Department of Internal Medicine and Department of Veterans Affairs Medical Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TNF-
), increases apoptosis in primary rat
alveolar type II epithelial cells. Intratracheal instillation of
TNF-
(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-
concomitantly
activated the p42/44 extracellular signal-related kinases and induced
nuclear factor-
B activation in the lung. Hypodiploid nuclei studies
revealed that intratracheal TNF-
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-
, or a
related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-
activation of sphingomyelin hydrolysis might activate the
mitogen-activated protein kinase and nuclear factor-
B pathways
without increasing programmed cell death in type II cells.
tumor necrosis factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-; 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-
also appears to stimulate cellular
proliferation (12, 46). Many of the biological effects of TNF-
involve signaling through activation of multiple mitogen-activated protein (MAP) kinases and the transcription factor nuclear factor (NF)-
B, which regulate the expression of several inflammatory genes
(14, 50). Similiar to the effects of TNF-
, 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-
B
activation appears to inhibit signals for cell death (1). NF-
B
exists in the cytosol as an inactive protein composed of two
heterodimeric subunits (p50 and p65/RelA) bound to an inhibitory
complex, I
B. Phosphorylation of I
B after TNF-
and Fas-receptor
activation results in degradation of I
B, which is necessary to
release NF-
B to the nucleus where it can trigger the transcription
of
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-- and Fas/Apo
ligand-induced apoptosis. Ceramide also possibly serves as an inducer
of NF-
B and MAP kinase activity (18, 35). In studies related to
apoptosis, TNF-
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--induced apoptosis was blocked by a synthetic
acid sphingomyelinase inhibitor. The authors concluded that ceramide
was necessary but not sufficient for TNF-
-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-
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- induces apoptosis in alveolar type II epithelial cells and whether this effect in vivo occurs via the sphingomyelin hydrolysis pathway. We focused on TNF-
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-
increases lung ceramide at the expense of
sphingomyelin hydrolysis. However, ceramide resulting from
TNF-
-induced sphingolipid turnover in vivo or in vitro was not
sufficient to induce apoptosis in alveolar type II epithelial cells.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. The
sphingomyelin, sphingosine, ceramide (type III and type IV), and
phospholipid standards; myelin basic protein (MBP); and
n-octyl--D-glucopyranoside
were purchased from Sigma (St. Louis, MO). Human TNF-
(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-
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- intratracheally. Each experiment typically consisted of two
control and two TNF-
-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-
-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--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 · min1 · 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-B DNA binding
activity. Type II cells harvested from control and
TNF-
-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-
B oligonucleotides were
labeled with
[
-32P]ATP, and
electrophoretic mobility gel shift assays were performed exactly as
previously described (6).
MAP kinase activity. We determined
whether TNF- 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
-glycerophosphate, 20 mM
p-nitrophenyl phosphate, 20 mM sodium
orthovanadate, 2 mM dithiothreitol, 20 µM ATP, 5 µCi of
[
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phospholipid analysis. Intratracheal
instillation of TNF- 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-
treated) nmol/mg protein 10 min after TNF-
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-
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-
selectively alters
the content of a major membrane-associated lipid, sphingomyelin, in the
lung.
|
Effect of TNF- on sphingomyelin
degradation products. TNF-
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-
increased ceramide content nearly twofold (Fig. 2;
P < 0.05). Consistent with its
effect on ceramide, TNF-
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-
treated,
respectively). These changes, however, did not reach significance (Fig.
2B). These results show that TNF-
induces a key bioactive sphingomyelin degradation product, ceramide, in
the lung and in adult type II cells.
|
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- had a significant effect on microsomal neutral sphingomyelinase
activity. As shown in Fig. 3B, in four separate experiments, TNF-
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-
treated, respectively). These observations suggest that the
decrease in lung sphingomyelin content after in vivo TNF-
administration is due, at least in part, to activation of an enzyme
involved in the sphingomyelin hydrolysis pathway.
|
Sphingolipid biosynthesis. The
activities of serine palmitoyltransferase and sphingomyelin synthase
were assayed to determine whether TNF- decreased sphingomyelin by
regulating sphingolipid biosynthesis. TNF-
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-
-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-
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).
|
NF-B DNA binding
activity. In some systems, TNF-
-ceramide signaling
results in coordinate or independent activation of the NF-
B pathway
in addition to stimulation of the cell death pathway (10). Activation
of the NF-
B pathway has also been shown to block the effects of
TNF-
on apoptosis (1). Type II cells harvested from both control and
TNF-
-treated animals expressed a low baseline level of DNA binding
activity that gradually increased up to 15 min (Fig.
5). TNF-
-exposed cells, however, showed
significantly greater NF-
B activity compared with control cells at
baseline and up to 30 min. Maximal NF-
B binding activity was
observed between 5 and 15 min. The activity then decreased in both
control and TNF-
-treated cells to baseline levels. These results
indicate that TNF-
stimulates NF-
B translocation to the nucleus.
|
MAP kinase activity. We determined
whether TNF- 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-
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.
|
Hypodiploid nuclei analysis of alveolar type II
epithelial cells. In addition to activation of the
NF-B and MAP kinase pathways, TNF-
has been shown to be a potent
inducer of apoptosis in several primary cells and cell lines.
Therefore, we determined whether TNF-
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-
exhibited
almost identical numbers of cells that were shown to be apoptotic
compared with cells isolated from rats administered diluent. Four hours
after TNF-
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-
-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-
-induced
sphingomyelin hydrolysis does not induce type II cell apoptosis in
vivo.
|
To assess the direct effects of TNF- 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-
, 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-
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-
does not accelerate programmed cell death in type II cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stress signals such as TNF- 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-
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-
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-
or Fas-receptor activation do not induce
apoptosis in these cells above control levels. Yet, in these studies,
TNF-
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- 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--induced apoptosis in vivo. Studies (29, 45) have demonstrated
TNF-
-inducible hepatocyte apoptosis after intravenous administration
in mice and that these effects of TNF-
are attenuated by
pretreatment with interleukin-1
or are altered in mice lacking the
TNF-
receptor. Another investigation (11) showed that cultured fibroblasts isolated from mice defective in expression of an
RNA-activated protein kinase were resistent to TNF-
-induced
apoptosis. Recently, Haimovitz-Friedman et al. (17) demonstrated that
intraperitoneal administration of lipopolysaccharide (LPS) and
intravenous TNF-
induced endothelial apoptosis in mice in a number
of tissues including the lung. LPS and TNF-
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-
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--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-
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-
-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-
actions in vivo might be required to induce apoptosis, but other downstream signaling events such as activation of
the interleukin-1
-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-
B
activation. The fact that TNF-
activated NF-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beg, A. A.,
and
D. Baltimore.
An essential role for NF-B in preventing TNF-
-induced cell death.
Science
274:
782-784,
1996
2.
Beg, A. A.,
W. C. Sha,
R. T. Bronson,
S. Ghosh,
and
D. Baltimore.
Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B.
Nature
376:
167-170,
1995[Medline].
3.
Bingisser, R.,
C. Stey,
M. Weller,
P. Groscurth,
E. Russi,
and
K. Frei.
Apoptosis in human alveolar macrophages is induced by exotoxin and is modulated by cytokines.
Am. J. Respir. Cell Mol. Biol.
15:
64-70,
1996[Abstract].
4.
Bligh, E. G.,
and
W. J. Dyer.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37:
911-917,
1959.
5.
Bradford, M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
6.
Carter, A. B.,
M. M. Monick,
and
G. W. Hunninghake.
Lipopolysaccharide-induced NF-B activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent.
Am. J. Respir. Cell Mol. Biol.
18:
384-391,
1998
7.
Chinnaiyan, A. M.,
C. G. Tepper,
M. F. Seldin,
K. O'Rourke,
F. C. Kischkel,
S. Hellbardt,
P. H. Krammer,
M. E. Peter,
and
V. M. Dixit.
FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis.
J. Biol. Chem.
271:
4961-4965,
1996
8.
Chmura, S. J.,
E. Nodzenski,
M. A. Beckett,
D. W. Kufe,
J. Quintans,
and
R. R. Weichselbaum.
Loss of ceramide production confers resistance to radiation-induced apoptosis.
Cancer Res.
57:
1270-1275,
1997[Abstract].
9.
Coroneos, E.,
Y. Wang,
J. R. Panuska,
D. J. Templeton,
and
M. Kester.
Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades.
Biochem. J.
316:
13-17,
1996[Medline].
10.
Dbaibo, G. S.,
L. M. Obeid,
and
T. A. Hannun.
Tumor necrosis factor-alpha (TNF-alpha) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF-alpha from activation of nuclear factor-kappa B.
J. Biol. Chem.
268:
17762-17766,
1993
11.
Der, S. D.,
Y. L. Yang,
C. Weissmann,
and
B. R. G. Williams.
A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.
Proc. Natl. Acad. Sci. USA
94:
3279-3283,
1997
12.
Dubaybo, B. A.,
G. Bayashi,
and
G. J. Rubeiz.
Changes in tumor necrosis factor in postpneumonectomy lung growth.
J. Thorac. Cardiovasc. Surg.
110:
396-404,
1995
13.
Fuchs, H. J.,
R. Debs,
J. S. Patton,
and
H. D. Liggitt.
The pattern of lung injury induced after pulmonary exposure to tumor necrosis factor- depends on the route of administration.
Diagn. Microbiol. Infect. Dis.
13:
397-404,
1990[Medline].
14.
Gamard, C. J.,
G. S. Dbaibo,
B. Liu,
L. M. Obeid,
and
Y. A. Hannun.
Selective involvement of ceramide in cytokine-induced apoptosis.
J. Biol. Chem.
272:
16474-16481,
1997
15.
Goillot, E.,
J. Raingeaud,
A. Ranger,
R. I. Tepper,
R. J. Davis,
E. Harlow,
and
I. Sanchez.
Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway.
Proc. Natl. Acad. Sci. USA
94:
3302-3307,
1997
16.
Guinee, D.,
M. Fleming,
T. Hayashi,
M. Woodward,
J. Zhang,
J. Walls,
M. Koss,
V. Ferrans,
and
W. Travis.
Association of p53 and WAF1 expression with apoptosis in diffuse alveolar damage.
Am. J. Pathol.
149:
531-538,
1996[Abstract].
17.
Haimovitz-Friedman, A.,
C. Cordon-Cardo,
S. Bayoumy,
M. Garzotto,
M. McLoughlin,
R. Gallily,
C. K. Edwards,
E. H. Schuchman,
Z. Fuks,
and
R. Kolesnick.
Lipopolysaccaride induces disseminated endothelial apoptosis requiring ceramide generation.
J. Exp. Med.
186:
1831-1841,
1998
18.
Hannun, Y. A.
Functions of ceramide in coordinating cellular responses to stress.
Science
274:
1855-1859,
1996
19.
Hannun, Y. A.,
and
R. M. Bell.
Functions of sphingolipids and sphingolipid degradation breakdown products in cellular regulation.
Science
243:
500-507,
1989[Medline].
20.
Hannun, Y. A.,
and
L. M. Obeid.
Mechanisms of ceramide-mediated apoptosis.
Adv. Exp. Med. Biol.
407:
145-149,
1997[Medline].
21.
Herry, I.,
M. Bonay,
F. Bouchonnet,
M. P. Schuller,
D. Lecossier,
A. Tazi,
D. H. Lynch,
and
A. J. Hance.
Extensive apoptosis of lung T-lymphocytes maintained in vitro.
Am. J. Respir. Cell Mol. Biol.
15:
339-347,
1996[Abstract].
22.
Higuchi, M.,
S. Singh,
J. P. Jaffrezou,
and
B. B. Aggarwal.
Acid sphingomyelinase-generated ceramide is needed but not sufficient for TNF-induced apoptosis and nuclear factor-B activation.
J. Immunol.
156:
297-304,
1996[Abstract].
23.
Ito, A.,
and
K. Horigome.
Ceramide prevents neuronal programmed cell death induced by nerve growth factor deprivation.
J. Neurochem.
65:
463-466,
1995[Medline].
24.
Jarvis, W. D.,
R. N. Kolesnick,
F. A. Fornari,
R. S. Traylor,
D. A. Gewirtz,
and
S. Grant.
Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway.
Proc. Natl. Acad. Sci. USA
91:
73-77,
1994[Abstract].
25.
Jayadev, S.,
B. Liu,
A. E. Bielawska,
J. Y. Lee,
F. Nazaire,
M. Y. Pushkareva,
L. M. Obeid,
and
Y. A Hannun.
Role for ceramide in cell cycle arrest.
J. Biol. Chem.
270:
2047-2052,
1995
26.
Kazzaz, J. A.,
J. Xu,
T. A. Palaia,
L. Mantell,
A. M. Fein,
and
S. Horowitz.
Cellular oxygen toxicity.
J. Biol. Chem.
271:
15182-15186,
1996
27.
Kolesnick, R. N.,
A. Haimovitz-Friedman,
and
Z. Fuks.
The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation.
Biochem. Cell Biol.
72:
471-474,
1994[Medline].
28.
Kresch, M. J.,
C. Christian,
F. Wu,
and
N. Hussain.
Ontogeny of apoptosis during lung development.
Pediatr. Res.
43:
426-431,
1998[Abstract].
29.
Leist, M.,
F. Gantner,
I. Bohlinger,
G. Tiegs,
P. G. Germann,
and
A. Wendel.
Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models.
Am. J. Pathol.
146:
1220-1234,
1995[Abstract].
30.
Longo, C. A.,
D. Tyler,
and
R. K. Mallampalli.
Sphingomyelin metabolism is developmentally regulated in rat lung.
Am. J. Respir. Cell Mol. Biol.
16:
605-612,
1997[Abstract].
31.
Mallampalli, R. K.,
and
G. W. Hunninghake.
Expression of immunoreactive cytidine 5'-triphosphate:cholinephosphate cytidylyltransferase in developing rat lung.
Pediatr. Res.
34:
502-511,
1993[Abstract].
32.
Mallampalli, R. K.,
S. N. Mathur,
L. J. Warnock,
R. G. Salome,
G. W. Hunninghake,
and
F. J. Field.
Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung.
Biochem. J.
318:
333-341,
1996[Medline].
33.
Mallampalli, R. K.,
M. E. Walter,
M. W. Peterson,
and
G. W. Hunninghake.
Betamethasone activation of CTP:cholinephosphate cytidylyltransferase is lipid dependent.
Am. J. Respir. Cell Mol. Biol.
10:
48-57,
1994[Abstract].
34.
Merrill, A. H., Jr.,
and
D. D. Jones.
An update of the enzymology and regulation of sphingolipid metabolism.
Biochim. Biophys. Acta
1044:
1-12,
1990[Medline].
35.
Modur, V.,
G. A. Zimmerman,
S. M. Prescott,
and
T. M. McIntyre.
Endothelial cell inflammatory responses to tumor necrosis factor .
J. Biol. Chem.
271:
13094-13102,
1996
36.
Nicoletti, I.,
G. Migliorati,
M. C. Pagliacci,
F. Grignani,
and
C. Riccardi.
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J. Immunol. Methods
139:
271-279,
1991[Medline].
37.
Ohta, H.,
F. Ruan,
S. Hakomori,
and
Y. Igarashi.
Quantification of free sphingosine in cultured cells by acylation with radioactive acetic anhydride.
Anal. Biochem.
222:
489-494,
1994[Medline].
38.
Polunovsky, V. A.,
B. Chen,
C. Henke,
D. Snover,
C. Wendt,
D. H. Ingbar,
and
P. B. Bitterman.
Role of mesenchymal cell death in lung remodeling after injury.
J. Clin. Invest.
92:
388-397,
1993[Medline].
39.
Preiss, J. E.,
C. R. Loomis,
R. M. Bell,
and
J. E. Niedel.
Quantitative measurement of sn-1,2-diacylglycerols.
Methods Enzymol.
141:
294-300,
1987[Medline].
40.
Rosner, B.
Fundamentals of Biostatistics. Belmont, CA: Wadsworth, 1995, p. 314-318.
41.
Santana, P.,
L. A. Pena,
A. Haimovitz-Friedman,
S. Martin,
D. Green,
M. McLoughlin,
C. Cordon-Carlo,
E. H. Schuchman,
Z. Fuks,
and
R. Kolesnick.
Acid-sphingomyelinase deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Cell
86:
189-199,
1996[Medline].
42.
Scavo, L. M.,
R. Ertsey,
C. J. Chapin,
L. Allen,
and
J. A. Kitterman.
Apoptosis in the development of rat and human fetal lungs.
Am. J. Respir. Cell Mol. Biol.
18:
21-31,
1998
43.
Schobersberger, W.,
G. Hoffmann,
P. Hobisch-Hagen,
G. Bock,
H. Volkl,
G. Baier-Bitterlich,
B. Wirleitner,
H. Wachter,
and
D. Fuchs.
Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2.
FEBS Lett.
397:
263-268,
1996[Medline].
44.
Slife, C. W.,
E. Wang,
R. Hunter,
S. Wang,
C. Burgess,
D. C. Liotta,
and
A. H. Merrill, Jr.
Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum.
J. Biol. Chem.
266:
14486-14490,
1989
45.
Speiser, D. E.,
E. Sebzda,
T. Ohteki,
M. F. Bachmann,
K. Pfeffer,
T. W. Mak,
and
P. S. Ohashi.
Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo.
Eur. J. Immunol.
26:
3055-3060,
1996[Medline].
46.
Sugarman, B. J.,
P. E. Hass,
and
J. S. Figari.
Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vivo.
Science
236:
943-945,
1985.
47.
Suzuki, A.,
M. Iwasaki,
M. Kato,
and
N. Wagai.
Sequential operation of ceramide synthesis and ICE cascade in CPT-11-initiated apoptotic death signaling.
Exp. Cell Res.
233:
41-47,
1997[Medline].
48.
Tepper, C. G.,
S. Jayadev,
B. Liu,
A. Bielawska,
R. Wolff,
S. Yonehara,
Y. A. Hannun,
and
M. F. Seldin.
Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity.
Proc. Natl. Acad. Sci. USA
92:
8443-8447,
1995[Abstract].
49.
Uhal, B. D.,
I. Joshi,
A. L. True,
S. Mundle,
A. Raza,
A. Pardo,
and
M. Selman.
Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L819-L828,
1995
50.
Vietor, I.,
P. Schwenger,
W. Li,
J. Schlessinger,
and
J. Vilcek.
Tumor necrosis factor-induced activation and increased tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in human fibroblasts.
J. Biol. Chem.
268:
18994-18999,
1993
51.
Watts, J. D.,
M. Gu,
A. J. Polverino,
S. D. Patterson,
and
R. Aebersold.
Fas-induced apoptosis of T cells occurs independently of ceramide generation.
Proc. Natl. Acad. Sci. USA
94:
7292-7296,
1997
52.
Wendt, C. H.,
V. A. Polunovsky,
M. S. Peterson,
P. B. Bitterman,
and
D. H. Ingbar.
Alveolar epithelial cells regulate the induction of endothelial cell apoptosis.
Am. J. Physiol.
267 (Cell Physiol. 36):
C893-C900,
1994
53.
Wertz, P. W.,
and
D. T. Downing.
Ceramidase activity in porcine epidermis.
FEBS Lett.
268:
110-112,
1990[Medline].