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Apr 2, 2003 - Unité de Défense Innée et inflammation/INSERM E336, Institut Pasteur, Paris, France; and Centro de Biotecnologia, Instituto Butantan, ... and Medical Research) via a “poste vert” fellowship and by the ... 25 Rue du Dr. Roux, 75015 Paris, France. E-mail: ..... in green, and an arrow indicates the cell nucleus.

Surfactant Protein-A and Phosphatidylglycerol Suppress Type IIA Phospholipase A2 Synthesis via Nuclear Factor-␬B Yong-Zheng Wu, Samir Medjane, Sophie Chabot, Flavia Saldanha Kubrusly, Isaias Raw, Michel Chignard, and Lhousseine Touqui Unite´ de De´fense Inne´e et inflammation/INSERM E336, Institut Pasteur, Paris, France; and Centro de Biotecnologia, Instituto Butantan, Sa˜o Paulo, Brazil

Correspondence and requests for reprints should be addressed to Lhousseine Touqui, Unite´ de De´fense Inne´e et Inflammation/INSERM E336, Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France. E-mail: [email protected]

for producing SP-C and for secreting phospholipids into the airspace (1, 2). Data from a wide range of studies have shown that besides its mechanical property in preventing distal airways and alveoli from collapsing, pulmonary surfactant also exhibits immunomodulatory functions and plays a key role in host defense against infection (3–6). For example, surfactant lipids suppress a variety of immune cell functions, most notably lymphocyte proliferation (3, 7). Both SP-A and SP-D improve phagocytosis of Escherichia coli and Streptococcus pneumoniae by polymorphonuclear neutrophils (8), and SP-C (9) and SP-A (10) can recognize and bind endotoxin. In addition, SP-A and SP-D each modify the in vivo response to instilled endotoxin, leading to decreased lung injury and reduced inflammatory cell recruitment (11, 12). Indeed, endotoxin treatment induced a higher tumor necrosis factor-␣ (TNF-␣) and nitric oxide production in SP-A⫺/⫺ mice when compared with the wild type, and these processes were reduced by instillation of purified SP-A into SP-A⫺/⫺ animals (11). LeVine and Whitsett demonstrated increased transmigration of inflammatory cells and cytokine expression (TNF-␣ and interleukin-6) in the alveolar space of SP-A⫺/⫺ and SP-D⫺/⫺ mice when compared with wild-type hosts (13). Taken as a whole, these data demonstrate that surfactant-associated hydrophilic proteins play a key role in the modulation of inflammation. PLA2 belongs to a large family of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position: this reaction liberates free fatty acids and lysophospholipids, both of which are involved in the pathophysiologic changes observed in a number of inflammatory processes (14). Several classes of PLA2 (including intracellular and secretory enzymes) have been cloned and characterized (15). This present work concerns secretory type IIA phospholipase A2 (sPLA2-IIA), one of the key enzymes that may potentially play a role in the pathogenesis of inflammatory diseases because its presence is observed in sera of patients with bacterial infection and in the airspaces of animals with endotoxin-induced acute lung injury (16, 17). We have previously shown that alveolar macrophages are the major source of sPLA2-IIA in a guinea pig model of endotoxin-induced acute lung injury (17). Conversely, surfactant inhibits expression of sPLA2-IIA in alveolar macrophages in vitro and in an in vivo acute lung injury model (18, 19). However, the mechanism(s) by which surfactant inhibits sPLA2-IIA expression in alveolar macrophages and the relative importance of surfactant phospholipids and proteins in this inhibition have not yet been clearly elucidated. The aim of the present study was to investigate the regulation of sPLA2-IIA expression by surfactant components— notably SP-A and phospholipids—and to identify the signaling pathway(s) involved.

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

METHODS

Am J Respir Crit Care Med Vol 168. pp 692–699, 2003 Originally Published in Press as DOI: 10.1164/rccm.200304-467OC on July 25, 2003 Internet address: www.atsjournals.org

(More detailed information about methods is provided in the online supplement.)

We previously showed that surfactant inhibits the synthesis of type IIA secretory phospholipase A2 (sPLA2-IIA) by alveolar macrophages. These cells have been identified as the main source of this enzyme in an animal model of acute lung injury. The aim of the present study was to identify the surfactant components involved in the inhibition of sPLA2-IIA expression in alveolar macrophages and the signaling pathways that mediate this inhibition. Our results show that various surfactant preparations can inhibit sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages. Both the surfactant protein (SP)-A and the surfactant phospholipid fraction inhibit this expression. The surfactant phospholipid dioleylphosphatidylglycerol (DOPG) abolishes sPLA2-IIA expression, whereas dipalmitoylphosphatidylcholine does not. Chromatographic analysis and confocal microscopy revealed that phosphatidylglycerol was rapidly incorporated and metabolized by alveolar macrophages and that its metabolites accumulate in the cytosol. Nuclear factor-␬B (NF-␬B) modulates sPLA2-IIA expression in endotoxin-activated alveolar macrophages, and surfactant preparations, surfactant phospholipid fraction, SP-A, and DOPG indeed suppressed NF-␬B activation. In summary, our results show that SP-A and DOPG play a role in the surfactant-mediated inhibition of sPLA2-IIA expression in alveolar macrophages and that this inhibition occurs via a downregulation of NF-␬B activation. Keywords: surfactant; phospholipase A2; nuclear factor-␬B; alveolar macrophages

Pulmonary surfactant is a lipoprotein complex whose principal known function is to reduce surface tension at the air–liquid interface, thus preventing the alveoli from collapsing. It is composed of a mixture of phospholipids and four surfactant proteins (SP) denoted SP-A, SP-B, SP-C, and SP-D (1, 2). The surfactant phospholipids are mainly phosphatidylcholine and phosphatidylglycerol species, and further analysis has shown that dipalmitoylphosphatidylcholine and dioleylphosphatidylglycerol (DOPG), in particular, play a major role in maintaining the biophysical properties of the surfactant film (2). Whereas SP-A, SP-D, and SP-B are synthesized and secreted by both alveolar type II cells and airway Clara cells, the former cell type is solely responsible

(Received in original form April 2, 2003; accepted in final form July 9, 2003) Y.-Z.W. was supported by the INSERM (the French National Institute for Health and Medical Research) via a “poste vert” fellowship and by the “Socie´te´ de Secours des Amis des Sciences” charity.

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Materials

Statistical Analysis

Male Hartley guinea pigs were obtained from Elevages Saint-Antoine (Pleudaniel, France) and were cared for in accordance with Pasteur Institute guidelines in compliance with the European animal welfare regulations. The fluorescent phospholipid 1-oleoyl-2-[12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycerol-3-[phospho-rac(1-glycerol)] (NBD-PG) was purchased from Avanti Polar Lipids (Alabaster, AL). The Sephadex LH-60 column was bought from Pharmacia (Uppsala, Sweden).

All data are presented as the means ⫾ SEM, and statistical analysis was performed using one-way analysis of variance with SPSS 8.0 software. For multiple comparisons, the Student–Neumen–Keuls post hoc test was used. Values of p less than 0.05 were considered significant.

Preparation of Surfactant Briefly, lavage-surfactant preparation (LSP) was prepared from cellfree bronchoalveolar lavage fluids. Both Curosurf and tissue-surfactant preparation (TSP) were prepared from whole, minced lung tissues using two distinct methods (see the details in References 20 and 21). All preparations were devoid of hydrophilic proteins SP-A and SP-D but did contain hydrophobic proteins SP-B and SP-C.

Isolation of Surfactant Phospholipid Fraction and SP-A According to the modified method of van Iwaarden and coworkers (22) and as described previously (23), surfactant phospholipid fraction (SPF) and SP-A were extracted and purified from pulmonary surfactant obtained from guinea pig bronchoalveolar lavage fluids.

Macrophage Isolation, Incubation Procedure, and Measurement of sPLA2-IIA Activity Macrophages were isolated by bronchoalveolar lavage of guinea pigs. After a 20-hour incubation in RPMI 1640 culture medium containing 1% antibiotics and 3% fetal calf serum in the presence or absence of endotoxin, sPLA2-IIA activity in both the supernatant and cells was measured as described previously, using a Jobin et Yvon JY3D spectrofluorimeter equipped with a Xenon lamp: excitation and emission wavelengths were 345 and 398 nm respectively, with a slitwidth of 4 nm (17).

Expression of sPLA2-IIA and TNF-␣ Messenger RNAs RNA was extracted from alveolar macrophages, electrophoresed, transferred to a blot support, and then hybridized with ␣-32P-deoxycytidine triphosphate–labeled guinea pig sPLA2-IIA, guinea pig TNF-␣, or mouse ␤-actin complementary DNAs (cDNAs). After hybridization, blots were imaged using autoradiography film.

Nuclear Extracts and Electrophoretic Mobility Shift Analysis The nuclear protein extracted from control or endotoxin-stimulated alveolar macrophages was analyzed using a gel shift assay after incubation with ␥-32P-adenosine triphosphate–labeled nuclear factor-␬B (NF-␬B) double-stranded oligonucleotides corresponding to a NF-␬B– binding site consensus sequence: 5⬘-GATCATGGGGAATCCCCA-3⬘. Supershift analysis was performed by using polyclonal anti-p50 or antip65 antibodies. Before binding with the labeled probe, nuclear protein was incubated with 2 ␮g of polyclonal antibodies. The migration was performed on a 5% polyacrylamide gel, which was then dried and exposed to film for 2 to 12 hours.

Thin-Layer Chromatography Analysis of PG Metabolism After 4 or 20 hours incubation of alveolar macrophages with 20 ␮g/ml of NBD-phosphatidylglycerol (PG), lipids were extracted from both pooled supernatant and cultured alveolar macrophages and were then separated on silica gel thin-layer chromatography plates using water– acetic acid–methanol–chloroform (1:3:45:65 vol/vol) as a solvent system. Lipids were subsequently identified under UV light by comparison with known fluorescent lipid standards.

Confocal Microscopy Alveolar macrophages seeded on slides (1 ⫻ 106/well) were treated with 20 ␮g/ml of fluorescent lipid (NBD-PG) in the presence or absence of endotoxin. After incubation for either 4 or 20 hours, cells were fixed with freshly prepared 4% paraformaldehyde and then mounted in Mowiol. The localization of fluorescent lipids was detected using a Zeiss LSM 510 confocal microscope.

RESULTS In previous studies, we have shown that Curosurf (a seminatural surfactant prepared from whole, minced, porcine lung tissue) inhibits sPLA2-IIA expression in isolated alveolar macrophages (18) and in a guinea pig model of endotoxin-induced acute lung injury (19). Because surfactant composition and properties are likely to vary according to the method of isolation and extraction, in the present study we compared the effect of Curosurf with that of TSP and LSP, two other seminatural surfactant preparations. Like Curosurf, TSP is also extracted from minced porcine lung tissue (although using a different protocol), whereas LSP is prepared from cell-free pig bronchoalveolar lavage fluids. Our results (Figure 1) show that TSP and LSP surfactant preparations both inhibit sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages to a similar extent as Curosurf did. This effect is accompanied by inhibition of TNF-␣ expression (Figure 1). Nonetheless, it could be argued that the inhibition of sPLA2IIA expression could be due to putative binding of endotoxin to surfactant preparations, thus preventing access of the endotoxin to its receptor on the alveolar macrophage. We therefore performed experiments in which endotoxin was added to alveolar macrophage cultures 1 hour before their incubation with surfactant preparations. No significant difference in the inhibitory effect was observed, regardless of whether surfactant was added to alveolar macrophages before or after endotoxin stimulation (unpublished observations). Next, we examined the effect of various surfactant components on endotoxin-induced sPLA2-IIA expression in alveolar macrophages. SPF (a guinea pig surfactant preparation from which all SP had been removed) was incubated with alveolar macrophages before endotoxin stimulation. This surfactant phospholipid preparation inhibited sPLA2-IIA expression, indicating that phospholipids play a major part in this process. This prompted us to examine the role of specific surfactant phospholipids, and our results show that DOPG is able to inhibit endotoxin-induced sPLA2-IIA expression in alveolar macrophages, whereas dipalmitoylphosphatidylcholine (the major phospholipid component in surfactant) had no effect (Figure 2). Other surfactant phospholipids, including dioleylphospatidylcholine, sphingomyelin, and distearoylphosphatidylethanolamine, equally had no significant effect on this expression (Figure 3). Furthermore, our findings show that DOPG is able to inhibit TNF-␣ expression in endotoxin-stimulated alveolar macrophages (Figure 2). It was thus of a great interest to investigate whether DOPG acts by itself on sPLA2-IIA and TNF-␣ expression or via its metabolites. We examined the metabolism and localization of this phospholipid in alveolar macrophages using fluorescent PG (i.e., PG labeled with NBD at the sn-2 position). Confocal microscopy analysis showed that after 4 hours of incubation, NBDPG had been rapidly taken up by alveolar macrophages and had accumulated in the cytosol (Figure 4), although after 20 hours of incubation the intensity of cytosolic fluorescence had begun to decrease. Thin-layer chromatography analysis showed that almost all the NBD-PG was internalized by alveolar macrophages after 20 hours incubation compared with the 4-hour time point (Figure 5). The analysis also showed that most of NBDPG was converted into NBD-labeled fatty acid, together with an unidentified component migrating at the solvent front. In addition, a nonnegligible proportion of fluorescent fatty acid

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Figure 1. Effect of various surfactant preparations on secretory type IIA phospholipase A2 (sPLA2-IIA) activity and sPLA2-IIA and tumor necrosis factor-␣ (TNF-␣) messenger RNA (mRNA) levels in endotoxin-stimulated alveolar macrophages. Cells were incubated in the presence or absence of Curosurf, tissue-surfactant preparation (TSP), and lavage-surfactant preparation (LSP) (all at 500 ␮g/ml) for 1 hour before endotoxin addition (25 ␮g/ml). Measurements and examinations were performed 20 hours after endotoxin stimulation. (A ) Shows total sPLA2-IIA activity measured in the cells and in the culture medium; (B ) total RNA was extracted from alveolar macrophages and northern blots were performed using specific ␣-32P-deoxycytidine triphosphate–labeled guinea pig sPLA2-IIA and TNF-␣ complementary DNAs. Mouse ␤-actin was used as internal standard.

was released into the culture medium. Interestingly, significant fluorescence comigrating with phosphatidylcholine was also detected in the cells after incubation for 20 hours. Addition of endotoxin to alveolar macrophages had no detectable effect on NBD-PG’s localization or metabolic profile, and control experiments showed that NBD-PG remained intact after 4 and 20 hours incubation with cell-free RPMI medium (Figure 5). Next, we investigated the effect of SP-A on endotoxin-induced sPLA2-IIA expression. Figure 2 shows that SP-A purified from guinea pig surfactant markedly reduced sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages. SP-A was also able to inhibit basal sPLA2-IIA expression in alveolar macrophages (Figure 2A). In addition, our results showed that inhibition of sPLA2-IIA synthesis by SP-A was accompanied by a marked decrease in TNF-␣ expression in endotoxin-stimulated alveolar macrophages (Figure 2B). We finally investigated the signaling pathways involved in the inhibition of sPLA2-IIA and TNF-␣ expression by surfactant. Figure 6 shows that the NF-␬B inhibitors caffeic acid phenethyl ester and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132)

Figure 2. Effect of different surfactant components on sPLA2-IIA activity and sPLA2-IIA and TNF-␣ mRNA levels in endotoxin-stimulated alveolar macrophages. Cells were incubated with surfactant phospholipid fraction (SPF) (500 ␮g/ml), dioleylphosphatidylglycerol (DOPG) (20 ␮g/ ml), dipalmitoylphosphatidylcholine (DPPC) (100 ␮g/ml), or SP-A (20 ␮g/ml) for 1 hour before endotoxin stimulation (25 ␮g/ml). Twenty hours later, sPLA2-IIA activity (A ) and sPLA2-IIA and TNF-␣ mRNA levels (B ) were analyzed as indicated in METHODS. Insert: alveolar macrophages were incubated 20 hours in the presence or absence of SP-A (20 ␮g/ ml) before the measurement of sPLA2-IIA activity.

inhibit sPLA2-IIA and TNF-␣ expression, and that this effect occurs at the transcriptional level. We confirmed that this phenomenon was due to inhibition of endotoxin-induced NF-␬B translocation in alveolar macrophages (Figure 7) and examined whether surfactant and its components suppress sPLA2-IIA and TNF-␣ expression by inhibiting NF-␬B activation. Figure 8 shows that Curosurf and TSP significantly reduced endotoxin-induced NF-␬B activation, as did LSP (though to a lesser extent). SP-A, SPF, and DOPG also inhibited NF-␬B complex formation, in contrast to dipalmitoylphosphatidylcholine, which had no effect (Figure 9). Supershift studies revealed that antibodies directed against NF-␬B’s p50 and p65 subunits displaced the NF-␬B band, thus confirming that these complexes belong to the NF-␬B family. Moreover, the intensity of the NF-␬B p50/p65 complex in endotoxin-stimulated alveolar macrophages was decreased by surfactant (Figure 10).

DISCUSSION Previous studies have reported that instillation of surfactant preparations such as Exosurf, Survanta, Curosurf, and Alveofact

Wu, Medjane, Chabot, et al.: Surfactant Downregulates sPLA2-IIA

Figure 3. Effect of different phospholipids of surfactant components on sPLA2-IIA activity in endotoxin-stimulated alveolar macrophages. Cells were incubated with dioleoylphosphatidylcholine (DOPC) (120 ␮g/ml), sphingomyelin (10 ␮g/ml), or distearoylphosphatidylethanolamine (DSPE) (30 ␮g/ml) for 1 hour before endotoxin stimulation (25 ␮g/ml). Twenty hours later, sPLA2-IIA activity was analyzed as indicated in METHODS.

Figure 4. Localization of NBD-labeled fluorescent PG in alveolar macrophages by confocal microscopy. Cells adhered on a glass slide at a concentration of 1 ⫻ 106 cells/well/2 ml were incubated for 4 or 20 hours with 20 ␮g/ml of NBD-labeled PG in the presence or absence of endotoxin (25 ␮g/ml). The fluorescence of NBD-labeled PG is shown in green, and an arrow indicates the cell nucleus.

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Figure 5. Analysis of the metabolism of NBD-labeled fluorescent PG in alveolar macrophages. Adhered alveolar macrophages (3 ⫻ 106 cells/ ml) were incubated for 4 or 20 hours with 20 ␮g/ml of NBD-labeled PG in the presence or absence of endotoxin (25 ␮g/ml). NBD-labeled PG was also incubated with RPMI medium as control. After incubation, phospholipids were extracted from the cells (C) and culture medium (M) as described in METHODS. Next, thin-layer chromatography was performed using a water–acetic acid–methanol–chloroform (1:3:45:65, vol/vol) solvent system. AM corresponds to alveolar macrophage and A indicates the solvent front. The positions of various compounds were localized by using corresponding commercial standards and were as follows: B ⫽ fatty acid; D ⫽ PG; and E ⫽ phosphatidylcholine.

improved lung function in animal models of acute lung injury (19, 24–26) and in patients with respiratory distress syndrome (27–31). This beneficial effect is due to a reduction in surface tension and a resultant promotion of the stability of terminal bronchi. Over the last decade, however, an accumulation of evidence suggests that the beneficial effect of this therapy can be attributed not only to the biophysical properties of surfactant (32) but also to its ability to modulate the inflammatory reaction (3, 7). This latter phenomenon involves a variety of cells, mediators (such as cytokines), and enzymes (including PLA2) (33). We have recently reported that the porcine surfactant Curosurf inhibits expression of proinflammatory sPLA2-IIA in isolated guinea pig alveolar macrophages (18) and in a guinea pig model of acute lung injury (19). Because the biological and mechanical properties of surfactant preparations likely vary as a function of the isolation and extraction procedures used, in the present study we compared the effect of Curosurf on endotoxin-induced sPLA2IIA expression in guinea pig alveolar macrophages to that elicited by other surfactant preparations (TSP and LSP). Even though all three surfactant preparations were isolated from the same animal species (the domestic pig), Curosurf and TSP were both extracted from minced lung (but using different techniques), whereas LSP was extracted from cell-free bronchoalveolar lavage fluid. Our data show that all these surfactant preparations (and SPF) inhibited endotoxin-induced TNF-␣ and sPLA2-IIA expression to a similar extent, indicating that the method of isolation and extraction of these preparations may not necessarily interfere with their biological activities, at least in our experimental conditions. The fact that SPF (a protein-free guinea pig surfactant preparation) downregulates sPLA2-IIA expression in alveolar macrophages

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Figure 7. Effects of NF-␬B inhibitors on NF-␬B complex formation. Alveolar macrophages were incubated with MG132 (100 nM) or caffeic acid phenethyl ester (CAPE) (10 ␮M) for 1 hour before endotoxin stimulation (25 ␮g/ml). Two hours later, nuclear extracts were obtained and gel shift analysis was performed (A). The histogram shows the quantification of NF-␬B complex formation (B).

Figure 6. Impairment by nuclear factor-␬B (NF-␬B) inhibitors of endotoxin-induced sPLA2-IIA and TNF-␣ expression in alveolar macrophages. Cells were incubated with MG132 (100 nM) or caffeic acid phenethyl ester (CAPE) (10 ␮M) for 1 hour before endotoxin stimulation (25 ␮g/ ml). Twenty hours later, sPLA2-IIA activity (A ), and sPLA2-IIA and TNF-␣ mRNA levels (B ) were measured as indicated above.

suggests that surfactant phospholipids play a major role in this process. Of the surfactant phospholipids we tested individually, only DOPG was able to inhibit endotoxin-induced sPLA2-IIA. In contrast, dipalmitoylphosphatidylcholine (the major phospholipid component of surfactant) failed to interfere with this expression, even when used at concentrations 25 times higher than DOPG. Other phospholipids including dioleylphospatidylcholine, phosphatidylethanolamine, and sphingomyelin, also had no significant effect on this expression. This clearly indicates that DOPG plays a major role in the inhibition of sPLA2-IIA expression by surfactant phospholipids and that this inhibition is not due to a nonspecific action at the cell membrane level. This conclusion prompted us to investigate the metabolism and localization of DOPG in alveolar macrophages in the presence or absence of endotoxin by using fluorescent PG (labeled with NBD at the sn-2 position). Our experiments showed that NBD-PG was rapidly taken up by alveolar macrophages and accumulated in the cytosol after just 4 hours of incubation. Thinlayer chromatography analysis showed that most of the NBDPG internalized by alveolar macrophages was converted into a metabolite migrating at the solvent front and, to lesser extent, a compound comigrating with phosphatidylcholine. However, the exact chemical nature of these metabolites and the metabolic

pathways involved in their formation remain unknown. On the other hand, a part of the NBD-PG was converted into fatty acid, suggesting that a PLA2-like enzyme is involved in the metabolism of NBD-PG by alveolar macrophages—but whether sPLA2-IIA itself is involved remains to be seen. Addition of endotoxin to alveolar macrophages modifies neither the localization nor the metabolic profile of NBD-PG. Although the mechanisms by which PG crosses the plasma membrane (via passive diffusion and/or through transporterdependent process) have yet to be fully described, our studies clearly show that PG is metabolized into several compounds including fatty acid, which then accumulates in the cytosol. It is of note that a nonnegligible portion of this fatty acid was released in the culture medium within 20 hours. If the results with NBDPG can be extrapolated to DOPG, it can be suggested that the latter is cleaved into oleic acid within the alveolar macrophage. This is of great interest because our previous studies have shown that oleic acid is able to downregulate the expression of sPLA2IIA in guinea pig alveolar macrophages (34). Taken as a whole, our results suggest that the observed inhibitory effect of DOPG is probably mediated (at least in part) by oleic acid. Our results also show that SP-A inhibits endotoxin-induced sPLA2-IIA expression in alveolar macrophages, indicating that this collectin plays a role in the regulation of sPLA2-IIA synthesis by surfactant. The inhibitory effect cannot be due to putative contamination of SP-A by endotoxin because the latter has been shown to stimulate sPLA2-IIA expression in our cell system even at low concentrations (14). SP-A also inhibits expression of sPLA2-IIA in the absence of endotoxin (Figure 2A). The fact that SPF (a surfactant phospholipid preparation from which SP-B and SP-C proteins were removed) inhibited sPLA2-IIA

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Figure 9. Inhibition by different surfactant components of endotoxin-induced NF-␬B activation. Alveolar macrophages were incubated with SPF (500 ␮g/ml), DOPG (20 ␮g/ml), DPPC (100 ␮g/ml), or SP-A (20 ␮g/ml) for 1 hour before endotoxin stimulation (25 ␮g/ml). Two hours later, nuclear extracts were obtained and gel shift analysis (A) was performed. (B) Shows the quantification of NF-␬B complex formation.

Figure 8. Inhibition by various surfactant preparations of endotoxininduced NF-␬B activation. Alveolar macrophages were incubated with different surfactant preparations (500 ␮g/ml) for 1 hour before endotoxin stimulation (25 ␮g/ml). Two hours later, nuclear extracts were obtained and gel shift analysis (A ) was performed; (B ) shows the quantification of NF-␬B complex formation.

expression with similar potency to that of Curosurf, LSP, and TSP (all of which contain SP-B and SP-C) suggests that SP-B and SP-C do not play a significant role in the inhibition of sPLA2IIA expression. However, the participation of SP-D in the regulation process remains to be determined. We next examined the signaling pathways by which surfactant inhibits sPLA2-IIA expression. We have previously shown that in guinea pig alveolar macrophages, endotoxin-induced sPLA2IIA synthesis occurs via an autocrine/paracrine process mediated by TNF-␣ (17). Expression of both sPLA2-IIA and TNF-␣ is modulated by a process involving NF-␬B activation (35, 36). Our present work shows that the surfactant preparations SPF, DOPG, and SP-A abolish endotoxin-induced NF-␬B activation and the resultant sPLA2-IIA and TNF-␣ expression in endotoxin-stimulated alveolar macrophages. Here again, dipalmitoylphosphatidylcholine had no effect. Observation of an inhibitory effect of SP-A fits with the fact that this protein has been shown to modulate the expression of proinflammatory cytokines in various cell types. Depending on the cell system and the experimental protocol, SP-A can either stimulate or inhibit production of such cytokines. Indeed, SP-A enhanced TNF-␣ expression in an endotoxin-stimulated THP-1 monocytic cell line (37) and

directly increased the production of this cytokine in alveolar macrophages (38). SP-A has been also shown to activate NF-␬B in the THP-1 monocytic cell line (39). In contrast, another study showed that SP-A inhibits the production of TNF-␣ by endotoxin-stimulated macrophages (40). There are several possible explanations for these discrepancies. It is likely that the structural and/or biological functions of SP-A will vary according to the purification method used. Furthermore, SP-A preparations can occasionally be contaminated by endotoxin, which may lead to artifactual effects. SP-A can also enhance the binding of endotoxin by alveolar macrophages in a dose-dependent manner (41). Previous studies have also reported the existence of specific SPA–binding sites in different cell systems (42–45), although it remains to be examined whether SP-A acts via these receptors or, in contrast, is incorporated by cells and then degraded to fragments that mediate its biological effects. Interestingly, a recent study showed that SP-A activates macrophages via the endotoxin receptor TLR4 (43), which may explain certain discrepancies found in the literature. In summary, our results show that pulmonary surfactant inhibits the expression of sPLA2-IIA and TNF-␣ in endotoxinstimulated alveolar macrophages. The surfactant components SP-A and DOPG play a major role in this inhibition via a process involving impairment of NF-␬B activation. Because alveolar macrophages are the major source of sPLA2-IIA and TNF-␣ in acute lung injury, and in light of the fact that NF-␬B plays a pivotal role in the induction of inflammatory reactions, our work suggests that SP-A and DOPG may potentially be of particular benefit in the treatment of acute lung injury.

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Figure 10. Suppression by the TSP of the translocation of NF-␬B p50/ p65 complex. Nuclear extracts were obtained after incubation of alveolar macrophages with endotoxin (25 ␮g/ml) in the presence or absence of TSP (500 ␮g/ml) for 2 hours. Supershift analysis was performed using antibodies specific for p50 and p65 before binding with a ␥-32Pdeoxyadenosine triphosphate end–labeled NF-␬B consensus sequence.

Conflict of Interest Statement : Y-Z.W. has no declared conflict of interest; S.M. has no declared conflict of interest; S.C. has no declared conflict of interest; F.S.K. has no declared conflict of interest; I.R. has no declared conflict of interest; M.C. has no declared conflict of interest; L.T. has no declared conflict of interest. Acknowledgment : The authors are grateful to Dr. Freek van Iwaarden for his advice on and help with SPF isolation. Guinea pig TNF-␣ cDNA was a kind gift from Dr. M. L. Watson (University of Bath, Bath, UK).

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