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Published ahead of print on July 3, 2003, doi:10.1164/rccm.200206-618OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1043-1050, (2003)
© 2003 American Thoracic Society

Functional Identification of the Alveolar Edema Reabsorption Activity of Murine Tumor Necrosis Factor-{alpha}

Nadia Elia, Maxime Tapponnier, Michael A. Matthay, Jürg Hamacher, Jean-Claude Pache, Marie-Anne Bründler, Martin Totsch, Patrick De Baetselier, Lucie Fransen, Norimasa Fukuda, Denis R. Morel and Rudolf Lucas

Division of Anesthesiological Investigations and Division of Clinical Pathology, University Medical Center, Geneva, Switzerland; Cardiovascular Research Institute, San Francisco, California; Laboratory of Cellular Immunology, Flemish Institute for Biotechnology, University of Brussels, Campus Rode, Sint-Genesius-Rode, Belgium; Innogenetics, Industriepark Zwijnaarde, Ghent, Belgium; and Biochemical Pharmacology, University of Konstanz, Konstanz, Germany

Correspondence and requests for reprints should be addressed to Rudolf Lucas, Ph.D., Biochemical Pharmacology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany. E-mail: Rudolf.Lucas{at}uni-konstanz.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) activates sodium channels in Type II alveolar epithelial cells, an important mechanism for the reported fluid resorption capacity of the cytokine. Both TNF-{alpha} receptor–dependent and –independent effects were proposed for this activity in vitro, the latter mechanism mediated by the lectin-like domain of the molecule. In this study, the relative contribution of the receptor-dependent versus receptor-independent activities was investigated in an in situ mouse lung model and an ex vivo rat lung model. Fluid resorption due to murine TNF-{alpha} (mTNF-{alpha}) was functional in mice that were genetically deficient in both types of mTNF-{alpha} receptor, establishing the importance of mTNF-{alpha} receptor–independent effects in this species. In addition, we assessed the capacity of an mTNF-{alpha}–derived peptide (mLtip), which activates sodium transport by a receptor-independent mechanism, to reduce lung water content in an isolated, ventilated, autologous blood-perfused rat lung model. The results show that in this model, mLtip, in contrast to mTNF-{alpha}, produced a progressive recovery of dynamic lung compliance and airway resistance after alveolar flooding. There was also a significant reduction in lung water. These results indicate that the receptor-independent lectin-like domain of mTNF-{alpha} has a potential physiological role in the resolution of alveolar edema in rats and mice.

Key Words: edema • cytokine • sodium transport

Several studies indicate that active salt transport drives reabsorption of edema fluid from the distal air spaces of the lung (reviewed in Matthay and coworkers [1]). Indeed, specific inhibitors of sodium transport, such as amiloride, have been shown to inhibit alveolar liquid clearance in the lungs of different species, including humans, when injected intratracheally. During the course of diseases such as the acute respiratory distress syndrome (ARDS) and pneumonia, the alveolar space as well as the interstitium are sites of intense inflammation, with exudation of protein-rich pulmonary edema fluid (2, 3). During this inflammatory process, proinflammatory substances such as tumor necrosis factor-{alpha} (TNF-{alpha}) are produced locally (3).

Although intravascular TNF-{alpha} has been implicated in the pathophysiology in lungs subjected to different inflammatory models, including acute lung injury (4–6) and ischemia–reperfusion injuries (7), this cytokine was also reported to exert potentially favorable effects by upregulating the rate of alveolar fluid clearance in vivo in rats with Pseudomonas aeruginosa pneumonia (8) or during intestinal ischemia–reperfusion (9), as well as in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage (10). Indeed, TNF-{alpha} is able to activate sodium channels in alveolar epithelial cells (8, 11), and this effect could have favorable consequences for fluid clearance from the lung.

Apart from interacting with its specific receptors, TNF-{alpha} also participates in immune functions through lectin-like interactions that occur independently from TNF-{alpha} receptor signaling (12). TNF-{alpha} was shown to contain a lectin-like domain (the tip domain), spatially distinct from its receptor-binding sites and mediating its lytic activity in salivarian trypanosomes (12). Although the lectin-like activity of TNF-{alpha} (13, 14) mediates its membrane conductance-increasing activity in macrophages and endothelial cells (15), the in vitro-mediated Na+ channel-activating effect of the cytokine in the human A549 Type II alveolar epithelial cell line seemed to include both TNF-{alpha} receptor–dependent as well as –independent components (11). Indeed, antibodies directed against both TNF-{alpha} receptors were able to block the ion channel-activating effect of TNF-{alpha} in these cells. On the other hand, a TNF-{alpha} mutant that still efficiently binds to both TNF-{alpha} receptors and activates TNF receptor 1–mediated activities to the same extent as does the wild-type molecule, but that lacks the lectin-like activity (16, 17), failed to increase Na+ currents (11). These results thus indicated that both TNF receptor–dependent and –independent activities of TNF could be implicated in the activation of sodium transport.

In this study, using mice genetically deficient in both TNF receptors, we investigated whether a murine TNF-{alpha} (mTNF-{alpha}) receptor–dependent or receptor–independent mechanism predominates in mediating mTNF-{alpha}–stimulated edema fluid resorption in this species in situ. In addition, using an isolated, ventilated, and autologous blood-perfused rat lung model, we compared the potency of the mTNF-{alpha} tip peptide, a 17-amino acid peptide mimicking the lectin-like domain of the cytokine (12), which lacks the proinflammatory activities of TNF-{alpha} (15), with that of the native protein. These experiments were performed in the absence or presence of amiloride, a selective inhibitor of Na+ channels. In the latter study, we evaluated changes in lung weight, pulmonary mechanics and hemodynamics, as well as histology over time. Taken together, the results indicate a predominant role for receptor-independent activities of TNF-{alpha} in the capacity of the cytokine to enhance alveolar fluid resorption in mice and rats.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
TNF-{alpha} and TNF-{alpha}-derived Peptides
Escherichia coli-derived recombinant murine TNF-{alpha} (further referred to as mTNF-{alpha} in text) (specific activity, 2 x 108 IU/mg; Innogenetics, Ghent, Belgium) was synthesized as described previously (12, 16). This mTNF was free of LPS and a single preparation was used in all experiments. Long tip peptide 99–115 (Ltip peptide) was synthesized with the use of Fmoc-{alpha}-amino group protection (18) and purified with a C18 reversed-phase high-performance liquid chromatography column.

To retain the original mTNF-{alpha} conformation as much as possible, Ltip peptide was circularized. Ser-99 of the mTNF-{alpha} sequence was replaced by cysteine, and Cys-100 was replaced by glycine, so that the disulfide bridge could be formed between Cys-99 and Cys-115 in the peptide. As a control peptide, a scrambled tip peptide was used, consisting of the same amino acid composition as the tip peptide, but in a random order.

  • Long tip (Ltip) peptide: CGPKDTPEGAELKPWYC
  • Scrambled tip peptide: CGTKPWELGPDEKPAYC

Amiloride hydrochloride hydrate (referred to as amiloride throughout the text) was purchased from Sigma-Aldrich (Buchs, Switzerland).

Animals and Animal Preparation
Mice.
Male C57BL/6 TNF-{alpha} receptor 1 (TNF-{alpha}R1)/TNF-{alpha} receptor 1 (TNF-{alpha}R2) double-knockout mice (19) (n = 8, 20–30 g) and C57BL/6 wild-type mice (n = 22, 20–30 g) were purchased from Jackson Laboratory (Bar Harbor, ME) or from the animal facilities at the University of Konstanz (Konstanz, Germany). These animals were housed in air-filtered, temperature-controlled units with food and water. All procedures were approved by the University of California at San Francisco Committee on Animal Research and the University of Konstanz Committee on Animal Research.

In situ mouse model.
Mice were killed by an overdose of pentobarbital sodium (200 mg/kg, intraperitoneal). A tracheotomy was done with a 20-gauge trimmed Angiocath plastic needle (BD Infusion Therapy Systems, Sandy, UT). The lungs were inflated with a continuous positive airway pressure of 7 cm H2O with 100% oxygen throughout the experiments. Body temperature was maintained at 37–38°C by an infrared lamp placed 30 cm above the body (Fisher, Santa Clara, CA). The lamp was cycled on and off to maintain the core temperature. A temperature probe (Yellow Springs Instrument, Yellow Springs, OH) was inserted via a 0.5-cm incision into the abdominal cavity to monitor the core temperature throughout the experiment. These methods have been reported in prior studies (1, 20). The instillate consisted of 5% bovine serum albumin (Sigma, St. Louis, MO) with Ringer's lactate that was adjusted to be isosmolar with mouse plasma (1, 20). mTNF-{alpha} (0.5 µg/mouse) or Ltip peptide (12.5 µg/mouse) was included in the instillate and applied into the distal lung. We added 0.1 µCi of 131I-labeled albumin (Merck-Frost, Montreal, PQ, Canada) to the instillate as a labeled alveolar protein tracer. Groups studied were as follows:

  • Group 1: Basal alveolar fluid clearance was measured in wild-type mice (n = 3) and in TNF-{alpha}R1/TNF-{alpha}R2 double-knockout mice (n = 3).
  • Group 2: Effect of wild-type TNF-{alpha} (0.5 µg/mouse)-stimulated alveolar fluid clearance was measured in wild-type mice (n = 5) and in TNF-{alpha}R1/TNF-{alpha}R2 double-knockout mice (n = 5) (19).
  • Groups 3 and 4: In an independent experiment, the effect of mLtip peptide on alveolar fluid clearance (12.5 µg/mouse; n = 8; Group 3) was also assessed versus C57BL/6 control mice (n = 6; Group 4). As in our previous studies, alveolar fluid clearance over 15 or 30 min was measured by the increase in the final concentration of the alveolar protein tracer compared with the initial instilled tracer protein concentration (1, 20).

Rats.
For the ex vivo experiments, 36 Sprague-Dawley rats of both sexes (300 to 350 g) were purchased from Biological Research Laboratories (Füllinsdorf, Switzerland). Under isoflurane anesthesia, the animals were tracheotomized with a 14-gauge polyethylene cannula, using sterile techniques, and mechanically normoventilated (40% oxygen in air) with a constant volume-cycled rodent ventilator (tidal volume, 7 ml/kg; positive end-expiratory pressure [PEEP], 2.5 cm H2O; respiratory rate, 70–80/min). The femoral artery was cannulated for blood sampling and continuous arterial blood pressure monitoring and the femoral vein was cannulated for fluid replacement. The experimental protocol was reviewed and approved by the Ethics Committee for Animal Research and by the Veterinary Office of the University Medical Center of the University of Geneva (Geneva, Switzerland).

Isolated lung preparation.
The rats were fully anticoagulated by intravenously administered heparin (1.5 IU/g body weight). Twenty milliliters of blood was then gently withdrawn via the arterial cannula while continuously replacing the collected blood by an intravenous constant rate infusion of dextran-40 (Macrodex, 10% in normal saline) to maintain a constant intravascular volume and a mean systemic blood pressure above 50 mm Hg to minimize lung ischemic lesions during this normovolemic hemodilution procedure. This resulted in the collection of 20 ml of diluted blood with a hematocrit of about 21%, which served as priming volume for the isolated perfusion circuit. Through a median sternotomy, the heart–lung block was cannulated with polyethylene catheters, one placed into the main pulmonary artery via the right ventricular outflow track and advanced to a point immediately below its bifurcation, a second in the left ventricle for collection of effluent blood, and a third catheter in the left atrium for continuous left atrial pressure recording. After pulmonary artery cannulation, the lungs were immediately flushed with 30 ml of cold (10°C) low-potassium dextran solution from a height of 30 cm to minimize the warm ischemic time period until ex vivo reperfusion.

Experimental groups.
The heart–lung block was dissected free of adjacent tissue, extracted, weighed, and assigned to one of four groups. Each received a pretreatment consisting of either 0.9% NaCl (n = 9), TNF-{alpha} (5 µg; n = 7), Ltip peptide (1 mg; n = 5), scrambled tip peptide (1 mg; n = 4), amiloride (600 µg; n = 6), or amiloride plus Ltip peptide (600 µg + 1 mg; n = 5), through intratracheal instillation of 200-µl aliquots placed in an air-filled 1-ml syringe that was emptied over 1 second through a 5-cm-long 28-gauge catheter placed into the trachea with its distal tip 1 cm above the carena. The pretreatment was administered after weighing the heart–lung block, that is, 5–10 minutes before starting reperfusion. Because the mouse Ltip peptide has a significantly lower sodium channel-activating activity compared with TNF-{alpha} (15), we treated the lungs with 1 mg of the peptide or of the control scrambled peptide, and with 5 µg of TNF-{alpha}.

Reperfusion method.
The heart–lung block was suspended by the ligatured aorta in a thermostated and humidified Plexiglas chamber maintained at 37°C, from an isometric force displacement transducer (Grass Telefactor FT03; Astro-Med, West Warwick, RI) to continuously measure weight changes. The lungs were ventilated (model 683; Harvard Apparatus, South Natick, MA) with room air mixed with 5% CO2 at a respiratory rate of 50 breaths/minute, a tidal volume of 7 ml/kg, and a PEEP of 2.5 cm H2O. Respiratory gases were continuously monitored with a Datex monitor (Ultima; Datex/Instrumentarium, Helsinki, Finland). The circuit was primed with the rat's own diluted blood. The lung was then perfused via the pulmonary artery cannula from an arterial reservoir placed at a fixed height to induce a mean pulmonary arterial pressure (Ppa) of 17.5 mm Hg. Pulmonary blood flow (PBF) was continuously monitored with a transit-time flow meter (T-201 CDS; Transonic Systems, Ithaca, NY) placed on the pulmonary inflow. Effluent blood was drained through the left ventricle cannula, whose distal extremity was placed at a sufficient height to obtain a of 7.5 ± 2 mm Hg, which produced West's Zone 3 conditions (Ppa > > airway pressure [Paw]). The blood dripping from the cannula was collected in a 10-ml cylinder. A peristaltic pump (Ismatec pump; Glattburg, Zurich, Switzerland) was used to recirculate the effluent blood to the pulmonary arterial reservoir. All material used was sterilized.

Assessment of lung function.
Ppa and were measured with calibrated pressure transducers (model 156-PC 06-GW2; Honeywell, Zurich, Switzerland) zeroed at the level of the lung hilus. Paw was measured with a calibrated pressure transducer (model Z46169; Gould, Valley View, OH). Tidal volume and airflow were measured with a pneumotachograph (Godart V, Type 17212; Gould) allowing the continuous breath-by-breath assessment of dynamic lung compliance [Cdyn = tidal volume/(peak Paw – PEEP)], and expiratory airway resistance [Raw = (peak Paw – PEEP)/maximal expiratory air flow]. An inspiratory and expiratory quasi-static pressure–volume (P–V) curve was performed at the end of the 2-hour reperfusion period by inflating and then deflating the lungs at a constant rate (0.3 ml/second), using an automated infusion pump, from functional residual capacity (ambient airway pressure without PEEP) with 7 ml of air. Pulmonary vascular resistance was calculated by dividing (Ppa – ) by PBF. Airflow, tidal volume, pressures, PBF, pulmonary vascular resistance, as well as weight changes ({Delta}W) were continuously recorded and stored at a sampling rate of 50 Hz via an analog–digital interface converter (Biopac, Santa Barbara, CA) on an AST microcomputer (AST, Limerick, Ireland). A sigh was applied in all groups every 15 minutes by blocking expiration during three breaths to minimize atelectasis formation. The pH and temperature of the perfusate were measured with a pH meter (691 pH meter; Metrohm, Herisau, Switzerland). The blood pH was maintained between pH 7.3 and 7.5 and, if necessary, corrected with 8.4% sodium bicarbonate or a change in the inspired CO2 as required by the blood gas analysis. Every 30 minutes, a blood sample was collected for blood gas analysis as well as for hematocrit and electrolyte concentration (model 505; Acid Base Laboratory, Radiometer, Copenhagen, Denmark).

Inclusion criteria.
Immediately after the start of reperfusion, the isolated lung preparation had to fulfill three technical inclusion criteria during the first 10 minutes of reperfusion to be included in the study: (1) between 5 and 10 mm Hg; (2) peak Paw < 15 cm H2O; and (3) PBF > 10 ml/minute. Once included, a stabilization period of 15 to 30 minutes was allowed for respiratory and hemodynamic variables to become constant and for the preparations to become isogravimetric. The lungs that were not isogravimetric within 30 minutes of perfusion were excluded. The included lungs were then flooded with a gentle intratracheal instillation of 2 ml of normal saline and the recorded variables were measured for the subsequent 2 hours.

Characteristics of the isolated rat lung preparation.
The weight of the heart–lung block at the beginning of the reperfusion period was similar between the six groups (NaCl, 3.49 ± 0.41 g; TNF-{alpha}, 4.03 ± 0.63 g; Ltip peptide, 3.50 ± 0.43 g; scrambled tip peptide, 4.05 ± 0.39 g; amiloride, 4.43 ± 0.15 g; amiloride + Ltip peptide, 4.02 ± 0.08 g; p = NS). Ppa, , and PEEP were maintained within 10% of baseline values during the 2-hour perfusion period and were not different between groups (two-way analysis of variance with repeated measure design; Table 1) . The characteristics of the perfusate did not significantly change in terms of blood temperature, pH, sodium concentration, or hematocrit throughout the experiment and were comparable between the groups (Table 1).


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  		TABLE 1. Overall means of physiologic data during 2-HOUR isolated lung perfusion period

 
Histologic analysis.
Random samples of upper and lower lobes were fixed for 24 hours in 10% buffered formalin solution and embedded in paraffin. The 4-µm-thick sections were then stained with hematoxylin–eosin and examined under light microscopy by pathologists blinded to the lung's protocol group assignment. Ten fields were examined in each lung at x100 and scored 1 to 4, using the following criteria: alveolar edema, interstitial/septal edema, and perivascular edema; alveolar and perivascular/septal hemorrhage; and alveolar and septal inflammatory cell infiltration (with 1 meaning normal and 4 indicating the most severe injury). Specimens were scored for injury severity by a pulmonary pathologist blinded to the treatment group. Additional lung samples were cut into 1-mm3 blocks for electron microscopy, and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 12 hours. Postfixation was achieved in osmium tetroxide and the blocks were then thoroughly washed and dehydrated through graded alcohols and propylene oxide, before being embedded in Epon.

Statistical Analyses
Group data are presented as mean values ± SD. A two-tailed t test was used to compare between two single groups if normal distribution was the case. A two-way analysis of variance was used to compare data at each time point between and within groups (repeated measures design), followed by Duncan's multiple comparison test if the analysis of variance resulted in a p value less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} Mediates Fluid Resorption in the in Situ Mouse Lung Model by Means of a TNF-{alpha} Receptor–independent Mechanism
Wild-type TNF-{alpha} in C57BL/6 mice increased alveolar fluid clearance by 24% (p < 0.05) over 15 min in the in situ mice compared with controls (Figure 1) . TNF-{alpha}R1/R2-/- C57BL/6 mice showed an equivalent increase in alveolar fluid clearance under control conditions as well as in the presence of TNF-{alpha}, indicating that receptor–independent activities of TNF-{alpha} affect the fluid clearance in this model in mice.



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  		Figure 1. Effect of TNF-{alpha}R1/TNF-{alpha}R2 double knockout on basal and TNF{alpha}-stimulated alveolar fluid clearance in mice. *p < 0.05 versus basal clearance.

 
Ltip Peptide Induces Significant Weight Loss in Flooded Perfused Rat Lungs
To validate the hypothesis that receptor-independent effects predominate in the fluid resorption capacity of TNF, we compared the effects of an intratracheal pretreatment with (1) Ltip peptide, a 17-amino acid TNF-{alpha}-derived peptide that does not bind to the TNF-{alpha} receptors, mimicks the Na+ channel-activating effect of TNF-{alpha}, but lacks its proinflammatory activities, such as intercellular adhesion molecule-1 upregulation, E-selectin induction, or interleukin-6 induction in microvascular endothelial cells (15), or (2) a scrambled peptide displaying the same amino acids but in a random order, with (3) the native protein in a volume-controlled isolated blood-perfused rat lung model. On the basis of relative potencies in activating sodium currents in vitro (15), the lungs were treated with 5 µg of TNF-{alpha} and with 1 mg of the peptides.

During the first 15 minutes of reperfusion, lung weight increased similarly in the four treatment groups, because of the filling of the pulmonary vasculature imposed by the respective predetermined vascular (Ppa and ) and airway pressure gradients (Paw and PEEP) (NaCl group, +0.69 ± 0.46 g; TNF-{alpha} group, +0.87 ± 0.60 g; mLtip peptide group, +0.80 ± 0.61 g; scrambled tip peptide group, +0.71 ± 0.50 g). After stable perfusion conditions for 15 minutes, alveolar flooding by intratracheal instillation of 2 ml of normal saline (time 0 in Figure 2) produced a further acute increase in lung weight of 1.9 g without modifying the characteristics and pulmonary hemodynamics of the perfusate. In contrast, there was a marked change in dynamic lung mechanics observed similarly in NaCl, TNF-{alpha}, Ltip peptide, and scrambled tip peptide groups, demonstrated by a twofold increase in peak insufflation pressure, a 60% reduction in Cdyn, and a moderate increase in expiratory Raw (Figure 2).



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  		Figure 2. Effect of intratracheal pretreatment with saline (open circles, n = 9), TNF-{alpha} (solid circles, n = 7), Ltip peptide (solid triangles, n = 5) or scrambled tip peptide (open inverted triangles, n = 4) on weight changes (A) and lung mechanics (B–D) in isolated rat lungs before and after alveolar flooding (at time 0) with 2 ml of normal saline and subsequent reperfusion for 2 hours. Data points represent mean values ± SE; *p < 0.05 compared with saline group.

 
After alveolar flooding, lung weight increased slightly, although not significantly, over time in NaCl-treated lungs (mean weight gain, +0.28 ± 0.09 g). In contrast, lungs pretreated with the Ltip peptide progressively decreased in weight (p < 0.001), and were significantly different from saline-treated lungs after 45 minutes of reperfusion (Figure 2). At 2 hours, the lungs in this group had lost approximately half of the intratracheally instilled saline. In contrast, lungs pretreated with TNF-{alpha} or scrambled tip peptide showed no significant change in weight over time. Moreover, mLtip peptide (12.5 µg/mouse, intratracheal) also significantly increased alveolar fluid clearance by 49% in the in situ flooded mouse lung model 30 min after instillation, as compared with controls (Figure 3) .



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  		Figure 3. Effect of intratracheal pretreatment with Ltip peptide on alveolar liquid clearance in the in situ flooded mouse lung model (12.5 µg/mouse, administered intratracheally; n = 8; *p = 0.018 compared with saline group [n = 6]). Data represent means ± SE.

 
To investigate whether the beneficial effect of the Ltip peptide treatment involves the activation of sodium currents, animals were pretreated with the rather specific sodium channel inhibitor amiloride. As shown in Figure 4 , in the animals pretreated with amiloride or with amiloride plus Ltip peptide, there was already a significant decrease in Cdyn before alveolar flooding. Instillation of 2 ml of saline produced a further decrease in Cdyn that was associated with a severe rise in Raw. Thereafter, a progressive and major increase in lung weight was observed in both amiloride groups. These results thus indicate that an inhibition of epithelial sodium channel activity inhibits the beneficial effect of the Ltip peptide.



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  		Figure 4. Effect of intratracheal pretreatment with amiloride (open diamonds, n = 6), Ltip peptide (solid triangles, n = 5) or amiloride plus Ltip peptide (solid squares, n = 5) on weight changes (A) and lung mechanics (B–D) in isolated rat lungs before and after alveolar flooding (at time 0) with 2 ml of normal saline and subsequent reperfusion for 2 hours. Data points represent mean values ± SE; *p < 0.05 compared with Ltip peptide group.

 
Improvement of Respiratory Parameters on Ltip Treatment
Peak Paw, Cdyn, and Raw of the NaCl-pretreated group remained stable after alveolar flooding, showing no significant change over time (Figure 2). Similarly, for the TNF-{alpha}- and scrambled tip peptide-pretreated lungs there was no difference from the saline-treated group. In contrast, Ltip peptide-pretreated lungs showed a progressive improvement in lung mechanics following alveolar flooding during reperfusion (p < 0.001 for all three measured variables) (Figure 2). These changes were statistically significant compared with NaCl-treated lungs after 15 minutes for Cdyn, and at 60 minutes for peak Paw, but did not reach statistical significance for Raw. Animals receiving Ltip peptide with amiloride did not improve lung mechanics as observed in animals receiving Ltip peptide alone (Figure 4).

Figures 5A and 5B show the quasi-static airway P–V curves obtained at the end of the study. In NaCl-treated lungs, alveolar flooding produced a severe decrease in the slope of the initial inspiratory limb of the curve (i.e., a reduced static inspiratory lung compliance), up to a sharp inflection point situated at 22.6 ± 1.9 cm H2O, obtained after only 1.5 ml of inflated volume. Thereafter, a continuing rise in inflation pressure with volume opened the flooded and collapsed lung abruptly. The expiratory limb of the P–V curve was not altered compared with control normal lungs (data not shown). Pretreatment of the lungs with TNF-{alpha} did not significantly influence the initial slope of the P–V curve, but shifted the second part to the left (lower inflection point, 20.0 ± 0.8 cm H2O; p < 0.05 compared with the NaCl group). Pretreatment with Ltip peptide consistently ameliorated the whole inspiratory curve, with a lower inflection point at 17.2 ± 1.6 cm H2O (p < 0.05 compared with both the NaCl and TNF-{alpha} groups) obtained at an inflation volume of 3.4 ± 0.9 ml, denoting an improved static inspiratory lung compliance. The expiratory part of the curve was not influenced by TNF-{alpha} or Ltip pretreatment, indicating that once fully recruited, the lung recovers its normal elastic recoil properties manifested during the deflation curve, independent of the amount of alveolar fluid.



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  		Figure 5. Effect of intratracheal pretreatment with saline (open circles, n = 4), TNF-{alpha} (solid circles, n = 4) or Ltip peptide (solid triangles, n = 5) (A and B), and with amiloride (open diamonds, n = 6), Ltip peptide (solid triangles, n = 5), or amiloride plus Ltip peptide (solid squares, n = 5) (C and D) on quasi-static inspiratory and expiratory pressure–volume curves in isolated rat lungs at the end of lung reperfusion for 2 hours after alveolar flooding with 2 ml of normal saline. Data points represent mean values ± SE; *p < 0.05 compared with saline group in (A) and (B), compared with Ltip peptide group in (C) and (D).

 
Figures 5C and 5D show P–V curves for amiloride groups compared with the already depicted Ltip peptide group.

The amiloride and the amiloride plus Ltip peptide groups produced a severe decrease in the slope of the initial inspiratory limb of the curve, demonstrating the absence of improvement in static lungs compliance when sodium channels are blocked by amiloride. Furthermore, amiloride alone or in the presence of Ltip peptide produced a significant rightward shift of the expiratory P–V curve, most probably secondary to an important interstitial edema produced by amiloride.

Histology
Histologic examination of the lungs showed significant differences in the overall lung injury score between TNF-{alpha}-treated (but not Ltip peptide-treated) and saline-treated lungs (saline, 1.936 ± 0.474; TNF-{alpha}, 2.492 ± 0.540*; Ltip peptide, 2.161 ± 0.442; *p < 0.05 versus saline). The differences were due mainly to a more pronounced septal and alveolar edema observed in TNF-{alpha}–treated animals (Table 2) . These findings support the antiedema property of Ltip peptide as compared with TNF-{alpha}. Analysis of TNF-{alpha}-pretreated lungs by means of electron microscopy revealed prominent numerous endothelial flaps (Figure 6A , open arrow) protracting in the vascular lumen around an erythrocyte (Figure 6A, E), numerous cytoplasmic blebs of the pneumocyte I (Figure 6A, solid arrow), and tubular myelin surfactant (SFigure 6A, S) in the alveolar space. This particular microscopic pattern was not found in Ltip peptide-treated lungs (Figure 6B) and was also less evident in scrambled tip peptide-treated or saline-treated lungs (Figures 6C and 6D), as compared with TNF-{alpha} instillation (Figure 6A). In all analyzed lungs, there was an increased number of pinocytic vesicles in endothelial cells and pneumocytes.


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  		TABLE 2. Mean ± sd of histologic lung injury scores

 


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  		Figure 6. Electron micrographs illustrating increased alveolar epithelial surface and epithelial blebs in representative rat lungs pretreated with TNF-{alpha} (A), Ltip peptide (B), scrambled tip peptide (C), or normal saline (D) followed by alveolar flooding with 2 ml of normal saline and subsequent lung reperfusion for 2 hours. (A) Prominent numerous endothelial flaps (open arrow) protracting in the vascular lumen around an erythrocyte (E). Numerous cytoplasmic blebs of the pneumocyte I (solid arrow). Tubular myelin surfactant (S) in the alveolar space. (B) Absence of significant endothelial flaps (open arrow) or epithelial blebs (solid arrow); slight edema of the pneumocytes Type I (solid arrow) and endothelial cells (open arrow). (C) Endothelial flaps (open arrow) and epithelial blebs (solid arrow) less evident than in (A). (D) Slight focal edema of the pneumocytes I (solid arrow) and endothelial cells (open arrow); minimal amount of flaps or blebs. (A–D) Original magnification, x9,800.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Active Na+ transport across the alveolar epithelium in vivo was proposed to help the resorption of fetal fluid after birth and to keep the adult alveolar spaces free of fluid, especially when alveolar permeability to plasma proteins has been increased (1, 21). Epithelial Na+ channels represent the rate-limiting step in Na+ absorption (21, 22). Different types of channels have been described on alveolar epithelial Type II and fetal distal lung epithelial cells. The results of in vivo and in vitro studies indicate that Na+ ions in the alveolar lining fluid passively diffuse into fetal distal lung epithelial and alveolar epithelial Type II cells through nonselective cationic channels and Na+ selective, amiloride-sensitive channels located in their apical membrane. The favorable electrochemical driving force for Na+ influx is maintained by the ouabain-sensitive basolateral Na+/K+-ATPase that also transports Na+ into the interstitial space (22).

Hydrostatic pulmonary edema is a common complication of congestive heart failure, resulting in substantial morbidity and mortality (23, 24). Moreover, acute pulmonary edema or pulmonary reimplantation response frequently occurs after lung transplantation (25), and is caused by ischemic vascular injury of the allograft, resulting in increased permeability of the lung after reperfusion and in turn leading to interstitial and alveolar edema. The majority of patients with acute lung injury or ARDS also have a dramatically decreased edema resorption capacity, correlating with morbidity and higher mortality (2).

ß2-Adrenergic agonists, such as terbutaline, have been shown to resolve hydrostatic edema efficiently in both sheep and rat models (26). However, long-term ß2-adrenoceptor agonist therapy leads to a desensitization of ß2-adrenoceptor-mediated cardiovascular and noncardiovascular effects in humans in vivo (27, 28) and may lead to tachyphylaxis in patients with asthma (29). In these patients there should be an evaluation of alternative agents. The TNF-{alpha}-derived Ltip peptide could represent such an alternative, because it is not likely to interfere with ß2-adrenoreceptors and it may activate sodium channels not only in Type II, but also perhaps in Type I, alveolar epithelial cells. Moreover, in contrast to ß2-adrenergic agonists, the sodium uptake activating effect of TNF-{alpha} cells in vitro cannot be inhibited by the Na+/K+-ATPase blocker ouabain (15).

The sodium uptake-activating effect of TNF-{alpha} in A549 cells was suggested to imply both TNF-{alpha} receptor–dependent and –independent activities. Indeed, on the one hand antibodies directed against TNF-{alpha}R1 and TNF-{alpha}R2 efficiently blocked this effect, but on the other hand, a mouse TNF-{alpha} mutant lacking its lectin-like activity, which still efficiently mediates most of the receptor-mediated effects, completely lacked the sodium channel-activating effect in vitro and when given to rats (11). The results in this study in an in situ mouse model and an ex vivo rat lung model indicate that TNF-{alpha} receptor–independent effects predominate in the fluid resorption activity of the cytokine in these species. Indeed, (1) in mice that lacked both TNF-{alpha} receptors (19), mouse TNF-{alpha} had the same efficiency in increasing fluid resorption as in wild-type animals and (2) the Ltip peptide, which does not activate either TNF-{alpha} receptor type, efficiently induced weight loss in the ex vivo flooded perfused rat lung, without exerting the TNF-{alpha} receptor-mediated proinflammatory activities that lead to leukocyte sequestration (15).

Physiologically relevant parameters were measured as indirect evaluation of edema clearance in the model of isolated, ventilated, and blood-perfused rat lung. After alveolar flooding, peak inspiratory pressure immediately increased in the lungs in all groups and provided an indirect indication of the volume of edema remaining in the alveoli. Increased intratracheal pressures during mechanical, constant volume ventilation can reflect bronchoconstriction, atelectasis formation, pulmonary edema, or restricted lung volume that appears after alveolar flooding. The isolated perfused lung is subject to atelectasis and we therefore chose to apply a sigh every 15 minutes. We have no reason to suspect a bronchoconstrictive phenomenon in this model, which lacked physiological innervation, and the amelioration of intratracheal pressure in the Ltip peptide-treated group suggests an effect of the Ltip peptide on the amount of alveolar edema. The dynamic compliance and airway resistance are other indirect but physiologically relevant measurements that demonstrated the efficacy of Ltip peptide pretreatment compared with both TNF-{alpha} and control pretreatments. The change in lung weight is another method for measuring lung edema clearance. The advantage of using an isolated lung was the ability to continuously measure lung weight throughout the experiment.

The weight loss associated with Ltip peptide pretreatment correlated with reduced intratracheal pressures, the partial recovery of baseline lung dynamic compliance, and therefore with an increase in alveolar fluid clearance. The continuous monitoring of all clinical parameters showed a continuous amelioration of the lung mechanics of the Ltip-treated group throughout the entire experiment. The recovery induced by the Ltip molecule seemed to be time dependent. This observation suggested a rate-limiting step in the fluid reabsorption, which was shown to be Na+ channel–dependent in previous studies, as well as in our study, in which amiloride completely blocked the Ltip effect (11, 15).

One major observation of this study was that Ltip peptide showed an edema reabsorption effect, whereas mTNF-{alpha} was only slightly different from NaCl. This result could be explained by the balance between, on the one hand, the receptor-mediated activities of wild-type mTNF-{alpha}, which can exert a negative effect on lung fluid clearance as suggested by others (30, 31), and, on the other hand, the beneficial effect of its lectin-like domain. It thus seems likely that the receptor-independent, potentially beneficial effects of mTNF-{alpha} can be masked by the inflammatory response induced by TNF-{alpha} receptor activation, which promotes edema formation by means of the increased expression of adhesion molecules leading to increased neutrophil sequestration (30). Alternatively, TNF-{alpha} directly triggers endothelial cell activation and barrier dysfunction, both implicated in the pathogenesis of pulmonary edema associated with acute lung injury syndromes (31), and was shown to inhibit the expression of epithelial sodium channel in Type II alveolar epithelial cells (32). In our experiments, mTNF-{alpha} led to a significant swelling of the alveolar epithelium, although not significantly reducing lung weight in the flooded rat lungs. Although we observed a tendency toward more infiltrated leukocytes in histology in the TNF-{alpha}-treated lungs, there was no significant difference from the control and Ltip-treated groups. This indicates that the lack of stimulation of edema resorption by TNF-{alpha} in our studied blood-perfused flooded rat lung model relates to direct effects of the cytokine on the alveolar epithelium or endothelium, rather than to sequestered cells. Moreover, the positive effect of Ltip peptide on lung weight cannot be explained solely by an eventual reduction of inflammation, as compared with control or TNF-{alpha}-treated lungs, in view of the complete inhibition of this effect by the sodium channel blocker amiloride, indicating that this peptide exerts its effect mainly by means of activating liquid clearance. In view of our observation that TNF-{alpha} can stimulate edema reabsorption in a Krebs–Henseleit-perfused isolated rat lung model (C. Braun et al., unpublished data) and that TNF-{alpha} increases alveolar fluid clearance in an in situ flooded rat lung model (8), this indicates that, depending on the setting used, TNF-{alpha} can at the same time stimulate edema resorption, mainly by means of receptor-independent activities, or promote edema formation, probably by means of its receptor-mediated effects.

In conclusion, this study indicates for the first time that receptor-independent activities of mTNF-{alpha} predominate in its edema reabsorption activity in an ex vivo flooded rat lung and an in situ flooded mouse lung model. Therefore, it should be further investigated whether administration of the tip peptide, derived from mTNF-{alpha}, in the air spaces of the lung may represent a novel therapy to accelerate the resolution of alveolar edema.


   Acknowledgments
 
The authors thank Mrs. Jennifer Hantson, Mr. Manuel Jorge-Costa, and Ms. Simone Poess for excellent technical assistance; Dr. Jutta Schlepper-Schäfer, Dr. Roger Lijnen, and Dr. Albrecht Wendel for critically reading the manuscript; and Mrs. Tessa James for editorial assistance.


   FOOTNOTES
 
Supported by National Institutes of Health grant HL51854 (M.A.M.) and by grant 32-68267.02 from the Swiss National Science Foundation.

N.E. and D.R.M. contributed equally to this work.

Conflict of Interest Statement: N.E. has no declared conflict of interest; M.T. has no declared conflict of interest; M.A.M. has no declared conflict of interest; J.H. has no declared conflict of interest; J-C.P. has no declared conflict of interest; M-A.B. has no declared conflict of interest; M.T. has no declared conflict of interest; P.D.B. has no declared conflict of interest; L.F. is an employee of Innogenetics, NV, and is co-inventor of a patent related to this publication; D.R.M. has no declared conflict of interest; R.L. is co-inventor of a patent concerning the peptide discussed in this study, the patent has been taken by Innogenetics and in 2003 he received an industrial grant from Innogenetics of 12,500 Euros for the follow-up of the study presented in this manuscript, previously he only received the peptides from the company and no financial support.

Received in original form June 27, 2002; accepted in final form June 25, 2003


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 RESULTS
 DISCUSSION
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