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* Unité de Défense Innée et Inflammation, Institut Pasteur, Institut National de la Santé et de la Recherche Médicale E 336, Paris, France;
Laboratoire de Biochimie, Unité de Recherche Asociée Centre National de la Recherche Scientifique, Faculté de Médecine St. Antoine, Paris, France;
Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France;
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195; and
¶ Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of secreted phospholipases A2 (sPLA2s) in a variety of inflammatory diseases including ARDS is now clearly established (3, 4). The sPLA2s form a growing family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the production of free fatty acids and lysophospholipids that serve as precursors for a variety of lipid-derived mediators involved in numerous biological activities (5, 6). To date, 10 different sPLA2s, referred to as groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII, have been characterized in mammals (7, 8, 9, 10, 11, 12). Of particular interest, levels of sPLA2 activity in bronchoalveolar lavage fluids (BALF) of patients with ARDS often correlate positively with the severity of the disease (13). However, the type of sPLA2 involved in this disorder remains unclear. While sPLA2-IIA is able to efficiently hydrolyze surfactant phospholipids, thereby contributing to surfactant alteration as observed in ARDS (14), the involvement of the other cloned sPLA2s in this process is not yet defined, and their roles in ARDS lung disease remain uncertain. In this study we investigated the potential pathological contribution of the newly cloned sPLA2s in ARDS by analyzing their ability to hydrolyze surfactant phospholipids in vitro.
Alteration of surfactant phospholipids occurs very early during the development of ARDS. Therefore, the development of inhibitors of sPLA2 preventing hydrolysis of surfactant phospholipids could represent an auspicious strategy for ARDS treatment. Indeed, despite recent advances in intensive care, the ARDS mortality rate still exceeds 40%, and there is no effective therapy apart from mechanical ventilation and other supportive measures (15). Interestingly, we previously showed that surfactant protein A (SP-A), which is the major surfactant-associated protein, inhibits the in vitro sPLA2-IIA activity through a direct protein-protein interaction (14). SP-A, which is a member of the C-type lectin superfamily and contains a COOH-terminal carbohydrate recognition domain (CRD) (16), plays an important role in pulmonary host defense and seems to inhibit inflammation following lung infection. Decreased SP-A levels in BALF are observed in patients with ARDS or at risk of developing ARDS (17, 18, 19). Thus, increased sPLA2 activity associated with decreased SP-A might promote the hydrolysis of pulmonary surfactant observed in ARDS. The aim of the present investigation was also to examine the effects of SP-A on sPLA2 activity in vivo and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SP-A and GM-CSF knockout mice (SP-A-/- and GM-CSF-/-) were bred from the C57BL/6/129Sv F2 homozygous SP-A-/- and GM-CSF-/- mice as previously described (20, 21). Recombinant guinea pig sPLA2-IIA (r-GP sPLA2-IIA) was produced in our laboratory (22). The recombinant mouse sPLA2s were expressed and purified as described previously (9, 10, 23). The rabbit polyclonal anti-rat SP-A Ab was a gift from F. X. McCormack (University of Cincinnati, Cincinnati, OH). Fatty acid-free BSA, phosphatidylcholine (PC), and phosphatidylglycerol (PG) standards were purchased from Sigma-Aldrich (St. Louis, MO).
Preparation of pulmonary surfactant
Animals were killed by i.p. administration of a lethal dose of sodium pentobarbital (Sanofi, Libourne, France). The trachea of mice was cannulated, and BALF collection was performed with a syringe by multiple cycles of instillation and aspiration with unitary 0.5 ml of saline to provide 4 ml of BALF for pulmonary surfactant isolation. Cell-free BALF was obtained after centrifugation (300 x g for 15 min), and pulmonary surfactant was isolated by centrifugation of cell-free BALF at 19,500 x g for 20 min.
SDS-PAGE and immunoblot analysis
SDS-PAGE was performed according to the method described by Laemmli (24). Twelve percent gels were loaded with 5 µg of total pulmonary surfactant phospholipids from the different mice or, as a control, 1 µg of purified human SP-A in the presence of 5% (v/v) 2-ME. Proteins were transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and probed with a rabbit polyclonal anti-SP-A Ab (1/50,000). Membrane was then probed with anti-rabbit IgG HRP-linked donkey Ab (1/2,500), washed, incubated with ECL detection reagent (Amersham Pharmacia Biotech, Little Chalfont, U.K.), and exposed to Kodak X-OMAT AR film (Kodak-Pathé, Paris, France). Protein standards (Bio-Rad, Hercules, CA) with molecular masses in the range of 6,500–175,000 were used.
Incubation of sPLA2s with pulmonary surfactant
The sPLA2 assay buffer contained 100 mM Tris, 150 mM NaCl, and 10 mM CaCl2 (pH 8). Surfactant was suspended in this buffer and then submitted to ultrasonication for 5 min (150 W) using an MSE sonifier (Annemasse, France). After adjusting the phospholipid concentration to 2 mM, surfactant was incubated for 1 h at 37°C with the indicated sPLA2s in the presence of 0.1% BSA (final volume, 50 µl). To analyze the role of SP-A in SP-A+/+ mice on fatty acid release by r-GP sPLA2-IIA, surfactant (2 mM phospholipid concentration) from SP-A+/+ mice was preincubated with 5 µg of rabbit polyclonal anti-SP-A IgG or control rabbit IgG in a final volume of 50 µl overnight at room temperature. Surfactant was then incubated for 1 h at 37°C with 1 µg/ml r-GP sPLA2-IIA, and fatty acid release was analyzed as described below.
Fatty acid analysis
Fatty acids were extracted by Dole’s procedure modified by Tsujishita et al. (25) and methylated with diazomethane. The methylated derivatives were separated by gas chromatography on a capillary column containing Supelcowax 10 bonded phase (diameter, 0.32 mm; 30 m long; Supelco, Bellefonte, PA) on a gas chromatograph (5890 series II; Hewlett-Packard, Palo Alto, CA) and detected by mass spectrometry (10-10C; Nermag, Reuil, France).
Analysis of pulmonary surfactant PC and PG
Lipids were extracted from isolated surfactant according to the method described by Bligh and Dyer (26). The separation of phospholipids was achieved by TLC using silica gel plates (Merck, St. Louis, MO) and a water/acetic acid/methanol/chloroform (65/45/3/1, v/v) solvent system. The surfactant PC and PG spots were localized by reference to corresponding standards and extracted from silica gel plates, and then their phosphorus contents were measured according to the method reported by Böttcher et al. (27).
Administration of sPLA2-IIA to mice
SP-A-/- or SP-A+/+ 7-wk-old male mice, weighing 25–30 g, were instilled through an intratracheal catheter with either 20 µg of group IIA recombinant mouse sPLA2 (mGIIA sPLA2) dissolved in 50 µl of saline or the same volume of saline containing 5 mM CaCl2. One hour after treatment, mice were killed by an overdose of sodium pentobarbital. BALF was collected, and pulmonary surfactant was isolated as described above. There were no significant differences in the total volume of saline infused into the lungs or in the volume recovered after the lavage procedure among any experimental groups. Mice were cared for in accordance with Pasteur Institute guidelines in compliance with the European animal welfare regulations.
Measurement of respiratory functions
The respiratory function of freely moving mice was measured by barometric plethysmography using whole-body plethysmography (Buxco Electronics, USA) according to the manufacturer’s instructions. In brief, anesthetized mice received intratracheal instillation of 20 µg of mGIIA sPLA2 or equivalent volume of saline. Then, each animal was placed in a main chamber and pressure difference between this chamber and a reference chamber were measured with a differential pressure transducer connected to amplifier and recorded with BioSystem XA analyzer software (Buxco Electronics, Birmingham, U.K.). Enhanced pause (Penh) correlated very closely with pulmonary resistance and was calculated as follows: Penh = (Te - Tr)/Tr(PEP/PIP), where Te is the expiratory time (seconds), Tr is the relaxation time (time of the pressure decay to 36% of total box pressure at expiration), PEP is the peak expiratory pressure (milliliters per second), and PIP is the peak inspiratory pressure (milliliters per second).
Statistical analysis
Results are expressed as the mean ± SEM for the indicated number of independently performed experiments. Comparisons between values were analyzed by Student’s t test for unpaired data, and p < 0.05 was considered significant.
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Results |
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We previously demonstrated the inhibitory effect of SP-A on sPLA2-IIA activity on synthetic PG substrates in vitro (14). To assess the relationship between SP-A and surfactant phospholipid hydrolysis by sPLA2-IIA, surfactants purified from wild-type mice, mice deficient in SP-A (SP-A-/- mice), or mice with increased alveolar SP-A (GM-CSF-/- mice) were used as substrates. In GM-CSF-/- mice, the increased SP-A content results from an alveolar accumulation of surfactant-associated proteins and phospholipids caused by impaired clearance by alveolar macrophages (28). The phospholipid concentrations of the different surfactants from SP-A+/+, SP-A-/-, and GM-CSF-/- mice were adjusted to 2 mM, and the concentration of SP-A was estimated by immunoblotting. As expected, SP-A was undetectable in the pulmonary surfactant from SP-A-/- mice, and its level was considerably increased in GM-CSF-/- mice compared with SP-A+/+ mice (Fig. 1A).
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Although the targeted disruption of the murine SP-A gene does not affect the level and composition of surfactant phospholipid (20), we aimed to confirm whether the increase in fatty acid release by sPLA2-IIA was indeed due to the absence of SP-A rather than to any potential difference in the properties of surfactant of SP-A-/- mice. Thus, SP-A+/+ surfactant was incubated overnight at room temperature with a polyclonal anti-SP-A Ab to neutralize SP-A; sPLA2-IIA (1 µg/ml) was added for 1 h at 37°C, and the release of fatty acids was measured. Hydrolysis of surfactant phospholipids increased significantly when the surfactant was preincubated with the anti-SP-A Ab, confirming the inhibitory effect of SP-A on sPLA2-IIA activity (Fig. 2).
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Recently, novel sPLA2s have been cloned, but their biological roles remain unknown (6). In this study we tested in vitro their ability to hydrolyze surfactant phospholipids using surfactant from SP-A-/- mice (Fig. 3). As previously found (14), sPLA2-IIA hydrolyzed surfactant phospholipids, and PG was the most preferred substrate for this enzyme. By contrast, the other type II sPLA2s, including IIC, IID, IIE, and IIF enzymes, were ineffective in hydrolysis. Both sPLA2-V and sPLA2-X hydrolyzed PC and PG. In summary, only sPLA2-IIA, sPLA2-V, and sPLA2-X hydrolyzed surfactant phospholipids.
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Our above in vitro studies showed that sPLA2-IIA hydrolyzed surfactant phospholipids and that SP-A reduced susceptibility of surfactant to hydrolysis. However, sPLA2-IIA activity depends not only on the chemical nature of phospholipids, but also on their physical state (30). For instance, sPLA2-IIA is very active toward micellar lipids as in our in vitro assay (31). Because surfactant phospholipids form in vivo a monolayer film that may be differentially hydrolyzed by sPLA2-IIA, we examined the ability of sPLA2-IIA to hydrolyze surfactant phospholipids in vivo in SP-A-/- and SP-A+/+ mice. In these experiments mGIIA sPLA2 was instilled intratracheally into the lungs of SP-A-/- and SP-A+/+ mice, and surfactant phospholipid hydrolysis was analyzed (Fig. 5). The ratio of PC/PG increased following the administration of sPLA2-IIA for SP-A-/- mice (Fig. 5A). While no change in PC levels was observed following treatment with sPLA2-IIA, the PG content decreased significantly (13.5 ± 4 and 38.4 ± 7% for SP-A+/+ and SP-A-/- mice, respectively, compared with corresponding control animals that were treated with saline (Fig. 5B). These data indicate that sPLA2-IIA is able to efficiently hydrolyze PG under in vivo conditions. Secondly, we observed that the surfactant PG content decreased markedly in SP-A-/- mice compared with wild-type mice after administration of sPLA2-IIA. Collectively, these results demonstrate that surfactant-associated SP-A reduces the hydrolysis of surfactant PG by sPLA2-IIA in vivo.
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To determine whether the decrease in PG leads to a defect of the surfactant function, we examined the effect of intratracheal administration of mGIIA sPLA2 on respiratory functions expressed as an increase in Penh. The latter is an index of airway resistance resulting from lung injury. In sPLA2-IIA-treated mice, Penh increased after 3 h, peaked within 
4 h, and then returned to basal levels by 6 h (data not shown). The increase in lung resistance was significantly higher in SP-A-/- mice than in wild-type mice. These results suggest that PG hydrolysis by sPLA2-IIA leads to an impairment of lung surfactant function, and that SP-A is effective in protecting the lung from injury induced by mouse sPLA2-IIA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study shows that the release of fatty acid, resulting from in vitro surfactant hydrolysis by sPLA2-IIA, is inversely correlated with the level of SP-A present in pulmonary surfactant (Fig. 1). Moreover, when SP-A present in surfactant from wild-type mice is neutralized with an anti-SP-A Ab, a significant increase in the release of fatty acids is observed (Fig. 2). Taken together, these data suggest that the absence of SP-A exacerbates the susceptibility of surfactant to degradation by sPLA2-IIA.
Mammalian sPLA2s are distinguished by their structural and enzymatic properties (6, 23). Subtle differences in their sequences markedly affect their substrate interfacial binding and rate of hydrolysis (32, 33). Here we analyzed the efficiencies of various sPLA2s, including recently cloned sPLA2s, to hydrolyze surfactant. Although most of these enzymes are expressed in the lung (6, 10), their enzymatic activities toward surfactant phospholipids have never been investigated. This present study shows that in addition to sPLA2-IIA, sPLA2-V and sPLA2-X are able to hydrolyze in vitro surfactant phospholipids. In these conditions PG is the most preferred substrate for sPLA2-IIA, while sPLA2-V and sPLA2-X hydrolyze PC and PG with similar rates (Fig. 3). By contrast, the other analyzed sPLA2s (IIC, IID, IIE, and IIF) are inefficient in inducing surfactant phospholipid hydrolysis.
When comparing the hydrolysis of surfactant phospholipids from SP-A-/- and SP-A+/+ mice, we observed that surfactant-associated SP-A could inhibit sPLA2-IIA and sPLA2-X activities, but failed to interfere with that of sPLA2-V (Fig. 4). These findings are of great interest because it has been shown that sPLA2-IIA and sPLA2-X bind to the CRD domain of the M-type receptor (34, 35, 36), and a structurally similar CRD domain is also present in SP-A (37). Moreover, the binding of sPLA2 to this receptor leads to an inhibition of catalytic activity (38). Surfactant-associated SP-A may therefore act as an endogenous inhibitor of sPLA2-IIA and sPLA2-X by interacting with these sPLA2s through its CRD domain.
Our results may have important pathophysiological relevance, as patients with ARDS have elevated sPLA2 activity and decreased SP-A concentrations in their BALF (17, 18, 19). However, no direct causal relationship has been demonstrated between the alteration in SP-A levels and the acute destruction of surfactant phospholipids. This led us to investigate the physiological consequence of the inhibition of sPLA2-IIA activity by surfactant-associated SP-A under in vivo conditions. We first checked whether mouse sPLA2-IIA was able to hydrolyze surfactant phospholipids under in vivo conditions in mice. Several studies have shown that intratracheal administration of sPLA2s can induce lung injury with interstitial and alveolar edema, accumulation of inflammatory cells, and free fatty acid release (14, 39, 40), which are pathological features typical of those seen in the lungs of ARDS patients. However, these studies have often used heterologous sPLA2s from snake venoms, which have much higher ability than mammalian sPLA2s to hydrolyze phospholipids on packed monolayer structures. Moreover, none of these studies used mice as an animal model. In the present study no hydrolysis of surfactant PC was observed in sPLA2-IIA-treated mice. This is not surprising, since sPLA2-IIA, which bears basic residues resulting in positive charges of its interfacial binding surface, cannot bind well to PC vesicles, as described for PG (33, 41, 42).
In contrast to PC, we observed a marked decrease in surfactant PG, particularly in sPLA2-IIA-treated SP-A-/- mice. This finding is consistent with findings that sPLA2-IIA is >100-fold more active on anionic, rather than zwitterionic, phospholipids (9, 30). Remarkably, hydrolysis of PG occurs as early as 1 h after sPLA2-IIA administration, indicating that this enzyme could account for surfactant abnormalities observed at early phases in ARDS (17). In fact, alterations in the relative composition of the surfactant phospholipids are seen in patients with ARDS (17, 18, 43, 44). A decrease in PG levels (by >80% in three of these studies) and a moderate reduction of PC levels were observed. Taken together, these data support a role for sPLA2-IIA in the alterations in PG levels occurring during the early phase of ARDS.
Therefore, it should be of great interest to know whether PG hydrolysis contributes to the pathophysiologic events encountered in ARDS. Surfactant from sPLA2-IIA-treated SP-A-/- mice showed an increase in the PC/PG ratio (Fig. 5A). Interestingly, Hite et al. (45) observed PG hydrolysis by sPLA2 in asthmatic patients. They demonstrated a positive correlation between the PC/PG ratio and surfactant dysfunction, and they concluded that the hydrolysis of surfactant PG by sPLA2 may be a mechanism contributing to surfactant dysfunction. Moreover, we showed here that intratracheal administration of mouse sPLA2-IIA leads to a time-dependent increase in Penh, demonstrating that the enzyme has a deleterious effect on lung function (Fig. 6). In this study we did not demonstrate a direct relationship between the decrease in PG content and the impairment of lung function. However, as PG has a relatively high content of unsaturated fatty acids, it may be functionally important because of its capacity to increase monolayer film fluidity and adsorption (46). It has also been shown that PG prevents the collapse of the surfactant film (47). Thus, surfactant PG hydrolysis by sPLA2-IIA may contribute to surfactant dysfunction. The pathophysiological relevance of these results is linked to the fact that the surface activity of the surfactant film from patients with ARDS is abnormal, including a reduced rate of absorption, higher surface compressibility, and lower film stability index (17, 48).
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This report also demonstrates a deleterious effect of sPLA2-IIA, sPLA2-V, and sPLA2-X on pulmonary surfactant, suggesting a possible involvement of these enzymes in the pathogenesis of ARDS. Our findings support a potential role for SP-A in the protection of surfactant in the early phases of ARDS. Our results corroborate the view that surfactant replacement therapy for ARDS might be enhanced by addition of SP-A.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lhousseine Touqui, Unité de Défense Innée et Inflammation, Institut National de la Santé et de la Recherche Médicale E 336, Institut Pasteur 25 rue du Dr. Roux, 75015 Paris, France. E-mail address: touqui{at}pasteur.fr
3 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; CRD, carbohydrate recognition domain; mGIIA sPLA2, group IIA recombinant mouse sPLA2; PC, phosphatidylcholine; Penh, enhanced pause; PG, phosphatidylglycerol; r-GP sPLA2-IIA, recombinant guinea pig sPLA2-IIA; SP-A, surfactant protein A; sPLA2, secreted phospholipase A2.
Received for publication January 13, 2003. Accepted for publication May 16, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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