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The Journal of Immunology, 2005, 175: 3402-3408.
Copyright © 2005 by The American Association of Immunologists

Dichotomal Role of TNF in Experimental Pulmonary Edema Reabsorption

Clemens Braun*, Jürg Hamacher*, Denis R. Morel{dagger}, Albrecht Wendel1,* and Rudolf Lucas1,*,{ddagger}

* Department of Biochemical Pharmacology, University of Konstanz, Konstanz, Germany; {dagger} Department of Anesthesiological Investigations, University Medical Center, Geneva, Switzerland; and {ddagger} Department of Pharmaceutical and Medical Biotechnology, University of Applied Sciences, Krems, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Distinct from its receptor binding sites, TNF carries a lectin-like domain, situated at the tip of the molecule, which specifically binds oligosaccharides, such as N,N'-diacetylchitobiose. In view of the apparently conflicting data concerning TNF actions in pulmonary edema, we investigated the contribution of, on the one hand, the receptor binding sites and, in contrast, the lectin-like domain of the cytokine on pulmonary fluid reabsorption in in situ and in vivo flooded rat lungs. Receptor binding sites were blocked with the human soluble TNFR type 1 construct (sTNFR1), whereas the lectin-like domain was blunted with the oligosaccharide N,N'-diacetylchitobiose. We observed that in situ, TNF failed to stimulate alveolar liquid clearance, but did so together with the sTNFR1, and this activity was neutralized by N,N'-diacetylchitobiose. In vivo TNF inhibited liquid clearance, but activated it when complexed with the sTNFR1. A TNF-derived peptide mimic of the lectin-like domain activated fluid reabsorption in flooded lungs, and this activity was blunted by cotreatment with TNF. Our results thus indicate that in these models the receptor binding sites of TNF inhibit, whereas its lectin-like domain activates, edema reabsorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human genome encodes only ~30,000 proteins, which indicates the importance of alternative levels of protein complexity in mammals. The 51-kDa homotrimeric protein TNF is mainly known for its receptor-mediated proinflammatory functions in the systemic inflammatory response and apoptosis induction on a cellular level (reviewed in Refs.1 and 2). TNF also induces endothelial cell activation and barrier dysfunction (reviewed in Ref.3), both of which are implicated in the pathogenesis of pulmonary edema that is often associated with acute lung injury. Regarding pulmonary dysfunction, the cytokine as such can promote edema formation by means of 1) TNFR-dependent up-regulation of chemokine production (4, 5) and adhesion molecule expression (6, 7, 8), leading to neutrophil attraction and sequestration; 2) decrease in transendothelial electrical resistance across human pulmonary artery endothelial cells and rearrangement of microtubules (9); or 3) upon induction of reactive oxygen intermediates (10). Moreover, TNF down-regulates the expression of the epithelial sodium channel in type II alveolar epithelial cells, which is crucial in edema reabsorption, and thus can inhibit edema reabsorption (11). In contrast, apart from its role in edema formation and inhibition of edema reabsorption, blockade of TNF was found to increase the amount of edema formation in both the pulmonary and pancreatic microvascular beds (12). Moreover, the cytokine increased lung liquid clearance (LLC)2 in rodent models of inflammation, such as a rat pneumonia model (13), a rat intestinal ischemia-reperfusion model (14), and a model of severe bronchial allergic inflammation, associated with endothelial and epithelial leakage (15). The TNF-mediated activation of alveolar fluid clearance was suggested to involve an increased sodium uptake in type II alveolar epithelial cells by means of a catecholamine-independent mechanism (13, 14, 16).

The lectin-like domain of human TNF (hTNF) is located at a distance from the receptor binding sites at the tip of the molecule. This structural element recognizes specific oligosaccharides, such as N,N'-diacetylchitobiose and branched trimannoses (17, 18). This domain of TNF, moreover, mediates the trypanolytic activity of the cytokine (19) and activates amiloride-sensitive sodium transport in mammalian cells (20). The functions of the lectin-like domain of TNF can be mimicked by a synthetic circular peptide that contains 17 aa (Tip peptide (Tip)). In a blood-perfused flooded rat lung model, the murine Tip peptide was recently shown to activate edema reabsorption, in contrast to native murine TNF, which did not increase the fluid clearance (21). In view of the apparently conflicting data regarding the effects of TNF on pulmonary edema, we therefore investigated the relative contributions of receptor-dependent vs lectin-like activities of hTNF on edema reabsorption by means of blocking each functional domain with, on the one hand, a human soluble TNFR type 1 construct (sTNFR1) construct and, in contrast, the oligosaccharide N,N'-diacetylchitobiose.

Our results demonstrate a predominant role for the lectin-like domain of hTNF in the activation of edema reabsorption in situ and in vivo. This activity is counteracted by receptor-mediated activities of the cytokine, thus indicating a dichotomal role of TNF in LLC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Substances

The human circularized Tip peptide (hTip) and the mutated human Tip peptide (mtTip) were synthesized as described previously (19) and were purchased from EMC Echaz Microcollections. Human TNF was a gift from Dr. D. Maennel (University of Regensburg, Regensburg, Germany) and had a specific activity of 2 x 107 IU/mg. The PEGylated sTNFR1 construct (reviewed in Ref.22) was a gift from Dr. R. Kelly (Amgen, Thousand Oaks, CA). The anti-hTNF Ab infliximab (Remicade; Centocor) was purchased from Essex Pharma. N,N'-diacetylchitobiose and terbutaline were purchased from Sigma-Aldrich.

Animals

Specific pathogen-free female Wistar rats were used in the in situ and inflammation experiments, with a body weight (BW) of 235 ± 15 g. These animals were obtained from Harlan-Winkelmann. Specific pathogen-free male Sprague-Dawley rats for the in vivo experiments, 300 ± 25 g BW, were obtained from Charles River Wiga Deutschland. The animals were held in the animal facility of University of Konstanz. All animals received humane care in accordance with the national animal health guidelines and the legal requirements in Germany. Rats were kept at a temperature of 24°C in 55% humidity with 12-h light, 12-h dark cycles, and regular chow (Altromin C 1310) and water were provided ad libitum. Experiments were performed in the period from February to July in the years 2002–2003. The protocols were approved by the committee on animal experiments at the regional board of Freiburg, Germany.

In vivo flooded rat lung model

Animal preparation. Male Sprague-Dawley rats were anesthetized by an i.p. injection of 100 mg/kg sodium pentobarbital (Narcoren; Merial) and were placed in the supine position on a heating plate to maintain a core body temperature of 37°C. A tracheotomy was performed, a 14-gauge catheter (Insyte; i.v. catheter; BD Biosciences) was inserted in the trachea, and ventilation was started. A vascular catheter (PE50 tubing; BD Biosciences) was placed into the right carotid artery to monitor blood pressure and to obtain samples for arterial blood gas measurements. Another vascular catheter was placed in the right jugular vein to monitor central venous pressure and administer medications. Mean arterial pressure and central venous pressure were continuously recorded (Isotec transducers (Quest Medical) and CFBA amplifiers (Hugo Sachs Electronik-Harvard Apparatus)), and data were recorded on a PO-NEH-MAH computer-based data acquisition system (PO-NEH-MAH).

Experimental protocol. The rats were monitored for ~40 min to obtain stable baseline hemodynamics and lung mechanics. To obtain a mean blood pressure between 100 and 120 mm Hg, sodium pentobarbital was administered i.v. if needed. Muscle relaxation was maintained with i.v. administered pancuronium bromide (1 mg/kg/h; Pancuronium Inresa; Inresa Arzneimittel). After the stabilization period, 8 ml/kg BW prewarmed saline (37°C) were instilled intratracheally. Blood gases were measured 5, 10, 20, 35, 50, 65, and 80 min after fluid instillation (ABL50; Radiometer). The volume of removed blood was substituted with saline. At the end of the experiments, rats were killed by exsanguination. During the experiment, a forced oscillating technique (FOT) lung mechanic measurement was every 10 min performed to assess lung mechanics.

Ventilation and lung mechanic measurement. Animals were ventilated with a flexiVent piston-ventilator (SCIREQ; Scientific Respiratory Equipment). Tidal volume was set at 2 ml, because tidal volume during spontaneous ventilation in conscious rats has been reported to be 7.0 ± 0.4 ml/kg (23). The fraction of inspired oxygen was 1.0, and the positive end-expiratory pressure was set at 5 cm H2O. The respiratory rate was chosen to maintain normoventilation, reflecting a PaCO2 between 35 and 45 mm Hg.

FOT. Lung mechanics were measured with the flexiVent ventilator as described previously (24). FlexiVent software version 3.2 calculated respiratory input impedance (Zrs) from data collected by the flexiVent. Zrs is usefully interpreted by fitting the so-called constant phase model to measurements of Zrs over a frequency range of 0.25–19.625 Hz. As results from this calculation, the parameters Rn, G, and H were evaluated in these experiments. The parameter Rn (Newtonian resistance) is a frequency-independent component of respiratory resistance and reflects the airway resistance. The parameter G reflects viscous energy dissipation in the tissues and is therefore related to tissue resistance, whereas H reflects energy storage in the tissues and is related to tissue elasticity (24, 25). Three replicates of an 8-s perturbation for each time point were averaged and normalized on the baseline value before instillation of the fluid. Ten seconds before each FOT measurement, a single deep breath was initiated, with a maximal tidal volume of 4.5 ml, but limited to a pulmonary inspiratory pressure of 24 cm H2O.

Determination of the wet to dry weight ratio and LLC. After the animal was killed by exsanguination, the heart-lung block was excised. Adjacent blood or tissue was removed carefully. Lung lobes were removed, and the wet weight was determined. Lungs were dried in an oven at 55°C until the weight was constant, and the wet weight to dry weight ratio was calculated. To calculate LLC in six sham control experiments, rats received an air, instead of a fluid, instillation. The wet to dry weight ratios were determined (4.94 ± 0.08 SEM), and the theoretical initial wet to dry weight ratio, as if the rats would have received a fluid instillation, were calculated (15.54 ± 0.52 SEM). The measured wet to dry weight ratio was defined as 100%, and the calculated initial wet to dry ratio was defined as 0% liquid clearance.

Rat in vivo model to test inflammatory properties

Female Wistar rats were anesthetized by halothane in a case via an evaporator (flow, 2 l/min oxygen; 4% halothane). Then the animals were intubated using a Seldinger technique and a 14-gauge venous catheter (Insyte; i.v. catheter; BD Biosciences). The rat was held in the neck in an upright position. A microsprayer that nebulizes fluids (Penn-Century) was used to instill 200 µl of saline with or without testing substances intratracheally next to the bifurcation. After 8 h, the animals were anesthetized with an i.p. injection of sodium pentobarbital (160 mg/kg BW) and were killed by trans-section of the abdominal aorta. Subsequently, a bronchoalveolar lavage (BAL) was performed by flushing the lungs twice with 5 ml of ice-cold saline. The BAL fluid (BALF) was used to determine total and differential cell counts and mediator measurement in the supernatant. The supernatant was obtained by centrifugation for 10 min at 600 x g at 4°C.

Total and differential cell counts. Recruitment of inflammatory cells associated with TNF and Tip peptide instillation was examined. Total cell count was determined by counting the cells in the BALF with a standard Neubauer counting chamber. Cytospin preparations were made, and the dried cells were stained with standard May-Grunwald-Giemsa stain. From each experiment two cytospin slides were made, and digital photographs were taken from several different sections (Nikon Coolpix 995 through a special ocular with a Zeiss Axiovert 50). Two hundred cells were counted using standard morphological criteria for the different cell types.

Measurement of cytokine-induced neutrophil chemoattractant 3 (CINC-3). Supernatants from the BALFs were stored at –20°C. Rat CINC-3 was assessed with a commercially available ELISA (catalogue no. DY525; R&D Systems Europe), performed according to the manufacturer’s instructions.

Measurement of alveolar liquid clearance (ALC) in the rat in situ model

ALC was measured by the increase in total protein concentration 30 min after the intratracheal instillation of a 5% bovine albumin Ringer lactate solution, as similarly performed by others (13, 26, 27, 28, 29, 30, 31, 32, 33). Osmolality was adjusted with sodium chloride to be iso-osmolar with rat plasma (305 ± 3 mosmol/kg). The pH of the solution was 7.0. The in situ model used in this study was similar to that described previously (13). Briefly, female Wistar rats were anesthetized by i.p. injection of pentobarbital sodium (100 mg/kg BW) and were placed in the supine position on an adjustable heating plate. A tracheotomy was performed, and a 14-gauge venous catheter was inserted into the trachea. Continuous positive airway pressure with 8 cm H2O and 100% oxygen was maintained throughout the experiment. The rats were exsanguinated, and 10 ml/kg BW prewarmed (37°C) 5% albumin solution were instilled intratracheally. A sample of the instilled solution served as baseline sample. At the end of the experiment, lung fluid samples were obtained by holding the rat head down and collecting what was pouring out of the tracheal cannula, assisted by gentle pressure on the thorax. At least 600 µl of fluid were collected. The samples were centrifuged for 10 min at 3000 x g, and supernatants were kept for the measurement of total protein concentration with a standard pierce bicinchoninic acid microassay. Thoracic temperature was measured with a thin thermometer probe through the esophagus (GMH3230; Greisinger Electronic). The heating plate was adjusted to maintain a thoracic temperature between 37.0 and 38.5°C. ALC was calculated according to the following equation, reported previously (33): ALC = ((VI – VF)/VI) x 100, where VI is the initial instilled fluid volume, and VF is the final alveolar fluid volume calculated from the increase in protein concentration in the final alveolar fluid. It was shown that protein transport or diffusion over an intact alveolar epithelium in both directions in different in situ, ex vivo, and in vivo models is negligible (28, 31, 33, 34).

Statistics

Data in the figures are given as the mean ± SEM. Data were analyzed by one-way ANOVA with the GraphPad PRISM 3 software package. Dunnett’s multiple comparison test was used for correction of multiple comparisons. It was tested vs saline controls unless otherwise indicated. Grubb’s test was used for detecting outliers. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Activity of TNF on edema reabsorption in the presence of the sTNFR1 construct in vivo and in situ

In view of the apparently conflicting data concerning the inhibitory vs activating role of TNF on the reabsorption of lung edema, we investigated the respective contributions of the receptor-binding and lectin-like domains in an in situ and an in vivo flooded rat lung model. In the in situ flooded rat lung, ALC, i.e., the amount of fluid transported over the epithelium out of the alveolar space, was measured 30 min after the intratracheal instillation of Ringer’s lactate with 5% BSA, with or without the test substances, by means of assessing the evolution of the BSA concentration. The 30 min point was previously shown by others to be optimal for this setting (16). In this in situ model, we could assess the TNF-modulated fluid transport over the intact epithelial barrier without interference by blood, blood pressure, or infiltrating cells, which can possibly contribute to edema formation or to inhibition of edema reabsorption. In contrast, in the in vivo flooded rat lung model, LLC, i.e., the amount of fluid that is actually cleared from the lungs, including interstitium, vessels, and lymphatics, was measured. To that purpose, rat lungs were flooded with 8 ml/kg saline, and LLC was estimated by the change in the wet-to-dry lung weight ratio 80 min after flooding. In this model, apart from edema reabsorption, edema formation and inflammation can occur. As shown in Fig. 1, TNF failed to stimulate liquid clearance in the in situ flooded rat lung, as was also found in an isolated Krebs-Henseleit buffer (data not shown) or blood-perfused (21) flooded rat lung preparation. However, coinstillation of TNF with the sTNFR1 construct in a molar ratio of 260:1 to TNF led to activation of ALC (Fig. 1). This switch was neutralized in the presence of N,N'-diacetylchitobiose (Fig. 1), a sugar that neutralizes the activity of the lectin-like domain of TNF (17, 18). Unlike the sTNFR1 construct, the humanized neutralizing anti-hTNF mAb infliximab, at a molar ratio of 30:1 to TNF, i.e., a concentration sufficient to inhibit all hTNF bioactivity in the WEHI 164 assay in vitro (data not shown), did not induce this switch.



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  		FIGURE 1. Impact on ALC in an in situ flooded rat lung model. A, Blocking the receptor binding site of hTNF (5 µg/lung; n = 7) with a sTNFR1 construct (1 mg/lung; n = 6) or blocking the lectin-like domain with N,N'-diacetylchitobiose (Chi; 650 µg/lung; n = 5), saline (n = 16), TNF plus sTNFR1 (+sTNFR1; n = 6), or TNF plus N,N'-diacetylchitobiose and sTNFR1 (+Chi/sTNFR1; n = 6). B, Blockade of the receptor binding site of hTNF with the mAb infliximab (Infl; 450 µg/lung; n = 3) or TNF plus infliximab (+Infl; n = 3). Substances were preincubated with TNF for 30 min at room temperature.

 
To better evaluate the physiological relevance of the previous findings, we next assessed the action of hTNF on LLC. However, in view of the differential effects of the sTNFR1 construct vs infliximab on the in situ flooded lung model, we first compared their efficacies to inhibit TNF-mediated changes in respiratory parameters in the in vivo flooded rat lung in supplementary lung function measurements with the FOT. As such, we assessed the parameter G, which essentially reflects tissue resistance. Changes in parameter G reflect changes in either tissue physical properties or regional small airways heterogeneity. Therefore, this lung mechanic parameter is probably the most adequate to evaluate lung edema. In untreated lungs, tissue resistance increased initially upon flooding of the rat lung, due to the distribution of fluid into interstitial compartments of the lung. This distribution seems to already be maximal in the control experiments, because there was no difference among the saline (1.81 ± 0.11; n = 17), sTNFR1 construct (1.88 ± 0.27; n = 5), and infliximab (2.32 ± 0.29, n = 3) experiments. Compared with the control groups, TNF treatment led to an attenuation of the initial increase in tissue resistance, and this was blunted in the presence of either the sTNFR1 construct or infliximab (Fig. 2). These data thus indicate that the receptor binding sites of TNF are causally involved in the decreased tissue resistance. As shown in Fig. 3, upon intratracheal instillation of hTNF (0.5 or 5 µg/rat) in the flooded rat lung in vivo, basal LLC was inhibited by ~20%. This action of TNF in the in vivo model is in contrast to what was found in the in situ model, and the inhibition of LLC could at least partially reflect the proinflammatory component of the cytokine. In the presence of the sTNFR1 construct in a molar ratio of 2600:1 to TNF, a concentration ratio required to neutralize all TNF activity in the WEHI 164 cytotoxicity assay (data not shown), a switch from inhibition to activation of LLC was observed (Fig. 3). In contrast, a 3-fold lower dose of the sTNFR1 construct, which did not lead to a full neutralization of TNF activity in vitro (data not shown), neutralized the negative effect of TNF, but did not induce the switch toward an activation of LLC. Unlike the sTNFR1 construct, the humanized neutralizing anti-hTNF mAb infliximab, at a molar ratio of 30:1 to TNF, did not induce this switch. In control experiments, the sTNFR1 construct or infliximab alone had no measurable effect.



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  		FIGURE 2. Effect of blockade of receptor binding sites of hTNF (5 µg/lung; n = 5) with either infliximab (Infl; 450 µg/lung; n = 3) or a sTNFR1 construct (10 mg/lung; n = 5) on the tissue resistance (parameter G). FOT lung mechanics were measured in vivo 5 min after intratracheal fluid instillation. The parameter G is related to tissue resistance and was normalized. A, sTNFR1 was instilled alone or after preincubation with TNF (+TNF; n = 4). B, The anti-TNF mAb infliximab was instilled alone or after preincubation with TNF (+Infl; n = 3). The dotted line represents the saline baseline value. Significance was determined by one-way ANOVA and Dunnett’s multiple comparison test vs sTNFR1 and infliximab, respectively.

 


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  		FIGURE 3. Action on LLC capacity 80 min after alveolar fluid instillation. Blockade of the receptor binding site of hTNF (5 µg/lung; n = 6) after treatment with infliximab (Infl; 450 µg/lung; n = 4), or the sTNFR1 construct (3 and 10 mg/lung; n = 4 and 6) saline controls (n = 14). TNF was incubated for 30 min at room temperature with either infliximab (+Infl; n = 4) or the sTNFR1 construct (+sTNFR1; 3 and 10 mg/lung; n = 5 and 6) before instillation. *, vs saline group; #, vs TNF group.

 
The results from the in situ and in vivo flooded rat lung experiments thus indicated that the lectin-like domain of TNF activates, whereas the receptor binding sites inhibit, LLC. Moreover, in contrast to the neutralizing mAb infliximab, which probably inhibits the activities of both the receptor binding sites and the lectin-like domain of TNF, the sTNFR1 construct specifically blunts the activities transmitted by the receptor binding sites, but does not interfere with the activity of the lectin-like domain.

A peptide mimicking the lectin-like domain of hTNF activates fluid reabsorption in situ and in vivo

To substantiate our hypothesis that the lectin-like domain of hTNF activates edema reabsorption, we assessed the activity of the hTip peptide, which mimics this domain. Like the {beta}2-adrenergic agonist terbutaline (10–4 M), the hTip peptide (125 µg/rat in vivo and 1 mg/rat in situ) activated fluid reabsorption in the in situ (Fig. 4A) and in vivo (Fig. 4B) flooded rat lung models. In the latter model, TNF had an inhibitory effect on LLC at both 0.5 and 5 µg/rat intratracheally (Fig. 4B). In vivo, the activity of the hTip peptide was blocked after adding a 5-fold molar excess of N,N'-diacetylchitobiose to the instillate (data not shown). The instillation of a control peptide (mtTip), in which three critical amino acids for the sodium transport activating activity of TNF were mutated, showed no effect on the LLC (data not shown).



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  		FIGURE 4. A, Impact of the coinstillation of TNF together with terbutaline or hTip on ALC after 30 min. Rat lungs were flooded with Ringer’s lactate containing 5% BSA. Protein content was measured before and 30 min after intratracheal application of the fluid: saline (n = 16), hTNF (5 µg/lung; n = 7), terbutaline (Ter; 10–4 M; n = 9), terbutaline plus TNF (n = 6), Tip (1 mg/lung; n = 14), and Tip plus TNF (n = 6). B, Action of the TNF-derived Tip peptide on LLC 80 min after alveolar instillation of saline (n = 14) or saline containing terbutaline (Ter; 10–5 M; n = 11), hTNF (0.5 µg/rat, n = 4; 5 µg/rat, n = 6), or Tip (125 µg/lung; n = 13).

 
The hTip peptide shows no proinflammatory activity in rat lungs compared with hTNF

To investigate inflammatory properties, rats received an instillation of 200 µl of saline alone or containing hTip peptide, terbutaline, or hTNF. After 8 h, the animals were killed, and BALs were performed to assess differential cell counts. The number of granulocytes was slightly increased in all groups, indicating a modest inflammation as a consequence of fluid instillation. However, this increase was far greater in the hTNF-treated lungs compared with that in lungs treated with saline, hTip peptide, or terbutaline (Fig. 5A). These data are in agreement with the increased production of the neutrophil chemotactic agent, CINC-3, in the BALF of hTNF-treated animals, which was not seen in hTip peptide- or terbutaline-treated animals (Fig. 5B).



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  		FIGURE 5. Assessment of possible proinflammatory properties of the TNF-derived Tip peptide. Two hundred microliters of saline (n = 6) or saline containing terbutaline (Ter; 10–5 M; n = 4), Tip (125 µg/lung; n = 6), or hTNF (5 µg/lung, n = 7) were applied intratracheally to rats. Eight hours after instillation, BALs were performed, and differential cell counts (A) and CINC-3 measurements (B) were assessed. AM, alveolar macrophage; Neu, neutrophil.

 
Receptor binding sites of TNF oppose activation of reabsorption by lectin-like domain

The combined treatment of hTNF and the hTip peptide or terbutaline blunted the fluid reabsorption-activating capacity of these agents in the in situ flooded rat lung (Fig. 4A). Thus, hTNF is able to intervene in edema reabsorption with a mechanism shared by the hTip peptide and terbutaline. The duration of the in situ experiments implies a fast onset of TNF action on fluid transport.

Taken together, our results indicate that hTNF possesses two spatially distinct domains with opposite actions on edema reabsorption: the receptor binding sites, which inhibit edema reabsorption and mediate proinflammatory properties, and the lectin-like domain, which, in contrast, activates pulmonary fluid clearance without sharing the proinflammatory activity of the original cytokine (Fig. 6).



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  		FIGURE 6. Dichotomal role of TNF in edema reabsorption. The receptor binding sites, shown in green, inhibit edema reabsorption, an activity that can be specifically blocked by the sTNFR1 construct. In contrast, the lectin-like domain of the molecule, shown in yellow, activates LLC and can be specifically inhibited by N,N'-diacetylchitobiose or mimicked by a 17-aa peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Pulmonary alveolar edema is a life-threatening condition resulting from an imbalance between forces driving fluid into the airspaces and the physiological mechanisms involved in its removal. The epithelial sodium channel was shown to exert a crucial role in pulmonary fluid reabsorption (reviewed in Ref.35). Recently, TNF was shown to directly decrease the expression of this sodium channel in vitro (11). Moreover, the initiation of the neutrophil migration process to the lungs is attributed to TNFR signaling (5). This process, which contributes to the formation of pulmonary edema, involves regulatory mechanisms such as complement activation, cytokine regulation, chemokine production, and activation of adhesion molecules and their respective counterreceptors (reviewed in Ref.36). In contrast to its role in the formation of edema or the inhibition of edema reabsorption, TNF was shown to increase fluid reabsorption during bacterial infection (13) and ischemia reperfusion (14) models in rats. Although antagonistic Abs against the TNFRs were shown to interfere with the sodium uptake-activating effect of TNF in human A549 cells (16), in a flooded mouse model in situ, the edema reabsorption-stimulating activity of murine TNF was shown to be identical in wild-type and double-TNFR knockout mice, indicating that receptor-independent activities of TNF predominate in this activity (21). In line with this assumption, a TNF lectin-deficient mutant, which still efficiently binds to both TNFRs, completely lacked the ability to activate sodium uptake in A549 cells (16).

In view of the apparently conflicting data concerning the inhibitory vs activating roles of TNF on liquid clearance, our results indicate that hTNF possesses at least two spatially distinct domains with opposing actions on experimental edema reabsorption. Beyond the experimental situation examined in this study, the observed switch from the LLC-inhibiting effect of hTNF to an LLC-activating one upon complexation with sTNFR1 could be pathophysiologically relevant in inflammatory conditions such as bacterial pneumonia as well as cardiogenic and septic shock. Indeed, in all these pathological situations, increased concentrations of both TNF and its soluble receptors were found within the lungs (37). The apparently contradictory results concerning the outcome of TNF treatment on edema reabsorption between the in situ and in vivo flooded rat lung models (null effect vs inhibitory effect, respectively) could reflect differences between experiments with living animals or "dead" organs. Likewise, we previously found null effects in ex vivo blood-perfused (21) or Krebs-Henseleit buffer-perfused (data not shown) flooded rat lung models. Furthermore, in the ex vivo blood-perfused flooded rat lung, there was no significant increase in alveolar leukocytes in TNF-treated lungs compared with the saline control (21). Although we observed increased neutrophil BALF counts in the TNF-treated animals in vivo at 8 h after treatment, we cannot exclude that their infiltration already occurs at much earlier time points, as was reported in other inflammation models (38, 39) and a human endotoxin inhalation study (40). Therefore, we propose that the negative TNF action on edema reabsorption in vivo could at least partially be caused by the presence of infiltrating neutrophils. Our observation that we need higher concentrations of sTNFR1 construct in vivo (10 mg/rat) than in situ (1 mg/rat) to switch the TNF activity toward activation of fluid reabsorption would support the hypothesis that the receptor-dependent activities of TNF have more components in vivo than in situ, and that this could be linked to infiltrating cells. Studies accessing the roles of both TNFRs in the activity of TNF on edema reabsorption and formation would provide valuable information about the implicated mechanism. One possible explanation for the observation by others that under certain conditions TNF is able to stimulate edema reabsorption in an in situ flooded rat lung (13), whereas we found it not to do so in the time period in which the experiments were performed, could be explained by a variation in the presence of factors antagonizing receptor-mediated TNF actions in the treated animals, caused either by differences in the induction of inflammation due to the protocols used or by variations in basal levels of these agents (41, 42).

The sTNFR1 was found not to interfere with the lectin-like activity of TNF (19); however, as demonstrated with the 1F3F3 neutralizing rat anti-mTNF mAb (43), mAbs, such as infliximab may well interfere with the function of the lectin-like domain by means of either steric hindrance or conformational changes in TNF. In fact, previous studies have observed that the neutralization of endogenous TNF with mAbs in a pneumonia model (13) or in an intestinal ischemia-reperfusion model (14) in rats led to decreased LLC. This might be interpreted such that the function of the lectin-like domain in the activation of edema reabsorption was indeed affected by these Abs. In contrast, in the presence of sTNFRs, as occurs during infection, the positive activity of the lectin-like domain on edema reabsorption seems to dominate, which could provide an explanation for the observation that a positive TNF action was reported mainly in infection models. It should be noted in this study that local TNF concentrations in the lungs of rats with LPS-induced lung injury can reach values > µg/ml, taking into account the dilution factor of the BAL (44). Moreover, in rat adjuvant arthritis studies, administration of up to 10 mg/kg, three times weekly, of the polyethylene glycolated sTNFR1 construct had been used (45). Therefore, with the 1 mg/rat in situ and 10 mg/rat in vivo doses used in our study, we are within this range. Also, the sTNFR2 construct etanercept (Enbrel) is clinically used in relatively high doses (0.4 mg/kg in children up to 0.7 mg/kg in adults, twice weekly). If we would use comparable amounts of sTNFR1 construct as of infliximab (i.e., a 30:1 ratio), we would not obtain sufficient inhibition of the TNFR-mediated effects, as indicated by our own findings in the WEHI 164 cytotoxicity assay (data not shown). In a comparable study, the inhibitory effect of Etanercept and infliximab on TNF-induced E-selectin expression was investigated. At a molar ratio of 30:1 to TNF, Etanercept only weakly inhibited E-selectin expression, whereas full neutralization occurred with infliximab (46). Our observation that the preincubation of TNF with N,N'-diacetylchitobiose is able to block TNF-mediated Na+ transport (20) as well as TNF-mediated activation of edema reabsorption indicates that TNF is binding directly to sugar groups of a still unknown receptor on alveolar epithelial cells. We are currently investigating this possibility. Moreover, the assessment of sodium channel expression in our models could provide data about the effect of TNF on their regulation. In clinical terms, in some indications patients may benefit from treatment with the sTNFR1 construct instead of infliximab, because the favorable activity of the lectin-like domain of TNF on edema reabsorption is preserved (e.g., patients with idiopathic pneumonia syndrome after allogeneic hemopoietic stem cell transplantation (47)). However, it should be stressed that we used the TNF-neutralizing substances as a tool to investigate compartmentalized, short-term actions of TNF in intact lungs, rather than as a systemically acting, therapeutic agent. To obtain high intra-alveolar concentrations, we applied the substances intratracheally instead of by the classically used i.v. or s.c. route. Moreover, because in our flooded rat lung models an intact alveolar-epithelial barrier is present, we cannot necessarily extrapolate our findings to an acute lung injury setting, in which these barriers are damaged. Therefore, more detailed studies should be performed in lung injury-related pulmonary edema models before the conclusion can be drawn that the sTNFR1 construct has advantages over infliximab.

Our study provides evidence that sTNFRs, apart from neutralizing (48) and stabilizing (49) TNF activity or controlling thresholds of innate immune activation (50), may essentially change even the activity of the cytokine. The concept proposed in this study of a dichotomal role of TNF in edema reabsorption (Fig. 6) extends the general hypothesis that sTNFRs block all known TNF activities to a more detailed molecular understanding. Our data suggest that, as is the case with the recently reported complement receptor 3 (51), TNF is an example of a "moonlighting protein" (reviewed in Ref.52), with differential activities mediated by its receptor-binding or lectin-like domains, which opens the possibility to design and develop more sophisticated therapeutic regimens to overcome the deleterious fluid accumulation in some major lung pathologies.


   Acknowledgments
 
We thank Drs. L. Casaer, S. Meija, and R. Kelly (Amgen) for the generous gift of the sTNFR1; Dr. J. Paal (Altana-Pharma) for his kind help with the molecular graphics, as well as Drs. M. Matthay, J.-F. Pittet (University of California, San Francisco, CA), and S. von Aulock (University of Konstanz, Konstanz, Germany) for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 Address correspondence and reprint requests to Drs. Rudolf Lucas and Albrecht Wendel, Medical and Pharmaceutical Biotechnology, University of Applied Sciences, Piaristengasse 1, A-3500, Krems, Austria. E-mail address: rudolf.lucas{at}imc-krems.ac.at Back

2 Abbreviations used in this paper: LLC, lung liquid clearance; ALC, alveolar liquid clearance; BAL, bronchoalveolar lavage; BALF, BAL fluid; BW, body weight; CINC-3, cytokine-induced neutrophil chemoattractant 3; FOT, forced oscillation technique; hTip, human circularized Tip peptide; mtTip, mutant Tip peptide; hTNF, human TNF; sTNFR1 construct, human soluble TNFR type 1 construct; Tip, human Tip peptide; Zrs, respiratory input impedance. Back

Received for publication October 8, 2004. Accepted for publication June 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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