not change significantly, SP-A was increased ∼4.0-fold (P < 0.001), ∼2.9-fold (P < 0.01), and ∼2.1-fold (P < 0.001) at 15 min, 45 min, and 4 h, respectively, and Chol was increased ∼1.8-fold (P < 0.001), ∼1.4-fold (P < 0.01), and ∼1.4-fold (P < 0.01), respectively, at these times.
Although DSPs were decreased ∼0.5-fold at 15 min (P< 0.005), the DSP-to-PL ratio was not significantly altered (Table 3). Although SP-A and SP-B did not change, Chol was increased ∼2.0-fold at 45 min (P < 0.01) and 4 h (P< 0.001).
Neither DSPs nor the DSP-to-PL ratio changed significantly (Table 3). However, at 15 min, SP-A and SP-B were increased ∼2.3-fold (P < 0.01) and ∼1.6-fold (P < 0.05), respectively. Chol was increased ∼2.0-fold (P< 0.001), ∼2.8-fold (P < 0.005), and ∼2.4-fold (P < 0.001) at 15 min, 45 min, and 4 h, respectively.
DSPs did not change significantly; however, the DSP-to-PL ratio was decreased ∼0.3-fold (P < 0.005) by 4 h (Table3). Although SP-B did not change significantly, SP-A was decreased ∼0.3-fold at 45 min and 4 h (both P < 0.05). In contrast, Chol was increased ∼1.3-fold (P < 0.05) and 2.2-fold (P < 0.001) at these times.
Despite a 1.5-fold increase in DSPs by 4 h (P < 0.005), the DSP-to-PL ratio was decreased ∼0.3-fold at all times (allP < 0.001; Table 3). Although SP-A and SP-B did not change significantly, Chol was increased ∼2.0-fold (P< 0.001), ∼2.4-fold (P < 0.005), and ∼2.4-fold (P < 0.001) at 15 min, 45 min, and 4 h, respectively.
Although DSPs were decreased ∼0.6- and ∼0.4-fold at 45 min and 4 h, respectively (both P < 0.01), the amount of DSPs per cell in fact increased by ∼2.9- and ∼2.3-fold, respectively (both P < 0.05), at these times because the number of cells harvested by lavage was dramatically reduced (Table3). Neither Chol, SP-A, nor SP-B changed significantly.
Although OA infusion dramatically increased lysophosphatidylcholine (LPC) in all fractions (P < 0.05 in all cases), it caused relatively few other changes in PL composition (Table4). Sphingomyelin tended to increase in all fractions. Microsomal phosphatidylinositol decreased at all time points (all P < 0.005), whereas microsomal phosphatidylserine increased (all P < 0.001).
Static compliance progressively deteriorated over the 4 h. Total lung capacity was also reduced (all P < 0.05), whereas opening pressure increased (all P < 0.001; Fig. 4).
We have found that OA infusion profoundly disrupts both the lung endothelium and epithelium, resulting in acute increases in lung fluid, total alveolar protein, and a marked deterioration in PaO2 despite an approximately twofold increase in V˙e. Although alveolar lung water and Alb flux tended to normalize during the experiment, suggesting that the alveolocapillary membrane rapidly regains its sieving properties, total alveolar protein remained elevated and lung compliance progressively deteriorated. Paradoxically, the total alveolar DSP pool increased. However, OA infusion acutely altered the composition of alveolar and subcellular surfactant fractions, dramatically increasing the levels of Chol and the alveolotoxin LPC while markedly reducing the DSP-to-PL ratio. Our findings illustrate that ALI can set in motion a sequence of events that worsen the respiratory failure irrespective of the surfactant pool size and the integrity of the alveolocapillary barrier per se.
Although OA-induced ALI was originally adopted as a model of fat embolism, long-bone fractures are primarily associated with the release of triolein rather than of OA (44). Moreover, although OA causes profound lung injury, its esterified form, triolein, does not. In fact, blood contains very little free fatty acid. Rather than forming pathological emboli, OA appears to act by binding to the endothelium and creating a lipid peroxidation chain reaction within the membrane. In addition, OA induces thromboxane and prostaglandin release, causing secondary pulmonary hypertension. The aim of this study was not to determine whether OA infusion models fat embolism syndrome but rather to use the model to study the early response of the lung to a profound increase in alveolocapillary permeability.
The permeability of the normal alveolocapillary barrier and the location and nature of the leakage sites remain polemic. Although the tight junctions of the endothelium (effective radii of ∼6.5–7.5 nm), but not those of the alveolar epithelium (effective radii of ∼0.5–0.9 nm), are thought to be permeable to molecules the size of Alb (atomic radius ∼3.5 nm) (46), a more recent study (9) suggests that the alveolocapillary barrier has functional pores with radii of 1, 40, and 400 nm, which account for 68, 30, and 2%, respectively, of total fluid flux into the alveoli. Indeed, empirical evidence indicates that the lung epithelial barrier only effectively restricts the passage of species > 200 kDa (5). Consequently, a gradation in protein concentration and composition is formed, going from the plasma to the interstitium and to the ELF (18).
Consistent with this, in our control rats, the intravenous125I-Alb took several hours to reach a steady state, whereas 99mTc-DTPA equilibrated in all compartments within minutes (data not shown). Thus whereas the percentage of99mTc-DTPA reflects the relative fluid volume and the percentage of 51Cr-RBCs reflects that of the vasculature, the percentage of 125I-Alb reflects the amount of Alb flux in 10 min. Because plasma proteins reach the alveoli by both convection and passive diffusion (18), this will depend on the concentration gradient, the perfusion pressure, and the permeability of the membrane (18).
We measured the labels in lavage fluid and in a resected lobe. Because the wet-to-dry weight ratio of the resected lobe was similar and directly related to that of the remaining lung, we assume that the OA infusion caused a relatively homogeneous injury, consistent with the work of others (44). If we accept that the lung endothelium is considerably more permeable than the epithelium and that the interstitial fluid compartment is normally considerably larger than that of the alveolus, then the radiolabel changes in the lavage fluid must reflect relative epithelial flux, whereas those in the resected lobe must primarily reflect that in the endothelium. Unlike some other ALI models that are associated with systemic damage, OA infusion did not alter compartmentalization in other tissues during the study.
Consistent with latent increases in thromboxane- and prostaglandin-induced pulmonary venous constriction (44), vascular volume was not reduced until 45 min. Because the percentage of51Cr-RBCs remained unaffected in other tissue beds and because the HR and BP remained constant, the decreased pulmonary vascular volume was probably associated with increased pulmonary capillary pressure (PCP).
OA infusion induced an acute increase in lung fluid as reflected in increases in both the wet-to-dry weight ratio and the percentage of99mTc-DTPA in the resected lobe. Because vascular volume did not change until 45 min, we believe that this initial increase in lung water reflects an increase in permeability rather than an increase in PCP. We have no direct measure of the distribution of lung fluid between the alveoli and the interstitium. However, because the relative change in the percentage of 99mTc-DTPA in the lavage fluid exceeded that in the resected lobe, OA infusion must have induced a proportionally greater change in the permeability of the epithelium than that of the endothelium, consistent with the former normally being the major barrier to the movement of fluid and solutes across the membrane (21).
The relative percentage of 99mTc-DTPA in the lavage fluid 4 h after OA infusion was less than that found after 15 min. Because the percentage of 99mTc-DTPA and the wet-to-dry weight ratio remained elevated in the resected lobe, this must reflect an increase in alveolar water clearance and possible resolution of the alveolar epithelial permeability. A recent study (31) has shown that the active transport of sodium by the type II cell provides the major driving force for the secondary removal of alveolar fluid. Sodium uptake occurs on the apical surface through amiloride-sensitive channels in response to active pumping into the interstitium by Na+-K+-ATPase in the basolateral membrane. How these pumps are regulated remains unknown; however, catecholamines, growth factors, and proinflammatory cytokines, including tumor necrosis factor-α, all increase sodium uptake and alveolar fluid clearance (31). The sodium pumps are remarkably resilient to injury (32). Consequently, the extent of edema in ALI depends on the balance between restoration of the tight junctions and maintenance of the fluid clearance mechanism.
The rate of Alb flux into the alveoli (1–3%/min) in our control rats was similar to that reported by others (21). Consistent with an acute increase in alveolocapillary permeability and a rapid influx of proteinaceous edema fluid, OA infusion induced an acute increase in 125I-Alb flux and total protein in the lavage fluid. Although we have no direct measure of the distribution of the 125I-Alb between the alveoli and the interstitium, the relative change in the percentage of 125I-Alb in the lavage fluid greatly exceeded that in the resected lobe, again emphasizing the importance of the epithelium in partitioning water and solutes (21).
Importantly, the acute increase in alveolar 125I-Alb flux markedly diminished during the experiment. Because the relative percentage of 99mTc-DTPA in the lavage fluid also diminished, whereas total protein did not, it is possible that rising alveolar plasma protein levels may have lessened the concentration gradient, favoring 125I-Alb influx. However, whereas the relative percentage of 99mTc-DTPA at 4 h was only ∼0.3-fold less than that at 15 min, 125I-Alb flux was reduced ∼4.2-fold over the same period. Because PCP probably increased during this period, we believe that restoration of alveolocapillary permeability rather than changes in either the colloid osmotic or hemodynamic forces is the most likely explanation.
It is often asserted that inflammation is not initially involved in OA-induced ALI because neutrophils only appear in the alveoli after several hours (44). However, the alveoli are not the initial site of injury. Although neutrophils were only recruited into the alveoli after 4 h, OA infusion rapidly decreased the number of alveolar macrophages. In addition, the number of leukocytes in the lung increased approximately threefold after just 5 min. We do not know whether the cells were activated; however, taken together, these changes may reflect early recruitment of inflammatory cells to the endothelium, the site of initial injury.
We found severe hypoxia by 15 min. Because V˙ewas increased approximately twofold, the deterioration in gas exchange must reflect increased ventilation-perfusion ratio mismatch due to either shunting, with collapse of some alveoli and overinflation of others, or possibly increased physiological dead space due to alterations in regional perfusion and lung fluid. Certainly, in most of the OA-treated animals, the distal air spaces appeared translucent, and we presume that the alveoli were either collapsed or completely fluid filled.
Although the control rats sighed approximately once every 3 min, sighing was initially inhibited, only returning to normal frequency after 2 h before increasing ∼60% by 4 h. The reflex for sighs is thought to be initiated in the lungs and involves the vagus nerve and muscarinic cholinergic receptors localized at the lower levels of the brain stem (47). Nicholas et al. (35) have previously shown that sighing increases surfactant turnover and improves lung compliance. Possibly the increase in its frequency during the latter part of the study may be a response to the deteriorating surfactant function and lung compliance.
Changes in Surfactant Composition
Microsomes represent a reproducible preparative artifact arising from shearing the endoplasmic reticulum into vesicles and, as such, include organelles involved in surfactant synthesis and recycling. Even though our microsomal fraction was derived from all lung cells, alveolar type II cells have a particularly high metabolic activity, and Barr et al. (2), Power et al. (40), and others (8) have shown that the microsomal content reflects alveolar type II cell surfactant synthesis and is characteristically high in DSPs.
OA infusion acutely, but differentially, increased microsomal SP-A, DSPs, and Chol. It is unlikely that this reflects increased recovery of the fraction because SP-B was not increased. Moreover, the levels were not altered when isolated ventilated lungs were perfused for 15 min with medium containing an equivalent amount of OA or when OA was added to lung homogenate (data not shown). Therefore, we presume that our findings reflect an initial acute increase in the synthesis of these components. Certainly, surfactant synthesis can occur extremely rapidly (1, 24). Because LB-A and alveolar SP-A did not change, possibly the transient increase in microsomal SP-A was associated with increased intracellular trafficking and sorting of PLs (7).
Lamellar body fractions.
Although lamellar bodies are generally regarded as the intracellular storage organelles for surfactant, those isolated by the traditional method of Duck-Chong (18a) can be subfractionated into classic-appearing lamellar bodies (LB-A) and a vesicular subfraction (LB-B) that may be a hybrid of releasable surfactant and surfactant destined for recycling (39).
In view of the increased microsomal DSP pool, it seems unlikely that the reduced LB-A DSPs reflect decreased LB-A formation. It also seems unlikely that OA infusion enhanced intracellular LB-A degradation or diminished recovery because LB-A recovery was not altered when isolated ventilated lungs were perfused for 15 min with medium containing an equivalent amount of OA or when OA was added to the lung homogenate (data not shown). Therefore, the rapid fall in LB-A DSPs probably reflects an acute increase in surfactant release.
Although OA infusion initially decreased LB-A DSPs, Chol progressively increased. Although alveolar Chol is purported to be derived primarily from serum lipoproteins via lamellar bodies (22), Davidson et al. (11) have recently reported that lamellar bodies, in fact, contain little Chol, and it is largely limited to the outer membrane (Orgeig S, Barr H, and Nicholas TE, unpublished data). Our present study describes large pools of microsomal Chol, particularly after OA infusion, consistent with the notion that lung de novo synthesis is an important source of alveolar surfactant Chol. Moreover, the microsomal and alveolar Chol-to-DSP ratios were appreciably greater than those in either LB-A or LB-B, supporting the concept that Chol and DSPs are differentially handled (27).
It is now clear that the majority of newly synthesized SP-A is secreted independent of lamellar bodies (15, 24, 38), and the same may also be true of SP-B (49). Interestingly, SP-A and SP-B were increased in LB-B after OA infusion, further supporting an intracellular role for this fraction in surfactant homeostasis because the protein levels were constant in the alveoli.
We have also monitored surfactant composition in two fractions harvested from the alveoli: Alv-1, an aggregated form rich in tubular myelin, and Alv-2, which is poor in tubular myelin (40). Our interest in these fractions is based on the apparent precursor-product relationships between lamellar bodies and these two alveolar subfractions (27, 36, 40).
Nicholas et al. (35) have previously suggested that Alv-1 might contain the controlled variable in surfactant homeostasis, that is, an intermediate surfactant pool that reflects a balance between supply from the type II cell and the physiological requirements of the gas-liquid interface. If so, and if the rapid fall in LB-A 15 min after OA infusion reflects acute surfactant release, then the constant amount of Alv-1 DSPs must reflect accelerated turnover of this fraction and the establishment of a new steady state comprising increased surfactant turnover. Indeed, we have observed greatly increased turnover of radiolabeled DSPs in other ALI models (data not shown). The progressive increase in Alv-2 DSPs could then reflect increased conversion of precursor fractions and/or decreased reuptake.
Although Jones et al. (27) have previously shown that the DSP specific activity time curves for Alv-1 and Alv-2 support a precursor-product relationship between lamellar bodies and these compartments, those for Chol do not (27). Moreover, Chol has a considerably longer alveolar half-life than DSPs (27). Therefore, the marked increase in alveolar Chol after OA infusion could reflect increased trafficking or decreased reuptake independent of the kinetics of the DSPs. Indeed, although microsomal and Alv-2 Chol were increased approximately twofold within 15 min, similar increases in LB-A, LB-B, and Alv-1 were not observed until 4 h. This acute change strongly suggests that Alv-2 Chol is largely derived independent of lamellar bodies.
In contrast to the traditional concept of surfactant trafficking from lamellar bodies to the alveoli, it has become increasingly apparent that other pathways exist. Not only is newly synthesized SP-A secreted largely independent of lamellar bodies (15, 24, 38), but Nicholas et al. (36) have shown in radiolabeled isolated perfused lungs ventilated with a high Vt that Alv-2 PL specific activity increases, with little change in that of Alv-1 and no change in that of lamellar bodies. This indicates either that Alv-2 can be supplied by a pool of surfactant independent of lamellar bodies or that some lamellar body PL converts directly to Alv-2. In the present study, numerous inverse relationships emerged between surfactant components, such as that between the amount of SP-B in Alv-1 and the amount of DSPs in LB-A (P < 0.001;r S = −0.556; n = 41 preparations). These relationships can only be explained by differential handling of the surfactant components and could include differences in the synthesis, packaging, secretion, extracellular processing, or recycling of the lipids and protein.
Proportion of DSPs.
The DSP-to-PL ratio in Alv-1 gradually decreased ∼20% during the study. Because DSPs remained constant, this must reflect an increase in the unsaturated phospholipids (USPs). Moreover, even though OA infusion increased Alv-2 DSPs by ∼45% during the experiment, the DSP-to-PL ratio was reduced by ∼33% after just 15 min, reflecting a large and acute increase in the proportion of USPs. Although an increase in cellular debris as a consequence of lung injury would seem a likely source for the increased USPs, such an increase would be expected to be most prevalent in the dense Alv-1 fraction rather than in the lighter Alv-2 fraction.
There are a number of potential sources of the additional USPs. Adult type II cells can synthesize fatty acids de novo at high rates (3). The unsaturated species, palmitoleic, oleic, and linoleic acids, comprise a large (∼60%) proportion of fatty acids in blood and can be obtained by lung lipoprotein lipase-induced degradation of very low density lipoprotein (30), chylomicron (10), triacylglyceride, or lesser amounts as free fatty acids. Fatty acids can also be obtained via hydrolysis of PLs after their uptake from the alveoli (3). Den Breejen et al. (13) estimate that only ∼45% of surfactant dipalmitoylphosphatidylcholine (DPPC) results from de novo synthesis and that the remainder arises from deacylation-reacylation remodeling of USPs. However, all of these sources seem unlikely because surfactant PL synthesis is largely microsomal (4), and our microsomal, LB-A, and LB-B DSP-to-PL ratios remained constant.
Recently, Jiang et al. (25) showed that the lung is the major tissue expressing plasma phospholipid transfer protein (PLTP), a molecule that transfers PLs between lipoprotein particles. High-density lipoprotein (HDL) contains ∼50% of the plasma PLs, ∼70% of which is unsaturated phosphatidylcholine (PC) (14). Because PLTP increases the turnover of [3H]PC ether-labeled HDL in the lung (41) and hypoxia acutely increases PLTP mRNA and PLTP activity (26), ALI may facilitate the trafficking of HDL from the interstitium to the alveoli (26). However, because HDL particles are only ∼10 nm in diameter, it is more likely that the increased alveolar USPs reflect OA-induced high-permeability edema. Plasma contains ∼2 g/l of PL, only ∼2% of which is disaturated (43). If we assume that the volume of the ELF in the rat is proportional to that in humans (18) and that the ∼5.5-fold increase in alveolar water and total protein is due solely to the influx of plasma, then the influx of plasma USPs could account for an increase of ∼480 μg USP/rat, which is essentially all of the alveolar increase in the OA-treated animals.
Although USPs may compromise the ability of surfactant to lower surface tension and control fluid balance, they also lower the phase transition temperature of DPPC and enhance the surface adsorption (48), changes that may be beneficial in view of the increased respiratory rate in the OA-treated animals. Finally, USPs also inhibit superoxide production by lung neutrophils, possibly protecting the lung against the oxidative burst during inflammation (51).
Although surfactant is released in response to a number of physiological stimuli including circulating agonists and physical distortion of the type II cell (34), nothing is known about the response of the surfactant system to these stimuli in ALI. Swimming-induced hyperpnea increases alveolar DSPs to an extent similar to that induced by OA infusion. However, although such swimming increases Vt by ∼300%, alveolar levels are normally proportional to Vt (34), and OA infusion increased Vt by a maximum of only ∼35%. And although the sigh frequency increased 2 h after OA infusion, it was all but abolished before this. Although there would almost certainly have been a strong stress-induced β-adrenoagonist stimulus, at least in normal lungs, secretagogues alone are unable to mimic the pattern of release in vivo (34).
Because lung compliance is largely dependent on the surface tension at the gas-liquid interface, then the progressive deterioration in static lung compliance and the increase in opening pressure would suggest that the minimum surface tension must have increased in the OA-treated rats. Consistent with the deterioration in static lung compliance, total lung capacity was greatly reduced in these animals. Taskar et al. (45) recently showed that surfactant dysfunction makes the lung vulnerable to the shear forces associated with breathing either through excessive stretch of the nondependent lung (overinflation) or by shearing the collapsed alveoli through their repeated opening and closing during tidal breaths. Certainly, the appearance of the lungs was consistent with this.
LPC is a detergent-like molecule that in the alveoli directly interferes with the surfactant monolayer, dramatically increasing surface tension (23). Even low amounts potentiate the inhibition of surfactant by plasma proteins as well as being toxic to the epithelium (23). Although surfactant normally contains only trace amounts of LPC, alveolar LPC is greatly elevated in patients with ALI (6). Moreover, the ratios of DSP to LPC and PC to LPC are directly related to the ratio of PaO2 to inspired O2 fraction and indirectly to lung mechanics (6).
Phospholipase A2 (PLA2) is a family of enzymes that hydrolyzes the fatty acyl group of PL in the sn-2 position to form lyso-PL. They can be divided into two classes: secretory (Ca2+ dependent) and cytoplasmic. The secretory forms can be further divided into two distinct subclasses: types I and II. All three forms have been identified in the lung (33). Lung secretory PLA2 has been localized to the microsomal fraction (20), and a novel Ca2+-independent form has recently been characterized in lamellar bodies (37). PLA2 activity is also present in macrophages, fibroblasts, and endothelial cells where it plays a role in the conversion of arachidonate to prostanoids and leukotrienes and may act as a secretory enzyme during septic shock, increasing lung permeability (19, 29). Although PLA2 is secreted by some types of bacteria and plasma levels correlate well with lung dysfunction in patients with gram-negative septic shock, LPC is elevated in patients with ALI irrespective of the nature of the initiating insult (23). Kim et al. (28) failed to demonstrate an increase in PLA2 activity specific for PC. Consequently, the source of alveolar LPC in ALI is unknown.
In the present study, we found a marked increase in the amount of LPC in all fractions. Intracellular PLA2 is essential for the reutilization of surfactant, degrading PC to generate LPC that is then acylated by microsomal LPC acyltransferase to DPPC (3). Only the intracellular forms exhibit PL substrate specificity for the fatty acid moiety (28). The highly toxic LPC is rapidly recycled from the normal alveoli, with a half-life of <10 min compared with ∼85 min for PC (42), although ALI reduces LPC clearance (42). Normally, the activity of LPC acyltransferase in type II cells is much higher than that of the other enzymes involved in recycling and synthesis so that little LPC resides in the cell (3) or lamellar bodies (39). Because LPC was also acutely elevated in microsomal, LB-A, and LB-B, this study illustrates for the first time that, at least in OA-induced ALI, alveolar LPC arises through impaired PC remodeling.
In summary, our findings are consistent with the following scenario: OA infusion acutely damages both the lung endothelium and epithelium, leading to a rapid influx of fluid and plasma proteins into the alveoli and a concomitant deterioration in gas exchange. The increased oncotic pressure generated by the plasma proteins in the alveoli further exacerbates the alveolar flooding. Even though the alveolocapillary barrier rapidly begins to regain its sieving properties, the plasma protein concentration in the ELF rises because plasma proteins are only slowly cleared from the alveoli (half-life < 24 h) in relation to water (minutes to hours). Because the relationship between surfactant inhibition by plasma proteins depends on their relative concentrations, the work of breathing progressively increases. In addition, hypoxemia induces a strong stress-induced β-adrenoagonist stimulus, hyperventilation, and increased sighing, all of which enhance surfactant release. In response to these stimuli, the type II cell develops a hierarchy for survival where surfactant release and Na+ clearance, acting to reduce the work of breathing and maintain lung fluid balance, attain a greater priority at the expense of remodeling PC. Consequently, increased LPC is secreted into the alveoli via lamellar bodies. In addition, direct damage to the type II cells impairs LPC reuptake, leading to a worsening cycle of surfactant dysfunction and respiratory failure.
We gratefully acknowledge Darren Peter (Division of Medical Imaging, Flinders University of South Australia, Bedford Park, Australia) for radiolabeling diethylenetriamine pentaacetic acid and rat leukocytes.
This research was supported by Grants 950054 and 981251 from the National Health and Medical Research Council of Australia and by a grant from the Australian Adult Respiratory Distress Syndrome Association.
Address for reprint requests and other correspondence: I. Doyle, Dept. of Human Physiology, Flinders Univ. of South Australia, Bedford Park, South Australia 5042, Australia (E-mail:).
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.
- Copyright © 2000 the American Physiological Society