Effect of surfactant on pulmonary expression of type IIA PLA2 in an animal model of acute lung injury

Yongzheng Wu, Monique Singer, Françoise Thouron, Mounia Alaoui-El-Azher, and Lhousseine Touqui

Unité de Défense Innée et Inflammation, Unité Associée Pasteur/Institut National de la Santé et de la Recherche Médicale U485, Institut Pasteur, 75015 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A2 (sPLA2-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA2-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA2-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 µg/kg) induced an increase in pulmonary expression of sPLA2-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA2-IIA than AM from saline-treated animals. This ex vivo sPLA2-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA2-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.

phospholipase A2; alveolar macrophage; lipopolysaccharide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is a clinical and pathophysiologically complex syndrome of an acute life-threatening lung injury characterized by severe dyspnea, arterial hypoxemia, decreased lung compliance, and diffuse bilateral infiltrates (4, 21). Numerous predisposing factors can be involved in the etiology of this complex disease, including pneumonia and bacterial sepsis. The mechanisms involved in the development of ARDS remain uncertain and, despite years of intensive research, no specific therapy has been proven to be effective in preventing or reversing the disease. Consequently, mortality remains unacceptably high at 30-50% (13, 26).

Pathophysiological features of ARDS involve, among other parameters, an alteration of pulmonary surfactant (12, 19). The latter is a lipid-protein complex that lowers surface tension along the alveolar epithelium, thereby promoting alveolar stability and preventing collapse of alveoli (27). Destruction of surfactant results in an increase in the surface tension at the air-liquid interface, which results in alveolar and peripheral tracheal collapse (7). However, the mechanisms concerned with surfactant alterations have not been elucidated clearly (3). Using an animal model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), our group recently showed that lung tissues produce a secretory type IIA phospholipase A2 (sPLA2-IIA) and that alveolar macrophages (AM) are the major cell source of this enzyme (2). PLA2 belongs to a widely distributed class of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position, leading to the concomitant release of fatty acids and lysophospholipids. In mammals, PLA2 forms a large family of enzymes that can be schematically divided into two major classes: high-molecular-weight intracellular PLA2 and low-molecular-weight secreted PLA2, including sPLA2-IIA. The latter is found at high levels in the circulation and locally in the tissues and has been suggested to play a role in a number of inflammatory diseases (25). Our recent study showed that sPLA2-IIA catalyzes the hydrolysis of surfactant phospholipids and suggested that this process can contribute to the loss of surface tension-lowering properties of surfactant (1).

On the other hand, it is clearly established that, besides its mechanical properties, surfactant also exhibits anti-inflammatory and immunoregulatory functions (15, 30). Indeed, surfactant has been shown to inhibit a number of cell functions, including lymphocyte proliferation (29), synthesis of sPLA2-IIA, and secretion of tumor necrosis factor-alpha (TNF-alpha ) by AM (5, 10, 23). Our previous study suggested that in normal animals the in vivo synthesis of sPLA2-IIA might be repressed by surfactant that blocks the expression of this enzyme by AM (9). These inflammatory cells may escape from the inhibitory effect of endogenous surfactant once they are removed from the lung or in conditions where the concentrations of surfactant in alveoli are reduced, which may result in an increase in the synthesis and secretion of sPLA2-IIA, leading to the exacerbation of inflammation. Thus we hypothesized that inhibition of the synthesis of sPLA2-IIA by exogenous surfactant may be an additional or alternative mechanism that might account for the clinical benefit of surfactant therapy.

The aim of the present studies was to investigate the effect of intratracheal administration of a seminatural surfactant, Curosurf, on the pulmonary synthesis of sPLA2-IIA in a guinea pig model of LPS-induced ALI. We also examined whether application of Curosurf was able to reduce the lung inflammation accompanying sPLA2-IIA expression in this model.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animal and Experimental Procedure

Animal groups. Male Hartley guinea pigs (Saint Antoine, Pleudaniel, France; 500 ± 50 g) were kept on a regular 12:12-h light-dark cycle and at room temperature. Food and water were given ad libitum.

Two sets of experiments were performed. In the first set of experiments, Curosurf (or its control saline) was applied 30 min after LPS administration, and protein concentration, cell counts, sPLA2-IIA activities, and TNF-alpha levels were measured 8 h later in bronchoalveolar lavage (BAL). Bronchial reactivity was followed throughout the 8 h after LPS instillation. At the end of the experiment, AM were collected as indicated below and cultured for an additional 24 h. Then sPLA2-IIA activity and mRNA levels were measured at the indicated time intervals (see below). In the second set of experiments, Curosurf (or its control saline) was applied 8 h after LPS administration, and protein concentration, cell counts, sPLA2-IIA activities, and TNF-alpha levels were measured 24 h later.

In all these studies, four different groups of guinea pigs were used. The same number of animals was used in the 8- and the 24-h protocol: 1) LPS group (n = 9), 2) LPS + saline group (n = 9), 3) LPS + Curosurf group (n = 8), and 4) saline group (n = 7).

Experimental procedure. Animals were preanesthetized via intramuscular injection of ketamine hydrochloride (50 mg/kg body wt) and xylazine (2 mg/kg body wt). LPS (from Escherichia coli 055:B5; Difco, Detroit, MI) was dissolved in 0.9% saline. Then a polyethylene tube was introduced into the trachea. After orotracheal intubations, LPS (330 µg/kg) or the equivalent volume of saline was instilled through the intratracheal catheter. After 30 min or 8 h, Curosurf (16 mg/animal) or an equivalent volume of saline was administered rapidly through an orotracheal catheter. After the drugs were instilled into the lung, the hind part of the guinea pig was lowered and rotated slowly to distribute drugs to all lung lobes and to peripheral sections of the lung. This instillation methodology has been shown to provide surfactant distribution to most lung fields (11, 20). After the surfactant or saline instillation, the catheter was removed and the animals were left at room temperature and given water and food until they were killed.

At 8 or 24 h after intratracheal administration of LPS, guinea pigs were killed with an overdose of pentobarbital sodium (40 mg/kg). BAL was performed with 100 ml of saline containing streptomycin (25 µg/ml) and penicillin (25 µg/ml). Aliquots (10 ml) were injected with a plastic syringe through a polyethylene catheter inserted into the trachea. The first 10 ml of BAL were centrifuged at 475 g for 10 min. Then corresponding cell-free BAL fluids (BALF) were centrifuged at 10,000 g for 20 min to remove surfactant and were used for the measurement of sPLA2-IIA activity, TNF-alpha levels, and protein concentration. The remaining 90 ml were also centrifuged, and their pellets were pooled with those of the first 10 ml for measurement of total BAL leukocytes. The latter were counted on a hemocytometer, and a differential cell count was based on morphology and staining characteristics by using modified May-Grunwald-Giemsa-stained cytocentrifuge smears.

Preparation of Surfactant

The seminatural surfactant Curosurf was prepared in the Chiesi Laboratory (Geneva, Switzerland) from porcine lungs as described elsewhere (16) and was provided by Serono Laboratories (Boulogne, France). This surfactant is composed of 99% phospholipids (including 74.5% phosphatidylcholine, 6.9% lysophosphatidylcholine, 8.1% sphingomyelin, 3.3% phosphatidylinositol, 4.5% phosphatidylethalonamine, and 1.2% phosphatidylglycerol) and 1% hydrophobic proteins (surfactant proteins B and C) (18). Before administration, Curosurf was ultrasonicated for 10 min using a 150-W sonifier (MSE, Annemasse, France) at room temperature.

Macrophage Isolation and Culture

After centrifugation at 475 g for 10 min, the pellets of the BAL were washed twice with saline and then resuspended in RPMI 1640 culture medium containing 1% antibiotics and 3% fetal calf serum. Cells were adjusted at 3 × 106 cells/ml and then allowed to adhere in a 12-well culture dish for 1 h at 37°C in 5% CO2-95% air. After 1 h, the adherent cells consisted of 95-99% macrophages. The plates were then washed twice with medium and incubated with serum-free medium again for 24 h. The cell viability was checked by the trypan blue dye exclusion test and was always >90%. To control cell lysis, the release of lactate dehydrogenase activity was monitored at the indicated time using a commercial kit from Boehringer (Mannheim, Germany). After incubation, the culture dishes were kept in an ice bath, supernatants were harvested and centrifuged at 1,500 g for 5 min at 4°C to remove detached cells, and 200-µl aliquots were stored at -20°C until use. Adherent macrophages were resuspended in 500 µl of cold Hanks' buffered saline solution containing 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (2 µg/ml), aprotinin (2 µg/ml), and 2 mM EDTA and scraped using a rubber policeman. Cells were then lysed in an ice bath using an MSE sonifier and kept at -20°C until use.

Measurement of sPLA2-IIA Activity

sPLA2-IIA activity was measured using the fluorometric assay, as described previously by Radvanyi et al. (14). The activity of enzyme was measured by hydrolysis of the fluorescent phospholipid 1-hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphoglycerol. Briefly, the fluorescent phospholipid was dried under nitrogen and suspended in ethanol at 0.2 mM. Vesicles were prepared by mixing the ethanol solution of the fluorescent phospholipid with a buffer solution containing 50 mM Tris · HCl, 500 mM NaCl, and 1 mM EGTA (pH 7.5). After 2 min of vigorous agitation, 960 µl of substrate solution were mixed in the cuvette with 10 µl of 10% fatty acid-free BSA. Assays were performed using 50 µl of sample from macrophage homogenates, cell-free culture medium, or BALF, which were maintained in the ice bath throughout the assay. The reactions were then initiated with 10 µl of CaCl2 at a final concentration of 10 mM in 4 × 10-mm disposable plastic cuvettes. The fluorescence measurements were performed with a spectrofluorometer (model JY3D, Jobin and Yvon) equipped with a xenon lamp and monitored using excitation and emission wavelengths of 345 and 398 nm, respectively, with a slit width of 4 nm. The final ethanol concentration was <0.1% and had no effect on the assay.

Measurement of TNF-alpha Levels

The quantitation of TNF-alpha levels was based on the measurement of TNF-alpha bioactivity, which was determined by cytotoxicity on fibrosarcoma cells (WEHI 164, clone 13). The cells were grown in Dulbecco's medium supplemented with 10% fetal calf serum and antibiotics [1% (wt/vol) gentamicin and 1% (wt/vol) amphotericin B] in a humidified atmosphere of 5% CO2. Cells (106/ml) were incubated for 3 h in the presence of actinomycin D (1 µg/ml). Aliquots of this cell suspension (50 µl/well containing 5 × 104 cells) were plated in 96 wells and incubated for 24 h with 50-µl samples of TNF-alpha standard dilutions in triplicate. The plates were further incubated for 24 h with sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (50 µl/well) labeling mixture prepared as recommended by the manufacturer. Optical density was measured in an automatic reader with a test wavelength of 490 nm and a reference wavelength of 630 nm.

Protein Assay

Total protein was quantitated in the supernatant of BAL according to the method of Bradford (6) in 96-well microtiter plates at 595 nm with ovalbumin as a standard.

Measurement of Bronchial Reactivity

The bronchial response of freely moving guinea pigs to different treatments was measured by barometric plethysmography using whole body plethysmography (Buxco Electronics) according to the methods of Hamelmann et al. (8). Briefly, guinea pigs were placed in a main chamber, and the pressure difference between the main chamber containing the guinea pigs and a reference chamber was measured with a differential pressure transducer connected to an amplifier and recorded with BioSystem XA analyzer software (Buxco Electronics). During the respiratory cycle of the animals, volume and resultant pressure changes in the main chamber produced a pressure signal. Data were obtained at baseline and after the different treatments and recorded for 10 min. Enhanced pause (Penh), which is a dimensionless parameter representing airway flow limitation (8, 17) and correlated very closely with pulmonary resistance measured by a conventional two-chamber plethysmograph in ventilated animals (8), was calculated according to the manufacturer's instructions as follows: Penh=(TE-TR)/TR(PEP/PIP), where TE is expiratory time (s), TR is relaxation time (time of the pressure decay to 36% of total box pressure at expiration), PEP is peak expiratory pressure (ml/s), and PIP is peak inspiratory pressure (ml/s). After administration of the drugs, the main alteration of the pressure signal occurs during early expiration and leads to changes in the pressure signal in the main chamber. Penh can reflect pressure changes in the main chamber during inspiration and expiration (PIP and PEP), and these changes can be combined with the timing comparison of early and late expiration.

Expression of sPLA2-IIA and TNF-alpha mRNAs

Total RNAs were extracted from AM with the RNeasy kit (Qiagen) or from lung tissues as previously described (28), electrophoresed on a 1% agarose gel by the formaldehyde method, and then transferred onto nylon membranes (Amersham, Buckingham, UK). Full-length guinea pig sPLA2-IIA, guinea pig TNF-alpha , or mouse beta -actin cDNA was labeled with [alpha -32P]dCTP (ICN Biochemical, Orsay, France) by random priming. The blots were hybridized overnight with sPLA2-IIA or TNF-alpha cDNA at 65°C in Church buffer (7% SDS, 0.5 M NaPi, pH 7.2, 1 mM EDTA, 1% BSA) and washed in 2× saline-sodium citrate (SSC) and 1% SDS, 1× SSC and 0.2% SDS, and then 0.5× SSC and 0.2% SDS. Blots were washed and rehybridized with mouse beta -actin cDNA at 60°C as internal control, because Northern blot analysis showed that neither Curosurf nor LPS had any effect on beta -actin level. sPLA2-IIA and TNF-alpha mRNA levels were normalized to beta -actin. After hybridization, the blots were analyzed by autoradiography. Quantitation of signals was also achieved by a PhosphorImager using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Statistics

Values are means ± SE for all experiments, and statistical analysis was performed using one-way ANOVA on SPSS 8.0 software. Student-Newman-Keuls post hoc test was used to analyze multiple comparisons. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Curosurf on Pulmonary sPLA2-IIA Synthesis

Figure 1 shows that LPS instillation to guinea pigs induced a marked increase of sPLA2-IIA activity in the BALF, the higher level being observed 8 h after LPS challenge. When Curosurf was instilled into the lung 30 min after LPS, a marked decrease was observed in the level of sPLA2-IIA activity measured 8 h after LPS administration (Fig. 1A). A comparable loss in sPLA2-IIA activity was observed when Curosurf was applied 8 h after LPS instillation and sPLA2-IIA activity was measured in the following 24 h (Fig. 1B). In both cases, no significant difference was observed between LPS- and LPS + saline-treated animals. In addition, Curosurf had no significant effect on basal sPLA2-IIA activity: 0.740 ± 0.149 and 0.304 ± 0.184 nmol · ml-1 · min-1 in saline + Curosurf and saline groups, respectively.


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Fig. 1.   Effect of Curosurf on type II secretory phospholipase A2 (sPLA2-IIA) activity in bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-treated guinea pigs. A: 30 min after intratracheal instillation of LPS (330 µg/kg, n = 9) or saline (100 µl, n = 7), Curosurf (16 mg/animal, n = 8) or an equivalent volume of saline (n = 9) was administered intratracheally. At 8 h after LPS challenge, sPLA2-IIA activity was assayed in BALF. B: 8 h after intratracheal instillation of LPS (330 µg/kg, n = 9) or saline (100 µl, n = 7), Curosurf (16 mg/animal, n = 8) or an equivalent volume of saline (n = 9) was administered intratracheally. At 24 h after LPS challenges, sPLA2-IIA activity was assayed in BALF. * P < 0.05; ** P < 0.01.

Our results also showed that Curosurf instillation reduced the levels of sPLA2-IIA mRNAs in lung tissues from LPS-treated animals whether Curosurf was administered 30 min or 8 h after LPS (Fig. 2). This suggests that Curosurf inhibits the pulmonary synthesis of sPLA2-IIA, probably by interfering with its expression at a transcriptional level.


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Fig. 2.   Effect of Curosurf on expression of sPLA2-IIA mRNA in lung tissues. Total RNA from fresh lung tissue was extracted, and Northern blots were performed using guinea pig sPLA2-IIA cDNA as a probe. Mouse beta -actin cDNA was used as an internal control. Levels of sPLA2-IIA mRNA were quantitated using a PhosphorImager. Results are expressed as the ratio of sPLA2-IIA to beta -actin. A: total RNA was extracted from lung tissue 8 h after LPS instillation (n = 16). B: total RNA was extracted from lung tissue 24 h after LPS challenge (n = 16). * P < 0.05; ** P < 0.01. There is no significant difference between LPS and LPS + saline group.

Effect of Curosurf on Ex Vivo sPLA2-IIA Synthesis in AM

We previously established that AM constitute the major cell source of sPLA2-IIA in our ALI model (2). This led us to investigate the ex vivo effect of Curosurf on sPLA2-IIA synthesis by AM. Animals were treated with LPS, and AM were isolated after 8 h. AM were washed twice after 1 h of incubation and cultured for another 24 h without treatment. The results showed that sPLA2-IIA activity in the cells and cell-free supernatants increased progressively to reach a maximal level within 24 h. When animals received Curosurf (16 mg/animal) 30 min after LPS, the levels of sPLA2-IIA activity in isolated AM were significantly lower than in LPS + saline-treated animals (Fig. 3). This effect was accompanied by a marked decrease in the levels of sPLA2-IIA mRNAs in isolated AM (Fig. 4).


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Fig. 3.   Ex vivo effect of Curosurf on sPLA2-IIA synthesis in alveolar macrophages (AM). Curosurf (16 mg/animal, n = 8) or an equivalent volume of saline (200 µl/animal, n = 9) was administered 30 min after LPS challenge to guinea pigs. After 8 h, AM were isolated and cultured for 24 h without additional treatment. sPLA2-IIA activity was evaluated in cells (A) and cell-free supernatants (B) of AM. * P < 0.05 and ** P < 0.01 vs. corresponding times in LPS + saline group.



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Fig. 4.   Ex vivo effect of Curosurf on sPLA2-IIA mRNA expression in AM. Experimental procedure was similar to that described in Fig. 3. At the end of incubation, total RNA was extracted from AM, and 10 µg of total RNA were subjected to electrophoresis. After transfer to a nylon membrane, RNA was hybridized with [alpha -32P]CTP-labeled guinea pig sPLA2-IIA cDNA and labeled mouse beta -actin probe.

Effect of Curosurf on TNF-alpha Levels

Inasmuch as TNF-alpha is a crucial intermediate in LPS-induced sPLA2-IIA synthesis in AM (1, 17), we examined whether Curosurf instillation interferes with TNF-alpha secretion in the alveolar space in our ALI model. Our results showed that LPS challenge induced a marked increase in TNF-alpha levels in BALF and that administration of Curosurf led to a dramatic decrease in this level (see Table 3). Curosurf had no effect on the basal secretion of TNF-alpha : 35.70 ± 9.36 and 15.06 ± 4.33 pg/ml for saline + Curosurf and saline groups, respectively (n = 14). Northern blot analysis showed that Curosurf instillation led to a marked decrease of TNF-alpha mRNA levels in lung tissues (Fig. 5), suggesting that the loss observed in the BALF TNF-alpha levels is due to inhibition of the synthesis of this cytokine by Curosurf.


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Fig. 5.   Effect of Curosurf on expression of tumor necrosis factor-alpha (TNF-alpha ) mRNA in lung tissue. Total RNA from fresh lung tissue was extracted, and Northern blots were performed using guinea pig TNF-alpha cDNA as a probe. Mouse beta -actin cDNA was used as an internal control. Levels of TNF-alpha mRNA were quantitated using a PhosphorImager. Results are expressed as TNF-alpha -to-beta -actin ratio. A: total RNA was extracted from lung tissue 8 h after LPS instillation (n = 16). B: total RNA was extracted from lung tissue 24 h after LPS challenge (n = 16). ** P < 0.01. There is no significant difference between LPS and LPS + saline group.

Effect of Curosurf on Cell Counts and Protein Contents

Intratracheal instillation of LPS resulted in an increase in the total cell number in BAL. Administration of Curosurf 8 and 24 h after LPS reduced this increase (Tables 1 and 2). Differential cell counts in BAL revealed that after LPS instillation the percentage of polymorphonuclear cells (PMN) increased dramatically, whereas the percentage of macrophages decreased in parallel. Curosurf instillation, not only 30 min but also 8 h after LPS, significantly reduced PMN and increased macrophage percentages in BAL (Tables 1 and 2). In BALF obtained 8 and 24 h after LPS challenge, protein concentration was much higher than that in saline-treated animals. Administration of Curosurf significantly decreased the protein concentrations in LPS-treated guinea pigs (Table 3). In contrast, treatment of animals with saline after LPS (LPS + saline group) had no effect on protein concentration in BALF.

                              
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Table 1.   Effect of Curosurf on cell counts for 8 h after LPS infusion


                              
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Table 2.   Effect of Curosurf on cell counts for 24 h after LPS instillation


                              
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Table 3.   Effect of Curosurf on TNF-alpha levels and protein contents in BALF

Effect of Curosurf on Noninvasive Bronchial Reactivity

Finally, we investigated whether inhibition of sPLA2-IIA/TNF-alpha production by Curosurf is accompanied by changes of bronchial reactivity in LPS-treated animals. Our results showed that instillation of LPS was followed by a rapid increase in Penh, which reached its maximal value 2 h after instillation. Administration of Curosurf 30 min after LPS challenge markedly reduced the effect of LPS on Penh (Fig. 6).


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Fig. 6.   Effect of Curosurf on enhanced pause (Penh). Curosurf (16 mg/animal, n = 8) or an equivalent volume of saline (200 µl/animal, n = 9) was administered 30 min after LPS. Barometric plethysmography was used to observe the animals for 8 h, and parameters were recorded. Penh was calculated according to the manufacturer's instructions. LPS (n = 9) induced a sharp increase in Penh, and Curosurf markedly reduced this increase. * P < 0.05 and ** P < 0.01 vs. LPS + saline group (n = 9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the in vivo effect of the seminatural surfactant Curosurf on the pulmonary synthesis of sPLA2-IIA in an animal model of ALI based on intratracheal instillation of LPS. Our results showed that LPS induced an increase in sPLA2-IIA expression in lung tissues and that administration of Curosurf markedly reduced this expression. This was in agreement with our previous studies reporting that Curosurf downregulates the expression of sPLA2-IIA in cultured AM (5, 10), the major source of sPLA2-IIA in this experimental model of ALI (3). The present studies established that AM from LPS-treated animals produced ex vivo higher levels of sPLA2-IIA activity, which were markedly decreased in AM from Curosurf-treated animals. This suggests that the blockage of sPLA2-IIA expression in AM might be the mechanism by which Curosurf inhibits the pulmonary synthesis of this enzyme in our experimental model of ALI. Moreover, the inhibitory effect of Curosurf on sPLA2-IIA synthesis was observed, despite the fact that AM were washed several times before cell culture, suggesting that this inhibition occurred in an irreversible manner. We assume that Curosurf might not interfere with the access of LPS to AM. Indeed, the previous studies of Thomassen et al. (22) showed that human alveolar macrophages incubated with LPS for 1 h, washed, and then treated with the artificial surfactant Exosurf for 24 h were still able to inhibit cytokine secretion. This clearly indicates that the mechanism of action of surfactant may not be due to binding of LPS. This is in agreement with our experimental model in which animals were treated with LPS (>= 1 h) before Curosurf was administered. In this model, sPLA2-IIA levels in the lung reached its maximal values 8 h after LPS instillation (1). Thus it is likely that Curosurf interferes with the upregulation of sPLA2-IIA in the 1-h model. However, the mechanism of action might be different in the 8-h model, because Curosurf was instilled into animals after sPLA2-IIA synthesis was complete.

To further investigate the mechanism by which Curosurf inhibits sPLA2-II expression, we examined the effect of this surfactant on TNF-alpha secretion. Indeed, this cytokine has been shown to be an obligatory intermediate in LPS-induced sPLA2-IIA synthesis in AM (2, 24). The present study indicates that Curosurf instillation markedly reduced the levels of TNF-alpha in BALF and TNF-alpha expression in lung tissues from LPS-treated animals, suggesting that inhibition of TNF-alpha secretion might be the mechanism by which Curosurf downregulates the pulmonary synthesis of sPLA2-IIA.

The present studies also demonstrate that Curosurf exhibits a curative anti-inflammatory effect in our ALI model. Indeed, instillation of this surfactant 30 min or 8 h after LPS administration significantly reduced the total number of cells, PMN infiltration, and protein extravasation in BAL of LPS-treated guinea pigs. In addition, a marked inhibition of Penh was also observed after Curosurf instillation, suggesting that the latter reduced the bronchial reactivity induced by LPS. However, whether the ability of Curosurf to inhibit sPLA2-IIA/TNF-alpha production and its anti-inflammatory effects are related remains to be investigated.

Our previous studies (5, 9, 10) led us to suggest that the in vivo synthesis of sPLA2-IIA in guinea pig lung tissues is repressed by pulmonary factor(s) blocking the expression of this enzyme in AM. These cells can escape this inhibitory effect once they are removed from the lung. We have postulated that surfactant may be one of these factors. This hypothesis was based on the following findings: 1) freshly collected AM have no detectable sPLA2-IIA activity, 2) isolated AM synthesize sPLA2-IIA after prolonged in vitro incubation, and 3) addition of exogenous surfactant to AM in culture downregulates this synthesis, the phospholipids being the major surfactant component involved in this inhibition (5). The results of the present study strongly support this hypothesis. Finally, we have demonstrated that sPLA2-IIA hydrolyzes surfactant phospholipids in the present model of ALI (1). Taken together, these findings suggest that the alteration of surfactant (which occurs in pathophyiological conditions such as ALI) would result in the removal of its inhibitory effect on sPLA2-IIA synthesis. This would lead to the accumulation of this enzyme in alveoli via a vicious cycle: more sPLA2-IIA is produced, more surfactant phospholipids are hydrolyzed, and so on (24). The results of the present study show that instillation of exogenous surfactant is able to interrupt this vicious cycle accompanying lung inflammation. This suggested that application of surfactant may represent a promising approach for treatment of ALI or ARDS.


    ACKNOWLEDGEMENTS

We are grateful to B. B. Vargaftig and A. Brody for critically reading and commenting on the manuscript, Dr. J. Lefort for excellent technical assistance in measurement of Penh, and Dr. M. L. Watson (University of Bath, Bath, UK) for the gift of guinea pig recombinant TNF-alpha .


    FOOTNOTES

Y. Wu was supported by the Institut National de la Santé et de la Recherche Médicale (poste vert).

Address for reprint requests and other correspondence: L. Touqui, Unité de Défense Innée et Inflammation, Unité Associee Pasteur/INSERM U485, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France (E-mail: touqui{at}pasteur.fr).

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

10.1152/ajplung.00181.2001

Received 23 May 2001; accepted in final form 22 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arbibe, L, Koumanov k, Vial D, Rougeot C, Aaure G, Hatet N, Longacre S, Vargaftig BB, Wolf D, and Touqui L. Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction. J Clin Invest 102: 1152-1160, 1998[Abstract/Free Full Text].

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