Department of Internal Medicine and the Department of Veterans Affairs Medical Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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Tumor necrosis factor
(TNF)- is a major cytokine implicated in inducing acute and chronic
lung injury, conditions associated with surfactant
phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-
decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-
, we investigated
PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-
overexpression. Compared with controls, TNF-
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-
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
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INTRODUCTION |
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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- (TNF-
) 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-
also regulates surfactant
metabolism. For example, TNF-
inhibits the synthesis of the
surfactant apoproteins, induces PtdCho degradation, and decreases the
biosynthesis of PtdCho (3, 37, 40). Recently, we
demonstrated that TNF-
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-
also stimulates PtdCho secretion in primary alveolar
epithelial cells (5). Thus TNF-
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- 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-
release is a plausible mechanism in the setting of chronic alveolitis
where high TNF-
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-
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-, Miyazaki et al.
(28) generated a transgenic mouse expressing the murine
TNF-
gene driven by the human surfactant protein C promoter. In this model, TNF-
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-
on PtdCho metabolism. We hypothesized that chronic overexpression of TNF-
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-
transgenics and
wild-type littermate controls.
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MATERIALS AND METHODS |
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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% -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
-actin were detected by using the ECL Western
blotting detection system as instructed by the manufacturer. The
dilution factor for anti-CT and anti-
-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.
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RESULTS |
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Phospholipid and DSPtdCho analysis.
Transgenic mice exhibiting chronic expression of TNF- displayed
several abnormalities in phospholipid composition compared with control
littermates. Furthermore, a distinctly different pattern of expression
of phospholipids was identified in TNF-
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-
transgenics
(P < 0.05). There were no other significant
differences in other major phospholipids. However, in lung microsomes,
TNF-
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-
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-
transgenics exhibited a 55% increase in PtdCho (Fig.
1B, P < 0.05). The level of alveolar DSPtdCho
was 46% greater in the TNF-
transgenics compared with control
(control 1,510 ± 420 nmol phosphorus/mg protein, TNF-
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-
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-
transgenics compared
with control, which may reflect differences in surfactant synthesis,
secretion, or intraalveolar processing.
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Choline incorporation into PtdCho.
To investigate whether TNF- 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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Enzymes of PtdCho synthesis.
We next determined if the decrease in surfactant content in the lungs
of murine TNF- 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-
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-
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-
inhibits a key regulatory enzyme, CCT, in murine lungs, resulting in an overall decrease in PtdCho content.
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Immunoblot analysis.
We performed immunoblot analysis to determine if reduced activity of
CCT in TNF- transgenics was due to a decrease in enzyme mass.
Unexpectedly, in three separate studies, we observed that transgenic
mice harboring the TNF-
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
-actin were unchanged between
control and TNF-
transgenic mice (Fig. 3B). Thus when we
expressed our results as specific activity of the enzyme using
densitometric measurements of enzyme mass, the TNF-
transgenic mice
exhibited an even more substantial reduction in catalytic activity
relative to wild-type controls (Fig. 3C). Collectively,
these results suggest that TNF-
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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Sphingolipids.
The data above indicate that adequate amounts of CCT protein are
detected in TNF- 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-
has been shown to acutely increase the levels of these
bioactive mediators in the lung (20). Although we observed that TNF-
transgenic mice expressed no significant differences in
sphingosine content, ceramide levels were significantly increased in
TNF-
transgenic mice (control 1,540 ± 430 pmol/mg protein, TNF-
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-
transgenic mice.
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Fatty acids.
In addition to its effects on sphingolipid metabolism, TNF-
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-
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-
transgenics (Table 3). There were
no substantial differences between the groups with regard to levels of
saturated fatty acids. TNF-
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-
expression.
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DSPtdCho alveolar uptake.
We determined if elevated alveolar PtdCho was secondary to altered
reuptake of DSPtdCho. Compared with control littermates, TNF-
transgenics had a 90% reduction in radiolabeled DSPtdCho uptake
(control 86,800 ± 21,100 dpm/mg protein, TNF-
transgenic 8,900 ± 2,800 dpm/mg protein, P < 0.01, Fig.
4). These data suggest that alveolar
surfactant recycling is decreased in TNF-
transgenic mice,
consistent with the findings of increased alveolar DSPtdCho and
decreased microsomal DSPtdCho.
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DISCUSSION |
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The goal of the present study was to investigate whether long-term
expression of TNF- downregulates surfactant PtdCho synthesis. Because of the relatively short half-life of TNF-
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-
is overexpressed in alveolar epithelium to pursue our studies (28). We observed that surfactant
lipid synthesis was decreased in the lungs of TNF-
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-
transgenic mice. Although
TNF-
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-, the levels of DSPtdCho and phosphatidylglycerol were decreased
significantly in lungs of TNF-
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-
at the level of either lipid synthesis or degradation within
the cells. We did not exclude the possibility that TNF-
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-
usually occurs more rapidly than regulation of enzymes involved in PtdCho synthesis.
We observed that the decrease in PtdCho mass in TNF- 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-
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-
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-
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-
transgenics compared with control. The results with ceramide are
important because TNF-
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-
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-
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-
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- transgenic mice
compared with control. There are several potential explanations for
these data. First, chronic TNF-
expression might regulate surfactant
secretion, uptake, or intraalveolar processing. In this regard, Benito
and Bosch (5) showed that TNF-
stimulates surfactant
lipid secretion in primary type II cells via a protein kinase-dependent
mechanism. In these studies, chronic TNF-
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-
was not global but restricted to alveolar
epithelium in this model. Because alveolar macrophages are not only a
major source of TNF-
but also participate in DSPdCho uptake, TNF-
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-
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-
transgenic mice
display a unique phospholipid profile that will require additional characterization.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Mason for providing the TNF--overexpressing
transgenic mouse line.
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FOOTNOTES |
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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.
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