INTRODUCTION

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SPHINGOLIPIDS REPRESENT A biologically active class of lipids that consists of a hydrophobic component, called a ceramide, which is amide linked to a long-chain sphingoid base, such as sphingosine. Ceramide and sphingosine, and related phosphorylated derivatives, appear to have diverse biological effects, including control of cell growth and differentiation, regulation of membrane stability, apoptosis, and signal transduction (11). Ceramide and sphingosine are synthesized in cells via the de novo pathway or can represent breakdown products resulting from sphingomyelin hydrolysis (20). Sphingomyelin hydrolysis has emerged as a potentially important effector pathway for stimulatory factors associated with acute lung injury such as tumor necrosis factor- (TNF-), Fas/Apo ligand, and ionizing radiation (4, 5, 7, 13, 15, 29). In this pathway, TNF- activates a sphingomyelinase that hydrolyzes sphingomyelin to ceramide; ceramide can then rapidly deacylate to sphingosine. TNF- increases ceramide within cells by activating lysosomal or plasma membrane-associated sphingomyelinases that exhibit varying pH and cationic requirements (16).

There is mounting information that sphingolipid degradation products participate in the pathophysiology of acute lung injury, although their role requires further elucidation. Lung cells express high levels of key sphingolipid enzymes and sphingolipid products (14), and related glycolipids are elevated in acute lung injury (23, 31). In the swine model, sphingoid bases such as sphinganine are implicated as mediators of pulmonary edema and alveolar injury (10). One potential mechanism whereby these lipids might participate in lung injury is by altering surfactant, a proteolipid complex that is essential for maintaining alveolar stability. Recently, our laboratory (18, 25) demonstrated that in vivo TNF- administration increases parenchymal ceramide levels concomitant with diminished alveolar surfactant. Cell-permeable ceramides also inhibit synthesis of disaturated phosphatidylcholine, the major phospholipid component of surfactant in vitro (1, 30). Thus these data suggest that sphingolipid products are abundantly generated within lung tissue in response to deleterious inflammatory cytokines, which in turn might decrease the intra-alveolar surfactant pool size by altering its biosynthesis.

In addition to negative effects on surfactant synthesis, sphingolipids might also be generated within the alveolus in response to an inflammatory stimulus and subsequently affect surface activity of the surfactant film. The ability of surfactant to lower alveolar surface tension is clearly disrupted in lung injury because a capillary leak and intra-alveolar inflammatory events trigger the elaboration of various inhibitory factors that antagonize surfactant activity (6, 9). Thus, in the present study, we investigated the hypothesis that bioactive sphingolipids, generated in the alveolus in response to TNF-, directly regulate the biophysical properties of surfactant. Herein, we demonstrate the existence of a cell-free alveolar pool of sphingolipids that is regulated by TNF-. Furthermore, ceramide resulting from a TNF--inducible secretory sphingomyelinase is shown to potently counteract the surface tension lowering effects of natural surfactant. The results provide a novel pathway by which extracellular bioactive sphingolipids are generated in the alveolus that leads to destabilization of the surfactant film in the setting of acute lung injury.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. The sphingolipids including D-erythro-sphingosine, D,L-erythro-dihydrosphingosine (sphinganine), and D-erythro-C₂₀-sphingosine were purchased from Matreya (Pleasant Gap, PA). Cell permeable ceramides of varying N-acyl chain length, D-erythro-C₂-ceramide and D-threo-C₂-ceramide were also obtained from Metreya. O-phthalaldehyde, Evans blue dye (EB), and human lyophilized hemoglobin were purchased from Sigma Chemical (St. Louis, MO). High-performance liquid chromatography reagents were obtained from Fisher Scientific (Pittsburgh, PA). Human TNF- (1 µg = 1.1 × 10⁵ activity units) was obtained from Endogen (Minneapolis, MN). All solvents were of Optima grade (Fisher Chemical). Silica LK5D (250 mm × 20 × 20 cm) thin layer chromatography plates were purchased from Whatman International (Maidstone, England). Choline-(methyl-¹⁴C) sphingomyelin was purchased from DuPont New England Nuclear Chemicals (Boston, MA). Infasurf (calfactant, 35 mg phospholipid · ml¹ · 0.65 mg protein¹) was obtained from Forest Pharmaceuticals (St. Louis, MO).

Animals and lavage preparation. Animal procedures were performed as was described previously by using a TNF- acute lung injury protocol that has been shown to result in altered surfactant synthesis (18, 25). Adult male Sprague-Dawley rats weighing 250-300 g were obtained from Sasco (Boston, MA) and were anesthetized with pentobarbitol (75 mg ip). Each experiment consisted of two control and two TNF--treated animals. Additional animals were administered ceramide (D-erythro-C₂-ceramide, 20-80 µg) intratracheally. The trachea was intubated with a 20-gauge plastic catheter, and animals immediately received either 0.5 ml of diluent, 5 µg of TNF-, or ceramide intratracheally. Ten to thirty minutes after cytokine treatment, animals were euthanized, the chests were opened, the inferior vena cava was severed, and the right ventricle and lungs were perfused with normal saline (prewarmed at 37°C). The lungs were lavaged by instilling eight aliquots each of 8 ml of normal saline. Aliquots were pooled, and the lavage fluid was first subjected to centrifugation at 300 g for 10 min at 4°C to isolate macrophages, and the supernatants were spun again at 100,000 g for 60 min at 4°C to isolate a surfactant-enriched pellet. Lipid and enzyme analysis was then performed on the crude surfactant pellet. These procedures are in accordance with the protocols approved by the University of Iowa Animal Care and Use Committee.

Sphingolipid analysis. For sphingomyelin analysis, lipids were extracted from equal amounts of protein from alveolar pellets by using the method of Bligh and Dyer (2). The lipids were dried under nitrogen gas and resolved by using chloroform-methanol-acetic acid-water [50:30:6:4, vol/vol (27)] as a solvent on silica LK5D plates. After each plate was dried in a fume hood, the sample lanes and sphingomyelin standard lanes were briefly exposed to iodine vapors. Samples that comigrated with sphingomyelin standard were scraped from the silica gel, and the levels of lipid were quantitated by using the phosphorus assay (19). With the use of this system, sphingomyelin and phosphatidylcholine areas were effectively resolved, with retardation factor values of 0.36 and 0.55, respectively.

Sphingomyelinase activity. Cell-free lavage sphingomyelinase activity was assayed as described previously (17). Each assay (0.2-ml volume) contained 25 µmol Tris/glycine buffer (pH 7.4), 2.5 pmol MgCl₂, 50 nmol choline-(methyl-¹⁴C) sphingomyelin (specific activity of 400 counts · min¹ · nmol¹), 0.5 mg of human serum albumin, 0.1 mg of cutscum, and 50-100 µg of lavage protein or macrophage cell lysate. After a 1-h incubation at 37°C, the reaction was terminated with 1 ml of cold 10% tricloroacetic acid. After addition of BSA (100 ug), the mixture was centrifuged, and a 1-ml aliquot of the supernatant was extracted with an equal volume of anhydrous ether at 4°C. An aliquot of the aqueous phase was taken for scintillation counting. Lung sphingomyelinase activity was linear from 50 to 1,000 µg of added protein, and the reaction was linear with time up to 2 h. Recovery of the cleavage product, phosphocholine, was 77%.

Surface tension analysis. Dynamic surface-tension analysis was obtained by using a pulsating bubble surfactometer (General Transco, Lancaster, NY) (8). The bubble was pulsated at 37°C at a rate of 20 cycles/min for up to 5 min. A commercially available surfactant isolated from calf lung (Infasurf) was used to evaluate potential inhibitors of surfactant function. Before assay on the bubble machine, all surfactant samples were adjusted to final concentrations of 10 mg/ml phospholipid and 1.6 mM CaCl₂ by using a PBS buffer (140 mM NaCl, 5 mM CaCl₂, pH 7.0). Stock ceramide-ethanol solutions were dried under nitrogen, dissolved in PBS buffer, and then adjusted to final concentrations of 1-25 nM in the surfactant suspension. Likewise, stock hemoglobin was dissolved in PBS buffer and adjusted to ~30 µM (2 mg/ml) for study of surfactant inhibitory activity. Prepared surfactant samples were vortexed, briefly sonicated (10 W for 5-10 s), vortexed again, and then incubated at 37°C for 60 min before analysis. Aliquots of surfactant/inhibitor mixtures were assayed within 2 h of preparation. In separate studies, surface-tension analysis was conducted on surfactant-enriched pellets isolated from rats administered diluent, TNF-, or ceramide intratracheally. In these studies, all analysis was done on pellets after normalizing for lipid phosphorus content.

Lung permeability analysis. The extravasation of EB into lung tissue and lavage was used as an index of lung permeability (24). Briefly, rats were anesthetized with ketamine and xlyazine (91 and 9.1 mg/kg ip). EB (40 mg/kg) was then given by injection into a femoral vein 5 min before intratracheal adminstration of TNF- (5 µg), ceramide (80 µg), or vehicle (250 µl PBS). Rats were exposed to TNF-, ceramide, or vehicle for 30 min and then euthanized as described above. EB was immediately removed from the pulmonary circulation by perfusing the right ventricle and lungs with 100 ml/kg of 0.9% saline. Lungs were lavaged as described above but by using PBS. Lungs were removed, dried in a 37°C oven, and EB extracted by using 2 ml of formamide (24 h at 40°C). The amount of EB in lung tissue and lavage supernatant was quantified by measuring the absorbance at 620 nm on a Beckman DU 650 spectrophotometer (24). EB concentration in lavage and extracted lung samples was quantified by interpolation by using standard curves of EB in the concentration ranges of 0-2.5 and 0-25 µg/ml, respectively. Values were expressed as micrograms of dye per milliliter (lavage samples) and nanograms of dye per milligram of dry tissue (lung samples).

Statistical analysis. Data is expressed as means ± SE. Statistical analysis was performed by using the Student's t-test or ANOVA for multiple comparisons.

	RESULTS

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Sphingolipid analysis. The levels of surfactant-associated sphingomyelin relative to phosphatidylcholine are relatively small, and, accordingly, we observed low nanomolar amounts of sphingomyelin per milligram of protein in rat lavage (Table 1). However, there was an ~8- to 30-fold excess of sphingomyelin compared with levels of ceramide or related sphingoid bases, sphinganine or sphingosine in rat lavage. The intratracheal administration of TNF- tended to decrease sphingomyelin content by 34% but had no effect on sphinganine or sphingosine levels. TNF- significantly elevated alveolar ceramide levels over twofold compared with control (Table 1). These data suggest that bioactive sphingolipids are expressed at low levels within the alveolar compartment and that some of these lipids can be regulated extracellularly by inflammatory cytokines.

View this table: [in this window] [in a new window]   Table 1.   Effect of intratracheal TNF- on sphingolipid content in alveolar lavage

Sphingomyelinase activity. Intracellular ceramide is derived partly from hydrolysis of sphingomyelin via activation of sphingomyelinases (15). Sphingomyelinases are also activated by TNF- (15). Thus we assayed this enzyme in the cell-free lavage pellet to determine the mechanism for TNF- induction of ceramide (Table 2). Acidic and neutral sphingomyelinases were both expressed extracellularly; however, generally higher activities were observed when the enzyme was assayed under acidic pH conditions. Furthermore, expression of the extracellular acidic sphingomyelinase was four- to fivefold greater than specific activities of this enzyme within alveolar macrophages. This disparity between macrophages and extracellular acidic sphingomyelinase was enhanced after TNF- treatment (Table 2). TNF- increased acidic sphingomyelinase by 76% relative to control (P < 0.05). Collectively, these data suggest the presence of a secretable form of sphingomyelinase that exhibits basal and regulatable expression within the alveolar space.

View this table: [in this window] [in a new window]   Table 2.   Effect of intratracheal TNF- on sphingomyleinase activity in alveolar lavage

Surface tension analysis. We next examined whether ceramide directly inhibits the biophysical properties of surfactant (Fig. 1). We tested the ability of nanomolar amounts of C₂ ceramide (as detected in lavage) to inhibit a commercially available calf lung surfactant preparation (Infasurf). Infasurf (10 mg/ml) reduced minimum surface tension to <5 mN/m (Fig. 1). Ceramide administered in a dose-dependent manner significantly inhibited the surface tension lowering effect of Infasurf throughout 4 min of pulsation. Because naturally occurring ceramides can vary substantially with regard to composition, we tested compounds that harbored differences in the N-acyl groups (Fig. 2). All molecular species examined effectively opposed Infasurf's surface activity as did D-erythro and D-threo ceramide isomers (Fig. 2, inset). In head-to-head studies, we analyzed effects of ceramide with hemoglobin, a serum product that has been shown to reduce surface activity (32). The results show that nanomolar amounts of ceramide were comparable to low micromolar amounts of hemoglobin in antagonizing surface-activity of Infasurf (Fig. 3). Interestingly, there was no additive or synergistic effect of ceramide and hemoglobin on biophysical activity. Finally, in preliminary studies, we administered TNF- and ceramide (20 µg) intratracheally and isolated lavage surfactant pellets for analysis of surface tension. Although TNF- did not significantly alter the surface active properties of rat surfactant after 10 min of instillation, ceramide increased surface tension during adsorption (time = 0 min) from 30 ± 0.9 mN/m (control, _max) to 38 ± 0.5 mN/m (ceramide, _max) and from 22 ± 0.3 mN/m (control, _min) to 27 ± 0.2 mN/m (ceramide, _min) (P < 0.01). Collectively, the results suggest that extracellular ceramide associated with surfactant can potently inhibit lung surface activity in vitro and might inhibit surface activity in vivo.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Dose response effects of ceramide on surface tension-lowering ability of Infasurf. C2 ceramide at various concentrations (1-25 nmoles) was reconstituted with Infasurf (10 mg/ml), and surface tension was determined by using a pulsating bubble surfactometer over 5 min of pulsation. Values are means ± SE and represent minimum surface tensions of n = 3 separate experiments. * P < 0.05 at the 1 and 15 nmol ceramide dose vs. control, and P < 0.01 at the 25 nmol dose vs. control.  P < 0.01 at the 15 and 20 nmol ceramide dose vs. control. ** P  0.01 at the 1, 15, and 20 nmol ceramide dose vs. control.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effects of N-acyl chain length of ceramides on surface tension-lowering ability of Infasurf. Ceramides (25 nmol) harboring different N-acyl groups (C2 to C16 ceramides) and D-erythro and D-threo C2 ceramide isomers (inset) were reconstituted with Infasurf as in Fig. 1 and tested for inhibition of surface activity. Surface tension was determined after 2 min of pulsation. All data represent minimum surface tensions as determined by a pulsating bubble surfactometer. All studies represent n = 3 separate experiments. Statistical analysis was performed by using ANOVA.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Comparative and additive effects of inhibitors on surface tension-lowering ability of Infasurf. In comparative studies, hemoglobin (Hb; 30 µM), C2 ceramide (25 nmol), or Hb (30 µM) in combination with C2 ceramide (25 nmol) was reconstituted with Infasurf and tested for inhibition of surface activity. All data represent minimum surface tensions as determined by a pulsating bubble surfactometer. All studies represent n = 3 separate experiments. Statistical analysis was performed using ANOVA. * P < 0.05 vs. control.

Lung permeability analysis. To address whether TNF- or ceramide produces a physiologically relevant effect, we also assessed extravasation of EB as a marker of lung permeability. Indeed, TNF- produced a fourfold increase in dye extravasation in lung lavage compared with control (Fig. 4A; P = 0.01). Ceramide also increased dye levels in lavage approximately twofold compared with control, although these effects did not reach significance. When analysis was performed on lung tissue, however, ceramide increased dye extravasation over fourfold compared with control (Fig. 4B; P < 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of intratracheal administration of tumor necrosis factor (TNF)- and ceramide on pulmonary extravasation of Evans blue dye. Adult rats were administered intratracheal TNF- (5 µg), ceramide (80 µg), or vehicle (PBS) for 30 min after intravenous administration of Evans blue (40 mg/kg). Animals were euthanized, lungs were lavaged, and Evans blue dye was extracted from lung tissue. The amount of Evans blue in lung lavage (A) and lung tissue (B) was quantified spectrophotometrically. Values are expressed as µg dye/ml (lavage samples) and ng dye/mg dry tissue (lung samples). All studies represent n = 3 separate experiments. Statistical analysis was performed using ANOVA. * P < 0.05 vs. control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates for the first time that bioactive sphingolipid degradation products such as ceramide are extracellularly detectable in association with pulmonary surfactant and that ceramide potently modulates lung biophysical activity. The results also demonstrate the presence of an alveolar secretory sphingomyelinase that is constitutively active and regulated by TNF-. The results suggest that activation of this sphingomyelinase might at least partly account for the induction of alveolar ceramides that could play an important role in the early phase of pulmonary impairment that is observed with cytokine-mediated acute lung injury.

The present results linking ceramide with inhibition of surface tension lowering activity of surfactant are noteworthy because biophysical effects of this lipid product were rapid and of a greater magnitude than inhibitory effects observed with serum proteins (6, 9, 32). By using high picomolar to low nanomolar amounts of ceramides, as detected in rodent lavage, we observed significant inhibition of surface activity compared with micromolar amounts of serum products that have previously been identified to impair surface tension (Table 1 and Fig. 1) (9, 32). Similiar observations showing inhibitory effects of ceramide in the nanomolar range on surface activity of bovine lung surfactant extracts were observed by using a captive bubble system (personal communication, Dr. Fred Possmayer, University of Western Ontario, CA). Our observed effects were independent of ceramide chain length and were seen with both naturally occurring and synthetic species. The molecular basis for interference of surface tension lowering effects of natural surfactant by ceramide are not yet clear. We speculate that the N-acyl moiety within the ceramide molecule integrates into the hydrophobic disaturated phosphatidylcholine monolayer in the interface film, thereby enhancing the film's rigidity. In addition, these effects of ceramide on inhibition of surface-activity were coupled to increased permeability (Fig. 4), which is further suggestive of a pathophysiological role for this bioactive lipid on lung edema formation in vivo.

The identification of alveolar sphingolipids and their regulation by inflammatory cytokines represents a complementary and potentially novel extracellular pathway by which inhibitory lipids could influence alveolar function in lung injury. An initial event in sepsis-induced lung injury is release of cytokines, such as TNF-, in serum by circulating monocytes. The present results suggest that intra-alveolar TNF- secreted by macrophages or other inflammatory cells could serve as a stimulus for the release of extracellular sphingomyelinases. Zinc-activated, inducible, extracellular sphingomyelinases have been shown to be secreted within the peritoneum and potentially could have originated from several cell types within the lung (26, 28, 33). The alveolar sphingomyelinase detected in our studies, however, appears distinct in that it did not exhibit a zinc requirement for optimal activation (data not shown). The acidic pH of the alveolar hypophase would provide a suitable environment for the constitutive expression of this sphingomyelinase (21). Presumably, low-level activity of this extracellular enzyme system regulates a delicate balance between the alveolar pool of sphingomyelin substrate and ceramides. During alveolar inflammation, this balance could be shifted toward the generation of injurious sphingolipids that could accentuate pulmonary edema. In addition to affecting surfactant metabolism or biophysical activity, extracellular ceramides generated via sphingomyelinase activation could potentiate alveolar damage by inducing cellular apoptosis, altering barrier function, or regulating cell signaling (11, 12, 22). Our findings coupled with observations that higher-order glycolipids are present in the lungs of patients with acute pulmonary injury gives support to the idea that these lipids are of unique pathophysiological significance in surfactant-deficient lung disease (23).