Death in Murine Macrophages Divergent Signaling Pathways to Induce Cell Lipopolysaccharide and Ceramide

Lipopolysaccharide and Ceramide Use Divergent Signaling Pathways to Induce Cell Death in Murine Macrophages

This information is current as Viktor Lakics and Stefanie N. Vogel of September 28, 2021. J Immunol 1998; 161:2490-2500; ; http://www.jimmunol.org/content/161/5/2490 Downloaded from References This article cites 62 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/161/5/2490.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lipopolysaccharide and Ceramide Use Divergent Signaling Pathways to Induce Cell Death in Murine Macrophages1

Viktor Lakics and Stefanie N. Vogel2

Ceramide is a well-known apoptotic agent that has been implicated in LPS signaling. Therefore, we examined whether LPS- induced macrophage cytotoxicity is mediated by mimicking ceramide. Both LPS and the cell-permeable ceramide analogue, C2 ceramide, induced significant cell death in IFN-␥-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 n ⅐ with Lps macrophages. C2 ceramide plus IFN-␥ failed to activate release of nitric oxide (NO ), whereas LPS-induced cell death, Downloaded from ⅐ G but not C2-induced cytotoxicity, was blocked by an inhibitor of inducible NO synthase (iNOS), N -monomethyl-L-arginine. Macrophages from IFN regulatory factor-1 (؊/؊) mice shown previously to respond marginally to LPS plus IFN-␥ to express iNOS mRNA and NO⅐, 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. The Journal of Immunology, 1998, 161: 2490–2500. http://www.jimmunol.org/ ram negative bacterial LPS is strongly immunostimula- cytotoxicity was used to study the inheritance of LPS alleles in tory and can elicit a shock syndrome that is character- murine macrophages (11). Later studies showed that IFN-␥ (12, G ized by a drastic fall in blood pressure, fever, hypogly- 13) or protein synthesis inhibitors (14) potentiate LPS-induced cemia, diarrhea, and vasoconstriction, which often leads to death toxicity in vitro, and the process was described as apoptotic, based (1). At the cellular level, LPS primarily stimulates macrophages on morphologic and DNA fragmentation criteria (12, 13, 15). and other cell types to induce expression of an impressive number IFN-␥ has also been implicated as essential in LPS-induced tox- of genes and to release inflammatory agents such as cytokines (2). icity in vivo (16). A growing body of evidence also suggests the The intracellular pathways that mediate LPS-induced effects are involvement of inducible NO⅐ synthase (iNOS) in LPS plus IFN- not yet completely understood, but the involvement of certain src- ␥-induced apoptosis (12, 13, 15, 17). by guest on September 28, 2021 related kinases (3), protein kinase C (PKC)3 (4), mitogen-activated Recent studies suggest that the ceramide signaling pathway is protein kinases (p38, p42, p44 MAPK) (5, 6), G proteins (7), also involved in LPS effects (18–21). Ceramide is a lipid second NF-␬B (8), and stress-activated protein kinases (SAPK/JNK) (9) messenger produced by the action of sphingomyelinase (SMase) all seem to be important elements. At much higher concentrations and is thought to be involved in mediating the effects of various than required for intracellular signaling, LPS exerts direct toxic cytokines (TNF-␣, IL-1␤, IFN-␥), the neuronal growth factor, Fas- effects on cultured macrophages (10). In fact, direct LPS-induced 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) Department of Microbiology and Immunology, Uniformed Services University of the PKC-␨ (24). Cell-permeable ceramide analogues and pharmaco- Health Sciences, Bethesda, MD 20814 logic elevation of endogenous ceramide levels are known inducers Received for publication February 4, 1998. Accepted for publication April 27, 1998. of cellular stress and subsequent apoptosis (22, 25), but depending The costs of publication of this article were defrayed in part by the payment of page on the target cell type, necrosis has also been reported (26, 27). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Because LPS shows strong functional and structural resem- 1 This work was supported by National Institutes of Health Grant AI-18797 (S.N.V.). blance to ceramide, it has been suggested that endotoxin may stim- The opinions or assertions contained within are the private views of the authors and ulate cells directly by mimicking ceramide, via its interaction with should not be construed as official or necessarily reflecting the views of the Uniformed ceramide-activated protein kinase (CAPK), rather than through the Services University of the Health Sciences or the Department of Defense. Research was conducted according to the principles set forth in “Guide for the Care and Use of activation of SMase (28). Supporting this view, LPS can activate Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources, CAPK in partially purified membrane preparations (21), and cer- National Research Council DHEW Publication Number 85-23 (National Institutes of Health, Bethesda, MD). amide can induce expression of a subset of LPS-inducible genes in murine macrophages (19), as well as activate c-Raf (29). It is also 2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Depart- ment of Microbiology and Immunology, Uniformed Services University of the known that LPS-hyporesponsive C3H/HeJ mouse macrophages Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. E-mail address: are defective in both LPS and ceramide responsiveness (18, 30) [email protected] and uptake of LPS and ceramide (20). Moreover, using LPS, mac- 3 Abbreviations used in this paper: PKC, protein kinase C; NF-␬B, nuclear factor ␬B; ⅐ rophages can be tolerized against subsequent stimulation with SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; NO , nitric oxide; iNOS, inducible nitric oxide synthase; SMase, sphingolmyelinase; TNFR:Fc, SMase (19). It has also been reported that LPS plus IFN-␥-induced soluble TNF receptor-Fc conjugate; PPMP, DL-threo-1-phenyl-2-palmytoylamino-3- cell death in RAW 264.7 macrophages (31), as well as ceramide- G morpholino-1-propanol; L-NMMA, N -monomethyl-L-arginine; IRF-1, IFN regulatory factor-1; LDH, lactic dehydrogenase; CPP32, 32-kDa cysteine protease; mediated apoptosis in ALL-697 leukemia cells (32), are Bcl-2- ANOVA, analysis of variance. dependent. NF-␬B translocation to the nucleus is thought to be a

critical element in the activation of iNOS, and therefore LPS-in- injection. Thioglycollate-elicited macrophages were plated in RPMI 1640 duced apoptosis. However, controversy exists in the literature as to medium supplemented with 2% FCS (HyClone, Logan, UT), 2 mM glu- tamine, 30 mM HEPES, 0.4% sodium bicarbonate, and penicillin-strepto- the ability of ceramide analogues or SMase to trigger the nuclear ␮ ␬ mycin (100 IU/ml and 100 g/ml). After3hofadherence, cells were translocation of NF- B (33–37). washed two times and then treated with various agents for the indicated Although LPS-induced apoptosis has been associated with time periods, using the serum-free version of the above-mentioned medium iNOS activation, little is known about upstream events. In this at 37°C in 5% CO2 atmosphere. A variety of cell culture dishes was used study, the question of whether LPS induces cell death in peritoneal for different measurements. For cytotoxicity studies (lactic dehydrogenase (LDH) assay and trypan blue exclusion), 24-well plates (5.4 ϫ 105 cells/ macrophages by mimicking ceramide was examined. The cytotox- well, in a volume of 1 ml) (Corning, Corning, NY) and six-well plates (3 ϫ icity of ceramide analogues in a variety of cell types is well es- 106 cells/well, in a volume of 1.5 ml) (Corning) were used. For DNA tablished, but their possible toxic effects on peritoneal exudate analysis by FACS and DNA fragmentation experiments, cells were cul- macrophages has not yet been described. Here we characterize cell tured in 6-well plates as described above. To study phosphatidyl serine death induced by N-acetylsphingosine (C2 ceramide) in peritoneal externalization, cells were plated in teflon beakers (Pierce, Rockford, IL; 3 ϫ 106 cells/beaker in a volume of 1.5 ml) and because of the weak macrophages, showing differences compared with LPS-induced adherence of macrophages to Teflon beakers, the washing steps were omit- toxicity. In IFN-␥-treated mouse peritoneal macrophage cultures, ted. Although throughout this study we used serum-free medium for treat- LPS was found to induce both necrosis and apoptosis, whereas C2 ments, addition of 2% FCS did not significantly change the kinetics, the ceramide-induced cell death was predominantly necrotic. We also type (i.e., apoptotic vs necrotic), or the extent of cell death caused by ceramide or LPS (data not shown). show that C3H/HeJ macrophages are resistant to both LPS plus IFN-␥-induced apoptosis and necrosis, and less sensitive to C2 Evaluation of cell death ceramide plus IFN-␥-induced cell death, compared with macro- Downloaded from Cell death was evaluated qualitatively and quantitatively by phase contrast phages from fully LPS-responsive C3H/OuJ mice. IFN regulatory microscopy of trypan blue-stained cells, and quantified by the measurement factor-1 (IRF-1) knockout mice, whose macrophages have been of LDH leakage from damaged cells, according to the method of Wrob- shown previously to be refractory to LPS plus IFN-␥ to release lewski and La Due (43). Cell injury was expressed as a ratio of LDH NO⅐ (38) and whose T cells are refractory to certain inducers of activity released into the media and the total LDH activity after detergent apoptosis (39), failed to exhibit cytotoxicity or apoptosis in re- 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

␥ http://www.jimmunol.org/ sponse to LPS plus IFN- . Finally, we compared the involvement method, at least 200 cells were counted in triplicate wells, and the per- of iNOS and the role of soluble TNF-␣ in C2 ceramide- and LPS- centage of trypan blue-positive cells was calculated. Unless otherwise induced macrophage cytotoxicity. Taken collectively, the data stated, results were expressed as arithmetic means of triplicate samples Ϯ demonstrate that LPS-induced cytotoxicity is, in part, apoptotic SEM. Wherever statistically significant differences are mentioned in the ⅐ text, one-way analysis of variance (ANOVA), combined with Tukey’s test and NO -mediated and distinct from the pathway evoked by cer- was used at the significance level of p Ͻ 0.05. amide in murine macrophages. DNA analysis by flow cytometry Materials and Methods For selective and quantitative determination of apoptosis, flow cytometric Reagents DNA analysis described by Nicoletti et al. (44) was utilized. Accordingly, the percentage of apoptotic cells whose DNA content is lower than that of by guest on September 28, 2021 Escherichia coli K235 LPS was prepared using the method of McIntire et diploid cells is calculated. Briefly, cells were harvested by using a rubber ␥ al. (40). Murine rIFN- was kindly provided by Genentech (South San policeman, then centrifuged at 400 ϫ g for 10 min. The pellet was gently Francisco, CA). Recombinant soluble TNF receptor Fc conjugate (TNFR: resuspended in 1 ml hypotonic fluorochrome solution (50 ␮g/ml propidium Fc) was provided by Immunex (Seattle, WA). Annexin-V-propidium io- iodide, 0.1% sodium citrate, 0.1% Triton-X-100) in polypropylene tubes. dide apoptosis detection kit was purchased from Boehringer Mannheim Tubes were kept at 4°C in the dark, at least overnight, and then the red (Indianapolis, IN). N-acetylsphingosine (C2 ceramide), dihydro-C2 cer- fluorescence (620 nm) of individual nuclei was measured by using a amide, and DL-threo-1-phenyl-2-palmytoylamino-3-morpholino-1-propa- Coulter EPICS XL-MCL flow cytometer equipped with System II acqui- nol (PPMP) were obtained from Biomol Research Laboratories (Plymouth G sition software (Coulter, Hialeah, FL). The forward scatter and side scatter Meeting, PA). N -monomethyl-L-arginine (L-NMMA) was purchased from of particles were simultaneously measured. Cell debris were excluded from Sigma (St. Louis, MO). All reagents were solubilized according to the analysis by appropriately raising the forward scatter threshold. All mea- manufacturers’ instructions DMSO for ceramides and PPMP; cell culture surements were done under the same instrument settings, and at least ␮ medium for L-NMMA. The endotoxin content of 25 M C2 ceramide and 10,000 cells were measured in every sample. Because of DNA loss, apo- PPMP was measured by Limulus amebocyte lysate assay and found to be ptotic cells are represented by a distinct and quantifiable subdiploid peak in less than 0.05 ng/ml in both cases. All other chemicals were obtained from the fluorescence histogram. Sigma and were the highest analytical grade possible. Evaluation of apoptosis by measurement of phosphatidyl serine Animals externalization C3H/OuJ (LPS-responsive) and C3H/HeJ (LPS-hyporesponsive) mice (fe- male, 5 to 6 wk old) were purchased from The Jackson Laboratories (Bar The externalization of phosphatidyl serine in the cell membrane is one of Harbor, ME). Mice with a targeted mutation in the IRF-1 gene (IRF-1 the earliest events in apoptosis and seems to be a cell type- and inducer- (Ϫ/Ϫ) gene homozygous “knockout” mice and their heterozygotic (ϩ/Ϫ) independent process. Using simultaneous annexin-V-FITC and propidium littermates) were originally obtained from Dr. Tak Mak (Amgen Institute, iodide staining, the percentage of cells that underwent apoptosis and ne- Toronto, Canada) (41). Mice were housed in a virus Ab-free facility. The crosis was quantitatively determined. Fluorescein-labeled annexin-V and IRF-1 (Ϫ/Ϫ) colony was produced by mating IRF-1 (Ϫ/Ϫ) mice to either propidium iodide from Boehringer Mannheim were used for detection of IRF-1 (Ϫ/Ϫ) or IRF-1 (ϩ/Ϫ) mice. The IRF-1 wild-type (ϩ/ϩ) colony apoptosis according to the manufacturer’s instructions. Because the mac- was maintained by mating IRF-1 (ϩ/ϩ) mice that were derived from het- rophages adhere so strongly to tissue culture plates, Teflon beakers were erozygous matings to either IRF-1 (ϩ/ϩ) or IRF-1 (ϩ/Ϫ) mice. To prevent used for all of these experiments to minimize induction of cell death by the background of the IRF-1 (Ϫ/Ϫ) and (ϩ/ϩ) colonies from straying, all mechanical shearing. In brief, after incubation with various cell death- new breeding pairs were the progeny of IRF-1 (ϩ/Ϫ) to IRF-1 (ϩ/Ϫ) inducing agents, macrophages were detached by gentle aspiration from the Teflon beaker surface and centrifuged at 400 ϫ g for 10 min. The pellet 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 was then resuspended in 100 l labeling solution (140 mM NaCl, 10 mM HEPES, 5 mM CaCl ,1␮g/ml propidium iodide, 1/50 volume of annexin- a 300-bp sequence from genomic DNA. 2 V-FITC), and then incubated for 10 min in the dark at 4°C. Samples were Culture of peritoneal macrophages analyzed on a Coulter EPICS XL-MCL flow cytometer using 488-nm ex- citation and 525-nm bandpass filters for fluorescein detection and a filter of Mice were injected i.p. with 3 ml of 3% thioglycollate medium (Difco, 620 nm for PI detection, after electronic compensation of the instrument to Detroit, MI). Peritoneal exudate cells were collected by lavage 4 days after exclude overlap of the two emission spectra. Propidium iodide positive 2492 LPS- AND CERAMIDE-INDUCED CELL DEATH IN MURINE MACROPHAGES

(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 NO⅐ and TNF bioassay Secretion of NO⅐ by macrophages was measured by spectrophotometric determination of nitrite, the stable end product of NO⅐ 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 M) standard curve. To measure bioactive TNF-␣/␤ 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 ␮g/ml proteinase-K (Boehringer Mannheim), 10

mM Tris-HCl (pH 8), 10 mM EDTA, and 0.5% SDS and incubated for 3 h Downloaded from 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 ϫ g), supernatants were treated with 25 ␮g/ml RNase (Sigma) for 1 h, then DNA fragments were precipitated overnight by two volumes of absolute ethanol at Ϫ20°C. Samples were centrifuged for 30 min at 10,000 ϫ 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 http://www.jimmunol.org/ (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. Results LPS and ceramide induce cell death in peritoneal macrophages FIGURE 1. IFN-␥ potentiates C2 ceramide- and LPS-induced toxicity It is well documented that LPS can exert direct toxic effects on in peritoneal macrophages. C3H/OuJ macrophages were cultured on 24- peritoneal macrophages, macrophage cell lines, endothelial cells, well plates, treated with 1 ␮g/ml LPS for 48 h (top panel), or 25 ␮MC2 and many other cell types. Although early studies utilized very ceramide for 24 h (bottom panel), in the absence or presence of 50 IU/ml by guest on September 28, 2021 ␥ high LPS concentrations (20–50 ␮g/ml) to demonstrate direct IFN- . Cell death was measured on the basis of LDH release from the dead Ϯ macrophage cytotoxicity (10, 48, 49), simultaneous stimulation cells into the culture media. Bars represent arithmetic means SEM of triplicate samples from a representative experiment (n ϭ 3). *Statistically with IFN-␥ or cycloheximide has been showed to potentiate the # Ͻ ␮ significant difference compared with medium treated control. No signifi- toxicity of much lower LPS concentrations ( 1 g/ml) (12, 13). In cant difference compared with medium treated control (n ϭ 3, p Ͻ 0.05, an initial series of studies (Fig. 1) in which cell death was assessed one-way ANOVA, combined with Tukey’s test). by release of LDH into the culture supernatants in 24-well plates, we found that 1 ␮g/ml LPS alone was not significantly cytototoxic for LPS-responsive, C3H/OuJ macrophages, even after 48 h of cubation in the presence of IFN-␥ (Fig. 2B). The inactive ceramide incubation. The presence of IFN-␥ potentiated the cell death, as analogue, dihydro-C2 ceramide, plus IFN-␥ did not affect the vi- previously reported (12, 13). No measurable cell death was ob- ability of macrophages, even after 48 h (data not shown). Elevation served with LPS alone or LPS plus IFN-␥ at 24 h (data not shown). of intracellular ceramide levels by inhibition of glucosylceramide The cell-permeable ceramide analogue N-acetylsphingosine (C2- synthase with 25 to 50 ␮M PPMP (50), which inhibits the further ceramide) at a dose of 25 ␮M, a dose shown in previous studies to incorporation of ceramide into glycolipids, also induced cell death induce gene expression in these macrophages (19), was found to be in macrophages after 24 h of treatment, and similarly to C2 cer- directly cytotoxic by 24 h of exposure, and like LPS, ceramide- amide-induced cell death, this was potentiated by the presence of induced cell death was potentiated by the presence of IFN-␥. IFN-␥ (Table I). LPS-hyporesponsive C3H/HeJ macrophages were These findings were extended by experiments shown in Figures highly refractory to very high concentrations of LPS (i.e., 50 ␮g/ 2–4. Using both trypan blue exclusion and LDH as measurements ml) plus IFN-␥-induced cell death (Fig. 2A), and they were also of macrophage cell death, Figure 2 illustrates that both LPS- (Fig. less sensitive to C2 ceramide (25 ␮M) plus IFN-␥-induced cell 2A) and C2 ceramide- (Fig. 2B) induced cytotoxicity are dose- death (Fig. 2B), although at 50 ␮M, they were equivalently sen- dependent in the presence of IFN-␥. Even when very high con- sitive to C3H/OuJ macrophages. centrations of LPS (25–50 ␮g/ml) plus IFN-␥ were used, cell death LPS plus IFN-␥-induced cell death was highly dependent on the rarely exceeded 50% (as measured in the LDH assay), whereas 1 volume of medium used in cell culture wells. The data presented ␮g/ml LPS plus IFN-␥ evoked suboptimal, but statistically signif- in Figures 1 and 2 were measured in 24-well plates, with 5.4 ϫ 105 icant, cell death. IFN-␥ alone failed to induce measurable cyto- cells in 1 ml of medium per well. Under conditions where the cells toxicity, even after 72 h of incubation (data not shown). LPS- were cultured in 6-well plates in a final volume of 1.5 ml medium hyporesponsive C3H/HeJ macrophages were highly refractory to (i.e., 3 ϫ 106 cells/well; the same cell density as in the 24-well LPS plus IFN-␥-induced cytotoxicity (Fig. 2A). Under identical plates, but in a relatively smaller volume of medium), the sensi- conditions, 25 and 50 ␮M C2 ceramide were also toxic, causing tivity of the macrophages to LPS plus IFN-␥ was greatly increased. ϳ40% and ϳ60% cell death, respectively, after only 24 h of in- Figure 3A illustrates that under these culture conditions, as little as The Journal of Immunology 2493 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. LPS plus IFN-␥- and C2 ceramide plus IFN-␥-induced cell death in primary macrophages. C3H/OuJ and C3H/HeJ peritoneal exudate macrophage cultures were prepared as described in Materials and Methods in 24-well culture plates in a final volume of 1 ml. Cell death was measured by trypan blue exclusion and the LDH activity released to the media by the injured cells. Bars represent the arithmetic means Ϯ SEM (triplicate wells) from a representative experiment. This experiment was repeated three times with similar results. A, LPS-induced cell death after 48 h of incubation in the presence of 50 IU/ml IFN-␥. B,C2 ceramide-induced cell death after 24 h of incubation in the presence of 50 IU/ml IFN-␥. *Statistically significant difference compared with 25 ␮M C2 ceramide plus IFN-␥-treated C3H/OuJ macrophages (n ϭ 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s test). **Statistically significant difference compared with IFN-␥-treated C3H/OuJ macrophages (n ϭ 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s test).

0.1 ng/ml LPS induced macrophage cytotoxicity in the presence of ditions of increased LPS sensitivity, the macrophages remained IFN-␥. Again, LPS-induced cytotoxicity was not evident at 24 h of comparably sensitive to C2 ceramide (data not shown). Figure 3B incubation, whether or not IFN-␥ was present. Under these con- demonstrates that the pattern of cytotoxicity induced by LPS plus IFN-␥ was paralleled by the release of NO⅐ into culture supernatants. Table I. PPMP induces cell death without concurrent nitrite release in peritoneal macrophage cultures Involvement of iNOS and autocrine TNF production in LPS- and % Cell Death ceramide-induced cell death (LDH release) Nitrite Release (␮M) A comparison of LPS- and C2 ceramide-induced cytotoxicity and Without IFN-␥ Without IFN-␥ NO⅐ production was next studied. As shown in Figure 3, Figure 4A Treatmenta IFN-␥ (50 U/ml) IFN-␥ (50 U/ml) again illustrates that LPS plus IFN-␥ induced both cell death and NO⅐ Medium 7.3 Ϯ 1.9 7.4 Ϯ 1.2 2.3 Ϯ 1.8 3.19 Ϯ 1.4 release in parallel in C3H/OuJ macrophages, whereas C3H/HeJ mac- PPMP (12.5 ␮M) 11.0 Ϯ 1.5 16.0 Ϯ 1.5* 2.64 Ϯ 2.2 6.9 Ϯ 0.6 rophages were highly refractory to LPS-induced cell death and pro- PPMP (25 ␮M) 19.6 Ϯ 1.6* 27.8 Ϯ 1.9* 2.5 Ϯ 2.1 6.7 Ϯ 0.3 ⅐ duced minimal levels of NO . In C3H/OuJ macrophages, L-NMMA, PPMP (50 ␮M) 35.1 Ϯ 1.0* 47.6 Ϯ 0.1* 2.8 Ϯ 2.3 6.4 Ϯ 0.8 an inhibitor of iNOS, blocked both cell death and NO⅐ release in a Cells were cultured and treated with PPMP for 24 h on 24-well plates in the ␥ ␮ ␥ response to LPS and IFN- . In contrast, 25 M C2 ceramide plus presence or absence of IFN- . Cell death and nitrite release into culture supernatants ⅐ were measured as described in Materials and Methods. Data represent arithmetic IFN-␥ did not induce NO release into the culture media (Fig. 4B), nor Ϯ ϭ means SEM of triplicate samples from a representative experiment (n 3). did L-NMMA protect C3H/OuJ macrophages from C2 ceramide plus * Statistically signiﬁcant difference, compared to the medium or IFN-␥-treated ␥ control, respectively (n ϭ 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s IFN- -induced cell death under conditions where it protected against test). LPS plus IFN-␥-induced cytotoxicity. Moreover, PPMP, an agent that 2494 LPS- AND CERAMIDE-INDUCED CELL DEATH IN MURINE MACROPHAGES

fragmentation studies, as well as quantitative ﬂow cytometric ap- proaches, were employed. After 48 h of treatment, 1 ␮g/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 ␮M gliotoxin (13, 51), resulted in much stronger DNA fragmentation, even after 8 h, compared with that induced by LPS plus IFN-␥. In contrast, 25 ␮M C2 ceramide plus IFN-␥ did not induce any measurable DNA fragmentation even after 24 h (Fig. 5), the time point when ϳ40 to 60% of the macrophages were dead (Figs. 1 and 2). To quantify macrophage apoptosis caused by LPS plus IFN-␥, the percentage of subdiploid nuclei, characteristic for apoptotic cell populations, was measured by ﬂow cytometry. After 48 h of incubation with 1 ␮g/ml LPS plus IFN-␥, which resulted ϳ70% macrophage death in 6-well plates (Fig. 4A), ϳ 20% of the cells exhibited subdiploid nuclei, in contrast to the Downloaded from positive control, 3 ␮M gliotoxin, which induced Ͼ90% 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 http://www.jimmunol.org/ 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). In contrast to gliotoxin, freeze-thawing of the cultures (Fig. 6B) or killing the macrophages by hypotonic lysis with distilled water (data not shown) did not produce a hypodiploid peak in FIGURE 3. LPS-induced cell death and NO⅐ release in the presence or the histogram, showing that this method distinguishes clearly absence of IFN-␥. LPS-induced cell death and NO⅐ release were measured between apoptosis and necrosis. In agreement with the DNA by guest on September 28, 2021 in the absence or presence of 50 IU/ml IFN-␥ at various LPS concentra- fragmentation studies (Fig. 5), 25 ␮M C2 ceramide plus IFN-␥ tions after 24 and 48 h of incubation. C3H/OuJ macrophage cultures were failed to elicit any apoptosis after 24 h of treatment, as mea- prepared as described in Materials and Methods in 6-well culture plates, in sured by flow cytometric DNA analysis (Fig. 6B, Table III). No a final volume of 1.5 ml. Cell death was measured by LDH activity re- increase in the percentage of subdiploid nuclei was measurable leased to the media by the injured cells (A), and nitrite concentration was even at a C2 ceramide concentration of 50 ␮M, in the presence assessed by Griess reaction (B). Data are from duplicate samples from a ␥ representative experiment (n ϭ 3). of IFN- (Fig. 6B). One of the earliest events in apoptosis is the externalization of phosphatidyl serine in the plasma membrane as a conse- quence of apoptotic injury. Besides DNA fragmentation, this elevates endogenous ceramide levels in cells, and also is toxic to mac- process is thought to be one of the hallmarks of apoptosis in a rophages, induced no significant nitrite release, even in the presence wide variety of experimental systems and, in fact, represents an of IFN-␥ (Table I). These findings suggest that NO⅐ is not involved in independent event from nuclear changes. Because DNA frag- ceramide-induced cytotoxicity. mentation and analysis of subdiploid nuclei failed to suggest Because TNF-␣, induced by LPS treatment, plays a key role in evidence for C2 ceramide plus IFN-␥-induced apoptosis, we development of sepsis syndrome in vivo, and because TNF-␣ is a also measured the binding of annexin-V to macrophages, con- well-known inducer of apoptosis, we investigated a potential role current with propidium iodide staining, in an attempt to detect for TNF-␣ in LPS plus IFN-␥- and C2 ceramide plus IFN-␥-in- possible phosphatidyl serine externalization. A relatively low duced cell death in vitro. Table II illustrates that LPS plus IFN-␥ ␮ induced a large accumulation of bioactive TNF. Although, inclu- dose of gliotoxin (0.5 M) elicited apoptosis by 24 h, as mea- sion of 1 ϫ 10Ϫ5 M soluble TNF-receptor (TNFR:Fc) eliminated sured by an increase in annexin-V binding to macrophages from ␮ the bioactive TNF-␣ induced by LPS plus IFN-␥, it was not pro- 11% to 75%. After4hoftreatment with 25 M C2 ceramide ␥ tective against the cell death induced under the same conditions. A plus IFN- , no increase was observed in the percentage of an- ␥ total of 25 ␮M C2 ceramide plus IFN-␥ failed to induce the release nexin-V binding cells (12% of C2 ceramide plus IFN- vs 11% of bioactive TNF-␣, as measured by TNF bioassay. TNFR:Fc con- of medium-treated control). Even after 24 h of incubation with jugate did not protect the cells against C2 ceramide plus IFN-␥- C2 ceramide plus IFN-␥, the proportion of annexin-V positive induced cell death (Table II). cells was only 13% vs 11% of medium-treated controls, indi- cating that the cell death caused by C2 ceramide plus IFN-␥ is ␥ LPS plus IFN- causes necrosis and apoptosis, whereas C2 not apoptotic. This is illustrated in Figure 7, A (medium- and ␥ ceramide plus IFN- induces necrosis only gliotoxin-treated cells) and B (medium and C2 ceramide plus To study whether the C2 ceramide plus IFN-␥- and LPS plus IFN- IFN-␥-treated cells), where the respective annexin-V-binding ␥-induced cell death were the result of apoptosis or necrosis, DNA histograms are shown in an overlayed fashion. The Journal of Immunology 2495 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Effect of an iNOS inhibitor, L-NMMA, on cell death and nitrite release induced by LPS and C2 ceramide, in the presence of IFN-␥. LDH and nitrite were measured from the very same wells of the cultures. Cell death values and nitrite levels were calculated as described in Materials and Methods. Bars represent the arithmetic means Ϯ SEM (triplicate samples) obtained in a representative experiment (n ϭ 3). A, LPS plus IFN-␥-induced cell death and NO⅐ release. Cells plated on 6-well plates were treated with 1 ␮g/ml LPS plus IFN-␥ (50 IU/ml) in the absence or presence of the iNOS inhibitor, 0.5 mM L-NMMA. Cell viability was measured in parallel with nitrite release 48 h later. *Statistically signiﬁcant difference compared with LPS plus IFN-␥-treated C3H/OuJ samples (n ϭ 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s test). B, C2 plus IFN-␥ induced cell death and NO⅐ release. Cells were plated on 24-well plates and treated with 25 ␮M C2 ceramide plus IFN-␥ (50 IU/ml) in the absence or presence of 0.5 mM L-NMMA, and then 24 h later cell viability was measured in parallel with nitrite release. #No statistically signiﬁcant difference compared with 25 ␮M C2 ceramide plus IFN-␥-treated samples (n ϭ 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s test).

LPS plus IFN-␥-induced cytotoxicity and apoptosis are (ϩ/ϩ) macrophages underwent apoptosis (ϳ30%, Fig. 8C)inre- IRF-1-dependent sponse to LPS plus IFN-␥, although necrotic cell death remained Ϫ Ϫ Previous studies have indicated that mice with a targeted mutation dominant. In contrast, IRF-1 ( / ) peritoneal macrophages did in the gene that encodes the IRF-1 possess macrophages that re- not show nuclear DNA loss as measured by the appearance of spond poorly to LPS plus IFN-␥ to induce iNOS gene expression subdiploid nuclei by FACS (Fig. 8C). These data demonstrate that ␥ and NO⅐ release (38). Moreover, the T cells of these mice have also both the LPS plus IFN- -induced cell death and apoptosis are IRF- been reported to be refractory to apoptosis induced by DNA dam- 1-dependent in peritoneal macrophages. age (e.g., ␥-irradiation, etoposide, adriamycin) (39). Therefore, we sought to evaluate the responsiveness of macrophages derived Discussion from IRF-1 (Ϫ/Ϫ) mice and IRF-1 (ϩ/ϩ) controls to LPS plus In addition to its systemic effects, LPS also exerts cellular toxicity that IFN-␥-induced cell death and apoptosis. Figure 8, A and B illus- may be particularly important as one of the causes of multiple organ trate that LPS plus IFN-␥ induces both cell death and NO⅐ release failure in sepsis. Indeed, LPS-induced endothelial cell death has been in parallel in IRF-1 (ϩ/ϩ) macrophages within 48 h, whereas proposed as a major cause of mortality in a murine model of endo- IRF-1 (Ϫ/Ϫ) macrophages were highly resistant to LPS-induced toxic shock (52). Although a variety of cell types such as T cells, B cell death and produced low levels of NO⅐. In IRF-1 (ϩ/ϩ) mac- cells, and neutrophils are reported to be sensitive to LPS toxicity in ⅐ rophages, L-NMMA blocked both cell death and NO release in vivo, little is known about whether macrophages undergo apoptosis response to LPS and IFN-␥. Also, as was observed in C3H/OuJ during the process of endotoxic shock. Recently, Ayala et al. (53) macrophage cultures (Fig. 6A), a signiﬁcant portion of IRF-1 showed increased peritoneal macrophage apoptosis in vivo in a cecal 2496 LPS- AND CERAMIDE-INDUCED CELL DEATH IN MURINE MACROPHAGES

Table II. LPS- and C2 ceramide-induced TNF release and cell death in peritoneal macrophages

% Cell Death TNF-␣ (LDH release) (IU/ml)

Treatment Expt. 1 Expt. 2 Expt. 1 Expt. 2

LPS-induced releasea Medium 13.1 10.0 Ͻ20 Ͻ20 IFN-␥ (50 IU/ml) 12.5 11.2 Ͻ20 Ͻ20 LPS (1 ␮g/ml) ϩ IFN-␥ 90.0 82.7 6826 4203 1 ϫ 10Ϫ5 M TNFR:Fc ϩ LPS ϩ IFN-␥ 87.2 75.2 93 150 C2 ceramide-induced releaseb Medium 14.7 9.6 Ͻ20 Ͻ20 IFN-␥ (50 IU/ml) 11.8 11.5 Ͻ20 Ͻ20 C2 (25 ␮M) ϩ IFN-␥ 42.0 35.1 Ͻ20 Ͻ20 TNFR:Fc (1 ϫ 10Ϫ5 M) ϩ LPS ϩ IFN-␥ 42.7 45.3 Ͻ20 Ͻ20

a Cells were cultured and treated in 6-well plates (1 ␮g/ml LPS ϩ IFN, 1 ϫ 10Ϫ5 M TNFR:Fc) for 48 h, and cell death was measured by LDH assay in parallel with measurement of TNF released to the culture medium. b Macrophages were cultured in 24-well plates for 24 h. Cell death was evaluated by LDH assay, as described in Materials and Methods, in parallel with measurement of TNF released to the culture medium. Data shown are means of triplicate samples from two independent experiments. Downloaded from ligation and puncture sepsis model (CLP), compared with sham-CLP In our studies, LPS- and ceramide-induced cytotoxicity was quan- animals. This increased apoptosis rate during sepsis appears to be tiﬁed by two different methods (trypan blue exclusion and LDH correlated with an “acquired immunosuppression” (dysfunction of release), and these assays gave comparable results (Fig. 2). High macrophages) in the late phase of sepsis. Moreover, it has also been concentrations of both LPS (25–50 ␮g/ml) and C2 (25–50 ␮M)

reported that treatment of macrophages from CLP mice in vitro with ceramide were directly toxic for peritoneal exudate macrophages http://www.jimmunol.org/ LPS can further increase apoptosis, and the process is NO⅐-dependent derived from C3H/OuJ mice. The presence of IFN-␥ potentiated (54). In spite of these in vivo and the aforementioned in vitro studies, this effect, but more strikingly for LPS than for C2 ceramide. Un- the exact contribution of the macrophage cell death to endotoxicity is der conditions of high cell number and low supernatant volume still not known. (e.g., 6-well plates), concentrations of LPS as low as 1 ng/ml could Involvement of ceramide pathway in signal transduction of cer- be demonstrated to be highly toxic in the presence of IFN-␥ (Fig. tain LPS effects has recently been suggested (21). It has also been 3A). This suggests that a factor(s) that accumulate(s) within the reported that in vivo, LPS-induced toxicity is mediated by TNF supernatant contribute(s) to LPS-induced cell death. Previous stud- release and subsequent ceramide generation in endothelial cells ies have demonstrated that LPS plus IFN-␥-induced cell death is (52). However, the possible role of the ceramide pathway in LPS- NO⅐-mediated (12, 13, 15, 17). Indeed, this was conﬁrmed in these by guest on September 28, 2021 induced cell death in macrophages remained to be elucidated. To studies by the demonstration that inactive arginine analogue, L- address this question, the in vitro toxicity of LPS and C2 ceramide NMMA, blocked LPS plus IFN-␥-induced cytotoxicity with a con- in peritoneal exudate macrophage cultures was compared in the current inhibition of NO⅐ release into the culture supernatant. Thus, presence of IFN-␥. In vivo, IFN-␥ has a very important role in the enhancement of LPS-induced cell death at low LPS concen- LPS-induced toxicity. In fact, injection of anti-IFN-␥ Abs has pro- trations by the presence of IFN-␥ is likely to be related to the tective effect of LPS-induced thymocyte cell death in vivo, show- well-characterized synergy between these two compounds for the ing that LPS and IFN-␥ may act in concert to induce toxicity (16). 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 NO⅐ release: C2 ceramide and PPMP (ϮIFN-␥) failed to stimulate release of NO⅐ 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 NO⅐ 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. Al- though C2 ceramide plus IFN-␥ induced massive cell death after 24 h FIGURE 5. Comparison of LPS plus IFN-␥- and C2 ceramide plus IFN- of incubation, even high concentrations of LPS (Ͼ10 ␮g/ml) plus ␥ -induced apoptotic DNA laddering. Cells were plated in 6-well plates and IFN-␥ did not induce cell death at the same time point. For LPS plus treated with medium (lane D, for 48 h), 1 ␮g/ml LPS plus IFN-␥ (lane E, IFN-␥-induced toxicity, at least 48 h of incubation was necessary, for 48 h), 1 ␮g/ml LPS plus IFN-␥ in the presence of 0.5 mM L-NMMA (lane F, for 48 h), 25 ␮M C2 ceramide plus IFN-␥ (lane B, for 24 h), or even with increased cell number/media volume ratio, which remark- ϳ ϳ 3 ␮M gliotoxin (lane C, for 8 h). DNA was extracted from macrophages ably increased the extent of cell death ( 70% compared with 30%), and subjected to electrophoresis (see Materials and Methods). As a m.w. but without an alteration in kinetics. This ﬁnding suggests important marker, HindIII digested ␭-phage DNA, was used (lane A). Similar data differences in the signal transduction and/or the effector mechanisms were obtained in another separate experiment. of cell death for these two compounds. The Journal of Immunology 2497

Table III. Statistical analysis of LPS- and C2 ceramide-induced apoptosis in the presence of IFN-␥

Number of % Subdiploid Independent Treatmenta Nuclei Experiments

Medium 6.8 Ϯ 0.7 9 LPS (1 ␮g/ml) ϩ IFN-␥ (50 U/ml) 18.5 Ϯ 1.5* 9 NMMA ϩ LPS ϩ IFN-␥ 10.4 Ϯ 2.5† 4 C2 (25 ␮M) ϩ IFN-␥ 9.8 Ϯ 1.9† 3

a DNA analysis experiments were done as described in Materials and Methods. Representative experiments were shown in Figure 6A and B. Data are arithmetic means Ϯ SEM from independent experiments. * Statistically signiﬁcant difference, (p Ͻ 0.05) compared to medium-treated control. † No statistically signiﬁcant difference compared to medium treated control (one- way ANOVA, combined with Tukey’s test).

It is well known that macrophages from Lpsd C3H/HeJ mice are

hyporesponsive to LPS effects in vivo and in vitro (2). In the in- Downloaded from terim, 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 http://www.jimmunol.org/ to LPS-induced cytotoxicity, even in the presence of IFN-␥, whereas longer (Ͼ24 h) incubation or high concentrations of C2 ceramide (50 ␮M) 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 Thie´blemont and by guest on September 28, 2021 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

FIGURE 6. Quantitative determination of apoptosis by ﬂow cytometric DNA analysis following propidium iodide staining. A, DNA analysis of LPS plus IFN-␥-induced cell injury. Macrophages from Lpsn (C3H/OuJ) and Lpsd (C3H/HeJ) mice were cultured in 6-well plates, treated with 1 ␮g/ml LPS alone, LPS plus IFN-␥, or 0.5mM NMMA plus LPS plus IFN-␥ for 48 h and 3 ␮M gliotoxin for 24 h. Bars represent percentage values of apoptotic cells with subdiploid nuclei that correspond to the “M2” marker in the histograms presented in B. At least 10,000 cells were analyzed at each point. Repeated experiments (at least n ϭ 3) gave similar results. B, DNA analysis of C2 ceramide plus IFN-␥-induced cell death. Cells were treated with medium, 50 U/ml IFN-␥,25or50␮M C2 ceramide plus IFN-␥ for 24 h, and 3 ␮M gliotoxin for 16 h. Freeze-thawing of macrophage cultures served as a positive control for necrosis. In the latter, macrophages were frozen at Ϫ20°C for 24 h, then were thawed and used for measurements. “M1” and “M2” markers represent the nonapoptotic diploid (viable or necrotic) and hypodiploid (apoptotic) nuclei, respectively. The numbers next to the M1 and M2 markers represent the percentage values of apoptotic and nonapoptotic cells in the graph. 2498 LPS- AND CERAMIDE-INDUCED CELL DEATH IN MURINE MACROPHAGES Downloaded from

FIGURE 7. C2 ceramide plus IFN-␥ does not induce apoptosis as as- http://www.jimmunol.org/ sessed by annexin-V staining in peritoneal macrophage cultures. C3H/OuJ macrophages were treated with medium, C2 ceramide plus IFN-␥ (4 and 24 h), or 0.5 ␮M gliotoxin (24 h). Phosphatidyl serine externalization was measured by annexin-V-FITC binding. Necrotic cells were gated out on the basis of their ability to uptake propidium iodide. A and B, Respective annexin-V binding histograms that are overlayed with medium-treated control. Note the increased annexin-V binding induced by gliotoxin (A, histogram is shifted, compared with medium treated control) and the lack of phosphatidyl serine externalization by 4 and 24 h of treatment with C2 by guest on September 28, 2021 ceramide plus IFN-␥ (B, completely overlapping histograms). M1 and M2 markers were used to calculate the percentages (see Results) for viable and apoptotic populations, respectively. Data are derived from a representative experiment (n ϭ 3). FIGURE 8. IRF-1 (Ϫ/Ϫ) macrophages are refractory to LPS plus IFN- ␥-induced cell death and apoptosis. Cells were treated with 50 IU/ml IFN-␥ alone and 1 ␮g/ml LPS plus IFN-␥ in the presence or absence of 0.5 mM space. In contrast, cytoplasmic content of necrotic cells can act as L-NMMA for 48 h. IRF-1 (Ϫ/Ϫ) and IRF-1 (ϩ/ϩ) macrophages were an additional inflammatory signal to enhance the uncontrolled in- cultured in 6-well plates in a volume of 1.5 ml, and cell death was mea- flammation that is characteristic of septic shock. To study further sured by LDH release (A). From the very same wells, apoptosis was as- ⅐ the cell death induced by LPS and C2 ceramide, we examined sessed by DNA-analysis (C) and NO release was also measured in parallel whether these compounds induced apoptosis or necrosis in perito- (B), as described in Materials and Methods. *Statistically significant dif- ␥ ϩ ϩ ϭ neal macrophages in vitro. It has been reported that LPS plus ference compared with LPS plus IFN- -treated IRF-1 ( / ) samples (n 3, p Ͻ 0.05, one-way ANOVA, combined with Tukey’s test). Data are IFN-␥ triggers apoptosis in peritoneal macrophages (12, 13) and from a representative experiment, repeated twice. 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. ptotic, even when ϳ70 to 80% cell death was induced by LPS plus 5). To quantify the extent of apoptosis, we used DNA analysis by IFN-␥ at 48 h. The necrotic cells still had intact nuclei as measured FACS. In contrast to previous reports (12, 13, 17), our data show by DNA analysis using FACS, so the cell death described here that LPS plus IFN-␥-induced cell death is predominantly necrotic, cannot be attributed to secondary necrosis. Moreover, in the case although a significant fraction of cells underwent apoptosis. A pos- of gliotoxin-treated macrophages, which are clearly apoptotic (ac- sible explanation for this contradiction is that in previous reports cording to DNA fragmentation, appearance of subdiploid nuclei, mentioned above, apoptotic cell death was demonstrated by mor- and increased annexin-V binding), the phagocytosis of apoptotic phologic evidence and DNA fragmentation with no precise quan- macrophages by neighboring cells did not interfere with the de- tification of apoptosis. Supportive of our findings, Albina et al. tection of apoptosis. The appearance of necrosis and apoptosis at (13) noted that only 10 to 15% of the cells showed apoptotic mor- the same time in response to cell death inducers has been reported phology at 48 h of incubation in their macrophage cultures treated in other cell types in vitro and in vivo (55–57). Similarly, in peri- with LPS plus IFN-␥. As an explanation, they hypothesized that toneal macrophage cultures, different subpopulations may exist although the process as a whole is apoptotic, this may be a result that respond differently to cell death inducers. Interestingly, both of efficient removal of apoptotic cells by neighboring macrophages the apoptotic and necrotic cell death of macrophages were antag- ⅐ or progression of the apoptosis into a phase of secondary necrosis. onized by the iNOS inhibitor, L-NMMA. Although NO -mediated Our results demonstrated that only 20 to 30% of cells were apo- cell death is generally considered as to be apoptotic (13, 15, 58), The Journal of Immunology 2499

NO⅐-induced necrosis has also been reported in oligodendrocytes distinct pathways can act simultaneously to induce apoptosis, de- (59) and in an epithelial cell line (60). As a possible mechanism for pending on the particular cell type and apoptosis inducer. NO⅐-induced apoptosis, direct DNA damage (61) and as a conse- Taken collectively, in this study we characterized cell death in- quence, induction of the tumor suppressor gene, p53, were sug- duced by C2 ceramide in peritoneal macrophages, showing the gested (15). Although p53 activation has been demonstrated in commonalties, as well as differences, with LPS-induced macro- response to LPS plus IFN-␥ treatment in RAW 264.7 cells, a yet phage cytotoxicity. In the presence of IFN-␥, LPS-induced cell unidentified p53-independent pathway is also likely to play a role death in peritoneal macrophages is mostly due to necrosis, with a in this process (15). It has also been reported that p53 activates significant fraction of cells undergoing apoptosis. Although differ- IL-1␤-converting enzyme-like proteases, specifically Caspase-3/ ing in extent, both C2 ceramide- and LPS-mediated cell death de- CPP32 (32-kDa cysteine protease), which is an important mediator pend on the presence of a normal Lps gene, suggesting a possible of apoptosis (61). As a mechanism for NO⅐-mediated necrosis, common early mediator in the signaling of cell death. In contrast, damage caused by free radicals, ATP depletion, and mitochondrial we demonstrated that LPS-induced, not C2 ceramide-triggered cell ⅐ membrane injury have been proposed (59). death, is mediated by NO in peritoneal macrophages in vitro. Sol- In contrast to LPS plus IFN-␥-induced cell death, C2 ceramide uble TNF-␣ was also ruled out as a possible mediator of ceramide- plus IFN-␥ caused purely necrotic cell death in peritoneal macro- or LPS-induced toxicity. In summary, our study suggests that al- phages as measured by DNA fragmentation, DNA analysis by though LPS may mimic certain ceramide effects, signal transduc- FACS, and annexin-V binding. This was very surprising, given the tion events that lead to cytotoxicity, as well as the downstream number of reports that demonstrate that ceramide is a potent in- mediators, diverge. Downloaded from ducer of apoptosis (22, 25). A growing body of evidence has implicated possible mediators and regulatory molecules that contrib- Acknowledgments ute to ceramide-induced apoptosis. Among others, ceramide- We thank Drs. Cindy Salkowski and Pierre Henkart for helpful discussions ␤ activated proteases (e.g., IL-1 -converting enzyme-like family during the preparation of this manuscript and Ms. Ranney McNally and proteases, especially Caspase-3/CPP32) (62), SAPK/JNK (63), Mr. M. J. Cody for maintenance and typing of IRF-1 mice. and cytosolic translocation of certain PKC isoforms (64) appear to http://www.jimmunol.org/ be important in mediating ceramide-induced apoptosis. Moreover, References Bcl-2 has regulatory role in this process (32). Much less is known 1. Weil, M. H., H. Shubin, and M. Biddle. 1964. Shock caused by gram-negative about the mechanisms of ceramide-induced necrosis. Recently, microorganisms. Ann. Intern. Med. 60:384. Arora et al (26) showed that ceramide analogues cause mitochon- 2. Vogel, S. N. 1992. Insights into the genetic and molecular basis of LPS respon- drial membrane permeability transition (MMPT), ATP depletion, siveness and macrophage differentiation. In Tumor Necrosis Factors: The Mol- ecules and Their Emerging Role in Medicine, 1st Ed. B. Beutler, ed. Raven, New and subsequent necrotic cell death. MMPT has been proposed as a York, p. 485. final common pathway of cell death. 3. Herrera-Velit, P., and N. E. Reiner. 1996. Bacterial lipopolysaccharide induces lyn Among other factors, TNF-␣ is believed to play fundamental the association and coordinate activation of p53/56 and phosphatidylinositol 3-kinase in human monocytes. J. Immunol. 156:1157. role in septic shock induced by LPS (2). Under in vivo conditions, 4. Fujihara, M., N. Connolly, and N. Ito. 1994. Properties of protein kinase C iso- by guest on September 28, 2021 LPS-induced TNF-␣ secretion and subsequent TNF-␣-induced ap- forms (␤II, ⑀, and ␨) in a macrophage cell line J774 and their roles in LPS- induced nitric oxide production. J. Immunol. 152:1898. optosis is one of the most important factors in LPS toxicity (52, 65, 5. Han, J., J.-D. Lee, L. Bibbs, and R. J. Ulevitch. 1994. A MAP kinase targeted by 66). To examine the role of TNF-␣ in LPS- and C2 ceramide- endotoxin and hyposmolarity in mammalian cells. Science 265:808. induced cell death in macrophages in vitro, we used a soluble TNF 6. Weinstein, S. L., J. S. Sanghera, K. Lemke, A. L. 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