TNF-alpha increases ceramide without inducing apoptosis in alveolar type II epithelial cells

Rama K. Mallampalli, Erik J. Peterson, Aaron Brent Carter, Ronald G. Salome, Satya N. Mathur, and Gary A. Koretzky

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


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-alpha (TNF-alpha ), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-alpha (5 µg) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-alpha concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-kappa B activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-alpha did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-alpha , or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-alpha activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-kappa B pathways without increasing programmed cell death in type II cells.

tumor necrosis factor-alpha


    INTRODUCTION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

CERAMIDE IS A BIOLOGICALLY active lipid product that consists of a long-chain sphingoid base that is amide linked to a fatty acid. Ceramide can be generated in tissues either via the de novo pathway by dehydrogenation of dihydroceramide or by hydrolysis of sphingomyelin (34). Removal of the phosphocholine head group by sphingomyelin hydrolysis is catalyzed by a lysosomal acid sphingomyelinase or a plasma membrane-bound, cation-dependent neutral sphingomyelinase. Ceramide formed in tissues can subsequently be phosphorylated to ceramide phosphate via ceramide kinase or undergo rapid deacylation to sphingosine. Ceramide and sphingosine appear to be potent second messengers implicated in the regulation of diverse cellular processes such as cell growth and differentiation, gene transcription, viral replication, ligand binding, and cell death (19, 27).

Apoptosis is a preprogrammed form of cell death characterized by DNA fragmentation and a series of distinct morphological changes that occurs in response to a variety of stress-related internal or external stimuli. Apoptotic cell death can be triggered by the inflammatory cytokine tumor necrosis factor (TNF)-alpha ; activation of the Fas/Apo antigen (CD95) receptor, a related member of the TNF-receptor superfamily; or ionizing radiation (27). Interestingly, aside from inducing apoptosis, TNF-alpha also appears to stimulate cellular proliferation (12, 46). Many of the biological effects of TNF-alpha involve signaling through activation of multiple mitogen-activated protein (MAP) kinases and the transcription factor nuclear factor (NF)-kappa B, which regulate the expression of several inflammatory genes (14, 50). Similiar to the effects of TNF-alpha , activation of MAP kinases, specifically the p42/44 extracellular signal-related kinases (ERKs), has been linked to stimulation of both cellular growth and induction of apoptosis (9, 15). Interference with MAP kinase pathways by ectopic expression of dominant-interfering mutant proteins, however, blocks apoptosis in some systems, supporting a key role for these kinases in mediating apoptotic cell death (15). In contrast, NF-kappa B activation appears to inhibit signals for cell death (1). NF-kappa B exists in the cytosol as an inactive protein composed of two heterodimeric subunits (p50 and p65/RelA) bound to an inhibitory complex, Ikappa B. Phosphorylation of Ikappa B after TNF-alpha and Fas-receptor activation results in degradation of Ikappa B, which is necessary to release NF-kappa B to the nucleus where it can trigger the transcription of kappa B-responsive elements. A recent study (2) indicated that mice deficient in the p65/RelA subunit are more prone to apoptosis during embryonic development.

Prior studies (14, 20, 27) suggested that ceramide, generated in response to activation of the sphingomyelin hydrolysis pathway, appears to be an important effector molecule of TNF-alpha - and Fas/Apo ligand-induced apoptosis. Ceramide also possibly serves as an inducer of NF-kappa B and MAP kinase activity (18, 35). In studies related to apoptosis, TNF-alpha and Fas ligand activate sphingomyelin hydrolysis and coordinately increase ceramide levels, which temporally precede the induction of programmed cell death (7, 48). In some systems, exposure of cells to exogenous cell-permeable ceramide analogs or exogenous sphingomyelinase also induces apoptosis (24, 25). The significance of ceramide in signaling the cell death pathway is further supported by recent genetic studies (8, 41) demonstrating that either acid sphingomyelinase- or neutral sphingomyelinase-deficient lymphoid cells and acid sphingomyelinase knockout mice fail to increase ceramide and undergo apoptosis in response to ionizing radiation. On restoration of sphingomyelinase activity by gene transfer, however, these responses are reversed (41).

Not all studies have been consistent with a role for ceramide in stimulating cell death. Higuchi et al. (22) observed that exogenous ceramide failed to trigger apoptosis in human myelogenous leukemic cells, although TNF-alpha -induced apoptosis was blocked by a synthetic acid sphingomyelinase inhibitor. The authors concluded that ceramide was necessary but not sufficient for TNF-alpha -induced apoptosis. Other studies either did not detect a Fas-mediated increase in ceramide content preceding apoptosis (51) or, alternatively, suggested that ceramide might actually protect cells from apoptosis (23). Furthermore, because essentially all studies to date have evaluated the effects of stress-related stimuli such as TNF-alpha on apoptosis in cultured cells, the significance of these agents and their effects on the sphingomyelin signaling cascade for cell death in vivo remains unclear.

In the present study, we investigated whether intratracheal administration of TNF-alpha induces apoptosis in alveolar type II epithelial cells and whether this effect in vivo occurs via the sphingomyelin hydrolysis pathway. We focused on TNF-alpha because of its important role in the pathogenesis of lung inflammation and injury. We evaluated the effect of this cytokine in alveolar type II epithelial cells because in addition to regulating alveolar fluid balance and surfactant homeostasis, these cells are critically involved in maintaining the integrity of the alveolar air-surface interface. Type II cells serve as stem cells for the repair of the alveolar epithelium in the setting of acute or chronic lung injury. A prior study (16) showed that in diffuse alveolar injury occurring in the adult human lung, alveolar type II epithelial cells become hyperplastic and undergo apoptosis. A recent study (49) also demonstrated that fibroblasts isolated from patients with chronic pulmonary fibrosis secrete a factor that induces apoptosis in alveolar epithelial cells. Herein, we demonstrate that TNF-alpha increases lung ceramide at the expense of sphingomyelin hydrolysis. However, ceramide resulting from TNF-alpha -induced sphingolipid turnover in vivo or in vitro was not sufficient to induce apoptosis in alveolar type II epithelial cells.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Materials. The sphingomyelin, sphingosine, ceramide (type III and type IV), and phospholipid standards; myelin basic protein (MBP); and n-octyl-beta -D-glucopyranoside were purchased from Sigma (St. Louis, MO). Human TNF-alpha (1 µg = 1.1 × 105 activity units) was obtained from R&D Systems (Minneapolis, MN). Escherichia coli strain N4830/PJW10 diacylglycerol kinase was from Calbiochem (La Jolla, CA). The antibodies to MAP kinase (p42/44 anti-ERK antibody) were from Zymed Laboratories (South San Francisco, CA). NF-kappa B oligonucleotides were obtained from Promega (Madison, WI). GammaBind sepharose was obtained from Pharmacia (Piscatawny, NJ). All solvents were of Optima grade (Fisher Chemical). Silica LK5D (250-mm × 20-cm × 20-cm) thin-layer chromatography plates were purchased from Whatman International (Maidstone, UK). DMEM was obtained from the University of Iowa (Iowa City) Tissue Culture and Hybridoma Facility. The [3H]ceramide [114.4 mCi/mM (53)] used for the ceramidase assay was a kind gift from Dr. Phillip Wertz (University of Iowa). All other radiochemicals were purchased from DuPont NEN (Boston, MA).

Animals and tissue preparation. Adult Sprague-Dawley rats weighing 250-300 g were obtained from Sasco (Boston, MA). The rats were anesthetized with phenobarbital sodium (5 µg ip). The trachea was intubated with a 20-gauge plastic catheter, and the animals immediately received either 0.5 ml of diluent or 5 µg of TNF-alpha intratracheally. Each experiment typically consisted of two control and two TNF-alpha -treated animals. The animals were then mechanically ventilated for 10 min with a Harvard model 683 ventilator with a tidal volume of 2.5 ml, inspired O2 fraction of 20%, and a rate of 50 breaths/min to allow for adequate intrapulmonary dispersal of the cytokine. After mechanical ventilation, the lungs were homogenized at 5 ml/g tissue in buffer A [150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4 (31)] at 4°C. Lung microsomes and cytosol were also prepared by sequential centrifugation at 10,000 g for 10 min and 125,000 g for 60 min. Microsomes were stored in buffer R [10 mM Tris · HCl, 0.25 M sucrose, and 0.1 mM PMSF, pH 7.4 (31)] at -80°C before use. In separate studies, primary alveolar type II epithelial cells were isolated with methods previously described (30). Purity of the type II cells was >90% as assessed by tannic acid staining, and cellular viability was >95% by trypan blue exclusion immediately after isolation. The yield of type II cells was 46 ± 8.3 × 106 and 44 ± 10.0 × 106 for the control and TNF-alpha -treated groups, respectively. In some studies, the cells were cultured in DMEM with 10% fetal calf serum (FCS) for various time intervals.

Phospholipid analysis. Lipids were extracted from equal amounts of protein from microsomes with the method of Bligh and Dyer (4). 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 (44)]. 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 individual standards were scraped from the silica gel, and the levels of the individual phospholipids were quantitated with a phosphorus assay (33).

Detection of sphingomyelin degradation products. The mass of ceramide was measured as described by Preiss et al. (39). Lipids were extracted from microsomal preparations with the method of Bligh and Dyer (4), dried under nitrogen gas, and solubilized in an aliquot of n-octyl-beta -D-glucopyranoside-cardiolipin solution. The composition of the lipid mixture, reaction with diacylglycerol kinase, and detection of ceramide were identical to previously described methods (30). The level of sphingosine was measured with an acylation method also as previously described (37). Quantitation of ceramide and sphingosine was made by running known amounts of each lipid standard through the entire assay procedure and subjecting the autoradiograms to densitometric analysis.

Enzyme assays. The activities of the sphingolipid hydrolases sphingomyelinase and ceramidase were assayed as previously described (32). The reaction mixture (0.2-ml volume) for sphingomyelinase contained 25 mmol Tris-glycine buffer (pH 7.4), 2.5 pmol MgCl2, 50 nmol [choline-methyl-14C]sphingomyelin (specific activity 400 counts · min-1 · nmol-1), 0.5 µg of human serum albumin, 0.1 mg of cutscum, and 50-100 µg of protein. After a 1-h incubation at 37°C, the reaction was terminated with 1 ml of cold 10% trichloroacetic acid. After the addition of BSA (100 µg), 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. Alkaline ceramidase activity was assayed in lung microsomes as previously described (32). The activities of the sphingomyelin biosynthetic enzymes serine palmitoyltransferase and sphingomyelin synthase were assayed exactly as previously described (30, 32). The optimal assay conditions with lung tissue have been previously described (32).

NF-kappa B DNA binding activity. Type II cells harvested from control and TNF-alpha -treated animals were incubated for up to 30 min and subsequently washed in phosphate-buffered saline. The cells were resuspended in lysis buffer (10 mM HEPES, 10 mM KCl, 2 mM MgCl2, and 2 mM EDTA) for 15 min on ice. The cells were lysed and centrifuged, and nuclear protein was extracted as previously described (6). NF-kappa B oligonucleotides were labeled with [gamma -32P]ATP, and electrophoretic mobility gel shift assays were performed exactly as previously described (6).

MAP kinase activity. We determined whether TNF-alpha activated one group of MAP kinases, the p42/44 ERKs, using an immune complex assay. Type II cells were washed in ice-cold phosphate-buffered saline and lysed with buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 µM aprotinin, 1 mM vanadate, 50 mM NaF, and 0.5 mM EGTA and centrifuged at 15,000 g. Typically, 300 µg of supernatant protein were transferred to a tube containing 3 µg/sample of rabbit anti-ERK antibody that was previously bound to GammaBind sepharose and incubated and rotated at 4°C overnight. The immunoprecipitates were washed three times with high-salt buffer (0.05 M Tris, pH 7.4, 0.05 M NaCl, and 1% Nonidet P-40) and three times with lysis buffer and incubated in 20 µl of a kinase reaction mixture containing 20 mM MgCl2, 25 mM HEPES, 20 mM beta -glycerophosphate, 20 mM p-nitrophenyl phosphate, 20 mM sodium orthovanadate, 2 mM dithiothreitol, 20 µM ATP, 5 µCi of [gamma -32P]ATP, and 10 µg of MBP. After 15 min at 25°C, the reaction was terminated by the addition of 40 µl of 2× sample buffer. The samples were boiled for 5 min and run on a 15% SDS-PAGE. The gel was dried and autoradiographed to visualize the 32P-labeled MBP, which was quantitated by densitometric analysis.

Detection of apoptosis by hypodiploid nuclei analysis. Detection of subdiploid apoptotic nuclei was achieved with the method of Nicoletti et al. (36). Briefly, 1 × 106 rat alveolar type II epithelial cells were washed once in Hanks' balanced salt solution (HBSS) with 5 mM EDTA and then fixed with 2 ml of 70% ethanol. The cells were stored at -20°C for at least 1 h. After being washed in HBSS, the cells were resuspended in 0.4 ml of HBSS containing 20 µg/ml of propidium iodide, 40 units of RNase A, and 200 units of RNase T1. Hypodiploid nuclei were detected by fluorescence-activated cell sorter analysis with a Becton Dickinson FACScan. Subdiploid nuclei were defined as cells displaying lower relative fluorescence than the G0/G1 peak.

Protein analysis. Protein concentration was measured with the Bradford method, with BSA as the protein standard (5).

Statistical analysis. The data are expressed as means ± SE. Statistical analysis was performed with Student's t-test or an ANOVA with a Bonferroni adjustment for multiple comparisons (40).


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Phospholipid analysis. Intratracheal instillation of TNF-alpha significantly decreased the content of sphingomyelin but did not alter the levels of other major phospholipids (Fig. 1). The content of sphingomyelin in lung microsomes decreased from 10.1 ± 1.3 (control) to 6.7 ± 0.9 (TNF-alpha treated) nmol/mg protein 10 min after TNF-alpha instillation (P < 0.05). Time-course studies revealed that sphingomyelin levels increased nearly 1.8-fold after 30 min of mechanical ventilation (Fig. 1, inset). TNF-alpha also decreased the content of sphingomyelin by 18, 34, and 44% compared with control values at 5, 10, and 30 min, respectively, after cytokine administration. These results indicate that TNF-alpha selectively alters the content of a major membrane-associated lipid, sphingomyelin, in the lung.


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Fig. 1.   Effect of tumor necrosis factor (TNF)-alpha on lung phospholipids. Levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) were determined in lung microsomes after intratracheal administration of TNF-alpha (5 µg) or diluent. Inset: content of SM assayed at different time intervals after TNF-alpha administration. Total lipids were extracted from equal amounts of tissue protein, and individual phospholipids were separated by TLC. Lipids were eluted from the gel and quantitated with a phosphorus assay. Results are means ± SE expressed as nmol phospholipid phosphorus/mg microsomal protein from 3 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals. * P < 0.05 vs. control.

Effect of TNF-alpha on sphingomyelin degradation products. TNF-alpha treatment increased ceramide levels in lung microsomes from 809 ± 291 to 1,411 ± 144 pmol/mg protein 10 min after intratracheal cytokine administration (P < 0.05; Fig. 2A). These changes in vivo were also observed in alveolar type II cells where TNF-alpha increased ceramide content nearly twofold (Fig. 2; P < 0.05). Consistent with its effect on ceramide, TNF-alpha tended to increase sphingosine, a product directly downstream from ceramide hydrolysis, in microsomes (553 ± 158 and 787 ± 330 pmol/mg protein for control and TNF-alpha treated, respectively). These changes, however, did not reach significance (Fig. 2B). These results show that TNF-alpha induces a key bioactive sphingomyelin degradation product, ceramide, in the lung and in adult type II cells.


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Fig. 2.   Effect of TNF-alpha on lung sphingolipid degradation products. Top: ceramide (A) and sphingosine (B) assayed in lung microsomes (M) after intratracheal administration of TNF-alpha (5 µg). Ceramide was also assayed in alveolar type II epithelial (T2) cells cultured for 4 h in presence of TNF-alpha (100 ng/ml). Lipids from equal amounts of microsomal or cellular protein were extracted and phosphorylated with [gamma -32P]ATP with diacylglycerol kinase for analysis of ceramide or acylated with [3H]acetic anhydride for sphingosine determination as described in METHODS. Resulting [gamma -32P]ceramide phosphate and C2 [3H]ceramide products were resolved with TLC and subjected to autoradiography for quantitative analysis of ceramide and sphingosine, respectively. Various amounts (nos. on top in pmol) of ceramide and sphingosine standards were also run through entire procedure and chromatographed. C, control. Bottom: densitometric analysis of autoradiograms performed to quantitate levels of endogenous sphingolipids in lung microsomes. Results are means ± SE from 3 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals. * P < 0.05 vs. control.

Sphingomyelin hydrolysis. Sphingomyelin catabolism was determined by assaying the sphingolipid hydrolases sphingomyelinase and ceramidase. Although no changes were detected in whole lung homogenates (Fig. 3A), TNF-alpha had a significant effect on microsomal neutral sphingomyelinase activity. As shown in Fig. 3B, in four separate experiments, TNF-alpha stimulated enzyme activity on average from 3.60 ± 0.39 to 5.86 ± 0.44 nmol · h-1 · mg protein-1 (range, 27-143% increase; P < 0.05). In contrast, the cytokine tended to decrease acid sphingomyelinase activity in the whole lung (P = 0.07; Fig. 3C) and had no significant effect on acid sphingomyelinase activity in lung microsomes (Fig. 3D). The cytokine did not alter the activity of ceramidase in the whole lung (data not shown) or in microsomes (0.60 ± 0.05 and 0.51 ± 0.07 nmol · h-1 · mg protein-1 for control and TNF-alpha treated, respectively). These observations suggest that the decrease in lung sphingomyelin content after in vivo TNF-alpha administration is due, at least in part, to activation of an enzyme involved in the sphingomyelin hydrolysis pathway.


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Fig. 3.   Effect of TNF-alpha on lung sphingomyelin hydrolysis. Activities of neutral (A and B) and acid (C and D) sphingomyelinase were assayed in lung homogenates (A and C) and lung microsomes (B and D) after intratracheal administration of TNF-alpha (5 µg). Each line represents a separate experiment consisting of 2 control and 2 TNF-alpha -treated animals.

Sphingolipid biosynthesis. The activities of serine palmitoyltransferase and sphingomyelin synthase were assayed to determine whether TNF-alpha decreased sphingomyelin by regulating sphingolipid biosynthesis. TNF-alpha did not alter the activity of serine palmitoyltransferase, the first committed enzyme required for sphingoid base synthesis (467 ± 152 and 538 ± 79 pmol · h-1 · mg protein-1 for control and TNF-alpha -treated lung microsomes, respectively; Fig. 4). The cytokine also did not affect the final enzyme in the biosynthetic pathway, sphingomyelin synthase (49.6 ± 7.2 and 61.0 ± 9 pmol · h-1 · mg protein-1 for control and TNF-alpha treated, respectively). These results indicate that changes in sphingomyelin and ceramide are not due to modulation of enzymes involved in sphingolipid biosynthesis as previously described in other systems (47).


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Fig. 4.   Effect of TNF-alpha on lung sphingomyelin biosynthesis. Activities of serine palmitoyltransferase (SPT) and sphingomyelin synthase (SMS) were assayed in adult rat lung microsomes after intratracheal administration of TNF-alpha (5 µg). Data are expressed as pmol · h-1 · mg membrane protein-1 for SPT activity and pmol · h-1 · mg-1 × 10 for SMS activity. Results are means ± SE from 3 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals.

NF-kappa B DNA binding activity. In some systems, TNF-alpha -ceramide signaling results in coordinate or independent activation of the NF-kappa B pathway in addition to stimulation of the cell death pathway (10). Activation of the NF-kappa B pathway has also been shown to block the effects of TNF-alpha on apoptosis (1). Type II cells harvested from both control and TNF-alpha -treated animals expressed a low baseline level of DNA binding activity that gradually increased up to 15 min (Fig. 5). TNF-alpha -exposed cells, however, showed significantly greater NF-kappa B activity compared with control cells at baseline and up to 30 min. Maximal NF-kappa B binding activity was observed between 5 and 15 min. The activity then decreased in both control and TNF-alpha -treated cells to baseline levels. These results indicate that TNF-alpha stimulates NF-kappa B translocation to the nucleus.


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Fig. 5.   Effect of TNF-alpha on expression of nuclear factor (NF)-kappa B. T2 cells were isolated and assayed for NF-kappa B nuclear binding activity as assessed by electrophoretic mobility shift assay at various time points after intratracheal administration of diluent (left) or TNF-alpha (5 µg; right). Shown is a representative autoradiogram of 4 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals. ns, Nonspecific band.

MAP kinase activity. We determined whether TNF-alpha induction of ceramide was associated with activation of the p42/44 ERKs because these MAP kinases are activated by ceramide (20, 35). Exposure of type II cells to the cytokine increased the levels of p42/44 ERK activity in cells cultured for 2, 4, and 6 h (Fig. 6A). Intratracheal TNF-alpha also increased expression of the p42/44 ERKs in freshly isolated cells and induced activity threefold in the lung (P < 0.05 vs. control value; Fig. 6B). In our preliminary studies, cell-permeable synthetic ceramides increase p42/44 ERKs activity in vitro (data not shown). Taken together, these results might suggest that ceramide resulting from sphingomyelin hydrolysis activates the MAP kinase pathway in type II cells in vivo.


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Fig. 6.   Effect of TNF-alpha on p42/44 extracellular signal-related kinase (ERK) activity. A: p42/44 ERK immunoprecipitated from equal amounts of supernatant protein obtained from T2 cells cultured for various times in presence and absence of TNF-alpha (100 ng/ml). Immunoprecipitates were then incubated in a kinase assay with myelin basic protein under phosphorylating conditions. B, left: p42/44 ERK activity assayed from equal amounts of protein from 3 separate cytosolic (Cyto) preparations obtained from rats given TNF-alpha (5 µg) or diluent (control) intratracheally and from freshly isolated T2 cells from rats treated similiarly. B, right: densitometric analysis of p42/44 ERK activity. * P < 0.05 vs. control cells by Student's t-test.

Hypodiploid nuclei analysis of alveolar type II epithelial cells. In addition to activation of the NF-kappa B and MAP kinase pathways, TNF-alpha has been shown to be a potent inducer of apoptosis in several primary cells and cell lines. Therefore, we determined whether TNF-alpha induction of ceramide correlated with the induction of apoptosis in type II cells. Cells were analyzed for hypodiploid nuclei formation at various time points after in vivo cytokine treatment. Apoptotic type II cell nuclei were defined as those exhibiting lower relative fluorescence than the G0/G1 peak. As shown in Fig. 7, alveolar type II cells isolated from rats 10 min after intratracheal TNF-alpha exhibited almost identical numbers of cells that were shown to be apoptotic compared with cells isolated from rats administered diluent. Four hours after TNF-alpha or diluent treatment, ~12% of the cells in each group were shown to be hypodiploid (Fig. 7B). No significant differences in the percentage of hypodiploid nuclei were subsequently observed between the control and TNF-alpha -treated groups at the 12- or 24-h time points of analysis. However, the number of apoptotic cells in each group gradually increased after time in culture because one-third of type II alveolar cells was shown to be hypodiploid after 24 h of culture. These results were confirmed with annexin V fluorescent binding, a method of detecting apoptotic cells through altered plasma membrane structure (data not shown). Moreover, as described in other cell types, ultraviolet radiation treatment significantly increased the number of apoptotic type II cells (41). Collectively, these results indicate that ceramide resulting from TNF-alpha -induced sphingomyelin hydrolysis does not induce type II cell apoptosis in vivo.


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Fig. 7.   Effect of TNF-alpha on apoptosis of T2 cells in vivo. T2 cells were isolated from rat lungs after intratracheal administration of TNF-alpha (5 µg) or diluent (control) and analyzed for apoptosis immediately after isolation (4 h) or subsequently cultured for up to 24 h in DMEM containing 10% FCS. Some T2 cells were also exposed to ultraviolet radiation (UV; 12 s of 12,000 J/m2) after 12 or 24 h of incubation. After culture, cells were collected, stained with propidium iodide, and analyzed for apoptosis with flow cytometric analysis (fluorescence-activated cell sorter). A: representative DNA histograms (right) and corresponding fluorescence emission analysis (left) in control and TNF-alpha - and UV-treated T2 cells after 24 h of culture. Right: ordinate, relative percentage of apoptotic cells; abscissa, DNA cell content. Area under M1 range represents hypodiploid nuclei. Left: ordinate, side scatter; abscissa, forward scatter. B: relative percentages of T2 cells that were determined to be apoptotic after intratracheal TNF-alpha or diluent and subsequent culture for various time intervals. Results are means ± SE from 3 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals. + P < 0.005 vs. respective 4-h group. * P < 0.05 vs. 24-h control and TNF-alpha -treated groups by ANOVA.

To assess the direct effects of TNF-alpha on apoptosis, separate studies were performed to assess apoptosis after type II cells were cultured in the presence of exogenous cytokine. A cell-permeable ceramide analog (C2 ceramide), TNF-alpha , or a monoclonal anti-CD95 antibody did not induce apoptosis in cells to a significantly greater extent than that in control cells after 12 (Fig. 8A) or 24 h of exposure (Fig. 8B). As observed with cells isolated after in vivo TNF-alpha administration, exposure to ultraviolet radiation significantly increased the number of apoptotic cells above the control level and the level of the cells exposed to the anti-CD95 antibody (Fig. 8B). These results indicate that ceramide resulting from direct exposure of cells to TNF-alpha does not accelerate programmed cell death in type II cells.


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Fig. 8.   Apoptosis of T2 cells in vitro. T2 cells were cultured in DMEM containing 10% FCS for 12 (A) or 24 (B) h in presence of C2 ceramide (10 µmol), anti-CD95 (Fas) ligand (200 ng/ml), or TNF-alpha (100 ng/ml) or were exposed transiently to UV radiation (12 s of 12,000 J/m2) after culture. Cells were then analyzed for apoptosis as described in Fig. 7. Results are means ± SE from 3 separate experiments, each consisting of 2 control and 2 TNF-alpha -treated animals. * P < 0.05 vs. all other groups by ANOVA.


    DISCUSSION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Stress signals such as TNF-alpha have been shown to stimulate sphingomyelin hydrolysis and ceramide accumulation and induce apoptotic cell death in several nonpulmonary in vitro systems (10, 24). To our knowledge, this is among the first studies to test this paradigm in the whole animal model. The present study demonstrates that TNF-alpha stimulates sphingomyelin hydrolysis by activating a magnesium-dependent neutral sphingomyelinase rather than the lysosomal hydrolytic enzyme. The ceramide generated in this manner was associated with activation of the p42/44 ERK and NF-kappa B pathways but was not sufficient to induce apoptosis in alveolar type II epithelial cells. Our in vitro studies also demonstrate that unlike ultraviolet radiation, other exogenous stress signals such as TNF-alpha or Fas-receptor activation do not induce apoptosis in these cells above control levels. Yet, in these studies, TNF-alpha increased ceramide content in the type II cells. Moreover, in the process of evaluating cell death in vitro, we observed that extended culture of type II cells is associated with a progressive increase in the number of cells that become apoptotic.

As in other organs, apoptosis is potentially highly relevant to the lung in the setting of a variety of fundamental processes such as organogenesis, immune cell regulation, and tissue injury. Apoptosis has been demonstrated in lung endothelium (52), neutrophils (52), mesenchymal cells (38), alveolar macrophages (3), alveolar lymphocytes (21), and alveolar type II cell lines (26, 43). Programmed cell death is also a feature of lung development, with notable species differences because apoptosis appears to be restricted to interstitial cells in human lung, whereas it occurs in both the mesenchyme and epithelium in the rat (28, 42). Human mesenchymal cells and alveolar type II epithelial cells have been observed to be apoptotic in acute lung injury (16, 38), and lymphocytes obtained from normal subjects and patients with chronic fibrotic lung disease appear to be more prone to apoptosis (21). Interestingly, the T lymphocytes in this latter study highly expressed the Fas receptor. Collectively, these latter observations suggest that programmed cell death may be an important process in the evolution or repair phase of lung disease. It is unclear from some studies, however, whether withdrawal of a death inhibitory factor or the presence of a stress signal is involved in initiating apoptotic cell death. The fact that TNF-alpha plays an integral role in the pathogenesis of both acute and chronic lung injury leads one to speculate that this cytokine might contribute, at least in part, to some of the cytopathic changes observed in human lung disease.

Few studies have prospectively investigated the mechanisms underlying TNF-alpha -induced apoptosis in vivo. Studies (29, 45) have demonstrated TNF-alpha -inducible hepatocyte apoptosis after intravenous administration in mice and that these effects of TNF-alpha are attenuated by pretreatment with interleukin-1beta or are altered in mice lacking the TNF-alpha receptor. Another investigation (11) showed that cultured fibroblasts isolated from mice defective in expression of an RNA-activated protein kinase were resistent to TNF-alpha -induced apoptosis. Recently, Haimovitz-Friedman et al. (17) demonstrated that intraperitoneal administration of lipopolysaccharide (LPS) and intravenous TNF-alpha induced endothelial apoptosis in mice in a number of tissues including the lung. LPS and TNF-alpha also produced elevated ceramide levels in the intestines of these animals. Although type II epithelial cells were not isolated in this study, the alveolar lining appeared intact after LPS treatment. In our study, we administered TNF-alpha intratracheally because macrophages located on the air side of the alveolar-capillary barrier could also potentially release large amounts of injurious cytokines directly into the alveolar lumen. By interacting with the apical side of type II cells rather than via the capillary endothelium, intratracheal instillation of the cytokine might represent a more direct and effective route to assess cytokine signaling in type II cells. However, our work corroborates the study of Haimovitz-Friedman et al. in that, despite induction of ceramide, limited, if any, apoptosis was observed in the alveolar epithelium regardless of the route of administration of the stress signal. We observed activation of a neutral sphingomyelinase, whereas stimulation of an acid sphingomyelinase was suggested in the former study. This discrepancy might be explained by differences in the stress signal or experimental conditions used or may represent organ-specific responses.

The observation that TNF-alpha -induced apoptosis was not seen in type II cells despite a functional sphingomyelin-ceramide pathway suggests several possibilities. First, we may have selected a dose of intratracheal TNF-alpha that was below the threshold beyond which significant inflammatory cellular influx and cellular necrosis occurs in lung tissue (13). It is possible that at the doses used in these studies, apoptosis occurred in other lung cells and that higher doses might have induced apoptosis in type II cells. This would suggest that type II cells, compared with other lung cells, might be relatively protected from TNF-alpha -induced apoptosis, in-line with their ability to secrete a heat-stable peptide that protects cells from programmed cell death (52). Apoptosis could also have occurred in type II cells after cytokine treatment, but it may not have been detected because of high cell turnover coupled with rapid elimination of these cells before analysis. Alternatively, during the process of isolation of type II cells, the experimental procedures used could have favorably altered the relative proportion of inhibitory to stress signals for apoptosis to prevent significant cell death by the cytokine. Finally, ceramide resulting from TNF-alpha actions in vivo might be required to induce apoptosis, but other downstream signaling events such as activation of the interleukin-1beta -converting enzyme cascade might also have been necessary (47). Regardless of these explanations, ceramide itself may have activated a p44/42 ERK in the lung or triggered NF-kappa B activation. The fact that TNF-alpha activated NF-kappa B in these studies is relevant because activation of this transcription factor may have served as a protective mechanism against type II cell apoptosis (1).

A novel observation from our in vitro studies is that a high proportion of alveolar type II epithelial cells are apoptotic after prolonged culture. Uhal et al. (49) reported that after isolation and culture of rat alveolar epithelial cells for 4 days in the presence of serum, only ~5% of cells were apoptotic. However, after 4 days in culture, the cells are exclusively of a type I phenotype, which perhaps may be less prone to apoptosis. Thus, on the basis of our present understanding of type II cells, it is reasonable to postulate that these cells are destined to undergo apoptosis or progress to a type I phenotype shortly after culture on plastic surfaces. The fact that nearly one-third of the cells cultured on plastic were shown to be apoptotic after 24 h of culture coupled with the likelihood that many of the remaining cells are also dedifferentiating underscores the importance of careful interpretation of data obtained on these cells after long-term culture. Future studies investigating the mechanisms that lead to apoptosis of cultured type II cells may be relevant to processes that govern programmed cell death in vivo. In this regard, it would be of interest to determine whether the use of various substrata that are currently in use to alter type II cell differentiation might also modulate apoptotic type II cell death.


    ACKNOWLEDGEMENTS

We thank Julie Weeks for technical assistance.


    FOOTNOTES

This study was supported by the Office of Research and Development, Department of Veterans Affairs; National Heart, Lung, and Blood Institute Grant HL-55584; and an Established Investigator Award from the American Heart Association (to R. K. Mallampalli).

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. §1734 solely to indicate this fact.

Address for reprint requests: R. K. Mallampalli, Pulmonary Division, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242.

Received 30 June 1998; accepted in final form 18 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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