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Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated, thioglycollate-elicited peritoneal
macrophages after 48 and 24 h, respectively. Ceramide-induced cell
death was neither accompanied by DNA fragmentation nor phosphatidyl
serine externalization, characteristics of apoptosis. In contrast, LPS
induced a significant fraction of cells to undergo apoptosis, as
demonstrated by DNA fragmentation and quantified by DNA analysis on
FACS, yet the majority of the cells died in a necrotic fashion. C3H/HeJ
Lpsd macrophages were resistant to LPS-induced
cell death and less sensitive to C2 ceramide-evoked cytotoxicity, when
compared with Lpsn macrophages. C2 ceramide plus
IFN- failed to activate release of nitric oxide (NO·),
whereas LPS-induced cell death, but not C2-induced cytotoxicity, was
blocked by an inhibitor of inducible NO· synthase
(iNOS),
NG-monomethyl-L-arginine.
Macrophages from IFN regulatory factor-1 (-/-) mice shown previously
to respond marginally to LPS plus IFN- to express iNOS mRNA and
NO·, were refractory to LPS plus IFN--induced
cytotoxicity and apoptosis. These data suggest that although LPS may
mimic certain ceramide effects, signal transduction events that lead to
cytotoxicity, as well as the downstream mediators,
diverge. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(8), and stress-activated protein kinases (SAPK/JNK) (9) all seem to be
important elements. At much higher concentrations than required for
intracellular signaling, LPS exerts direct toxic effects on cultured
macrophages (10). In fact, direct LPS-induced cytotoxicity was used to
study the inheritance of LPS alleles in murine macrophages (11). Later
studies showed that IFN- (12, 13) or protein synthesis inhibitors
(14) potentiate LPS-induced toxicity in vitro, and the process was
described as apoptotic, based on morphologic and DNA fragmentation
criteria (12, 13, 15). IFN- has also been implicated as essential in
LPS-induced toxicity in vivo (16). A growing body of evidence also
suggests the involvement of inducible NO· synthase (iNOS)
in LPS plus IFN--induced apoptosis (12, 13, 15, 17).
Recent studies suggest that the ceramide signaling pathway is also
involved in LPS effects (18, 19, 20, 21). Ceramide is a lipid second messenger
produced by the action of sphingomyelinase (SMase) and is thought to be
involved in mediating the effects of various cytokines (TNF-
,
IL-1ß, IFN-), the neuronal growth factor, Fas-ligand, and ionizing
radiation (22). There are at least three known targets for ceramide
action: 1) the ceramide-activated protein kinase (21), 2)
ceramide-activated protein phosphatase (23), and 3) PKC-
(24).
Cell-permeable ceramide analogues and pharmacologic elevation of
endogenous ceramide levels are known inducers of cellular stress and
subsequent apoptosis (22, 25), but depending on the target cell type,
necrosis has also been reported (26, 27).
Because LPS shows strong functional and structural resemblance to
ceramide, it has been suggested that endotoxin may stimulate cells
directly by mimicking ceramide, via its interaction with
ceramide-activated protein kinase (CAPK), rather than through the
activation of SMase (28). Supporting this view, LPS can activate CAPK
in partially purified membrane preparations (21), and ceramide can
induce expression of a subset of LPS-inducible genes in murine
macrophages (19), as well as activate c-Raf (29). It is also
known that LPS-hyporesponsive C3H/HeJ mouse macrophages are defective
in both LPS and ceramide responsiveness (18, 30) and uptake of LPS and
ceramide (20). Moreover, using LPS, macrophages can be tolerized
against subsequent stimulation with SMase (19). It has also been
reported that LPS plus IFN--induced cell death in RAW 264.7
macrophages (31), as well as ceramide-mediated apoptosis in ALL-697
leukemia cells (32), are Bcl-2-dependent. NF-B translocation
to the nucleus is thought to be a critical element in the activation of
iNOS, and therefore LPS-induced apoptosis. However, controversy exists
in the literature as to the ability of ceramide analogues or SMase to
trigger the nuclear translocation of NF-B (33, 34, 35, 36, 37).
Although LPS-induced apoptosis has been associated with iNOS
activation, little is known about upstream events. In this study, the
question of whether LPS induces cell death in peritoneal macrophages by
mimicking ceramide was examined. The cytotoxicity of ceramide analogues
in a variety of cell types is well established, but their possible
toxic effects on peritoneal exudate macrophages has not yet been
described. Here we characterize cell death induced by
N-acetylsphingosine (C2 ceramide) in peritoneal macrophages,
showing differences compared with LPS-induced toxicity. In
IFN--treated mouse peritoneal macrophage cultures, LPS was found to
induce both necrosis and apoptosis, whereas C2 ceramide-induced cell
death was predominantly necrotic. We also show that C3H/HeJ macrophages
are resistant to both LPS plus IFN--induced apoptosis and necrosis,
and less sensitive to C2 ceramide plus IFN--induced cell death,
compared with macrophages from fully LPS-responsive C3H/OuJ mice. IFN
regulatory factor-1 (IRF-1) knockout mice, whose macrophages have been
shown previously to be refractory to LPS plus IFN- to release
NO· (38) and whose T cells are refractory to certain
inducers of apoptosis (39), failed to exhibit cytotoxicity or apoptosis
in response to LPS plus IFN-. Finally, we compared the involvement
of iNOS and the role of soluble TNF- in C2 ceramide- and LPS-induced
macrophage cytotoxicity. Taken collectively, the data demonstrate that
LPS-induced cytotoxicity is, in part, apoptotic and
NO·-mediated and distinct from the pathway evoked by
ceramide in murine macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli K235 LPS was prepared using the
method of McIntire et al. (40). Murine rIFN- was kindly provided by
Genentech (South San Francisco, CA). Recombinant soluble TNF receptor
Fc conjugate (TNFR:Fc) was provided by Immunex (Seattle, WA).
Annexin-V-propidium iodide apoptosis detection kit was purchased from
Boehringer Mannheim (Indianapolis, IN). N-acetylsphingosine
(C2 ceramide), dihydro-C2 ceramide, and
DL-threo-1-phenyl-2-palmytoylamino-3-morpholino-1-propanol
(PPMP) were obtained from Biomol Research Laboratories (Plymouth
Meeting, PA).
NG-monomethyl-L-arginine
(L-NMMA) was purchased from Sigma (St. Louis, MO). All
reagents were solubilized according to the manufacturers’ instructions
DMSO for ceramides and PPMP; cell culture medium for
L-NMMA. The endotoxin content of 25 µM C2 ceramide and
PPMP was measured by Limulus amebocyte lysate assay and
found to be less than 0.05 ng/ml in both cases. All other chemicals
were obtained from Sigma and were the highest analytical grade
possible.
Animals
C3H/OuJ (LPS-responsive) and C3H/HeJ (LPS-hyporesponsive) mice (female, 5 to 6 wk old) were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice with a targeted mutation in the IRF-1 gene (IRF-1 (-/-) gene homozygous "knockout" mice and their heterozygotic (+/-) littermates) were originally obtained from Dr. Tak Mak (Amgen Institute, Toronto, Canada) (41). Mice were housed in a virus Ab-free facility. The IRF-1 (-/-) colony was produced by mating IRF-1 (-/-) mice to either IRF-1 (-/-) or IRF-1 (+/-) mice. The IRF-1 wild-type (+/+) colony was maintained by mating IRF-1 (+/+) mice that were derived from heterozygous matings to either IRF-1 (+/+) or IRF-1 (+/-) mice. To prevent the background of the IRF-1 (-/-) and (+/+) colonies from straying, all new breeding pairs were the progeny of IRF-1 (+/-) to IRF-1 (+/-) matings. The genotype of all IRF-1 mice was determined by PCR using the primers and methods described previously (42). The IRF-1 primers amplify a 300-bp sequence from genomic DNA.
Culture of peritoneal macrophages
Mice were injected i.p. with 3 ml of 3% thioglycollate medium (Difco, Detroit, MI). Peritoneal exudate cells were collected by lavage 4 days after injection. Thioglycollate-elicited macrophages were plated in RPMI 1640 medium supplemented with 2% FCS (HyClone, Logan, UT), 2 mM glutamine, 30 mM HEPES, 0.4% sodium bicarbonate, and penicillin-streptomycin (100 IU/ml and 100 µg/ml). After 3 h of adherence, cells were washed two times and then treated with various agents for the indicated time periods, using the serum-free version of the above-mentioned medium at 37°C in 5% CO2 atmosphere. A variety of cell culture dishes was used for different measurements. For cytotoxicity studies (lactic dehydrogenase (LDH) assay and trypan blue exclusion), 24-well plates (5.4 x 105 cells/well, in a volume of 1 ml) (Corning, Corning, NY) and six-well plates (3 x 106 cells/well, in a volume of 1.5 ml) (Corning) were used. For DNA analysis by FACS and DNA fragmentation experiments, cells were cultured in 6-well plates as described above. To study phosphatidyl serine externalization, cells were plated in teflon beakers (Pierce, Rockford, IL; 3 x 106 cells/beaker in a volume of 1.5 ml) and because of the weak adherence of macrophages to Teflon beakers, the washing steps were omitted. Although throughout this study we used serum-free medium for treatments, addition of 2% FCS did not significantly change the kinetics, the type (i.e., apoptotic vs necrotic), or the extent of cell death caused by ceramide or LPS (data not shown).
Evaluation of cell death
Cell death was evaluated qualitatively and quantitatively by phase contrast microscopy of trypan blue-stained cells, and quantified by the measurement of LDH leakage from damaged cells, according to the method of Wroblewski and La Due (43). Cell injury was expressed as a ratio of LDH activity released into the media and the total LDH activity after detergent treatment of cells in the very same wells. For comparison, in some cases, the percent cell death was also assessed by trypan blue exclusion. For this method, at least 200 cells were counted in triplicate wells, and the percentage of trypan blue-positive cells was calculated. Unless otherwise stated, results were expressed as arithmetic means of triplicate samples ± SEM. Wherever statistically significant differences are mentioned in the text, one-way analysis of variance (ANOVA), combined with Tukey’s test was used at the significance level of p < 0.05.
DNA analysis by flow cytometry
For selective and quantitative determination of apoptosis, flow cytometric DNA analysis described by Nicoletti et al. (44) was utilized. Accordingly, the percentage of apoptotic cells whose DNA content is lower than that of diploid cells is calculated. Briefly, cells were harvested by using a rubber policeman, then centrifuged at 400 x g for 10 min. The pellet was gently resuspended in 1 ml hypotonic fluorochrome solution (50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% Triton-X-100) in polypropylene tubes. Tubes were kept at 4°C in the dark, at least overnight, and then the red fluorescence (620 nm) of individual nuclei was measured by using a Coulter EPICS XL-MCL flow cytometer equipped with System II acquisition software (Coulter, Hialeah, FL). The forward scatter and side scatter of particles were simultaneously measured. Cell debris were excluded from analysis by appropriately raising the forward scatter threshold. All measurements were done under the same instrument settings, and at least 10,000 cells were measured in every sample. Because of DNA loss, apoptotic cells are represented by a distinct and quantifiable subdiploid peak in the fluorescence histogram.
Evaluation of apoptosis by measurement of phosphatidyl serine externalization
The externalization of phosphatidyl serine in the cell membrane is one of the earliest events in apoptosis and seems to be a cell type- and inducer-independent process. Using simultaneous annexin-V-FITC and propidium iodide staining, the percentage of cells that underwent apoptosis and necrosis was quantitatively determined. Fluorescein-labeled annexin-V and propidium iodide from Boehringer Mannheim were used for detection of apoptosis according to the manufacturer’s instructions. Because the macrophages adhere so strongly to tissue culture plates, Teflon beakers were used for all of these experiments to minimize induction of cell death by mechanical shearing. In brief, after incubation with various cell death-inducing agents, macrophages were detached by gentle aspiration from the Teflon beaker surface and centrifuged at 400 x g for 10 min. The pellet was then resuspended in 100 µl labeling solution (140 mM NaCl, 10 mM HEPES, 5 mM CaCl2, 1 µg/ml propidium iodide, 1/50 volume of annexin-V-FITC), and then incubated for 10 min in the dark at 4°C. Samples were analyzed on a Coulter EPICS XL-MCL flow cytometer using 488-nm excitation and 525-nm bandpass filters for fluorescein detection and a filter of 620 nm for PI detection, after electronic compensation of the instrument to exclude overlap of the two emission spectra. Propidium iodide positive (necrotic) cells were gated out, and results are shown as annexin-V binding histograms, where at least 10,000 cells were measured in each sample.
Measurement of NO· and TNF bioassay
Secretion of NO· by macrophages was measured by spectrophotometric determination of nitrite, the stable end product of NO· oxidation, as described previously (45). Supernatants (or their appropriate dilutions) from macrophage cultures were mixed with an equal volume of Griess reagent (one part 0.1% N-(1-naphthyl)-ethylenediamine in water and one part 1% sulfanilamide in 5% phosphoric acid), and the absorbance at 570 nm was measured and compared with a NaNO2 (1–100 µM) standard curve.
To measure bioactive TNF-/ß released into the cell culture medium,
a previously described, a standard TNF cytotoxicity assay was used
(46).
DNA isolation and electrophoresis
DNA isolation was performed by the method of Miller et al. (47), with minor modifications. Briefly, macrophages were cultured in 6-well plates and treated for 8 to 48 h, depending on the cell death-inducing agent, then cells were harvested and centrifuged, and pellets were rapidly frozen in liquid nitrogen. After thawing the pellets on ice, cells were resuspended in a buffer containing 200 µg/ml proteinase-K (Boehringer Mannheim), 10 mM Tris-HCl (pH 8), 10 mM EDTA, and 0.5% SDS and incubated for 3 h at 55°C. Chromosomal DNA and proteins were precipitated overnight at 4°C in the presence of 1 M NaCl. After centrifugation (30 min at 5000 x g), supernatants were treated with 25 µg/ml RNase (Sigma) for 1 h, then DNA fragments were precipitated overnight by two volumes of absolute ethanol at -20°C. Samples were centrifuged for 30 min at 10,000 x g, DNA pellets were air-dried and then resuspended in 10 mM Tris-HCl (pH 8), and 1 mM EDTA, and were subjected to horizontal gel electrophoresis (agarose, 2%). Images of ethidium bromide-stained gels were captured by the Eagle Eye gel documentation system (Stratagene, La Jolla, CA) and were processed by subtracting the background, defined by medium-treated samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well documented that LPS can exert direct toxic effects on
peritoneal macrophages, macrophage cell lines, endothelial cells, and
many other cell types. Although early studies utilized very high LPS
concentrations (20–50 µg/ml) to demonstrate direct macrophage
cytotoxicity (10, 48, 49), simultaneous stimulation with IFN- or
cycloheximide has been showed to potentiate the toxicity of much lower
LPS concentrations (<1 µg/ml) (12, 13). In an initial series of
studies (Fig. 1
) in which cell death was
assessed by release of LDH into the culture supernatants in 24-well
plates, we found that 1 µg/ml LPS alone was not significantly
cytototoxic for LPS-responsive, C3H/OuJ macrophages, even after 48
h of incubation. The presence of IFN- potentiated the cell death, as
previously reported (12, 13). No measurable cell death was observed
with LPS alone or LPS plus IFN- at 24 h (data not shown). The
cell-permeable ceramide analogue N-acetylsphingosine
(C2-ceramide) at a dose of 25 µM, a dose shown in previous studies to
induce gene expression in these macrophages (19), was found to be
directly cytotoxic by 24 h of exposure, and like LPS,
ceramide-induced cell death was potentiated by the presence of IFN-.
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–4. Using both trypan blue exclusion
and LDH as measurements of macrophage cell death, Figure 2 illustrates
that both LPS- (Fig. 2A) and C2 ceramide- (Fig. 2B) induced cytotoxicity are dose-dependent in the presence
of IFN-. Even when very high concentrations of LPS (25–50 µg/ml)
plus IFN- were used, cell death rarely exceeded 50% (as measured in
the LDH assay), whereas 1 µg/ml LPS plus IFN- evoked suboptimal,
but statistically significant, cell death. IFN- alone failed to
induce measurable cytotoxicity, even after 72 h of incubation
(data not shown). LPS-hyporesponsive C3H/HeJ macrophages were highly
refractory to LPS plus IFN--induced cytotoxicity (Fig. 2A). Under identical conditions, 25 and 50 µM C2 ceramide
were also toxic, causing 
40% and 60% cell death, respectively,
after only 24 h of incubation in the presence of IFN- (Fig. 2B). The inactive ceramide analogue, dihydro-C2 ceramide,
plus IFN- did not affect the viability of macrophages, even after
48 h (data not shown). Elevation of intracellular ceramide levels
by inhibition of glucosylceramide synthase with 25 to 50 µM PPMP
(50), which inhibits the further incorporation of ceramide into
glycolipids, also induced cell death in macrophages after 24 h of
treatment, and similarly to C2 ceramide-induced cell death, this was
potentiated by the presence of IFN- (Table I). LPS-hyporesponsive C3H/HeJ
macrophages were highly refractory to very high concentrations of LPS
(i.e., 50 µg/ml) plus IFN--induced cell death (Fig. 2A), and they were also less sensitive to C2 ceramide (25
µM) plus IFN--induced cell death (Fig. 2B), although at
50 µM, they were equivalently sensitive to C3H/OuJ macrophages.
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-induced cell death was highly dependent on the volume
of medium used in cell culture wells. The data presented in Figures 1
and 2 were measured in 24-well plates, with 5.4 x 105
cells in 1 ml of medium per well. Under conditions where the cells were
cultured in 6-well plates in a final volume of 1.5 ml medium (i.e.,
3 x 106 cells/well; the same cell density as in the
24-well plates, but in a relatively smaller volume of medium), the
sensitivity of the macrophages to LPS plus IFN- was greatly
increased. Figure 3A
illustrates that under these culture conditions, as little as 0.1 ng/ml
LPS induced macrophage cytotoxicity in the presence of IFN-. Again,
LPS-induced cytotoxicity was not evident at 24 h of incubation,
whether or not IFN- was present. Under these conditions of increased
LPS sensitivity, the macrophages remained comparably sensitive to C2
ceramide (data not shown). Figure 3B demonstrates that the
pattern of cytotoxicity induced by LPS plus IFN- was paralleled by
the release of NO· into culture supernatants.
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A comparison of LPS- and C2 ceramide-induced cytotoxicity
and NO· production was next studied. As shown in Figure 3, Figure 4A again illustrates
that LPS plus IFN- induced both cell death and NO·
release in parallel in C3H/OuJ macrophages, whereas C3H/HeJ macrophages
were highly refractory to LPS-induced cell death and produced minimal
levels of NO·. In C3H/OuJ macrophages,
L-NMMA, an inhibitor of iNOS, blocked both cell death and
NO· release in response to LPS and IFN-. In contrast,
25 µM C2 ceramide plus IFN- did not induce NO·
release into the culture media (Fig. 4B), nor did
L-NMMA protect C3H/OuJ macrophages from C2 ceramide plus
IFN--induced cell death under conditions where it protected against
LPS plus IFN--induced cytotoxicity. Moreover, PPMP, an agent that
elevates endogenous ceramide levels in cells, and also is toxic to
macrophages, induced no significant nitrite release, even in the
presence of IFN- (Table I). These findings suggest that
NO· is not involved in ceramide-induced
cytotoxicity.
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, induced by LPS treatment, plays a key role in
development of sepsis syndrome in vivo, and because TNF- is a
well-known inducer of apoptosis, we investigated a potential role for
TNF- in LPS plus IFN-- and C2 ceramide plus IFN--induced cell
death in vitro. Table II
illustrates that LPS plus IFN- induced a large accumulation of
bioactive TNF. Although, inclusion of 1 x 10-5 M
soluble TNF-receptor (TNFR:Fc) eliminated the bioactive TNF- induced
by LPS plus IFN-, it was not protective against the cell death
induced under the same conditions. A total of 25 µM C2 ceramide plus
IFN- failed to induce the release of bioactive TNF-, as measured
by TNF bioassay. TNFR:Fc conjugate did not protect the cells against C2
ceramide plus IFN--induced cell death (Table II).
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causes necrosis and apoptosis, whereas C2 ceramide
plus IFN- induces necrosis only
To study whether the C2 ceramide plus IFN-- and LPS plus
IFN--induced cell death were the result of apoptosis or necrosis,
DNA fragmentation studies, as well as quantitative flow cytometric
approaches, were employed. After 48 h of treatment, 1 µg/ml LPS
plus IFN- caused detectable apoptotic DNA fragmentation in
peritoneal macrophage cultures (Fig. 5).
No DNA laddering was observed after 24 h of treatment. Blocking
iNOS activity with L-NMMA eliminated the apoptotic DNA
laddering caused by LPS plus IFN- (Fig. 5). The positive control, 3
µM gliotoxin (13, 51), resulted in much stronger DNA fragmentation,
even after 8 h, compared with that induced by LPS plus IFN-. In
contrast, 25 µM C2 ceramide plus IFN- did not induce any
measurable DNA fragmentation even after 24 h (Fig. 5), the time
point when 40 to 60% of the macrophages were dead (Figs. 1 and 2).
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, the
percentage of subdiploid nuclei, characteristic for apoptotic cell
populations, was measured by flow cytometry. After 48 h of
incubation with 1 µg/ml LPS plus IFN-, which resulted 70%
macrophage death in 6-well plates (Fig. 4A), 20% of the
cells exhibited subdiploid nuclei, in contrast to the positive control,
3 µM gliotoxin, which induced >90% apoptosis within 24 h (see
Fig. 6A for a typical
experiment, and Table III for statistical
analysis). Without IFN-, LPS did not increase the percentage of
apoptotic cells, compared with medium-treated controls (Fig. 6A). Again, consistent with the DNA fragmentation
experiments (Fig. 5), L-NMMA blocked the appearance of
cells with subdiploid nuclei in LPS plus IFN--treated samples.
Macrophages from Lpsd mice proved to be resistant to
apoptotic insult by LPS plus IFN- (Fig. 6A).
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B) or killing the macrophages by hypotonic lysis with
distilled water (data not shown) did not produce a hypodiploid peak in
the histogram, showing that this method distinguishes clearly between
apoptosis and necrosis. In agreement with the DNA fragmentation studies
(Fig. 5), 25 µM C2 ceramide plus IFN- failed to elicit any
apoptosis after 24 h of treatment, as measured by flow cytometric
DNA analysis (Fig. 6B, Table III). No increase in the
percentage of subdiploid nuclei was measurable even at a C2 ceramide
concentration of 50 µM, in the presence of IFN- (Fig. 6B).
One of the earliest events in apoptosis is the externalization of
phosphatidyl serine in the plasma membrane as a consequence of
apoptotic injury. Besides DNA fragmentation, this process is thought to
be one of the hallmarks of apoptosis in a wide variety of experimental
systems and, in fact, represents an independent event from nuclear
changes. Because DNA fragmentation and analysis of subdiploid nuclei
failed to suggest evidence for C2 ceramide plus IFN--induced
apoptosis, we also measured the binding of annexin-V to macrophages,
concurrent with propidium iodide staining, in an attempt to detect
possible phosphatidyl serine externalization. A relatively low dose of
gliotoxin (0.5 µM) elicited apoptosis by 24 h, as measured by an
increase in annexin-V binding to macrophages from 11% to 75%. After
4 h of treatment with 25 µM C2 ceramide plus IFN-, no
increase was observed in the percentage of annexin-V binding cells
(12% of C2 ceramide plus IFN- vs 11% of medium-treated control).
Even after 24 h of incubation with C2 ceramide plus IFN-, the
proportion of annexin-V positive cells was only 13% vs 11% of
medium-treated controls, indicating that the cell death caused by C2
ceramide plus IFN- is not apoptotic. This is illustrated in Figure 7, A (medium- and
gliotoxin-treated cells) and B (medium and C2 ceramide plus
IFN--treated cells), where the respective annexin-V-binding
histograms are shown in an overlayed fashion.
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-induced cytotoxicity and apoptosis are
IRF-1-dependent
Previous studies have indicated that mice with a targeted mutation
in the gene that encodes the IRF-1 possess macrophages that respond
poorly to LPS plus IFN- to induce iNOS gene expression and
NO· release (38). Moreover, the T cells of these mice
have also been reported to be refractory to apoptosis induced by DNA
damage (e.g., -irradiation, etoposide, adriamycin) (39). Therefore,
we sought to evaluate the responsiveness of macrophages derived from
IRF-1 (-/-) mice and IRF-1 (+/+) controls to LPS plus IFN--induced
cell death and apoptosis. Figure 8,
A and B illustrate that LPS plus IFN- induces
both cell death and NO· release in parallel in IRF-1
(+/+) macrophages within 48 h, whereas IRF-1 (-/-) macrophages
were highly resistant to LPS-induced cell death and produced low levels
of NO·. In IRF-1 (+/+) macrophages, L-NMMA
blocked both cell death and NO· release in response to
LPS and IFN-. Also, as was observed in C3H/OuJ macrophage cultures
(Fig. 6A), a significant portion of IRF-1 (+/+) macrophages
underwent apoptosis (30%, Fig. 8C) in response to LPS
plus IFN-, although necrotic cell death remained dominant. In
contrast, IRF-1 (-/-) peritoneal macrophages did not show nuclear DNA
loss as measured by the appearance of subdiploid nuclei by FACS (Fig. 8C). These data demonstrate that both the LPS plus
IFN--induced cell death and apoptosis are IRF-1-dependent in
peritoneal macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Involvement of ceramide pathway in signal transduction of certain LPS
effects has recently been suggested (21). It has also been reported
that in vivo, LPS-induced toxicity is mediated by TNF release and
subsequent ceramide generation in endothelial cells (52). However, the
possible role of the ceramide pathway in LPS-induced cell death in
macrophages remained to be elucidated. To address this question, the in
vitro toxicity of LPS and C2 ceramide in peritoneal exudate macrophage
cultures was compared in the presence of IFN-. In vivo, IFN- has
a very important role in LPS-induced toxicity. In fact, injection of
anti-IFN- Abs has protective effect of LPS-induced thymocyte
cell death in vivo, showing that LPS and IFN- may act in concert to
induce toxicity (16). In our studies, LPS- and ceramide-induced
cytotoxicity was quantified by two different methods (trypan blue
exclusion and LDH release), and these assays gave comparable results
(Fig. 2). High concentrations of both LPS (25–50 µg/ml) and C2
(25–50 µM) ceramide were directly toxic for peritoneal exudate
macrophages derived from C3H/OuJ mice. The presence of IFN-
potentiated this effect, but more strikingly for LPS than for C2
ceramide. Under conditions of high cell number and low supernatant
volume (e.g., 6-well plates), concentrations of LPS as low as 1 ng/ml
could be demonstrated to be highly toxic in the presence of IFN-
(Fig. 3A). This suggests that a factor(s) that accumulate(s)
within the supernatant contribute(s) to LPS-induced cell death.
Previous studies have demonstrated that LPS plus IFN--induced cell
death is NO·-mediated (12, 13, 15, 17). Indeed, this was
confirmed in these studies by the demonstration that inactive arginine
analogue, L-NMMA, blocked LPS plus IFN--induced
cytotoxicity with a concurrent inhibition of NO· release
into the culture supernatant. Thus, the enhancement of LPS-induced cell
death at low LPS concentrations by the presence of IFN- is likely to
be related to the well-characterized synergy between these two
compounds for the induction of iNOS gene expression (45). In contrast,
the cytotoxic effects of C2 ceramide or elevation of endogenous
ceramide levels by PPMP (in the absence or presence of IFN-) appear
to be independent of NO· release: C2 ceramide and PPMP
(±IFN-) failed to stimulate release of NO· in C3H/OuJ
peritoneal exudate macrophages, and L-NMMA failed to
protect the cells from C2 ceramide-induced cytotoxicity. These
observations, coupled with the failure of the inactive dihydro-ceramide
analogue to trigger cytotoxicity, further support the notion that
macrophage cytotoxicity induced through the activation of the ceramide
pathway is mechanistically distinct from that induced by LPS.
In addition to the lack of evidence for participation of
NO· in ceramide-induced cytotoxicity, other important
differences in kinetics, the type of cell death, and the relative
sensitivity of Lpsd macrophages to LPS- vs C2
ceramide-induced cytotoxicity were observed. Although C2 ceramide plus
IFN- induced massive cell death after 24 h of incubation, even
high concentrations of LPS (>10 µg/ml) plus IFN- did not induce
cell death at the same time point. For LPS plus IFN--induced
toxicity, at least 48 h of incubation was necessary, even with
increased cell number/media volume ratio, which remarkably increased
the extent of cell death (70% compared with 30%), but without
an alteration in kinetics. This finding suggests important differences
in the signal transduction and/or the effector mechanisms of cell death
for these two compounds.
It is well known that macrophages from Lpsd C3H/HeJ
mice are hyporesponsive to LPS effects in vivo and in vitro (2). In the
interim, it has been demonstrated that not only are macrophages derived
from C3H/HeJ mice hyporesponsive to LPS, but also, they fail to respond
to soluble ceramide analogues and exogenously added SMase to induce
expression of a subset of LPS-inducible genes or to secrete cytokines
in response to SMase stimulation. Interestingly, C3H/HeJ macrophages
were profoundly refractory to LPS-induced cytotoxicity, even in the
presence of IFN-, whereas longer (>24 h) incubation or high
concentrations of C2 ceramide (50 µM) plus IFN- induced
cytotoxicity comparable to that observed in the normoresponsive
macrophages. Thus, in C3H/HeJ macrophages, the longer incubation time
or high concentrations of C2 ceramide can apparently circumvent the
mutation that protects these cells from cytotoxicity induced by high
concentrations of LPS, in the absence or presence of IFN-. It is
interesting to note that similar observation was made by
Thiéblemont and Wright (20) with respect to defective ceramide
uptake in C3H/HeJ macrophages; i.e., at longer incubation times, the
initial defect in ceramide uptake became normalized to that observed in
C3H/OuJ macrophages.
In sepsis, or in in vivo models of endotoxemia, varieties of cell types
such as thymocytes, B cells, and endothelial cells have been reported
to undergo apoptosis in response to bacterial LPS. Whether a cell death
process is apoptotic or necrotic has particular importance in vivo,
because in apoptosis, the cytoplasm of dying cells is preserved in
"membrane-packed" apoptotic bodies; therefore, the content of the
cell is not released into the extracellular space. In contrast,
cytoplasmic content of necrotic cells can act as an additional
inflammatory signal to enhance the uncontrolled inflammation that is
characteristic of septic shock. To study further the cell death induced
by LPS and C2 ceramide, we examined whether these compounds induced
apoptosis or necrosis in peritoneal macrophages in vitro. It has been
reported that LPS plus IFN- triggers apoptosis in peritoneal
macrophages (12, 13) and macrophage-like cells (15) through an
iNOS-dependent pathway. In agreement with these publications, LPS plus
IFN- induced detectable DNA fragmentation in our macrophage cultures
(Fig. 5). To quantify the extent of apoptosis, we used DNA analysis by
FACS. In contrast to previous reports (12, 13, 17), our data show that
LPS plus IFN--induced cell death is predominantly necrotic, although
a significant fraction of cells underwent apoptosis. A possible
explanation for this contradiction is that in previous reports
mentioned above, apoptotic cell death was demonstrated by morphologic
evidence and DNA fragmentation with no precise quantification of
apoptosis. Supportive of our findings, Albina et al. (13) noted that
only 10 to 15% of the cells showed apoptotic morphology at 48 h
of incubation in their macrophage cultures treated with LPS plus
IFN-. As an explanation, they hypothesized that although the process
as a whole is apoptotic, this may be a result of efficient removal of
apoptotic cells by neighboring macrophages or progression of the
apoptosis into a phase of secondary necrosis. Our results demonstrated
that only 20 to 30% of cells were apoptotic, even when 70 to 80%
cell death was induced by LPS plus IFN- at 48 h. The necrotic
cells still had intact nuclei as measured by DNA analysis using FACS,
so the cell death described here cannot be attributed to secondary
necrosis. Moreover, in the case of gliotoxin-treated macrophages, which
are clearly apoptotic (according to DNA fragmentation, appearance of
subdiploid nuclei, and increased annexin-V binding), the phagocytosis
of apoptotic macrophages by neighboring cells did not interfere with
the detection of apoptosis. The appearance of necrosis and apoptosis at
the same time in response to cell death inducers has been reported in
other cell types in vitro and in vivo (55, 56, 57). Similarly, in
peritoneal macrophage cultures, different subpopulations may exist that
respond differently to cell death inducers. Interestingly, both the
apoptotic and necrotic cell death of macrophages were antagonized by
the iNOS inhibitor, L-NMMA. Although
NO·-mediated cell death is generally considered as to be
apoptotic (13, 15, 58), NO·-induced necrosis has also
been reported in oligodendrocytes (59) and in an epithelial cell line
(60). As a possible mechanism for NO·-induced apoptosis,
direct DNA damage (61) and as a consequence, induction of the tumor
suppressor gene, p53, were suggested (15). Although p53 activation has
been demonstrated in response to LPS plus IFN- treatment in RAW
264.7 cells, a yet unidentified p53-independent pathway is also likely
to play a role in this process (15). It has also been reported that p53
activates IL-1ß-converting enzyme-like proteases, specifically
Caspase-3/CPP32 (32-kDa cysteine protease), which is an important
mediator of apoptosis (61). As a mechanism for
NO·-mediated necrosis, damage caused by free radicals,
ATP depletion, and mitochondrial membrane injury have been proposed
(59).
In contrast to LPS plus IFN--induced cell death, C2 ceramide plus
IFN- caused purely necrotic cell death in peritoneal macrophages as
measured by DNA fragmentation, DNA analysis by FACS, and annexin-V
binding. This was very surprising, given the number of reports that
demonstrate that ceramide is a potent inducer of apoptosis (22, 25). A
growing body of evidence has implicated possible mediators and
regulatory molecules that contribute to ceramide-induced apoptosis.
Among others, ceramide-activated proteases (e.g., IL-1ß-converting
enzyme-like family proteases, especially Caspase-3/CPP32) (62),
SAPK/JNK (63), and cytosolic translocation of certain PKC isoforms (64)
appear to be important in mediating ceramide-induced apoptosis.
Moreover, Bcl-2 has regulatory role in this process (32). Much less is
known about the mechanisms of ceramide-induced necrosis. Recently,
Arora et al (26) showed that ceramide analogues cause mitochondrial
membrane permeability transition (MMPT), ATP depletion, and subsequent
necrotic cell death. MMPT has been proposed as a final common pathway
of cell death.
Among other factors, TNF- is believed to play fundamental role in
septic shock induced by LPS (2). Under in vivo conditions, LPS-induced
TNF- secretion and subsequent TNF--induced apoptosis is one of
the most important factors in LPS toxicity (52, 65, 66). To examine the
role of TNF- in LPS- and C2 ceramide-induced cell death in
macrophages in vitro, we used a soluble TNF receptor (TNFR:Fc). A total
of 1 x 10-5 M TNFR:Fc neutralized most of the
bioactive TNF- induced by LPS plus IFN- (Table II). However,
TNFR:Fc did not protect peritoneal macrophages against LPS plus
IFN--induced cell death, suggesting that TNF- is not involved in
LPS cytotoxicity in vitro. Similar results were published by Yamamoto
et al. (17) and Amano et al. (14). In contrast to SMase stimulation of
macrophages (19), C2 ceramide failed to induce bioactive TNF- in
peritoneal macrophage cultures, and TNFR:Fc was not protective against
C2 ceramide plus IFN--induced cell death (Table II), suggesting that
TNF- does not mediate this toxicity.
Since IRF-1 was shown previously to be important in certain types of
apoptosis (39) and its role in induction of iNOS is well documented
(38), we next investigated the participation of IRF-1 in LPS plus
IFN--induced cell death and apoptosis. Our experiments with IRF-1
(-/-) mice show that not only the LPS plus IFN--induced apoptosis
(30% of the cells), but cell death in general, are IRF-1-dependent
(Fig. 8, A and C). Because a functional IRF-1
gene is essential for activation of iNOS (Fig. 8B), these
data further support the hypothesis that LPS plus IFN--induced cell
death is NO·-mediated. It is also interesting to note
that the IRF-1- and p53-dependent apoptotic pathways are considered to
be distinct in T lymphocytes (39). P53-dependent pathways in LPS plus
IFN--induced apoptosis were described in RAW 264.7 macrophages (58),
and here we show that this process in peritoneal macrophages is
IRF-1-dependent. This suggests that distinct pathways can act
simultaneously to induce apoptosis, depending on the particular cell
type and apoptosis inducer.
Taken collectively, in this study we characterized cell death induced
by C2 ceramide in peritoneal macrophages, showing the commonalties, as
well as differences, with LPS-induced macrophage cytotoxicity. In the
presence of IFN-, LPS-induced cell death in peritoneal macrophages
is mostly due to necrosis, with a significant fraction of cells
undergoing apoptosis. Although differing in extent, both C2 ceramide-
and LPS-mediated cell death depend on the presence of a normal
Lps gene, suggesting a possible common early mediator in the
signaling of cell death. In contrast, we demonstrated that LPS-induced,
not C2 ceramide-triggered cell death, is mediated by NO·
in peritoneal macrophages in vitro. Soluble TNF- was also ruled out
as a possible mediator of ceramide- or LPS-induced toxicity. In
summary, our study suggests that although LPS may mimic certain
ceramide effects, signal transduction events that lead to cytotoxicity,
as well as the downstream mediators, diverge.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; NF-B, nuclear factor B; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; NO·, nitric oxide; iNOS, inducible nitric oxide synthase; SMase, sphingolmyelinase; TNFR:Fc, soluble TNF receptor-Fc conjugate; PPMP, DL-threo-1-phenyl-2-palmytoylamino-3-morpholino-1-propanol; L-NMMA, NG-monomethyl-L-arginine; IRF-1, IFN regulatory factor-1; LDH, lactic dehydrogenase; CPP32, 32-kDa cysteine protease; ANOVA, analysis of variance.
Received for publication February 4, 1998. Accepted for publication April 27, 1998.
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-phage DNA, was used (lane A). Similar data were
obtained in another separate experiment.
