Pulmonary-specific expression of tumor necrosis factor-alpha alters surfactant lipid metabolism

James L. Carroll Jr., Diann M. McCoy, Stephen E. McGowan, Ronald G. Salome, Alan J. Ryan, and Rama K. Mallampalli

Department of Internal Medicine and the Department of Veterans Affairs Medical Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-alpha decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-alpha , we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-alpha overexpression. Compared with controls, TNF-alpha transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-alpha results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.

disaturated phosphatidylcholine; choline kinase; choline phosphotransferase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY SURFACTANT is a heterogeneous material that is essential for life, consisting of key hydrophobic proteins and lipids that maintains alveolar patency (35). Disaturated phosphatidylcholine (DSPtdCho) is the major surface-active lipid component of surfactant that is produced by the alveolar type II epithelial cell via the CDP-choline pathway. The initial step in this pathway involves phosphorylation of choline to cholinephosphate by the enzyme choline kinase (CK, EC 2.7.1.32). The second step involves conversion of cholinephosphate to CDP-choline. This latter step is a slow, energy-requiring reaction catalyzed by the regulatory enzyme CTP:phosphocholine cytidylyltransferase (CCT, EC 2.7.7.15.) (17). The terminal reaction coupling diacylglycerol to CDP-choline is catalyzed by choline-phosphotransferase (CPT, EC 2.7.8.2). To date, most of the interest in PtdCho synthesis has focused on the regulatory mechanisms for CCT because of its important role in controlling intracellular PtdCho content (19, 32).

Tumor necrosis factor-alpha (TNF-alpha ) is a small polypeptide cytokine released in the lung primarily by alveolar macrophages (6). In addition to diverse effects promoting the acute and chronic inflammatory response, TNF-alpha also regulates surfactant metabolism. For example, TNF-alpha inhibits the synthesis of the surfactant apoproteins, induces PtdCho degradation, and decreases the biosynthesis of PtdCho (3, 37, 40). Recently, we demonstrated that TNF-alpha inhibits PtdCho synthesis both in vitro and in vivo by inhibiting the activity of CCT (21, 36). In contrast to these negative effects of the cytokine on phospholipid synthesis, TNF-alpha also stimulates PtdCho secretion in primary alveolar epithelial cells (5). Thus TNF-alpha could simultaneously trigger several events directly related to PtdCho metabolism and processing. Presumably, the net effect of the cytokine on effector pathways such as synthesis, secretion, and intraalveolar processing of PtdCho will likely dictate the availability of surfactant lipid necessary to maintain the integrity of a stable surface-active film.

Although much of the prior work has improved our understanding of how TNF-alpha affects surfactant lipid metabolism, essentially all studies to date have investigated short-term exposures to the cytokine; long-term effects have not been addressed. Sustained or intermittent TNF-alpha release is a plausible mechanism in the setting of chronic alveolitis where high TNF-alpha exists in tissue as a receptor-bound or membrane-associated form (4). This process appears to occur in late-phase acute lung injury (4, 15). Long-term TNF-alpha exposure may also be a feature of chronic lung disorders such as bronchopulmonary dysplasia and idiopathic pulmonary fibrosis, which are also associated with surfactant abnormalities (29, 31).

To investigate long-term expression of TNF-alpha , Miyazaki et al. (28) generated a transgenic mouse expressing the murine TNF-alpha gene driven by the human surfactant protein C promoter. In this model, TNF-alpha mRNA was selectively expressed within the alveolar epithelium. Morphologically, transgenic mice exhibit lymphocytic alveolitis, and by 6 mo of age the mice had features typical of chronic interstitial fibrosis (28). In the present study, we used these mice to investigate long-term effects of TNF-alpha on PtdCho metabolism. We hypothesized that chronic overexpression of TNF-alpha will result in a decrease in surfactant synthesis. To test our hypothesis, we measured the sequential enzymatic steps involved in surfactant synthesis and key regulatory lipids in TNF-alpha transgenics and wild-type littermate controls.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Silica LK5D (0.25 mm × 20 cm × 20 cm) TLC plates were purchased from Whatman International (Maidstone, England). All radiochemicals were purchased from DuPont New England Nuclear Chemicals (Boston, MA). Immunoblotting membranes were obtained from Millipore (Bedford, MA). The enhanced chemiluminescence (ECL) Western blotting detection system was from Amersham Pharmacia Biotech (Piscataway, NJ). A rabbit polyclonal antibody to synthetic peptide corresponding to residues 164-176 was generated by Covance Research Products (Richmond, CA). Phospholipid standards and o-phthalaldehyde (OPA) were obtained from Sigma Chemical (St. Louis, MO). Fatty acid standards were from NuCheck Prep (Elysian, MN) and PUFA-2 from Supelco (Bellefonte, PA). Sphingolipid standards were purchased from Matreya (Pleasant Gap, PA). HPLC reagents were obtained from Fisher Scientific (Pittsburgh, PA).

Animals and tissue preparation. C57BL/6 transgenic mice were obtained from Dr. Robert Mason (Department of Medicine, University of Colorado). Littermates lacking the transgene according to PCR genotyping of tail clips were used for wild-type controls. Mice were anesthetized with pentobarbital sodium (10 mg ip). The trachea was intubated with a small plastic catheter. The lungs were lavaged sequentially with a solution of 6 mM glucose and 0.2 mM EGTA, and then with a solution containing 2 mM CaCl2 and 1.3 mM MgSO4, to obtain an total of 8 ml of fluid. Lavage fluid was centrifuged sequentially as described to obtain a crude surfactant pellet (36). In separate studies, lungs were resected after the heart, and major airways were dissected away from the lung parenchyma. Lungs were homogenized in a buffer containing 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, and 0.025% sodium azide. Homogenates were centrifuged sequentially as described to obtain microsomes for lipid and enzymatic analysis (18).

Phospholipid and DSPtdCho analysis. Lipids were extracted from equal amounts of protein using the method of Bligh and Dyer (7). The lipids were dried under nitrogen gas, applied in 50 µl of chloroform-methanol (2:1) to silica LK5D plates, and developed in chloroform-methanol-petroleum ether-acetic acid-boric acid (40:20:30:10:1.8 vol/vol/vol/vol/wt) (14). After each plate was dried in a fume hood, the sample lanes and phospholipid standard lanes were briefly exposed to iodine vapors. Samples that comigrated with the appropriate phospholipid standard were scraped from the silica gel and quantitatively assayed for phosphorus content (12). For DSPtdCho analysis, the phosphatidylcholine samples were reacted with osmium tetroxide and run in the second dimension. The levels of DSPtdCho were quantitated by phosphorus assay (19).

Choline incorporation into PtdCho. The trachea was cannulated with a 20-gauge intravenous catheter. A mixture of M199 medium and 0.67% noble agar (final concentration) heated to 37°C was instilled into the lungs. The mice were subsequently placed on ice to solidify the agar. The lungs were removed and manually cut into 1-mm slices. Each slice was placed onto a sterile filter paper resting on a stainless steel mesh in a six-well culture plate. Four milliliters cold medium (500 ml M199, 50,000 units penicillin G, 50,000 µg streptomycin sodium, 1,250 µg amphotericin B, and 50 ml fetal bovine serum) were added to each well (16). After an initial incubation period, the plates were pulsed with 1.25 µCi [methyl-3H]choline/1 ml M199 medium as described (23). After pulsing, lung slices were rinsed with 4 ml PBS, placed into polystyrene tubes, and homogenized in 1 ml PBS. Lipids were extracted from 50 µg protein using the method of Bligh and Dyer (7) and processed for determination of PtdCho as described above. PtdCho spots were scraped from the silica gels and quantitated by scintillation counting.

Enzymes of PtdCho synthesis. The activity of CK was assayed in cytosolic fractions as described (21). The reaction mixture (0.1 ml volume) contained 100 mM Tris · HCl buffer (pH 8.0), 10 mM magnesium acetate, 0.016 mM [14C]choline [specific activity ~7,000 disintegrations/min (dpm)/nmol], 10 mM ATP, and 50-100 µg of cell sample. After a 1-h incubation at 37°C, the reaction was terminated with 0.02 ml of cold 50% trichloroacetic acid. Twenty-microliter aliquots of the mixture were spotted on Whatman 3MM paper, and choline metabolites were resolved by using paper chromatography as described (21). The spots that comigrated with the radiolabeled standard choline phosphate were cut and used for scintillation counting.

The activity of CT was determined by measuring the rate of incorporation of [methyl-14C]phosphocholine into CDP-choline by a charcoal extraction method (21). All assays were performed without the inclusion of a lipid activator in the reaction mixture. Enzyme-specific activity is expressed as picomoles per minute per milligram of protein or picomoles per minute per unit of enzyme mass.

The activity of CPT was assayed as described (21). Each reaction mixture contained 50 mM Tris · HCl buffer (pH 8.2), 0.1 mg/ml Tween 20, 1 mM 1,2-dioleoylglycerol, 0.8 mM phosphatidylglycerol, 0.5 mM [14C]CDP-choline (specific activity 1,110 dpm/nmol), 5 mM dithiothreitol, 5 mM EDTA, 10 mM MgCl2, and 30-40 µg of sample. The lipid substrate was prepared by combining appropriate amounts of 1,2-dioleoylglycerol (1 mM) and phosphatidylglycerol (0.8 mM) in a test tube, drying under nitrogen gas, and sonicating on ice for 10 min before addition to the assay mixture. The reaction proceeds for 1 h at 37°C and is terminated with 4 ml of methanol-chloroform-water (2:1:7 vol/vol/vol). The remainder of the assay was performed exactly as described (27).

Immunoblot analysis. For immunoblot analysis, equal amounts of microsomal protein were used. Each sample was adjusted to give a final concentration of 60 mM Tris · HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% beta -mercaptoethanol and heated at 100°C for 5 min. Samples were then electrophoresed through a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. CCT and beta -actin were detected by using the ECL Western blotting detection system as instructed by the manufacturer. The dilution factor for anti-CT and anti-beta -actin was 1:1,000.

Fatty acid gas chromatography. Lipids were extracted from cell homogenate (1 mg protein) according to the method of Bligh and Dyer (7). Fatty acid methyl esters (FAME) were prepared by transmethylation in the presence of 10% boron trifluoride. The FAME derivatives were separated by gas liquid chromatography and detected using flame ionization detection. The GC column packing was 10% SP-2330 on 100/120 Chromosorb W AW (Supelco). The initial column temperature was held at 165°C for 8 min and then increased 3°/min to a final temperature of 210°C. The final temperature was maintained for 12 min. Individual fatty acids were identified by comparing the retention times with that of known standards.

Sphingolipid analysis. Sphingosine was extracted from microsomal samples (0.25-1 mg protein per sample) plus 200 pmole D-erythro-C20-sphingosine (an internal standard) according to the method of Bligh and Dyer. The chloroform layer was isolated and dried under nitrogen gas. The dried extracts were resuspended in 0.33 ml chloroform and 0.66 ml 0.1 M KOH in methanol and incubated at 37°C for 1 h. The samples were rinsed with 1 ml chloroform and 1 ml 1.0 M NaCl. The chloroform phase was washed with NaCl and dried under nitrogen gas. Ortho-phthalaldehyde derivatives were prepared by dissolving the dried samples in 50 µl methanol, followed by the addition of 50 µl OPA reagent (5 mg o-phthalaldehyde in 100 µl ethanol, 9.9 ml 3% boric acid, and 5 µl 2-mercaptoethanol), incubated at room temperature for 5 min, diluted with methanol-water (94:6 vol/vol), and quantitated by HPLC. Ortho-phthalaldehyde derivatives were separated on a Beckman Ultrasphere C-18 column, with methanol-water (94:6 vol/vol) mobile phase at a rate of 1 ml/min. The derivatives were detected using a Thermoseparation Products Spectra System FL3000 fluorescence detector at 340 nm excitation and 454-nm emission wavelengths. Ceramide (an N-acylated sphingosine) was extracted from cells and resolved from sphingosine using TLC before an acid hydrolysis step (converting it to sphingosine) before derivatization and HPLC as described above (26).

Alveolar uptake of DSPtdCho. A mixture of [choline-methyl-14C]DSPtdCho (1.7 µCi, 7.8 µg) and cold DSPtdCho (7.8 µg) was dried under nitrogen gas. Normal saline (90 µl) and 2% brilliant blue (10 µl) were added, and the mixture was sonicated on ice for 10 min. Mice were sedated with ketamine (2-3 mg ip). The trachea was exposed, and 20 µl of the [14C]DSPtdCho mixture was instilled under direct observation. After 10 min of spontaneous breathing and careful positional rotation, the mice were killed with pentobarbital sodium (10 mg ip). Microsomal fractions were obtained as described above. Lipids were extracted, DSPtdCho was isolated, and radioactivity within the DSPtdCho spots was analyzed by scintillation counting (43).

Statistical analysis. The data are expressed as means ± SE. Statistical analysis was performed by using Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phospholipid and DSPtdCho analysis. Transgenic mice exhibiting chronic expression of TNF-alpha displayed several abnormalities in phospholipid composition compared with control littermates. Furthermore, a distinctly different pattern of expression of phospholipids was identified in TNF-alpha transgenics and wild-type mice between the lung parenchyma and alveolar compartment. In whole lung homogenates, total PtdCho levels decreased from 122 ± 5 nmol/mg protein phospholipid phosphorus in controls to 58 ± 2 nmol/mg phospholipid phosphorus in TNF-alpha transgenics (P < 0.05). There were no other significant differences in other major phospholipids. However, in lung microsomes, TNF-alpha transgenics almost uniformly contained lower levels of several major phospholipids in the lung. Significantly lower levels of PtdCho, phosphatidylglycerol, and phosphatidylinositol were detected (Fig. 1A). Because PtdCho is the major lipid of eukaryotic membranes and surfactant, we assayed DSPtdCho content, a marker of surfactant lipid. This was also reduced by 36% (control 105 ± 8 nmol phospholipid phosphorus/mg protein, TNF-alpha transgenics 67 ± 3 nmol phosphorus/mg protein, P < 0.01, Fig. 1A insert). In contrast to these parenchymal lipids, analysis of bronchoalveolar lavage lipids revealed that TNF-alpha transgenics exhibited a 55% increase in PtdCho (Fig. 1B, P < 0.05). The level of alveolar DSPtdCho was 46% greater in the TNF-alpha transgenics compared with control (control 1,510 ± 420 nmol phosphorus/mg protein, TNF-alpha transgenics 2,210 ± 660 nmol phosphorus/mg protein, Fig. 1B insert), but this finding did not reach statistical significance. There were no significant differences in other alveolar phospholipids. Collectively, these studies suggest that chronic expression of TNF-alpha in this murine model results in an overall decrease in the synthesis of several major phospholipids. Moreover, a divergent pattern for PtdCho between the alveolar and lung parenchymal compartment was observed in TNF-alpha transgenics compared with control, which may reflect differences in surfactant synthesis, secretion, or intraalveolar processing.


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Fig. 1.   Distribution of phospholipids. A: pulmonary microsomal levels of phosphatidylcholine (PtdCho), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and sphingomyelin (SM) were determined in microsomal lipids extracted from equal amounts of protein, and separated by TLC. Lipids were extracted from TLC spots and quantitated by the phosphorus assay. Results are means ± SE expressed as nmol phospholipid phosphorus/mg protein, from 6 control and 6 tumor necrosis factor (TNF)-alpha transgenic mice *P < 0.01 vs. control. Inset: levels of disaturated PtdCho (DSPtdCho) were determined by two dimensional TLC and quantitated by the phosphorus assay. Results are means ± SE, expressed as percent change from wild type, *P < 0.01 vs. control; dagger nmol phospholipid phosphorus/mg protein. B: bronchoalveolar lavage phospholipid levels were determined as above. Results are means ± SE expressed as nmol phospholipid phosphorus/mg lavage protein, with 6 control and 6 TNF-alpha transgenic animals, *P < 0.05. Inset: levels of DSPtdCho determined as above, expressed as percent change from wild type, *P < 0.05 vs. control; dagger nmol phospholipid phosphorus/mg protein.

Choline incorporation into PtdCho. To investigate whether TNF-alpha mice had lower PtdCho biosynthetic capacity compared with wild-type controls, we prepared whole lung slices and measured radiolabeled incorporation of [3H]choline into the phospholipid (Fig. 2). Indeed, we observed that lung slices from wild-type controls exhibited a 3.8-, 4.8-, and 3.9-fold greater incorporation of [3H]choline into PtdCho at 2, 4, and 8 h of analysis, respectively.


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Fig. 2.   Radiolabeled choline incorporation into lung slices. Lung slices of wild-type (WT) and TNF-alpha transgenic mice were incubated for various times at 37°C and subsequently pulsed with [methyl- 3H]choline (1.25 µCi/ml). After the pulse, lipids were extracted from tissue homogenates and processed for determination of PtdCho. Radioactivity within PtdCho was determined by scintillation counting. Results are from 3 control and 3 transgenic mice at each time point, expressed as means ± SE, *P < 0.05, **P < 0.01. dpm, Disintegrations/min.

Enzymes of PtdCho synthesis. We next determined if the decrease in surfactant content in the lungs of murine TNF-alpha transgenics was due to diminished synthesis by assaying activities of enzymes in the CDP-choline pathway, the primary pathway for PtdCho synthesis. Compared with control littermates, TNF-alpha transgenic mice exhibited no differences in the activity of CK, the first committed enzyme of the CDP-choline pathway (Table 1). In contrast, the activity of CCT, the rate-limiting enzyme within this pathway, was reduced in TNF-alpha transgenics by 42% compared with control mice (P < 0.05). Interestingly, this reduced activity for CCT was associated with nearly a threefold induction of cholinephosphotransferase activity, the final enzyme in the pathway. These results suggest that chronic expression of TNF-alpha inhibits a key regulatory enzyme, CCT, in murine lungs, resulting in an overall decrease in PtdCho content.

                              
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Table 1.   Activities of enzymes of PtdCho synthesis

Immunoblot analysis. We performed immunoblot analysis to determine if reduced activity of CCT in TNF-alpha transgenics was due to a decrease in enzyme mass. Unexpectedly, in three separate studies, we observed that transgenic mice harboring the TNF-alpha gene exhibited a twofold increase in CCT protein compared with wild-type littermate controls (Fig. 3A). In contrast to expression of immunoreactive CCT, levels of beta -actin were unchanged between control and TNF-alpha transgenic mice (Fig. 3B). Thus when we expressed our results as specific activity of the enzyme using densitometric measurements of enzyme mass, the TNF-alpha transgenic mice exhibited an even more substantial reduction in catalytic activity relative to wild-type controls (Fig. 3C). Collectively, these results suggest that TNF-alpha transgenic mice have reduced CCT activity associated with an increase in enzyme mass. The enzyme protein, however, is largely physiologically inactive.


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Fig. 3.   Effect of TNF-alpha overexpression on cytidylyltransferase (CCT) expression. A: the amount of CCT was assayed using immunoblotting. Lanes were loaded with 100 µg microsomal protein from WT and TNF-alpha transgenic mice. CCT standard was derived from purified rat liver enzyme. An anti-CCT-specific rabbit polyclonal antiserum was used. B: beta -actin immunoblot with similar experimental conditions. C: the specific activity is expressed as enzyme activity/enzyme mass [(nmol · min-1 · mg protein-1)/(mass units/mg protein) = nmol · min-1 · mass unit-1]. Mass units were arbitrary values determined by densitometric analysis of 3 WT and 3 TNF-alpha transgenic lanes from the preceding immunoblot. Data are expressed as means ± SE, P < 0.05.

Sphingolipids. The data above indicate that adequate amounts of CCT protein are detected in TNF-alpha transgenic mice, but the enzyme is not present in an activated form. CCT is an enzyme that is primarily regulated by lipids (17). One class of lipids that have been shown to be inhibitory for PtdCho synthesis and CCT function is sphingolipids, such as sphingosine and ceramide (1, 38, 41). In addition, TNF-alpha has been shown to acutely increase the levels of these bioactive mediators in the lung (20). Although we observed that TNF-alpha transgenic mice expressed no significant differences in sphingosine content, ceramide levels were significantly increased in TNF-alpha transgenic mice (control 1,540 ± 430 pmol/mg protein, TNF-alpha transgenic 3,540 ± 220 pmol/mg protein, P < 0.01.) (Table 2). These results suggest that induction of ceramide may be a mechanism by which CCT activity is decreased in the TNF-alpha transgenic mice.

                              
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Table 2.   Lung sphingolipid content

Fatty acids. In addition to its effects on sphingolipid metabolism, TNF-alpha regulates the biosynthesis, uptake, and release of fatty acids from triglycerides (13, 24, 25, 30). Unsaturated fatty acids activate CCT (22, 39). Thus we hypothesized that TNF-alpha might decrease the availability of key fatty acids required for enzyme activation. As expected, palmitic acid was the major saturated fatty acid and oleic acid the major unsaturated species in both wild-type and TNF-alpha transgenics (Table 3). There were no substantial differences between the groups with regard to levels of saturated fatty acids. TNF-alpha transgenics, however, contained a significantly lower proportion of linoleic acid but greater amounts of arachidonic acid compared with control littermates. These results suggest that the relative abundance of selective unsaturated fatty acid species might also limit CCT activation in the setting of chronic TNF-alpha expression.

                              
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Table 3.   Lung fatty acid distribution

DSPtdCho alveolar uptake. We determined if elevated alveolar PtdCho was secondary to altered reuptake of DSPtdCho. Compared with control littermates, TNF-alpha transgenics had a 90% reduction in radiolabeled DSPtdCho uptake (control 86,800 ± 21,100 dpm/mg protein, TNF-alpha transgenic 8,900 ± 2,800 dpm/mg protein, P < 0.01, Fig. 4). These data suggest that alveolar surfactant recycling is decreased in TNF-alpha transgenic mice, consistent with the findings of increased alveolar DSPtdCho and decreased microsomal DSPtdCho.


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Fig. 4.   Uptake of alveolar DSPtdCho. Animals were exposed to [14C]DSPtdCho for 10 min after intratracheal instillation. After death, lipids were extracted from equal amounts of microsomal protein and separated by TLC. Radioactivity of the DSPtdCho spots was analyzed by scintillation counting, from 3 control and 4 TNF-alpha transgenic animals, expressed as dpm/mg protein. Results are means ± SE, *P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to investigate whether long-term expression of TNF-alpha downregulates surfactant PtdCho synthesis. Because of the relatively short half-life of TNF-alpha and limitations with repetitive in vivo administration, we felt that chronic exogenous administration of the cytokine would not be a suitable model system to test our hypothesis. Rather, we opted to use a well-characterized murine model in which TNF-alpha is overexpressed in alveolar epithelium to pursue our studies (28). We observed that surfactant lipid synthesis was decreased in the lungs of TNF-alpha transgenics as evidenced by reduced PtdCho (and DSPtdCho) content and decreased radiolabeled incorporation of choline into PtdCho, coupled with a decrease in the activity of the rate-regulatory enzyme CCT. A somewhat unanticipated finding in our studies, however, was that these changes in lung tissue were not reflected in alveolar lavage, as PtdCho and DSPtdCho were elevated in these transgenic mice. Our radiolabeling studies suggest that elevated alveolar lipids may be due to reduced reuptake of surfactant in TNF-alpha transgenic mice. Although TNF-alpha might affect other aspects of surfactant processing, the inhibitory mechanisms on PtdCho synthesis may be important in understanding cytokine signaling in the setting of chronic exposure.

Consistent with observations in other systems after acute exposure to TNF-alpha , the levels of DSPtdCho and phosphatidylglycerol were decreased significantly in lungs of TNF-alpha transgenics compared with wild-type littermate controls (2, 3, 21, 36). Phosphatidylglycerol, a minor component of surfactant lipid, may in fact originate from several different populations of pulmonary cells. However, although the decrease in lung DSPtdCho content is not specific to type II cells, DSPtdCho is the major surface-active lipid of surfactant, and it is reasonable to attribute changes in this lipid to regulation by TNF-alpha at the level of either lipid synthesis or degradation within the cells. We did not exclude the possibility that TNF-alpha might alter turnover of DSPtdCho in the transgenics. This may also be relevant because the cytokine has been shown to activate both phospholipase A2 and PtdCho-specific phospholipase C (11, 37). However, the kinetics of phospholipase activation by TNF-alpha usually occurs more rapidly than regulation of enzymes involved in PtdCho synthesis.

We observed that the decrease in PtdCho mass in TNF-alpha transgenics was secondary to a decrease in the activity of CCT. Although CPT activity was markedly elevated in these studies, overall it did not offset the reduction in CCT function, as PtdCho content in tissue remained decreased in the TNF-alpha transgenics compared with control. These results further support a rate-regulatory role for CCT within the biosynthetic pathway for PtdCho (33). CCT activity is controlled in cells by several mechanisms, including lipid regulation, reversible phosphorylation, and regulation at the level of enzyme protein and mRNA (17). A somewhat surprising and consistent finding was that TNF-alpha transgenics had increased CCT protein content compared with wild-type mice (Fig. 3A). Thus when our functional data are expressed as a ratio of specific activity of CCT per unit of enzyme mass, TNF-alpha transgenics expressed CCT activity that was markedly reduced compared with control mice (Fig. 3C). We next determined whether there might be concurrent alteration in the levels of regulatory lipids for the enzyme. CCT is inhibited by short-chain ceramides, sphingosine, and lysophosphatidylcholine, whereas activity is stimulated in the presence of phosphatidylglycerol, phosphatidylinositol, and unsaturated fatty acids (1, 8, 17). When we assayed levels of these lipids, ceramide content was increased and linoleic acid reduced in TNF-alpha transgenics compared with control. The results with ceramide are important because TNF-alpha has been shown to increase the levels of this bioactive lipid by triggering sphingomyelin hydrolysis, its generation from higher order sphingolipids including glucosylceramide, or via de novo synthesis (9). To our knowledge, decreases in linoleic acid in the setting of TNF-alpha exposure have not been described, and such changes together with reduced anionic lipids would be expected to decrease PtdCho synthesis and CCT activity. The induction of arachidonic acid in TNF-alpha transgenics, however, is well described after acute cytokine exposure in other systems and might serve as a proinflammatory mediator (2, 10, 34). Collectively, our studies strongly suggest that surfactant phospholipid synthesis in TNF-alpha transgenics is impaired as a consequence of decreased CCT activity. This reduction in CCT activity appears to be associated with a large pool of membrane-associated enzyme that is catalytically inhibited by ceramide and diminished availability of activating lipids.

In contrast to effects on lung PtdCho content, the alveolar levels of PtdCho were significantly increased in the TNF-alpha transgenic mice compared with control. There are several potential explanations for these data. First, chronic TNF-alpha expression might regulate surfactant secretion, uptake, or intraalveolar processing. In this regard, Benito and Bosch (5) showed that TNF-alpha stimulates surfactant lipid secretion in primary type II cells via a protein kinase-dependent mechanism. In these studies, chronic TNF-alpha expression appears to decrease pulmonary epithelial uptake of alveolar PtdCho. However, in these studies we did not specifically isolate the lamellar body fraction to determine if alveolar DSPtdCho is recovered within this intracellular surfactant storage compartment. Our preliminary studies do show parallel changes in recycling activity within the cytosol. This, coupled with the tenfold higher activity of DSPtdCho reuptake in microsomes, does suggest that recycling is a global phenomenon within all compartments of the lung. The regulatory mechanisms for this decrease require further elucidation. Second, reduced alveolar DSPtdCho levels in parallel to parenchymal levels may not have been observed because expression of TNF-alpha was not global but restricted to alveolar epithelium in this model. Because alveolar macrophages are not only a major source of TNF-alpha but also participate in DSPdCho uptake, TNF-alpha expression in these cells would be expected to add another level of control by stimulating intraalveolar lipid degradation (42). Finally, our alveolar lipid results might be attributed to secondary effects of TNF-alpha overexpression. For example, histologically the alveoli have increased numbers of lymphocytes, and it is possible that increased PtdCho in our lavage might, in part, originate from membranes recovered from these inflammatory cells after apoptosis or necrosis. Although each of these explanations might be relevant, the results indicate that TNF-alpha transgenic mice display a unique phospholipid profile that will require additional characterization.


    ACKNOWLEDGEMENTS

We thank Dr. Robert Mason for providing the TNF-alpha -overexpressing transgenic mouse line.


    FOOTNOTES

This research was supported by a GlaxoSmithKline Pulmonary Fellowship Award (to J. L. Carroll, Jr.); National Heart, Lung, and Blood Institute Grants RO1 HL-55584 and HL-68135; a Career Investigator Award from the American Lung Association; and a Merit Review from the Department of Veterans Affairs (R. K. Mallampalli). R. K. Mallampalli is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: R. K. Mallampalli, Pulmonary Div., Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: rama-mallampalli{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00120.2001

Received 29 March 2001; accepted in final form 5 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allan, D. Lipid metabolic changes caused by short-chain ceramides and the connection with apoptosis. Biochem J 345: 603-610, 2000[ISI][Medline].

2.   Arias-Diaz, J, Vara E, Garcia C, and Balibrea JL. Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins. J Clin Invest 94: 244-250, 1994[ISI][Medline].

3.   Arias-Diaz, J, Vara E, Garcia C, Gomez M, and Balibrea JL. Tumour necrosis factor-alpha inhibits synthesis of surfactant by isolated human type II pneumocytes. Eur J Surg 159: 541-549, 1993[ISI][Medline].

4.   Armstrong, L, Thickett DR, Christie SJ, Kendall H, and Millar AB. Increased expression of functionally active membrane-associated tumor necrosis factor in acute respiratory distress syndrome. Am J Respir Cell Mol Biol 22: 68-74, 2000[Abstract/Free Full Text].

5.   Benito, E, and Bosch MA. The inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes. Mol Cell Biochem 189: 169-176, 1998[ISI][Medline].

6.   Beutler, B, and Cerami A. Cachectin: more than a tumor necrosis factor. N Engl J Med 316: 379-385, 1987[ISI][Medline].

7.   Bligh, EG, and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917, 1959[ISI].

8.   Boggs, KP, Rock CO, and Jackowski S. Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step. J Biol Chem 270: 7757-7764, 1995[Abstract/Free Full Text].

9.   Bourteele, S, Hausser A, Doppler H, Horn-Muller J, Ropke C, Schwarzmann G, Pfizenmaier K, and Muller G. Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells. J Biol Chem 273: 31245-31251, 1998[Abstract/Free Full Text].

10.   Brekke, OL, Sagen E, and Bjerve KS. Tumor necrosis factor-induced release of endogenous fatty acids analyzed by a highly sensitive high-performance liquid chromatography method. J Lipid Res 38: 1913-1922, 1997[Abstract].

11.   Candela, M, Barker SC, and Ballou LR. Sphingosine synergistically stimulates tumor necrosis factor alpha-induced prostaglandin E2 production in human fibroblasts. J Exp Med 174: 1363-1369, 1991[Abstract].

12.   Chalvardjian, A, and Rudnicki E. Determination of lipid phosphorus in the nanomolar range. Anal Biochem 36: 225-226, 1970[ISI][Medline].

13.   De Bandt, JP, Lim SK, Plassart F, Lucas CC, Rey C, Poupon R, Giboudeau J, and Cynober L. Independent and combined actions of interleukin-1 beta, tumor necrosis factor alpha, and glucagon on amino acid metabolism in the isolated perfused rat liver. Metabolism 43: 822-829, 1994[ISI][Medline].

14.   Gilfillan, AM, Chu AJ, Smart DA, and Rooney SA. Single plate separation of lung phospholipids including disaturated phosphatidylcholine. J Lipid Res 24: 1651-1656, 1983[Abstract].

15.   Hashimoto, S, Kobayashi A, Kooguchi K, Kitamura Y, Onodera H, and Nakajima H. Upregulation of two death pathways of perforin/granzyme and FasL/Fas in septic acute respiratory distress syndrome. Am J Respir Crit Care Med 161: 237-243, 2000[Abstract/Free Full Text].

16.   Ikegami, M, Korfhagen TR, Bruno MD, Whitsett JA, and Jobe AH. Surfactant metabolism in surfactant protein A-deficient mice. Am J Physiol Lung Cell Mol Physiol 272: L479-L485, 1997[Abstract/Free Full Text].

17.   Kent, C. CTP:phosphocholine cytidylyltransferase. Biochim Biophys Acta 1348: 79-90, 1997[ISI][Medline].

18.   Mallampalli, RK, and Hunninghake GW. Expression of immunoreactive cytidine 5'-triphosphate: cholinephosphate cytidylyltransferase in developing rat lung. Pediatr Res 34: 502-511, 1993[Abstract].

19.   Mallampalli, RK, Mathur SN, Warnock LJ, Salome RG, Hunninghake GW, and Field FJ. Betamethasone modulation of sphingomyelin hydrolysis upregulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung. Biochem J 318: 333-341, 1996[ISI][Medline].

20.   Mallampalli, RK, Peterson EJ, Carter AB, Salome RG, Mathur SN, and Koretzky GA. TNF-alpha increases ceramide without inducing apoptosis in alveolar type II epithelial cells. Am J Physiol Lung Cell Mol Physiol 276: L481-L490, 1999[Abstract/Free Full Text].

21.   Mallampalli, RK, Ryan AJ, Salome RG, and Jackowski S. Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase. J Biol Chem 275: 9699-9708, 2000[Abstract/Free Full Text].

22.   Mallampalli, RK, Salome RG, and Hunninghake GW. Lung CTP:choline-phosphate cytidylyltransferase: activation of cytosolic species by unsaturated fatty acid. Am J Physiol Lung Cell Mol Physiol 265: L158-L163, 1993[Abstract/Free Full Text].

23.   Marino, PA, and Rooney SA. Surfactant secretion in a newborn rabbit lung slice model. Biochim Biophys Acta 620: 509-519, 1980[ISI][Medline].

24.   Marshall, MK, Doerrler W, Feingold KR, and Grunfeld C. Leukemia inhibitory factor induces changes in lipid metabolism in cultured adipocytes. Endocrinology 135: 141-147, 1994[Abstract].

25.   McDonagh, J, Fossel ET, Kradin RL, Dubinett SM, Laposata M, and Hallaq YA. Effects of tumor necrosis factor-alpha on peroxidation of plasma lipoprotein lipids in experimental animals and patients. Blood 80: 3217-3226, 1992[Abstract].

26.   Merrill, AH, and Wang E. Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s). J Biol Chem 261: 3764-3769, 1986[Abstract/Free Full Text].

27.   Miller, JC, and Weinhold PA. Cholinephosphotransferase in rat lung. The in vitro synthesis of dipalmitoylphosphatidylcholine from dipalmitoylglycerol. J Biol Chem 256: 12662-12665, 1981[Abstract/Free Full Text].

28.   Miyazaki, Y, Araki K, Vesin C, Garcia I, Kapanci Y, Whitsett JA, Piguet PF, and Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis. J Clin Invest 96: 250-259, 1995[ISI][Medline].

29.   Murch, SH, Costeloe K, Klein NJ, and MacDonald TT. Early production of macrophage inflammatory protein-1 alpha occurs in respiratory distress syndrome and is associated with poor outcome. Pediatr Res 40: 490-497, 1996[Abstract].

30.   Pelech, SL, Pritchard PH, Brindley DN, and Vance DE. Fatty acids promote translocation of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine synthesis. J Biol Chem 258: 6782-6788, 1983[Abstract/Free Full Text].

31.   Piguet, PF, Ribaux C, Karpuz V, Grau GE, and Kapanci Y. Expression and localization of tumor necrosis factor-alpha and its mRNA in idiopathic pulmonary fibrosis. Am J Pathol 143: 651-655, 1993[Abstract].

32.   Post, M, Batenburg JJ, Schuurmans EA, and Van Golde LM. The rate-limiting step in the biosynthesis of phosphatidylcholine by alveolar type II cells from adult rat lung. Biochim Biophys Acta 712: 390-394, 1982[ISI][Medline].

33.   Post, M, Batenburg JJ, Van Golde LM, and Smith BT. The rate-limiting reaction in phosphatidylcholine synthesis by alveolar type II cells isolated from fetal rat lung. Biochim Biophys Acta 795: 558-563, 1984[ISI][Medline].

34.   Robinson, BS, Hii CS, Poulos A, and Ferrante A. Effect of tumor necrosis factor-alpha on the metabolism of arachidonic acid in human neutrophils. J Lipid Res 37: 1234-1245, 1996[Abstract].

35.   Rooney, SA. The surfactant system and lung phospholipid biochemistry. Am Rev Respir Dis 131: 439-460, 1985[ISI][Medline].

36.   Salome, RG, McCoy DM, Ryan AJ, and Mallampalli RK. Effects of intratracheal instillation of TNF-alpha on surfactant metabolism. J Appl Physiol 88: 10-16, 2000[Abstract/Free Full Text].

37.   Schutze, S, Berkovic D, Tomsing O, Unger C, and Kronke M. Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C. J Exp Med 174: 975-988, 1991[Abstract].

38.   Sohal, PS, and Cornell RB. Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase. J Biol Chem 265: 11746-11750, 1990[Abstract/Free Full Text].

39.   Weinhold, PA, Rounsifer ME, Williams SE, Brubaker PG, and Feldman DA. CTP:phosphorylcholine cytidylyltransferase in rat lung. The effect of free fatty acids on the translocation of activity between microsomes and cytosol. J Biol Chem 259: 10315-10321, 1984[Abstract/Free Full Text].

40.   Whitsett, JA, Clark JC, Wispe JR, and Pryhuber GS. Effects of TNF-alpha and phorbol ester on human surfactant protein and MnSOD gene transcription in vitro. Am J Physiol Lung Cell Mol Physiol 262: L688-L693, 1992[Abstract/Free Full Text].

41.   Wieder, T, Orfanos CE, and Geilen CC. Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine. J Biol Chem 273: 11025-11031, 1998[Abstract/Free Full Text].

42.   Wright, JR. Clearance and recycling of pulmonary surfactant. Am J Physiol Lung Cell Mol Physiol 259: L1-L12, 1990[Abstract/Free Full Text].

43.   Young, SL, Wright JR, and Clements JA. Cellular uptake and processing of surfactant lipids and apoprotein SP-A by rat lung. J Appl Physiol 66: 1336-1342, 1989[Abstract/Free Full Text].


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