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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1516-1525, (2002)
© 2002 American Thoracic Society

Original Article

Endotoxin Induces Respiratory Failure and Increases Surfactant Turnover and Respiration Independent of Alveolocapillary Injury in Rats

Kate G. Davidson, Andrew D. Bersten, Heather A. Barr, Kay D. Dowling, Terence E. Nicholas and Ian R. Doyle

Department of Human Physiology, School of Medicine, Flinders University; Department of Critical Care Medicine and Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park, Australia

Correspondence and requests for reprints should be addressed to Dr. Ian R. Doyle, Department of Human Physiology, Flinders University, Bedford Park, South Australia, Australia 5042. E-mail: ian.doyle{at}flinders.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although endotoxin-induced acute lung injury is associated with inflammation, alveolocapillary injury, surfactant dysfunction, and altered lung mechanics, the precise sequence of these changes is polemic. We have studied the early pathogenesis of acute lung injury in spontaneously breathing anesthetized rats after intravenous infusion of Salmonella abortus equi endotoxin. The animals became hypoxic, and airway resistance, tissue resistance, lung elastance, and static compliance all deteriorated well before any change in alveolar neutrophils, macrophages, lung fluid (99mTc-labeled diethylenetriamine pentaacetic acid), or 125I-albumin flux, which were only appreciably increased at 8.5 hours. Lung elastance deteriorated before airway resistance, indicating that the compliance change was specific rather than caused by reduced lung volume. The subcellular and alveolar content of surfactant proteins A and B, cholesterol, disaturated phospholipids, and phospholipid classes remained normal in the face of a dramatic increase in the synthesis and turnover of 3H-disaturated phosphatidylcholine. Our findings indicate that the increase in surfactant disaturated phospholipid turnover reflects, at least in part, an approximately five-fold increase in "sigh frequency." We suggest that endotoxin has direct effects on tissue resistance and lung elastance independent of surfactant composition and that the initial respiratory failure results primarily from endotoxin-induced ventilation/perfusion mismatch independent of edema or alveolocapillary injury per se.

Key Words: endotoxin-induced acute lung injury • pulmonary surfactant • lung mechanics • alveolocapillary permeability • respiratory pathophysiology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A hallmark of acute lung injury (ALI) is a profound increase in alveolocapillary permeability in response to inflammatory insults mediated either directly, via the airways, or indirectly, via the blood. With the loss of this macromolecular barrier, alveoli are flooded with serum proteins that impair the function of surfactant. This creates hydrostatic forces that further exacerbate the condition, leading to fulminating alveolar edema and a concomitant deterioration in gas exchange and lung compliance.

Not withstanding this well-accepted scenario, the precise sequence of events leading to the initial respiratory failure remains poorly understood. We have recently shown that oleic acid-induced ALI is associated with increasing respiratory dysfunction irrespective of the surfactant pool size or the integrity of the alveolocapillary barrier (1). Similarly, Castiello and colleagues (2) and Fehrenbach and coworkers (3) have shown that endotoxin decreases lung compliance and changes surfactant content and structure without causing edema. Possibly, early respiratory failure in endotoxin-induced ALI can arise by mechanisms independent of alveolocapillary injury. Indeed, there is increasing evidence that the early changes in permeability are dissociated in time from the recruitment of neutrophils into the lungs (4, 5). Consistent with this, leukopenia attenuates, but does not prevent, endotoxin-induced lung injury in rats (6–8). Moreover, endotoxins induce phenotypic changes in intracellular surfactant structures in isolated perfused rat lungs (9).

In order to explore how endotoxin initially causes respiratory failure, we have compared the sequence of changes in alveolar and interstitial lung fluid, protein flux, and vascular volume with alveolar cytology and gas exchange in ALI induced in spontaneously breathing anesthetized rats by the intravenous infusion of Salmonella abortus equi endotoxin. Furthermore, as endotoxin has recently been reported to increase minute ventilation (V·E) in both animals (10) and humans (11), and as we have previously shown that hyperpnea increases surfactant turnover and enhances lung function (12–14), we have now examined whether endotoxin-induced changes in respiration alter surfactant turnover and lung mechanics.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Induction of Lung Injury
Male Sprague-Dawley rats (200–330 g) were anesthetized with intraperitoneal methohexital sodium and pentobarbital sodium (1) and were maintained by continuous arterial infusion of pentobarbital sodium (21 mg/kg/hour) in heparinized saline (2 U/ml) (2 ml/hour). The test rats were infused over approximately 2 minutes with intravenous S. abortus equi endotoxin (3 mg/kg). Each endotoxin-treated animal was matched with a saline control treated in an identical manner except for the induction of lung injury.

Determination of Variables
Blood gases, static lung compliance, alveolar cytology, systemic arterial blood pressure (BP), heart rate (HR), tidal volume (VT), breathing frequency, sigh frequency (deep breaths at least 2.5-fold more than resting VT), and the compartmentalization of 51Cr-sodium-labeled red blood cells (51Cr-RBCs), 125I-labeled human serum albumin (125I-Alb), and 99mTc-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) in lavage and tissue were determined (1). We also measured total phospholipid, disaturated phospholipids (DSPs), the phospholipid species, cholesterol, surfactant proteins A and B, and total protein content in microsomes (Mic), classic-appearing lamellar bodies (Lb-A), a vesicular subfraction (Lb-B), and alveolar fractions rich (Alv-1) and poor (Alv-2) in tubular myelin (1).

Disaturated Phosphatidylcholine Labeling and Lung Mechanics: Ex Vivo Studies
In a separate cohort, intravenous 3H-choline (20 µCi/kg body weight) was infused 3 hours before death. After endotoxin infusion, the animals either spontaneously breathed or were paralyzed with intraperitoneal pancuronium bromide (1 mg/kg) (David Bull Laboratories, Scottsdale, AZ). Paralysis was maintained by continuous arterial infusion of pancuronium bromide (0.12 mg/kg/h), and the animals were ventilated with air at VT = 7 ml/kg body weight with zero end-expired pressure using a constant phase waveform where TI/Ttot = 0.3 (flexiVent; SCIREQ Scientific Respiratory Equipment, Montreal, Quebec, Canada) and breathing frequency was adjusted to maintain normocapnea (15). At 2.5 and 4.5 hours after endotoxin infusion, the lungs were ventilated with 5% CO2/95% O2. The pulmonary artery and left atrium were catheterized, and without interrupting the circulation, the lungs perfused with Krebs bicarbonate solution containing 4.5% bovine serum albumin recirculated through a 50-ml reservoir equilibrated with 5% CO2/95% O2. The lungs were removed, placed in a humidified chamber at 37° C, and ventilated at 60 breaths per minute, VT = 7 ml/kg with zero end-expired pressure.

Lung elastance (Htis), tissue resistance (Gtis), and airway resistance (Raw) were recorded after 30 minutes by measuring the lung's impedance (Z) using the computer-controlled ventilator (16, 17). Impedance was measured during a 16-second of interruption of mechanical ventilation, during which 19 sinewaves with mutually prime frequencies (0.25 to 19.125 Hz) were applied, with a peak-to-peak volume excursion of 1.0 ml above the end-expiratory lung volume. The volume displacement of the ventilator's piston and cylinder pressure signals were low-band pass filtered at 30 Hz and were sampled at 128 Hz before acquisition.

The data were fitted to a constant phase model (16, 18) where Z = Raw + jI + (Gtis-jHtis)/(2{pi}f){alpha}, where I is inertance, j is the imaginary unit, f is frequency, and {alpha} = (2/{pi})arc tan (Htis/Gtis). Inertance was deleted because it was very small and had an insignificant effect on the other parameters.

At the end of an approximately 3-minute period of data acquisition, during which the lung was maintained at 60 breaths per minute, VT = 7 ml/kg body weight with zero end-expired pressure, surfactant was harvested and fractionated, the DSP extracted, and the 3H-disaturated phosphatidylcholine specific activity (3H-DSPC sp act) determined (19).

Statistics
Unless otherwise stated, all findings are described relative to the matching control (1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cardiorespiratory Variables
Endotoxin infusion caused a modest increase in both VT (all p < 0.05) (Figure 1A) and breathing frequency (all p < 0.05) (Figure 1B), with V·E maximally elevated approximately 1.7-fold (that is, 170% relative to the matching control V·E) at 4.5 hours (all p < 0.025) (Figure 1C). Endotoxin dramatically increased sigh frequency approximately 4.9, approximately 5.0, and approximately 5.6-fold at 2.5 hours, 4.5 hours, and 8.5 hours, respectively (all p < 0.0001) (Figure 1D). Whereas HR and mean BP in the control animals were approximately 350 bpm and approximately 100 mm Hg, respectively, endotoxin tended to increase HR (Figure 2A) while decreasing BP (Figure 2B) approximately 1.3-fold (p < 0.05) at 2.5, 4.5 (p < 0.01), and 8.5 hours (p < 0.01).



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  		Figure 1. The effect of endotoxin infusion on respiratory variables. Changes in VT (A), frequency (B), V·E (C), and the frequency of sighing (defined as periodic deep breaths approximately 2.5-fold greater that resting VT) (D) are shown. Except for the sigh frequency, the changes are expressed as the mean ± SE relative to time zero where VT, breathing frequency, and V·E were approximately 7 ml, 60 Hz, and 420 ml, respectively. Open symbols represent animals infused with Salmonella abortus equi, whereas the closed symbols represent matching control subjects. Significance is expressed relative to the matching control (*p at least < 0.05). The number of preparations is as in Table 1.

 

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  		TABLE 1. Changes in lung compartmentalization in response to endotoxin infusion

 


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  		Figure 2. The effect of endotoxin infusion on relative HR (A) and BP (B). Changes are expressed as the mean ± SE relative to time zero. Open symbols represent animals infused with Salmonella abortus equi, whereas the closed symbols represent matching control animals. The details are as in Figure 1.

 
Blood Gases
Although arterial PCO2 and pH did not change, endotoxin progressively decreased both arterial PO2 (4.5 hours, p < 0.025; 8.5 hours, p < 0.0005) (Figure 3) and HCO3- (4.5 hours, p < 0.02; 8.5 hours, p < 0.02) approximately 0.2-fold (that is, 20% less than the matching control arterial PO2 and HCO3-).



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  		Figure 3. The effect of endotoxin infusion on blood oxygenation (A) and blood bicarbonate (B). Open symbols represent animals infused with Salmonella abortus equi, whereas the closed symbols represent matching control animals. Details are as in Figure 1.

 
Pulmonary Compartmentalization
Pulmonary fluid volume
Endotoxin increased the 99mTc-DTPA percentage in the lavage fluid by approximately 1.5 (p < 0.0001) at 4.5 hours and 1.7-fold (p < 0.0025) at 8.5 hours (Table 1). In contrast, the wet/dry weight ratio and 99mTc-DTPA percentage in the resected lobe remained constant.

Pulmonary albumin flux
Whereas the 125I-Alb percentage in the resected lobe and lavage fluid was normal at 2.5 and 4.5 hours, by 8.5 hours, it was increased approximately 1.5-fold (p < 0.02) in the resected lobe and approximately 2.7-fold (p < 0.015) in the lavage fluid.

Total alveolar protein
Total protein in the lavage fluid was elevated approximately 1.3-fold (p < 0.05) at 8.5 hours.

Pulmonary vascular volume
There was no change in the 51Cr-RBC percentage in either the lavage fluid or the resected lobe during the study. Hematocrit also remained constant.

Systemic Compartmentalization
Whereas endotoxin did not alter the wet/dry weight ratio or the compartmentalization of 99mTc-DTPA and 51Cr-RBC in the resected liver, kidney, duodenum, diaphragm, and adduct magnus, by 8.5 hours, the 125I-Alb percentage was increased by approximately 1.7-fold, 2.0-fold, 1.9-fold, 2.5-fold, and 2.4-fold, respectively (all p < 0.0001) (Table 2).


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  		TABLE 2. Rat tissue compartmentalization

 
Cytology
The number of alveolar macrophages harvested after endotoxin was unchanged at 2.5 and 4.5 hours but was increased approximately 1.4-fold (p < 0.05) at 8.5 hours (Figure 4A) . Although the lavage fluid contained few neutrophils at 2.5 or 4.5 hours, by 8.5 hours, they were prevalent in some samples (Figure 4B).



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  		Figure 4. The effect of endotoxin infusion on alveolar differential cell numbers (nos). Changes in alveolar macrophage (A) and alveolar neutrophil (B) numbers are shown x106 cells per lung. Open symbols represent animals infused with Salmonella abortus equi, whereas the closed symbols represent matching control animals. Details are as in Figure 1.

 
Surfactant Analysis
The amount of surfactant was normalized to gram of dry lung weight.

Intracellular surfactant composition
Microsomal composition remained constant throughout the experiment with the exception of surfactant protein A, which was increased approximately 2.1-fold (p < 0.0025) at 2.5 hours, approximately 1.7-fold (p < 0.025) at 4.5 hours, and approximately 1.6-fold (p < 0.025) at 8.5 hours (see Table E1 in the online data supplement). Endotoxin infusion did not significantly alter the relative amounts of constituents in either Lb-A or Lb-B. However, in both the control and test animals, the amounts of cholesterol, DSP, surfactant protein A, and surfactant protein B in Lb-A and Lb-B were slightly decreased at 4.5 hours as compared with 2.5 hours. By 8.5 hours, the amount of these constituents tended to be increased above that at 2.5 hours.

Alveolar surfactant composition
With the exception of Alv-2 DSP, which was decreased approximately 0.5-fold (p < 0.005) at 8.5 hours, the composition of Alv-1 and Alv-2 remained constant throughout the experiment.

Alveolar macrophages
The surfactant composition of the alveolar macrophages also remained constant, both in absolute amounts and when expressed per cell (data not shown).

Phospholipid classes
Endotoxin had no significant effect on phospholipid composition in any of the fractions (see Table E2 in the online data supplement).

Static Compliance
Static compliance progressively deteriorated in the spontaneously breathing animals over the 8.5 hours. Total lung capacity was reduced approximately 0.1-fold (p < 0.025) at 4.5 hours and approximately 0.2-fold (p < 0.0025) at 8.5 hours (Figure 5) .



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  		Figure 5. The effect of endotoxin-infusion on static lung compliance. The lungs were isolated 2.5 (A, n = 6), 4.5 (B, n = 6), and 8.5 hours (C, n = 6) after endotoxin infusion and compliance determined by recording the relaxation pressures during inflation and deflation in 1 ml steps between 0 and 30 cm H2O. Because the animals varied slightly in weight, the lung volumes were corrected by normalizing to body weight. Pressures from the normalized compliance curves were then extrapolated at 1 ml increments and used to establish the mean static lung compliance in each group. Open symbols represent animals infused with Salmonella abortus equi, whereas the closed symbols represent matching control subjects. Remaining details are as in Figure 1.

 
Ex Vivo Studies
3H-DSPC sp act
Endotoxin increased the 3H-DSPC sp act in the spontaneously breathing animals relative to the matching control subjects at 2.5 hours approximately 1.4-fold (p < 0.05) in Mic, approximately 1.2-fold (p < 0.05) in Lb-A, approximately 1.9-fold (p < 0.005) in Alv-1, and approximately 2.2-fold (p < 0.0025) in Alv-2 (Figure 6) . At 4.5 hours, 3H-DSPC sp act was increased in these animals approximately 1.7-fold (p < 0.025) in Mic, approximately 1.4-fold (p < 0.005) in Lb-A, approximately 1.8-fold (p < 0.005) in Alv-1, and approximately 2.2-fold (p < 0.001) in Alv-2.



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  		Figure 6. The effect of endotoxin infusion on 3H-DSPC sp act in Mic (A), Lb-A (B), Lb-B (C), Alv-1 (D), and Alv-2 (E) isolated from spontaneously (top panel ) or ventilated (7 ml/kg VT and zero end expired pressure, bottom panel ) rats 2.5 and 4.5 hours after endotoxin infusion (Endo) and from matching control animals (Con). Significance is expressed relative to the matching control (*ventilated animals, {dagger}spontaneously breathing animals, p at least < 0.05, n = 7).

 
Endotoxin also increased the relative 3H-DSPC sp act in the ventilated animals at 2.5 hours approximately 1.9-fold (p < 0.001) in Alv-1 and approximately 1.7-fold (p < 0.001) in Alv-2. Although endotoxin decreased the relative 3H-DSPC sp act at 4.5 hours by approximately 0.8-fold (p < 0.05) in Alv-1 and approximately 0.7-fold (p < 0.05) in Alv-2, this was not due to a fall in the 3H-DSPC sp act of the endotoxin-treated animals, but rather to an increase in the 3H-DSPC sp act of all fractions (all p < 0.001) harvested from the matching control animals at 4.5 hours. The 3H-DSPC sp act of Lb-B at 4.5 hours was decreased approximately 30% (p < 0.025) in the ventilated animals.

Lung Mechanics
Raw
Whereas endotoxin did not statistically alter Raw in the spontaneously breathing animals at 2.5 hours, it was increased approximately 1.8-fold (p < 0.015) at 4.5 hours (Figure 7A) . In the ventilated animals, endotoxin did not statistically increase Raw relative to the matching control animals. However, ventilation increased Raw relative to the comparable spontaneously breathing animals approximately 2.1-fold (p < 0.001) at 2.5 hours and approximately 1.9-fold (p < 0.001) at 4.5 hours in the control animals, and approximately 2.8-fold (p < 0.001) at 2.5 hours and approximately 1.6 (p < 0.001) at 4.5 hours in the endotoxin-treated animals.



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  		Figure 7. Raw (A), Gtis (B), and Htis (C ) in lungs isolated from spontaneously (bars in background ) or ventilated (bars in foreground ) rats 2.5 and 4.5 hours after endotoxin infusion (Endo) and from matching control subjects (n = 7) (Con). The lung mechanics are shown after 30 minutes of ventilation (7 ml/kg VT and zero end-expired pressure). Significance is expressed relative to the matching control (*p at least < 0.05). Remaining details are as in Figure 6.

 
Gtis
In the spontaneously breathing animals, Gtis was increased approximately 1.4-fold (p < 0.015) at 2.5 hours and approximately 1.6-fold (p < 0.005) at 4.5 hours (Figure 7B). Although endotoxin did not increase Gtis in the ventilated animals relative to the matching control animals, ventilation increased Gtis in the control animals relative to the spontaneously breathing animals approximately 1.6-fold (p < 0.001) at 2.5 hours and approximately 1.5-fold (p < 0.001) at 4.5 hours.

Htis
Similarly, in the spontaneously breathing animals, Htis was increased approximately 1.3-fold (p < 0.05) at 2.5 hours and approximately 1.5-fold (p < 0.005) at 4.5 hours (Figure 7C). Although endotoxin did not increase Htis in the ventilated animals relative to the matching control animals, ventilation increases Htis in the control animals relative to the spontaneously breathing animals approximately 1.5-fold (p < 0.001) at 2.5 hours and approximately 1.4-fold (p < 0.001) at 4.5 hours.

Comparative Analysis
Lungs isolated 8.5 hours after endotoxin infusion often appeared normal, although in some cases, distinct regions of consolidation were evident (see Figure E1 in the online data supplement). In contrast to lungs isolated from rats 4.5 hours after oleic acid infusion (1), there was no overt edema, hemorrhage, or focal petechiae, even though the extent of respiratory dysfunction was similar in both models (1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have found that endotoxin infusion causes a marked early deterioration in arterial PO2 and a progressive deterioration in lung mechanics despite an approximately 1.7-fold increase in V·E and an approximately 5-fold increase in sigh frequency. We confirm the observations of Castiello and colleagues (2) and Fehrenbach and coworkers (3); the lungs did not become edematous, and alveolocapillary albumin flux only increased late in the experiment, with total alveolar protein only marginally increased at this time. Consistent with a delayed inflammatory response, albumin flux in systemic beds was also only increased late in the experiment. Because oxygenation and lung mechanics deteriorated before any increase in permeability or lung water or the appearance of alveolitis, it is likely that endotoxin initially causes ventilation/perfusion (V/Q) mismatch without alveolocapillary injury. Although endotoxin infusion greatly increased phosphatidylcholine synthesis and turnover in spontaneously breathing animals, its effects were variable and time-dependent in animals ventilated with constant resting VT. Our findings illustrate that respiratory dysfunction can precede the inflammation and alveolocapillary damage associated with endotoxin-induced ALI and that endotoxin alters surfactant turnover in a manner that, at least in part, depends on the pattern of respiration.

The Model
Intravenous infusions of endotoxins from gram-negative bacteria have been extensively used to model sepsis, the highest single risk factor for ALI. Although the timing and severity of the pathogenesis varies with the endotoxin and differences in the dosing regimen and animal species, we chose S. abortus equi because it causes reproducible respiratory dysfunction (20), consistent with many of the clinical features of sepsis-induced ALI, including severe hypoxia, pulmonary inflammation, high-permeability edema, and diffuse bilateral infiltrate. The times studied were chosen because they both immediately preceded and followed the respiratory dysfunction, which our preliminary studies showed occurred approximately 2.5 hours after endotoxin infusion. The aim of this study was not to model sepsis per se, but rather to study the pathophysiologic changes associated with the onset of the early respiratory dysfunction.

The Respiratory Failure
Gas exchange
Although arterial PCO2 and pH did not change, endotoxin progressively decreased both arterial PO2 and HCO3- in the spontaneously breathing animals. By 8.5 hours, the A-a gradient was approximately 26 mm Hg as compared with approximately 6 mm Hg in the matching control animals.

Static lung compliance, dynamic Gtis, Htis, and Raw
To standardize the ventilatory history and ameliorate any secondary physiologic effects associated with chest wall mechanics, Raw, Gtis, and Htis were measured in isolated lungs after ventilating with a constant "resting" VT for 30 minutes.

Consistent with the findings of Mora and colleagues (21), prior endotoxin infusion approximately doubled Gtis and Htis in lungs from spontaneously breathing animals. We now report that initially these parameters increase independent of Raw, which only rose after 4.5 hours. This suggests that a reduced parenchymal mass secondary to airway collapse was not the primary cause of the increase in Gtis and Htis at 2.5 hours (16). Rather, it is more likely that alveolar instability led to an increase in Raw secondary to ventilation of a smaller lung volume. In accordance with the increased Htis, endotoxin progressively deteriorated static compliance and total lung capacity.

Raw, Gtis, and Htis were also increased in lungs isolated from control rats paralyzed and ventilated with constant "resting" VT, relative to lungs isolated from the spontaneously breathing control animals. It is well-known that paralysis profoundly changes ventilation and perfusion (22), presumably because of the loss of interdependence associated with diminished respiratory muscle contraction. Indeed, the A-a gradient in the paralyzed control animals was approximately 16 mm Hg immediately before harvesting the lungs.

Potential Mechanisms for the Respiratory Failure
Alveolocapillary injury: pulmonary edema
It is well-accepted that the epithelium, rather than the endothelium, is the major barrier to the movement of water and solutes into the alveolus (23). Consistent with this, we have shown (1) that in control animals, intravenous 99mTc-DTPA equilibrates in all compartments within minutes and thus reflects the relative fluid volumes. In contrast, the 125I-Alb requires several hours to reach a steady state across the endothelium, and for all intents and purposes, does not normally breach the alveolar epithelium, whereas the 51Cr-RBC percentage reflects only the vascular volume. Consequently, under the conditions used, the 125I-Alb percentage reflects the amount of albumin flux in the 10-minute period selected. Because plasma proteins reach the alveolus by both convection and passive diffusion, the flux of albumin across the endothelium and epithelium will depend on its concentration gradient, the perfusion pressure, and the permeability of the membrane (23).

The 51Cr-RBC percentage in the resected lobe remained unaffected, suggesting that pulmonary capillary pressure did not change. Although the direct effects of the endotoxin and the vasoconstrictive effects of impaired oxygenation would be expected to increase pulmonary arterial pressure (24, 25), endotoxin also initially reduces cardiac output by decreasing contractility. Possibly, these opposing influences resulted in no net effect on pulmonary capillary pressure. Endotoxin infusion progressively increased the alveolar 99mTc-DTPA percentage but did not alter the total lung fluid in the whole resected lobe as reflected by both the normal wet/dry weight ratio and the 99mTc-DTPA percentage. We have no direct measure of the distribution of lung fluid between the alveolus and the interstitium. However, if we accept that the interstitial fluid compartment is normally much larger than that of the epithelial lining fluid, which probably comprises no more that a total of 40 µl in the rat lung (26), then the increase in 99mTc-DTPA percentage in the lavage fluid is consistent with increased permeability and the role of the epithelium as the major barrier to the movement of fluid and solutes into the alveolus (23).

Endotoxin infusion induced a late increase in 125I-Alb flux in both the resected lobe and in the lavage fluid. Again, although we have no direct measure of the distribution of the 125I-Alb between the alveolus and the interstitium, the relative change in 125I-Alb percentage in the lavage fluid exceeded that in the whole resected lobe, further emphasizing the importance of the epithelium in restricting water and solutes egress into the alveolus. There is little evidence that active transcytosis is a major pathway for proteins into or out of the alveolus (27). Because neither the wet/dry weight ratio nor the compartmentization of 99mTc-DPTA or 125I-alb changed in the resected lobe, we suggest that the late increase in alveolar albumin flux must be due to increased diffusion rather than increased convection. This, in turn, must reflect an increase in permeability.

Our finding contrasts with that of Wiener-Kronish and associates (28), who concluded that in sheep, the alveolar epithelium is significantly more resistant than the endothelium to the injurious effects of endotoxin. The difference between our findings may be related to the animals studied. Certainly, the structure and strength of the alveolocapillary barrier differs between species (29). However, unlike our technique, that used by Wiener-Kronish and associates dramatically alters the diffusion gradient because it relies on the alveolar instillation of 3 ml/kg serum or 5% albumin ringer's lactate solution.

Whatever the case, although alveolar edema encourages atelectasis, inhibits surfactant function, and increases the blood/gas diffusion barrier, edema was clearly not the initial cause of the respiratory failure in our model.

Surfactant dysfunction: impaired surface tension
We have previously shown that lung mechanics correlate with surfactant composition in patients with ALI (30), and numerous studies have shown altered surfactant composition and function in endotoxin models (2, 3, 21). However, the nature of the changes has been quite variable, again possibly reflecting differences in the dosing regimen, animal species, and the type of endotoxin. We have extensively examined surfactant composition in organelles involved in surfactant synthesis and recycling (Mic), intracellular storage (Lb-A), a hybrid possibly containing releasable surfactant and surfactant destined for recycling (Lb-B), and two fractions harvested from the alveolus. Endotoxin had little discernible effect on surfactant composition.

Our present findings are in marked contrast to our oleic acid-infusion ALI model, where a similar degree of respiratory dysfunction was associated with dramatic changes in 15 minutes in the composition of all five fractions (1). Alveolar cholesterol increased approximately 200% and caused the DSP/total phospholipid ratio to fall by approximately 25%, even though DSP increased by approximately 30% (1). Specifically, whereas we suggested that the increased intracellular and extracellular lysophosphatidylcholine may be a major factor contributing to the reduced lung compliance in that model, lysophosphatidylcholine was normal in this study. The same holds true of surfactant protein B, despite reports by Mora and associates (21) that late changes in endotoxin-induced lung dysfunction correspond closely with impaired surfactant function and reduced surfactant protein B expression. Finally, although total alveolar protein was increased at 8.5 hours, the increase appears insufficient to affect surface activity appreciably (31).

We did not test the surface properties of the surfactant but suggest that increased surface tension is still likely to have contributed to the initial respiratory failure. Indeed, it is difficult to explain the early increase in Htis in its absence. Moreover, endotoxin has been reported to bind to surfactant and increase the minimum surface tension (32) and to induce the secretion of a soluble inhibitor (33).

Cardiorespiratory response: V/Q mismatch
Endotoxin infusion can produce a complex physiologic response that in most species includes airway constriction and a dampened hypoxic response independent of cell-mediated inflammation (34). In addition, endotoxin causes systemic hypotension and pulmonary hypertension secondary to changes in vascular resistance, with little change in either right or left atrial pressure (34). Consistent with this, we found that endotoxin infusion decreased BP but had no effect on hematocrit. It seems likely that these effects, which are probably cyclooxygenase-mediated (35), caused alterations in regional perfusion and increased physiologic dead space, causing an initial V/Q mismatch and hence the severely impaired oxygenation that we observed after 2.5 hours.

Although the effects of endotoxin are exacerbated by neutrophil-mediated cyclooxygenase-2 expression, thromboxane release, and bronchoconstriction (36), endotoxin-induced V/Q mismatch can probably occur independent of recognition by blood leukocytes. Circulating amounts of the phospholipase A2 receptor and its expression on alveolar type II cells are markedly increased by endotoxin (37). Receptor-mediated phospholipase A2-induced release of arachidonic acid from membrane phospholipids may be the limiting step in prostacyclin and other eicosanoid production in macrophages (38), endothelial cells (39), and alveolar type II cells (40). In isolated perfused lungs, endotoxin induces cyclooxygenase-2, the formation of prostaglandins and thromboxane, and the activation of the thromboxane receptor on airway smooth muscle cells, and it enhances vasoconstriction and bronchoconstriction in response to antagonists (41). Endotoxin also constricts terminal bronchioles, increases Raw in isolated perfused lungs (42), and attenuates cell–cell communication between isolated endothelial cells through a signaling pathway that is tyrosine kinase–dependent (43). Finally, endotoxin evokes an immediate, potent, prolonged, but reversible nitric oxide–dependent, prostaglandin-independent vasodilation in blood-free suffused skeletal muscles in vivo (44).

Delayed Inflammation
We can only speculate as to the mechanism responsible for the delayed increase in permeability in our study. Neutrophils were elevated in some animals, but only well after the increase in alveolar 99mTc-DTPA flux. Moreover, whereas the macrophage and neutrophil numbers in the alveolus varied enormously between the different endotoxin-treated animals, possibly reflecting individual variations in inflammatory response, the lung permeability, as reflected by both the 99mTc-DTPA percentage and the 125I-Alb percentage, was uniformly elevated. Possibly, other cell types were involved.

CD14 is an important receptor for endotoxin expressed constitutively by monocytes, macrophages, and neutrophils and possibly also by epithelial (45) and endothelial cells (46). Transfer of the endotoxin–CD14 complex to toll-like receptors engages the toll-like receptor/MD-2 complex and stimulates an intracellular signaling pathway that uses a series of adaptor proteins and serine/threonine kinases that link to the protein kinase nuclear factor-{kappa}B–inducing kinase (47). Nuclear factor-{kappa}B subsequently induces key effector genes, including those for proinflammatory cytokines involved in both innate and adaptive host defense (47). However, we recently showed that endotoxin also stimulates the secretion of a range of cytokines from cultured alveolar type II cells (48), despite recent studies that suggest that lung epithelial cells do not express toll-like receptors (49, 50). Moreover, it has recently been suggested that the increase in microvascular permeability during lung inflammation is due to at least two distinct mechanisms, an early one related to the neutrophil influx and a delayed one occurring even with neutropenia (51). In addition, potential injury can also arise through lymphocyte, macrophage, platelet, complement, arachidonic acid metabolite, protease, and cyclic nucleotide-mediated pathways (35).

Regardless, whether the increased epithelial permeability was due to the direct cytotoxic effects of the endotoxin (52) or as a consequence of neutrophil recruitment and activation at this time, by 8.5 hours, alveolar fluid, as reflected by the 99mTc-DTPA percentage, was increased approximately 1.7-fold. This additional lung fluid would have contributed to V/Q mismatch and the continuing impairment in oxygenation.

Physiologic Compensation
Cardiorespiratory response
Although endotoxin may initially reduce cardiac output by decreasing contractility, an increase is common some hours later (approximately 4.5 hours) where a higher HR compensates for the lower stroke volume (53). Consistent with this, we found that HR increased by approximately 10%.

In our study, V·E increased, although it seems unlikely that the impaired oxygenation was sufficient to stimulate lung vagal C-fiber afferents and peripheral chemoreceptors (10). Preas and coworkers (11) have recently suggested that endotoxin increases respiratory drive directly via a cyclooxygenase-mediated pathway. We now report that endotoxin also dramatically increases the sigh frequency.

Surfactant turnover: the stimulus
Modeling precursor–product relationships between the various surfactant compartments are made difficult by the lack of precise pulse labeling, poor separation of the compartments, mixing, and recycling. Choline is rapidly incorporated into alveolar type II cells from plasma, probably reaching a steady state (54) and used in the synthesis of DSPC, the major component of surfactant. We (19) and others (55), have modified the approach of Zilversmit and associates to generate specific activity time curves consistent with DSP moving from Mics to lamellar bodies to tubular myelin-rich and then to tubular myelin-poor surfactant fractions. In this study, we infused the animals with 3H-choline 3 hours before death, having previously shown that the peak of 3H-DSPC lamellar body labeling occurs at this time, whereas in the alveolus, it occurs after approximately 8 hours (19). Consistent with this, at 3 hours, the 3H-DSPC sp act was higher in Lb-A and Lb-B than in the other fractions. However, the absolute amount of 3H-DSPC sp act in any given fraction will depend not only on the rate of trafficking between the fractions and the time course of the pulse, but also on the pool size and the extent to which the 3H-DSPC is diluted by unlabeled DSP from other sources. Not surprisingly, Mics, which are derived from all lung cell types, had the lowest 3H-DSPC sp act.

In lungs harvested from the spontaneously breathing animals, endotoxin infusion increased 3H-DSPC sp act in all fractions, except Lb-B, but to different extents. This must reflect an increase in DSP synthesis and turnover. Although Lb-A and Lb-B had the highest 3H-DSPC sp act, the increase was relatively modest (approximately 20% to 40%) as compared with that in Alv-1 and Alv-2 (approximately 80% to 125%). This is consistent with Lb-A and Lb-B 3H-DSPC being diluted by unlabeled DSP from reuptake or newly synthesized labeled DSP being secreted directly into the alveolus (14).

Romero and associates (56) have shown that up until approximately 2.5 hours, endotoxin increases phosphatidylcholine secretion from cultured rat alveolar type II cells in a dose-dependent manner. In apparent contrast, at later times synthesis and secretion are inhibited (56, 57). Bosch and colleagues (58) and Oldham and associates (59) have shown that endotoxin decreases phosphatidylcholine synthesis and secretion in rats in vivo, whereas Castiello and colleagues (2) and Fehrenbach and associates (3) found that it causes the accumulation of lamellar bodies and decreases the secretion of surfactant lipids. However, it should be cautioned that none of these researchers "de-gassed" the lungs prior to lavage. This markedly reduces surfactant recovery and could introduce artifacts in the endotoxin-treated animals where reduced compliance and atelectasis are likely.

We can only speculate as to the stimulus responsible for the increased surfactant turnover in our model. Surfactant is normally released in response to a number of physiologic stimuli, including circulating agonists and physical distortion of the type II cell (15). Certainly, endotoxin would cause a strong stress-induced release of epinephrine. Importantly, endotoxin also increased the alveolar 3H-DSPC sp act in animals ventilated with constant "resting" VT. Although this may suggest that ventilation and physical distortion of the type II cell is not a major determinant of surfactant turnover in our model, 3H-DSPC sp act was also increased in fractions harvested from the ventilated control subjects where less ß-adrenergic stimulation would be expected. The increased turnover in these control animals may reflect increased heterogeneity and regional ventilation associated with the paralysis and loss of alveolar interdependence (22). Whatever the case, taken together, our findings indicate that endotoxin exerts both direct and indirect respiratory mediated time-dependent effects on surfactant turnover.

The chicken or the egg?
We have previously shown that in isolated perfused lungs a single deep respiratory cycle analogous to a "sigh" releases surfactant and enhances static lung compliance (60). The reflex for sighs is thought to initiate in the lungs, possibly in response to changes in pressure and increased work of breathing detected by irritant receptors in the lower airways, and involve the vagus nerve and muscarinic cholinergic receptors localized at the lower brainstem (61). Therefore, two scenarios may explain the dramatic increase in sighing we observed: either sighing is increased in the endotoxin-treated rats as a compensatory response that stimulates surfactant turnover and alleviates the increased work of breathing possibly associated with the V/Q mismatch, or in the endotoxin-treated rats, surfactant function is flawed and the increased sighing represents a futile attempt to restore the ailing lung mechanics.

Our findings support the latter hypothesis as Raw, Gtis, and Htis remained elevated, but did not increase further in lungs isolated from the endotoxin-treated animals ventilated with constant "resting" VT.

Systemic Compartmentalization
The 125I-Alb percentage increased approximately two-fold late in the course of the experiment in all tissues examined, consistent with ALI being only one facet of systemic endotoxemia. Surprisingly, the increase in albumin flux in these beds occurred in the absence of edema, as reflected by both the normal wet/dry weight ratio and the 99mTc-DTPA percentage. Similar findings have been observed by Carati and associates in the guinea pig gut in response to ischemic injury (personal communication). Although the reason for this is unclear, there is an increasing realization that maintenance of fluid balance in tissue is more complex than previously thought. Fleck and coworkers (62) have shown that the rate of loss of albumin to the tissue spaces increases by more than 300% in patients with septic shock, despite much smaller increases in edema. Similarly, Hersch and associates (63) have shown that in the hindlimb of sheep, sepsis is not associated with an increase in prefemoral lymph flow despite an approximately three-fold increase in the flux of 125I-Alb. Indeed, lymph drainage may be decreased in sepsis because of reduced lymphatic contractility (64, 65). Possibly, the expected increase in systemic fluid movement with the increased endothelial permeability was offset by the decrease in systemic BP. Alternatively, Finn and coworkers (66) have shown that sepsis is associated with an approximately two-fold increase in total tissue protein catabolism. Possibly, the amino acids produced and normally destined for acute phase protein production, primarily by the liver, provide an osmotic force countering the solvent drag created by the plasma proteins diffusing into the interstitium. These and our present observations suggest that increased endothelial permeability alone will not lead to edema without changes in colloid balance and/or hydrostatic pressure.

Conclusion
Our findings illustrate that infusion of the endotoxin S. abortus equi in rats initially impairs oxygenation, deteriorates lung mechanics, and increases sighing and surfactant turnover independent of alveolar neutrophil or macrophage recruitment or changes in alveolocapillary permeability. We suggest that endotoxin initially causes V/Q mismatch independent of inflammation and alveolocapillary injury and increases surfactant turnover both directly and in a manner that depends on the pattern of respiration. Although we were unable to detect any changes in surfactant composition, Htis initially increased without a change in Raw. This must reflect alveolar instability, most likely due to increased surface tension. All mammals involuntarily sigh, a reflex that probably normally stimulates surfactant release in response to increased work of breathing. We suggest that sighing increases in endotoxin-induced ALI at least partially in response to impaired surfactant function.


   Acknowledgments
 
The authors gratefully acknowledge Darren Peter, Division of Medical Imaging, for radiolabeling the DTPA.

Supported by grants 950054 and 981251 from the National Health and Medical Research Council of Australia and grants from the Australian and New Zealand College of Anaesthetists, the Australian and New Zealand Intensive Care Society, and the Australian Adult Respiratory Distress Syndrome Association.


   FOOTNOTES
 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form December 11, 2000; accepted in final form February 19, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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