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and from patients with ARDS showed that the PI-PLC specific activity was decreased ..... the best-suited role for an extracellular PI-PLC activity in alveolar space ...
A Specific Phospholipase C Activity Regulates Phosphatidylinositol Levels in Lung Surfactant of Patients with Acute Respiratory Distress Syndrome Spyros Spyridakis1, George Leondaritis1‡, George Nakos2, Marilena E. Lekka3, and Dia Galanopoulou1 1

Department of Chemistry, University of Athens, Athens, Greece; 2University Hospital of Ioannina, Medical School, and 3Department of Chemistry, University of Ioannina, Ioannina, Greece

Lung surfactant (LS) is a lipid-rich material lining the inside of the lungs. It reduces surface tension at the liquid/air interface and thus, it confers protection of the alveoli from collapsing. The surfaceactive component of LS is dipalmitoyl-phosphatidylcholine, while anionic phospholipids such as phosphatidylinositol (PtdIns) and primarily phosphatidylglycerol are involved in the stabilization of the LS monolayer. The exact role of PtdIns in this system is not wellunderstood; however, PtdIns levels change dramatically during the acute respiratory distress syndrome (ARDS) evolution. In this report we present evidence of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid, which may regulate PtdIns levels. Characterization of this extracellular activity showed specificity for PtdIns and phosphatidylinositol 4,5bisphosphate, sharing the typical substrate concentration-, pH-, and calcium-dependencies with mammalian PI-PLCs. Fractionation of BAL fluid showed that PI-PLC did not co-fractionate with large surfactant aggregates, but it was found mainly in the soluble fraction. Importantly, analysis of BAL samples from control subjects and from patients with ARDS showed that the PI-PLC specific activity was decreased by 4-fold in ARDS samples concurrently with the increase in BAL PtdIns levels. Thus, we have identified for the first time an extracellular PI-PLC enzyme activity that may be acutely involved in the regulation of PtdIns levels in LS. Keywords: phospholipase C; phosphatidylinositol; phosphatidylinositol 4,5-bisphosphate; lung surfactant; acute respiratory distress syndrome

Broncoalveolar lavage (BAL) fluid is a valuable material for the diagnosis of lung disorders, as it represents alveolar fluid in dilution. It contains lung surfactant (LS), cells (cell fraction consists of almost 80% of alveolar macrophages) (1), lipid and peptide mediators, and various classes of proteins involved in the secretion and function of LS. LS is a lipid-rich material lining the inside of the lungs, and it serves in reducing surface tension. Thus, it protects alveoli from collapsing, especially at the end of expiration (2). In addition, it participates in the local host-defense system (3). The major lipid component of LS is the dipalmitoyl analog of phosphatidylcholine (DPPC), comprising 70% of total phospholipids. The anionic phospholipid phosphatidylglycerol, also involved as a surface-active agent, occurs in significant amounts (10%) (4). It has been suggested by in vitro studies that anionic phospholipids are required for stabilization of the LS monolayer (5). As phosphatidylglycerol alone could play this role, the structural function of other anionic phospho-

(Received in original form March 1, 2009 and in final form May 2, 2009) ‡

Present affiliation: Department of Pharmacology, Medical School, University of Thessaly, Larissa, Greece. Correspondence and requests for reprints should be addressed to Dia Galanopoulou, Laboratory of Biochemistry, Department of Chemistry, University of Athens, Zografou, 15771 Athens, Greece. E-mail: [email protected]

Am J Respir Cell Mol Biol Vol 42. pp 357–362, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2009-0078OC on June 2, 2009 Internet address: www.atsjournals.org

lipids like phosphatidylinositol (PtdIns), which is present in minor amounts in LS, is not well understood. However, due to the fact that PtdIns serves as a biochemical marker for nonmature fetal lung, it has been suggested that it might play an alternative physiologic role in LS (6): It is known that PtdIns interacts specifically with the LS protein SP-D, which participates in the innate immune response of the lung (7), yet the physiological importance of the PtdIns–SP-D interaction has remained obscure. The phospholipids in BAL fluid originate largely from the LS secreted from alveolar type II cells; however, in acute respiratory distress syndrome (ARDS), they could also originate from the circulation, passing through the alveolar–capillary membrane due to increased permeability (1) or from damaged cells. The phospholipid levels in BAL could also depend on the presence of phospholipases. In particular, members of the secreted phospholipase A2 (sPLA2) family have been shown to affect pulmonary function either directly, by hydrolyzing LS DPPC, or indirectly, through production of biologically active lipid mediators (8, 9). These phospholipases may serve as markers in inflammation and signaling and include PLA2 isoforms implicated in ARDS in humans (8), the type II PLA2 secreted by guinea pig alveolar macrophages (10), and the PAF-specific acetylhydrolase (8). In addition to PLA2 enzymes, a phospholipase C activity hydrolyzing phosphatidylcholine (PC-PLC) has been detected in BAL (11) but, so far, no PLC activity hydrolyzing PtdIns has been reported. Phosphoinositide-specific PLCs (PI-PLCs) constitute a family of enzymes that are specific for PtdIns and phosphoinositides and are present in all mammalian cell types (12–14). They are intracellular enzymes, although there is a report on an extracellular PI-PLC activity that is released from Swiss 3T3 cells in culture (15). In vivo and in response to activation of numerous cell surface receptors, PI-PLC hydrolysis of plasma membrane PtdIns(4,5)P2 generates inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol. These two second messengers function as master regulators of calcium and protein phosphorylation signaling and eventually regulate a variety of important cellular functions, including secretion, cell proliferation, differentiation, and immune cell activation (16, 17). The PI-PLC family contains several isoforms that exhibit similar enzymatic properties but differ in their tissue distribution, primary sequence, and mode of activation (14). In lung, at least six different isoforms, namely PLCd1, PLCb1, b3, PLCg1, PLCe, and PLCh, have been suggested or proved to be substantially expressed (14, 18–20); in particular, PLCb3 has been shown to be preferentially expressed in adult rat type II cells (18). The aim of the present study was to assess whether PtdInsspecific enzyme activities are present in BAL and whether these could be involved in the regulation of PtdIns levels. We have detected and characterized a PI-PLC activity, which is involved in the regulation of PtdIns levels in LS, and which was found to be decreased in BAL samples from patients with ARDS. This may indicate a possible PI-PLC implication in lung injury.

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MATERIALS AND METHODS Materials Phospholipid standards, PtdIns, polyphosphoinositide mixture, and boron trifluoride/methanol were obtained from Sigma (Steinheim, Germany, or St. Louis, MO); Silica gel 60H and 60G from Merck (Darmstadt, Germany); Dowex AG 1-x8 Poly-Prep columns, formate form, from Bio-Rad (Hercules, CA); [3H]PtdIns(4,5)P2 (specific activity 6.5 Ci/mmole) and [14C]palmitoyl-sn-1-DPPC (0.1 mCi/ml) from ARC (St. Louis, MO); [3H]Ins(1,4,5)P3 from Amersham (Buckinghamshire, UK; TRK1000). Fatty acid methyl ester standards were obtained from Chem Service (West Chester, PA). All other chemicals and solvents were of analytical grade.

Patients Ten mechanically ventilated patients were used in this study, six patients with ARDS and four patients as the control group. Standard criteria for ARDS diagnosis were: (1) acute hypoxemic respiratory failure requiring mechanical ventilation, (2) diffuse bilateral alveolar infiltrates on the chest roentgenogram, (3) refractory hypoxemia (PaO2/ FIO2 , 200 regardless of PEEP level), (4) pulmonary artery wedge pressure , 18 mm H2O or no clinical evidence for left atrial hypertension, and (5) recognized appropriate clinical setting or risk factor for the development of ARDS (21). The inclusion criteria for control subjects were: absence of cardiopulmonary disease, normal chest radiograph, and PaO2/FIO2 . 300 mm Hg. The patients’ demographic data and clinical and biological characteristics are shown in Table 1. The protocol was approved by the Ethics Committee of the University Hospital of Ioannina, and the patients or the next of kin gave an informed consent to the study.

Bronchoalveolar Lavage Bronchoalveolar lavage (BAL) was performed by fiberoptic bronchoscopy, as described in a previous study (1). In summary, patients were ventilated with a control mechanical ventilation mode. Six aliquots of 20 ml sterile normal saline at 378C were infused through the working channel of the bronchoscope. The first aspirated fluid, reflecting a bronchial sample, was discharged, while the others were collected in ice-cold tubes. BAL was filtered through cell strainer filters (70 mm Nylon, Falcon; Becton Dickinson, Franklin Lakes, NJ), for removal of mucus. Then, it was centrifuged at 500 3 g for 15 minutes at 48C to obtain a pellet corresponding to BAL cells. BAL fluid and BAL cells were kept at 2208C until analysis, which was performed on the same day. Upon thawing, cells were resuspended in saline and homogenized by sonication.

Fractionation of BAL Fluid and Protein and Phospholipid Determinations An aliquot of 20 to 25 ml of the 500 3 g supernatant (BAL fluid) was centrifuged at 30,000 3 g for 1 hour at 48C. The pellet of 30,000 3 g corresponded to large surfactant aggregates. The supernatant of 30,000 3 g corresponded to small surfactant aggregates and soluble fraction. Total protein was determined in BAL fluid and subfractions, as well as in BAL cell homogenates according to the method of Lowry and coworkers (22). Total phospholipids were extracted from the same fractions according to Schacht (23), to fully recover PtdIns, and lipid phosphorus was determined according to Bartlett as modified by Marinetti (24).

Isolation and Determination of PtdIns To quantitate PtdIns levels in BAL fluid from control subjects or patients with ARDS, total lipid extracts were chromatographed using two-dimensional thin layer chromatography (2-D TLC) with solvent systems: (1) chloroform/methanol/25% ammonium hydroxide (65:35:5, by vol), and (2) chloroform/acetone/methanol/acetic acid/ water (14:4:2:2:1, by vol) (25). PtdIns was identified by comparison to an authentic standard run in a separate plate, extracted from the silica gel by a modification of the Schacht method, and lipid phosphorus was determined as described above.

TABLE 1. DEMOGRAPHIC CHARACTERISTICS OF PATIENTS WITH ACUTE RESPIRATORY DISTRESS SYNDROME AND CONTROL PATIENTS No.

Age

Sex

Disease

Day of MV*

Outcome

1 2 3 4 5 6 Mean SD

56 49 73 60 42 70 58 12

F M F F M M

Sepsis Aspiration Sepsis Pneumonia Sepsis Sepsis

3 5 4 2 3 4

Alive Died Alive Alive Alive Died

1 2 3 4 Mean SD

38 46 47 75 51 16

M F M M

H/S CVA NM H/S

5 2 5 6

Alive Alive Alive Died

Definition of abbreviations: CVA, cerebrovascular accident; H/S, head and/or spine trauma; MV, mechanical ventilation; NM, neuromuscular disease. * Day of mechanical ventilation when BAL was performed.

PI-PLC Assay PLC was assayed at 378C using 5 to 12 mg of protein in a standard reaction mixture consisting of Tris-HCl 50 mM (pH 6.5), free CaCl2 10 mM (buffered with EGTA 3 mM), sodium deoxycholate 0.01% and [3H]PtdIns(4,5)P2 60 mM (4,000 cpm nmole-1) as a substrate, to a final volume of 50 ml (26). In the case of PtdIns, [3H]PtdIns (specific activity 4,500 cpm nmole-1) isolated from [3H]inositol-labeled Tetrahymena pyriformis cells was used as a substrate (25). The substrates were solubilized by sonication in Tris-HCl 50 mM (pH 6.5) and sodium deoxycholate 0.05%. Reactions started by the addition of either the substrate or the protein preparation and terminated after 15 minutes (BAL cells) or 30 minutes (BAL fluid) by the addition of 1 ml ice-cold chloroform/methanol (2:1, by vol) and 250 ml HCl 1N. After phase separation by centrifugation at 3,000 3 g for 3 minutes, aliquots of the aqueous phase were counted for radioactivity. In all experiments, control reactions were performed in the absence of protein or with addition of heat-inactivated BAL fluid or cell homogenate. The calcium dependency of PI-PLC was studied with EGTA-buffered Ca21 solutions as described previously (27). For kinetic analysis, PtdIns(4,5)P2 was used at 20 to 180 mM, while the concentration of deoxychholate was kept constant at 240 mM (28). The reaction was performed with 10 mg of BAL fluid protein at 10 mM free Ca21 for 30 minutes.

Analysis of PI-PLC Products Analysis of PI-PLC lipid products was performed by TLC using the following solvent systems depending on the substrate used: for PtdIns, chloroform/methanol/25% ammonium hydroxide/water (86:76:6:16, by vol); for PtdIns4,5P2, 1-propanol/acetic acid 2M (13:7, by vol); for PtdCho, chloform/methanol/water (65:35:4, by vol) for polar lipids and petroleum ether/diethylether/acetic acid (70:30:1, by vol) for neutral lipid analysis. The separation of water-soluble inositol phosphate products was performed by anion-exchange chromatography on Dowex AG 1-x8 columns. The aqueous phase of the PI-PLC assay was diluted with water (1:8, by vol), neutralized with dilute NH4OH (1:6, by vol), and loaded onto Dowex columns, and the products were eluted batchwise with increasing concentrations of ammonium formate in formic acid. Glycerophosphoinositol was eluted with ammonium formate 60 mM/ sodium borate 5 mM, while inositol monophosphate, inositol bisphosphate, and inositol trisphosphate were eluted with 10 to 12 ml of ammonium formate 0.2 M/formic acid 0.1 M, 12 to 14 ml of ammonium formate 0.4 M/formic acid 0.1 M, and 10 to 12 ml of ammonium formate 1 M/formic acid 0.1 M, respectively. In all samples, 2-ml fractions were collected and the radioactivity was assayed by liquid scintillation counting. Standard [3H]Ins(1,4,5)P3 and [3H]glycerophosphoinositol, derived from alkaline hydrolysis of [3H]PtdIns, were routinely used for the calibration of the columns.

Spyridakis, Leondaritis, Nakos, et al.: PI-PLC Activity in Lung Surfactant TABLE 2. PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C ACTIVITY IN BAL FLUID AND CELLS Specific Activity (nmole/min/mg) Sample

PtdIns(4,5)P2

PtdIns

BAL fluid BAL cells

0.362 6 0.199 0.056 6 0.047

0.15 6 0.03 0.04 6 0.02

Definition of abbreviations: BAL, bronchoalveolar lavage; PtdIns, phosphatidylinositol. PI-PLC activity was assayed as described in MATERIALS AND METHODS. Results are means 6 SD from four (PtdIns(4,5)P2) and two (PtdIns) BAL samples, each assayed in duplicate.

Fatty Acid Determination BAL fluid PtdIns and PtdCho were separated by TLC and their fatty acids profile were assayed by gas chromatography (GC) after derivatization to methyl esters using boron trifluoride/methanol (25).

Calcium Determination by Atomic Absorption Atomic absorption measurements were performed in an AA spectrometer model 2380 (Perkin Elmer, Waltham, MA) using salinesupplemented Ca21 solutions for the standard curves.

RESULTS An Extracellular PI-PLC Activity Hydrolyzing PtdIns and PtdIns(4,5)P2 Is Present in BAL Fluid

As shown in Table 2, use of [3H]PtdIns or [3H]PtdIns(4,5)P2 as exogenous substrate for a putative PtdIns-hydrolyzing activity resulted in the detection of this activity in both BAL fluid

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and BAL cells from mechanically ventilated patients without cardiopulmonary disease. The specific activity against both substrates was higher in BAL fluid (Table 2). PI-PLC specific activity values presented in Table 2 were calculated on the basis of radioactivity measurements of total [3H]inositol-labeled water-soluble products generated during the reaction. To determine whether the radioactivity of the products corresponded to the expected Ins(1,4,5)P3, when PtdIns(4,5)P2 was used as a substrate, the aqueous phase of the assay was analyzed by ion-exchange chromatography. This analysis confirmed the presence of a PI-PLC activity in BAL fluid, since the vast majority (z 70%) of water-soluble radioactivity was recovered in the equivalent fractions with standard [3H]Ins(1,4,5)P3 (Figure 1A). A minor amount of inositol bisphosphate, probably due to nonspecific hydrolysis of Ins(1,4,5)P3 by phosphatases, was also detected in the aqueous phase of the assay (Figure 1A). To exclude the presence of a PLA2 activity acting on PtdIns(4,5)P2 (29), we analyzed the lipid products of the assay using TLC to examine whether lyso-[3H]PtdIns(4,5)P2 has been produced during the reaction. As shown in Figure 1B, no lyso-PtdIns(4,5)P2 was detected; this confirmed that water-soluble products were exclusively inositol phosphates and not the putative deacylation products of PtdIns(4,5)P2. Similar results were obtained for the BAL cell activity. In addition, when [3H]PtdIns was used as a substrate, the only water-soluble product was InsP (data not shown), and lyso-PtdIns was not detected in the organic phase. Therefore, under our experimental conditions, we have not detected other activities hydrolyzing PtdIns(4,5)P2 and PtdIns in BAL fluid. Furthermore, to assess whether this PLC activity is a specific PI-PLC and not a nonspecific phospholipid-

Figure 1. Detection of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid using phosphatidylinositol (PtdIns)(4,5)P2 as a substrate. [3H]PtdIns(4,5)P2 was incubated withsaline (control) or 8 mg protein of BAL fluid (BAL) and (A) water-soluble and (B) lipid-soluble products of the reaction were analyzed as described in MATERIALS AND METHODS. (C) [14C]palmitic-labeled PtdCho was incubated with BAL fluid under identical conditions. Lipid-soluble products were chromatographed by TLC as described in MATERIALS AND METHODS for separation of diacylglycerol (DAG) (left panel) or PtdCho and lyso-PtdCho (right panel). Or, origin; MAG, monoacylglycerol; TAG, triacylglycerol; FA, fatty acids. (D) BAL fluid was fractionated into a 30,000 3 g pellet and supernatant fractions and protein content, total lipid phosphorus, and PtdIns(4,5)P2PLC activity were determined as described in MATERIALS AND METHODS. The results shown are means of duplicate samples from a representative experiment that was repeated with at least four different BAL samples.

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Figure 2. Characterization of BAL fluid PIPLC activity. Linearity of BAL fluid PI-PLC activity with respect to (A) incubation time and (B) protein. (C) pH-dependency of BAL fluid PI-PLC activity was assayed by varying pH values with acetic acid/sodium acetate (AcA) or Tris-HCl buffers. (D) BAL fluid PIPLC was assayed with increasing concentrations of PtdIns(4,5)P2 in the presence of a fixed amount of Triton X-100 and plotted against the molar ratio of PtdIns(4,5)P2 to Triton X-100. The results shown are means of duplicate samples from a representative experiment which was repeated with at least four different BAL samples.

hydrolyzing activity, we tested [14C]PtdCho as a substrate under identical conditions. In contrast to hydrolysis observed with both phosphoinositides, no [14C]diacylglycerol indicative of a possible PC-PLC activity was detected when TLC analysis of the organic phase of the reaction was performed (Figure 1C). These novel findings suggested that a PI-PLC activity may be actively or passively released from epithelial type II cells, alveolar macrophages, or other cell types in the alveolar space. Release of this type of enzyme from mammalian cells has been reported previously for cultured Swiss 3T3 cells (15), but there are no data concerning the mechanism of this release. Alternatively, PI-PLC in BAL fluid could be released from damaged cells during the course of mechanical ventilation on healthy lungs. However, determination of the activity of an abundant, soluble, cytosolic enzyme, namely LDH, gave no detectable levels in BAL fluid fraction. Moreover, the specific activity of PI-PLC was higher in BAL fluid compared with BAL cells, indicating an enrichment of activity in BAL fluid (Table 2). The above findings thus argue against a nonspecific, artefactual release of this activity in BAL fluid. Fractionation of BAL fluid into large surfactant aggregates (30,000 3 g pellet, which consist mainly of lamellar bodies and tubular myelin and contribute to the reduction of surface tension) and soluble fraction (30,000 3 g supernatant, which also contains small lipid vesicles with poor surface activity [30]), showed that the PI-PLC activity was distributed mainly in the soluble fraction and not in BAL large aggregates (Figure 1D). Thus, the enzyme activity is either part of the small vesicle fraction found in 30,000 3 g supernatant or a soluble activity; in either case, active secretion along with surfactant constituents from the same or other types of cells remains to be elucidated.

appear to be more active in pH 7.0 (32). Interestingly, the dependency of activity on the molar fraction of PtdIns(4,5)P2 in the presence of deoxycholate was represented by a sigmoidal curve (Figure 2D). This kinetic model, which was first developed for PLA2 (33), is common for activities functioning on lipid–water interfaces and it has been applied to secreted bacterial PLCs (34) and certain mammalian PI-PLC isoforms (28, 35, 36). It seems that the same model is valid for the BAL fluid PI-PLC described in this report. PI-PLCs exhibit a characteristic Ca21 and substrate specificity in vitro: at low Ca21 concentrations, they hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in preference

BAL Fluid PI-PLC Activity Shares Common Functional Characteristics with Intracellular PI-PLCs

We proceeded in characterizing further the PI-PLC activity of BAL fluid. As shown in Figures 2A and 2B, PtdIns(4,5)P2-PLC activity was linear for up to 60 minutes and 10 mg of protein, while the optimum pH was found to be 7.0 (Figure 2C). This value is slightly different from those previously reported for PIPLCs (optimum pH, 6.5) (31), although there are some activities (from rat liver and cerebral-cortical membranes) that

Figure 3. BAL fluid PI-PLC activity displays a typical Ca21 dependency. BAL fluid PI-PLC was assayed in the presence of EGTA or BAPTA, or, in the presence of the indicated free Ca21 concentrations with (A) PtdIns(4,5)P2 or (B) PtdIns as substrate. Results are means 6 SD from three to four different BAL samples, each assayed in duplicate.

Spyridakis, Leondaritis, Nakos, et al.: PI-PLC Activity in Lung Surfactant

to PtdIns. At high Ca21 concentrations, however, PtdIns hydrolysis increases (13, 31). The Ca21 dependency of BAL fluid PIPLC activity was studied with both PtdIns(4,5)P2 and PtdIns as substrates. As shown in Figure 3A, in the presence of the Ca21chelators EGTA (3 mM) or BAPTA (1 mM), low levels of activity were detected. Increase of the free Ca21 concentration to 1 mM or 10 mM resulted in enhanced activity against PtdIns(4,5)P2, which was maximal at 10 mM. Further increase of the Ca21 concentration above 100 mM, however, resulted in reduction of activity against PtdIns(4,5)P2 (Figure 3A). In contrast, when PtdIns was used as a substrate, maximal activity was observed at Ca21 concentrations above 100 mM (Figure 3B). Importantly, at all Ca21 concentrations, the specific activity against PtdIns(4,5)P2 was higher compared with the activity against PtdIns (Figure 3). Thus, the in vitro dependency on both Ca21 and substrate that we observed for the BAL fluid extracellular PI-PLC is typical of intracellular PI-PLC isoforms. Within cells, low physiological Ca21 levels support primarily the hydrolysis of the PtdIns(4,5)P2 and not that of PtdIns. Our atomic absorption measurements of Ca21 in BAL fluid from mechanically ventilated patients with healthy lungs showed a concentration of 3.3 to 4.2 ppm; this value corresponds to a nominal Ca21 concentration of 100 mM at diluted conditions. Thus, and taking into account that presently there is no evidence for the occurence of PtdIns(4,5)P2 in LS, we propose that the best-suited role for an extracellular PI-PLC activity in alveolar space is the regulation of the levels of PtdIns present in lung surfactant. BAL Fluid PI-PLC Activity Is Inversely Correlated with PtdIns Levels in Patients with ARDS

To investigate the possible functional role of the PI-PLC activity in the regulation of LS PtdIns levels, we proceeded in analyzing BAL fluid samples from patients with ARDS. In a previous study we had found elevated PtdIns levels in the BAL fluid of patients with ARDS (37). In this study, we also found that PtdIns levels were significantly increased in BAL fluid from patients with ARDS compared with our control group, while the total amount of phospholipids was not changed (Table 3). The examination of fatty acid profiles of both PtdIns and PtdCho fractions after separation by 2-D TLC using GC showed differences in the ARDS samples, similar to those reported previously by Schmidt and coworkers (38). Although we found no changes in the palmitate content of PtdIns, total C18 fatty acid content was elevated by almost 30%, while PtdCho C18 levels were doubled and its palmitate content was decreased by 20% (unpublished data). Thus, PtdIns, the physiological substrate of BAL fluid PI-PLC, was enriched in C18 fatty acids in ARDS samples. When we assayed the PI-PLC BAL fluid activity in ARDS samples, we found that it was reduced by 70% compared with the activity of control samples (Table 3). It is important to note that the reduction of PI-PLC activity that we observed correlates perfectly with the increase of PtdIns levels in LS during ARDS evolution. Taking together, these results support the notion that BAL fluid PI-PLC may be actually involved in the turnover of PtdIns levels in LS.

CONCLUSIONS In this report we present evidence for a previously undetected extracellular PI-PLC activity in BAL fluid. Our study showed that BAL PI-PLC shares common enzymatic characteristics with all known intracellular PI-PLC isoforms and corresponds to a soluble enzyme that most probably is released locally from

361 TABLE 3. TOTAL PHOSPHOLIPIDS, PHOSPHATIDYLINOSITOL CONTENT, AND PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C ACTIVITY IN BAL FLUID FROM CONTROL SUBJECTS AND FROM PATIENTS WITH ARDS

Control ARDS

Total Phospholipid Content (mg/ml)*

PtdIns Content (% of total phospholipids)†

PI-PLC Activity (nmole/min/mg)‡

1.44 6 0.27 1.57 6 0.33

1.3 6 0.2 4.2 6 0.4x

0.362 6 0.199 0.108 6 0.065x

Definition of abbreviations: ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; PI-PLC, phosphoinositide-specific phospholipase C; PtdIns, phosphatidylinositol. * Total phospholipids content was determined by lipid phosphorus determination after extraction of BAL fluid. Results are means 6 SD from four control and four ARDS samples. † PtdIns content was determined after separation by two-dimensional thin layer chromatography and lipid phosphorus determination. Results are means 6 SD from three control and three ARDS samples. ‡ PI-PLC was assayed with PtdIns(4,5P)2 as a substrate as described in MATERIALS AND METHODS. Results are means 6 SD from four (control) and six (ARDS) samples, each assayed in duplicate. x P , 0.05, Student’s t test.

epithelial type II cells, alveolar macrophages or other cell types in the alveolar space. The role of intracellular PI-PLC isoforms signaling in LSsecreting epithelial type II cells and alveolar macrophages has been well documented in several studies (18, 19, 39). PI-PLC b and g isoform expression has been related to activation of differentiation and secretion of LS in type II cells (18, 19). In addition, PI-PLCg is implicated in regulation of phagocytosis in macrophages (39) and protection of lung epithelium against oxidative stress induced by iNOS, thus preventing amplification and perpetuation of an uncontrolled, oxidant-induced, inflammatory cascade (40). Our results, although they do not directly address the isoform responsible for the BAL fluid PI-PLC activity, clearly suggest that this activity is involved in the regulation of LS PtdIns levels. As such, reduced PI-PLC activity levels were observed in BAL fluid of patients with ARDS, and this corresponded to an increase in PtdIns levels observed during the ARDS evolution as we have shown in a previous study (37). These findings point to a specific role for PtdIns in the development of surfactant dysfunction and possibly pathogenesis in ARDS. Considering the unique (among LS phospholipids) property of PtdIns in SP-D binding (7), it is tempting to speculate that alterations in PtdIns levels may impact directly on SP-D function. SP-D is involved in lung innate immunity, and it also plays an important role in surfactant homeostasis by conversion of PtdIns-rich newly synthesized surfactant into small lamellar bodies (41). Therefore, PI-PLC–dependent increase of PtdIns in ARDS samples may lead to surfactant dysfunction via SP-D interactions with PtdIns-rich surfactant. It is generally accepted that the acidic phospholipid PtdGro performs key functions in adsorption, spreading, and respreading of LS phospholipid layer. PtdIns reportedly serves as a cross-functional phospholipid with PtdGro (42), given also that they share a common biosynthetic pathway in LS-producing epithelial type II cells (43). However, the levels of these two phospholipids are differently regulated in several pathological conditions. For example, both PtdGro and PtdIns are decreased in endobronchial antigen challenge (44), while PtdIns is increased, with either no changes or decrease of PtdGro, in ARDS (37, 38; the present article) and cystic fibrosis in children (45). Therefore, it can be deduced that they may have distinct roles, and accordingly we propose that changes in PtdIns levels,

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or a decrease in PtdGro/PtdIns ratio, could be used as an additional biomarker together with several other parameters (1, 37) to better characterize ARDS evolution and particularly its early stages. Importantly, the PtdIns-specific PI-PLC activity that we have characterized in this report provides an alternative mechanism for uncoupling PtdIns and PtdGro regulation and possibly their functions in LS homeostasis. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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