INTRODUCTION

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Phospholipases A2 (PLA2s, phosphatide 2-acylhydrolase, EC 3.1.1.4) are widely distributed enzymes (1), abundant in pancreatic juice and in the venom of snakes and bees, where they serve digestive functions. They are present in mammalian cells and are involved in the turnover and remodeling of membrane phospholipids. These enzymes catalyze the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and play a key role in inflammation (1, 2). Based on their primary structure, mammalian PLA2s can be classified in two families: the intracellular and the secretory PLA2s (sPLA2) (3, 4). The secretory type II PLA2 (sPLA2-II) (5), the most studied enzyme in the sPLA2 family, is produced by a variety of inflammatory cells including guinea-pig alveolar macrophages (AM) (6, 7) and has been involved in various inflammatory diseases (2, 8). We have recently shown that macrophages are the major cell source of sPLA2-II synthesized by lung tissues in a guinea-pig model of acute lung injury and that tumor necrosis factor-alpha (TNF-) released in the air-lung interface plays a key role in this sPLA2-II synthesis (9). Accumulating evidence suggests that sPLA2-II may play a role in the development of acute respiratory distress syndrome (ARDS) (10). The latter is a syndrome clinically defined by arterial hypoxemia and bilateral pulmonary infiltrates on chest radiograph, disruption of endothelial barrier, and early alteration of pulmonary surfactant (11). Pulmonary surfactant is a lipid-protein complex synthesized by the alveolar type II epithelial cells, that lowers surface tension along the alveolar epithelium, thereby promoting alveolar stability. It is composed of approximately 10% proteins and 90% lipids, with unusually high proportions of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (12). Destruction of surfactant increases surface tension at the air-liquid interface, which results in alveolar collapse and deterioration of mechanical properties of the lung (11). Seminatural surfactant Curosurf has been shown to reduce mortality in premature infants with respiratory distress syndrome (13).

The beneficial effect of this therapy can be attributed not only to the biophysical properties (11) of surfactant but also to the modulation of the inflammatory reaction (14). We have recently reported that Curosurf inhibits the expression of the proinflammatory sPLA2-II by guinea-pig AM (18), but the mechanism involved in this inhibition has not been elucidated. Here, we show that phospholipid components of surfactant, mainly dioleylphosphatidylglycerol (DOPG), downregulate the expression of sPLA2-II in guinea-pig AM and that this effect occurs, at least in part, through the inhibition of TNF- secretion.

	METHODS

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Materials

Male Hartley guinea pigs were obtained from Elevages Lebeau (Gambais, France). RPMI 1640 culture medium and fetal calf serum (FCS) were from Jacques Boy (Reims, France). Hanks' balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺ was from GIBCO (Bethesda Research Laboratories, Gaithersburg, MD). N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), fatty acid-free bovine serum albumin (BSA), leupeptin, aprotinin, L-glutamine, 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (St. Louis, MO). Sodium pentobarbital was from Sanofi Laboratories. Fluorescent phospholipid (1-palmitoyl-2-[10-pyrenedecanoyl]-sn-glycero-monomethylphosphatidylglycerol) was from Interchim (Montluçon, France). Phospholipids (DPPC, DOPC, DPPG, DOPG, and sphingomyeline [SPH]) used for the preparation of artificial surfactant (AS) were from Sigma. Seminatural surfactant (Curosurf) from pig lung was a gift from Serono (Boulogne, France). Products for staining cytocentrifuge smears (modified May-Grünwald-Giemsa) were from Diff-Quik (Düdingen, Switzerland). [¹⁴C]phosphatidylglycerol ([¹⁴C]DOPG, 50 mCi/mmol) was a gift of F. Guerbette (Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Université Paris VI, Paris). 1,2 dipalmitoyl, L-3-phosphatidyl(N-methyl-[³H])choline ([³H]DPPC, 85 Ci/mmol) was from Amersham (Arlington Heights, IL). 1-O-Octadecyl-2-[³H]acetyl-sn-glycero-3-phosphocholine ([³H]acetyl-platelet-activating factor [PAF], 10 Ci/mmol) was from CEA (Saclay, France). Recombinant guinea pig TNF- (gp-TNF-) was produced and purified as previously described (19).

Preparation of Surfactant

AS was prepared in the laboratory as described by Rooney and coworkers (20). Briefly, DPPC, DOPC, DPPG, DOPG, and SPH were dissolved in chloroform, mixed and evaporated under a stream of nitrogen. The lipids were dispersed in HBSS to give a final concentration of 50 mg/ml with the following composition: DPPC (64%), DOPC (24%), DPPG (6%), DOPG (4%), and SPH (2%). This preparation was sonicated for 10 min and filtered through a 0.45-µm filter before the incubation with AM.

Seminatural surfactant (Curosurf) was prepared in Chiesi Laboratories (Geneva, Switzerland) from porcine lungs as described (21) and was provided by Laboratoires Serono (Boulogne, France).

Bronchoalveolar Lavage and Macrophage Isolation

Male Hartley guinea pigs weighing 600 to 1,000 g were anesthetized by the intravenous injection of sodium pentobarbital (20 mg/kg). Twenty successive bronchoalveolar lavages (BAL) were performed aseptically with 5-ml aliquots of saline, containing 25 µg/ml of streptomycin and 25 U/ml of penicillin, which were injected with a plastic syringe through a polyethylene cannula inserted into the trachea. The cell suspensions were centrifuged at 475 × g for 10 min at 25° C and the pellets were washed twice with saline and resuspended in RPMI 1640 culture medium containing 50 µg/ml of streptomycin, 50 U/ml of penicillin, 1% of L-glutamine (wt/vol), 0.7% of Hepes (wt/vol), 0.4% of BSA (wt/vol), and 10% of FCS (vol/vol), pH 7.2. Cells were adjusted at 3 × 10⁶ cells per milliliter. Differential counts were made on modified May-Grünwald-Giemsa-stained cytocentrifuge smears. The composition of the major cell types in the bronchoalveolar lavage fluids (BALF) comprised 85.7 ± 6.3% AM, 8.6 ± 2.3% eosinophils, and 5.7 ± 3.4% lymphocytes (mean ± SE, n = 25).

Macrophages Culture and Incubation Procedures

AM (1 ml) were allowed to adhere in 35-mm culture dishes during 1 h at 37° C in 5% CO₂/95% air. At this step, the cell population of adherent cells consisted of 95 to 99% macrophages after the first hour of adhesion. The plates were then washed three times with medium (37° C) and incubated with serum- and BSA-free RPMI 1640, in the presence or in the absence of AS, Curosurf, or phospholipid preparations, as detailed in the figures. In certain experiments, AM were incubated with gp-TNF- (50 nM) 30 min after the addition of surfactants or phospholipids.

The effect of surfactants and phospholipids on cell adherence was checked by counting the number of detached cells at different time intervals (5, 10, and 20 h). The cell viability was checked by the trypan blue dye exclusion test and was always above 90%. To control cell lysis, the release of lactate dehydrogenase (LDH) activity in the medium was measured at the time intervals indicated previously using a commercial kit from Boehringer (Mannheim, Germany).

Preparation of Cell Lysates

At the end of the incubations, the culture dishes were kept in an ice bath and supernatants were removed. Adherent macrophages were washed and resuspended with 1 ml of cold HBSS containing 0.5 mM PMSF, 2 µg/ml leupeptine, 2 µg/ml aprotinin, and 2 mM ethylenediaminetetraacetic acid (EDTA) and scraped using a rubber policeman. Cells were then lysed by ultrasonication (2 × 30 s, 150 watts) in an ice bath, using a MSE (Annemasse, France) sonifier and kept at 20° C until use.

Measurement of sPLA2-II Activity

The measurement of sPLA2-II activity was carried out using the fluorometric assay described by Radvanyi and coworkers (22) and shown to be selective for sPLA2 type. Furthermore, sPLA2 activity measured in the cell lysates of AM was totally blocked by the specific sPLA2-II inhibitor LY311727 (23) at 10 µM, indicating that sPLA2 activity measured in AM corresponds to a sPLA2-II activity (data not shown). Briefly, the fluorescent substrate phosphatidylglycerol (PG) was dried under nitrogen and suspended in ethanol at a concentration of 0.2 mM. Vesicles were prepared by mixing the ethanol solution of the fluorescent phospholipid with a buffer solution containing 50 mM Tris-HCl, 100 mM NaCl, and 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (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. Macrophage homogenates were maintained in an ice-cold bath throughout the experiment and aliquots (10 to 50 µl corresponding to 1 to 5% of the total homogenate) were introduced into the cuvettes and allowed to equilibrate at 37° C for 1 min. The reactions were then initiated with 10 µl of CaCl₂ at a 10 mM final concentration. The fluorescence measurements were performed with a Jobin et Yvon JY3D spectrofluorometer (Domont, France) equipped with a xenon lamp and all the reactions were carried out in 4 × 10 mm disposable plastic cuvettes. The fluorescence intensity was monitored using excitation and emission wavelengths of 345 and 398 nm, respectively, with a slit width of 4 nm. The final ethanol concentration was less than 0.1% and had no effect on the assay.

Measurement of Lysophospholipase and PAF-Acetylhydrolase Activities

These assays were performed on AM lysate prepared as described previously except that PMSF (which inhibits the activity of these enzymes) was omitted from the preparation. For measuring lysophospholipase activity, the substrate, lyso[³H]PC (1-palmitoyl-sn-glycero-3-phospho(N-methyl-[³H])choline), was prepared in the laboratory as previously described (6) and incubated with aliquots (0.5 ml) of AM lysates at 5 × 10⁴ cpm/ml in the presence of unlabeled lyso-PC at a final concentration of 50 µM. For measuring PAF-acetylhydrolase activity, the procedure was the same except that AM lysates were incubated with [³H]acetyl-PAF (5 × 10⁴ cpm/ml) instead of lyso[³H]PC in the presence of 10 µM unlabeled PAF (final concentration). Incubations were performed for 30 min at 37° C and then PAF-acetylhydrolase and lysophospholipase activities were measured as previously described (6).

Extraction and Analysis of sPLA 2-II Messenger RNA (mRNA) Levels

AM were isolated and cultured as previously indicated and then total RNA was prepared according to the method of Chomczynski and Sacchi (24). Total RNA (10 µg/lane) was electrophoresed on a 1% agarose gel with the formaldehyde method (25) and then transferred onto nylon membranes. The blots were hybridized at 68° C overnight as described by Church and Gilbert (26), using a ³²P-labeled (random priming) full-length guinea pig sPLA2-II complementary DNA (cDNA) (7) as a probe, and washed in 3× saline sodium citrate (SSC) and 5% sodium dodecyl sulfate (SDS), followed by 1× SSC and 1% SDS washes (1× SSC = 0.15 M NaCl; 0.015 M sodium citrate). Blots were washed off and rehybridized with rat -actin cDNA at 65° C, as internal control.

Uptake of Surfactant Phospholipids by AM

Labeled AS was prepared as previously indicated, except that radioactive DPPC or DOPG was added to surfactant preparations. Four different preparations were used: preparation A = surfactant + [³H]DPPC; preparation B = surfactant + [¹⁴C]DOPG; preparation C = DPPC + [³H]DPPC; preparation D = DOPG + [¹⁴C]DOPG. Phospholipids were dissolved in chloroform, mixed and evaporated under a stream of nitrogen. The composition and phospholipid concentration were identical to those described earlier. [³H]DPPC and [¹⁴C]DOPG were used at final concentrations of 80,000 and 25,000 cpm/ml, respectively. After ultrasonication, these preparations were incubated with AM at final concentration of 500 µg/ml and then 50-µl aliquots of medium were removed at different times intervals. The radioactivity was then measured by liquid scintillation counting.

Determination of TNF- Release

TNF- bioactivity was measured by cytotoxicity on fibrosarcoma cells (WEHI 164 clone 13 line, kindly provided by Dr. F. J. Zijlstra, Erasmus University, Rotterdam, The Netherlands). These cells were grown in Dulbecco's medium (Life Technologies, Cergy Pontoise, France) supplemented with 10% FCS (Boehringer, Mannheim, Germany) and antibiotics (1% wt/vol gentamicin and 1% wt/vol amphotericin B; Boehringer Mannheim) in a humidified atmosphere of 5% CO₂. Cells (10⁶/ml) were incubated for 3 h in the presence of 1 µg/ml actinomycin D (Sigma, St. Quentin Fallavier, France). Aliquots of this cell suspension (50 µl/well containing 5 × 10⁴ cells) were plated in 96-well, flat-bottom microtiter plates (Nunclon Delta, Roskilde, Denmark) and incubated for 24 h with 50-µl samples or TNF- standard dilutions (recombinant hTNF- provided by Dr. G. R. Adolf, Bender-Wien, Vienna, Austria) in triplicate. The plates were further incubated for 24 h with 50 µl/well XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) labeling mixture prepared as recommended by the manufacturer (Cell proliferation kit II XTT; Boehringer Mannheim, Germany). Optical density was measured in an automatic reader with a test wavelength of 490 nm and a reference wavelength of 630 nm (MR5000; Dynatech, Marnes-La Coquette, France).

Calculations and Statistical Analyses

Data are expressed as mean ± SE of separate experiments and statistical analyses were performed using unpaired Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of Curosurf and AS on sPLA2-II Expression by AM

We have previously shown that Curosurf, which contains essentially phospholipids, inhibits the synthesis of sPLA2-II by AM (18). To examine whether phospholipids play a role in this process, we investigated the effect of a protein-free AS on the synthesis of sPLA2-II by AM. Because in our experimental conditions (i.e., culture in serum-free medium) no sPLA2-II activity was detected in the supernatant of AM (data not shown), the measurements of sPLA2-II activity were performed on cell homogenates. The latter reached maximal values of 9.37 ± 2.09 nmoles/ml/min (mean ± SE, n = 9) within 20 h of AM culture. Figure 1 shows that incubation of AM with AS for 20 h reduced the level of cell-associated sPLA2-II activity in a concentration-dependent manner although less effectively than Curosurf (concentration that inhibits 50% [IC₅₀] = 500 and 250 µg/ml, respectively). At 500 µg/ml, Curosurf was two times more effective than AS in reducing sPLA2-II activity produced by AM (p < 0.01, n = 10). It could be argued that the observed inhibition might result from an interference of surfactant phospholipids with the assay of PLA2. Purified guinea-pig recombinant sPLA2-II, produced in the baculovirus system (27), was incubated with 500 µg/ml of AS or Curosurf and then aliquots were transferred to the cuvettes containing the fluorescent substrate for the measurement of sPLA2-II activity. The results show that, in our experimental conditions, surfactant had no effect on the activity of recombinant sPLA2-II (data not shown). We have also checked that neither Curosurf nor AS interfered with cell adherence or viability (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1.   Comparative effect of Curosurf and artificial surfactant on sPLA2-II production by AM. AM were incubated with increasing concentrations of AS or Curosurf for 20 h. Then the plates were washed twice and sPLA2-II activity was evaluated in AM sonicates as described in METHODS. The results show the sPLA2-II activity of AM treated with AS (open circles) or Curosurf (closed circles) and are expressed as percentage (mean ± SE, n = 10) of the activity in control cells. **p < 0.01, ***p < 0.001, Student's t test.

Incubation of AM with AS or Curosurf reduced the concentration of sPLA2-II mRNA, clearly indicating that the loss of sPLA2-II activity is caused by inhibition of the synthesis of this enzyme at a transcriptional level (Figure 2).

View larger version (34K): [in this window] [in a new window]   Figure 2.   Effect of surfactant and its phospholipid components on sPLA2-II mRNA expression. AM were incubated with AS (AS, 500 µg/ml), DPPC (320 µg/ml), or DOPG (20 µg/ml) or Curosurf (500 µg/ml) for 20 h, then the cells were washed and total RNA extracted. Ten micrograms of total RNA was electrophoresed, transferred to a nylon membrane, and hybridized sequentially with radiolabeled sPLA2-II and -actin probes. The figure shows sPLA2-II mRNA expression in control (1), AS- (2), DPPC- (3), DOPG- (4), and Curosurf- (5) treated cells and is representative of two independent experiments.

Effect of Surfactant Phospholipids on sPLA2-II Expression

These data indicate that phospholipid components of surfactant are responsible for the suppression of sPLA2-II expression by AM and led us to determine which phospholipid component is involved in this process. The results show that among surfactant phospholipids only DOPG had a signficiant effect in reducing sPLA2-II activity and mRNA expression (Figures 2 and 3). It should be noted that the proportion of DOPG with respect to other phospholipids in our AS preparation is similar to that reported for Curosurf (21). The inhibitory effect of DOPG is concentration-dependent, with a maximal effect obtained at 20 µg/ml (Figure 4), and is not due to a nonspecific membranous effect of DOPG since its structural analog, DPPG, had no significant effect on the level of sPLA2-II activity. Furthermore, DPPC, the major surfactant phospholipid component, does not modulate sPLA2-II activity and mRNA expression (Figures 2 and 3). We verified that these phospholipid preparations failed to interfere with the sPLA2-II enzymatic assay (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3.   Comparative effect of surfactant phospholipids on the production of sPLA2-II by AM. AM were incubated for 20 h with indicated phospholipids at the following concentrations (µg/ml): DPPC = 320, DOPC = 120, DPPG = 30, DOPG = 20, and SPH = 10. These concentrations correspond to the proportions of each phospholipid in AS (see METHODS). The results show the sPLA2-II activity of AM and are expressed as percentage (mean ± SE, n = 6) of the activity in control cells. ***p < 0.001, Student's t test.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Concentration-dependent effect of DOPG on the production of sPLA2-II by AM. AM were incubated for 20 h with indicated concentrations of DOPG. The results show the sPLA2-II activity of AM and are expressed as percentage (mean ± SE, n = 5) of the activity in control cells. *p < 0.05, ***p < 0.001, Student's t test.

Uptake of Surfactant Phospholipids by AM

These results led us to investigate the ability of AM to incorporate surfactant phospholipids. Our results show that AM incorporate DOPG in a time-dependent manner with a maximal value (50 to 75% of total added radioactivity) observed within 20 h. However, DPPC was incorporated by the cells to a much lower extent (Figure 5). The level of DOPG incorporation was similar, irrespective of whether DOPG was added to AM alone or in combination with other surfactant phospholipids.

View larger version (19K): [in this window] [in a new window]   Figure 5.   Uptake of DOPG and DPPC by AM. AM were incubated with [3H]DPPC and [14C]DOPG or AS containing one of these labeled phospholipids, as indicated in METHODS. After the indicated time intervals, 50-µl aliquots were taken off from the medium and their radioactivity was measured by liquid scintillation counting. The results show the incorporation of radioactive DOPG and DPPC in the indicated experimental conditions and are expressed as percentage (mean ± SE, n = 4) of total added radioactivity.

Effect of Surfactant and Phospholipids on Lysophospholipase and PAF-Acetylhydrolase Activities

To verify whether the inhibition of sPLA2-II expression was due to a nonspecific effect of surfactant, we examined the effect of surfactant and phospholipid preparations on the activities of lysophopholipase and PAF-acetylhydrolase. Table 1 shows that these enzymatic activities were not altered when AM were treated with Curosurf, AS, DOPG, or DPPC.

Effect of Surfactant and Phospholipids on TNF- Release

We have previously reported that the expression of sPLA2-II by AM is mediated by TNF- through an autocrine/paracrine process (9). We then examined whether the inhibition of sPLA2-II by surfactant and its components occurs through the suppression of TNF- secretion. Our results show that Curosurf, AS, and DOPG significantly reduced the secretion of TNF- by AM, whereas DPPC had no significant effect (Figure 6).

View larger version (13K): [in this window] [in a new window]   Figure 6.   Effect of Curosurf, AS, and phospholipids on TNF- release. AM were incubated with Curosurf (CS, 500 µg/ml), AS (500 µg/ml), DOPG (20 µg/ml), or DPPC (320 µg/ml). After 20 h incubation, aliquots were taken off from the medium and then centrifuged to remove detached cells. Then the cell-free supernatants were analyzed for TNF- bioactivity as described in METHODS. The results show the TNF- released (mean ± SE, n = 3) in the incubation medium and are expressed in ng/ml. ***p < 0.001, Student's t test.

Effect of Surfactant and Phospholipids on TNF--induced sPLA2 Expression

Incubation of AM with exogenous gp-TNF- led to a concentration-dependent increase in sPLA2-II synthesis with the maximal effect being observed at 50 nM of gp-TNF-. A 3- to 5-fold increase was observed in the activity of sPLA2-II, 20 h after the addition of 50 nM of gp-TNF- (data not shown). Preincubation of AM with Curosurf, AS, and DOPG 30 min before the addition of gp-TNF- (50 nM) markedly reduced the level of sPLA2-II activity (Figure 7).

View larger version (14K): [in this window] [in a new window]   Figure 7.   Effect of surfactant and phospholipids on TNF--induced sPLA2 expression. AM were incubated with Curosurf (CS, 500 µg/ ml), AS (500 µg/ml), DOPG (20 µg/ml), or DPPC (320 µg/ml), 30 min before the addition of gp-TNF- (50 nM). After 20 h incubation, the plates were washed twice and sPLA2-II activity was evaluated in AM lysates as described in METHODS. The results show the sPLA2-II activity of AM and are expressed as percentage (mean ± SE, n = 4) of control cells (treated with TNF alone). ***p < 0.001, Student's t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In our previous study (18), we reported that Curosurf inhibits the synthesis of sPLA2-II by guinea pig AM but the mechanisms involved in this inhibition have not been elucidated. Here, we report that AS, which is composed only of phospholipids, reproduced the inhibitory effect of Curosurf although less effectively than the latter. This clearly indicates that phospholipid components are responsible, at least in part, for the inhibitory effect of pulmonary surfactant on sPLA2-II expression by AM. Our data are in agreement with those of Hayakawa and coworkers (16) which reported that AS inhibits the oxidative metabolism of rabbit AM, at concentrations similar to those used here. In order to determine which phospholipid component contained in surfactant preparation accounts for the inhibition of sPLA2-II expression, the effect of individual surfactant phospholipids was investigated. The concentrations of phospholipids used in this study were derived from those of AS (500 µg/ml) giving maximal inhibition of sPLA2-II expression and correspond to the proportion of each phospholipid in whole surfactant. Our studies show that DOPG inhibits sPLA2-II synthesis by AM, whereas the other phospholipids had only a marginal effect. The inhibitory effect of DOPG occurs in a concentration-dependent manner, with a maximal inhibition being observed at 20 µg/ml. These findings suggest that DOPG is responsible for the major part of the effect of surfactant on sPLA2-II synthesis. The inhibitory effect of DOPG correlated well with its rapid uptake by AM, in contrast to DPPC which was only poorly taken up by these cells. This uptake was also observed when radioactive DOPG was added in combination with other surfactant phospholipids, indicating the existence of selective transfer of DOPG from the surfactant to AM. Whether DOPG acts on sPLA2-II expression directly or via its metabolic products remains to be investigated.

It should be noted that the observed inhibition of sPLA2-II expression might not be due to an alteration of all cellular protein synthesis by surfactant preparations because the latter failed to reduce the activities of lysophospholipase and PAF-acetylhydrolase (two phospholipid-metabolizing enzymes) in treated AM.

Because the induction of sPLA2-II synthesis by guinea-pig AM is mediated by TNF- through an autocrine/paracrine process (9), we examined whether inhibition of sPLA2-II expression is caused by an impairment of TNF- release by surfactant. Our studies show that both Curosurf and AS reduce the secretion of TNF- by AM, consistent with previous studies of Thomassen and coworkers (15) showing that synthetic surfactant Exosurf inhibits secretion of cytokines by human AM. The inhibitory effect of surfactant on TNF- secretion was mimicked by DOPG, but to a much lesser extent by DPPC. However, addition of an excess of exogenous gp-TNF- failed to reverse the inhibition of sPLA2-II expression by Curosurf, AS, or DOPG. This suggests that surfactant and its components interfere with a signaling pathway, such as nuclear factor kappa B (NF-B), involved in both stimulation of TNF- synthesis and TNF--induced sPLA2-II expression. Indeed, NF-B, is a ubiquitous transcription factor implicated in the upregulation of TNF- expression (28) and is an essential component of the cytokine signaling cascade involved in sPLA2-II gene regulation (29). This is in agreement with the fact that exogenous surfactant suppresses NF-B activation in human monocytic cells (30).

Our study suggests that in normal conditions in alveoli where AM are surrounded by the pulmonary surfactant, the expression of sPLA2-II is under supression and that, when these cells were washed from surfactant, they "escaped" the inhibitory effect. A deficiency or alteration of surfactant, such as in neonatal or adult respiratory distress syndrome (11), may thus result in an increase in the synthesis and secretion of sPLA2-II in alveoli. In agreement with this hypothesis, we have recently shown that sPLA2-II expression is accompanied by an important hydrolysis of surfactant phospholipids in an experimental model of acute lung injury (27). In this model, hydrolysis of surfactant phospholipids by sPLA2-II contributes to surfactant alteration, which in turn would trigger sPLA2-II production, thus leading to the installation of a vicious circle.

Because sPLA2-II has been shown to induce inflammatory response (10), an increase of its concentrations in lung tissues may exacerbate the pulmonary inflammation. Then, downregulation of sPLA2-II synthesis by surfactant may account for the clinical benefit of surfactant therapy in respiratory distress syndromes.