Role of Ceramide in Lipopolysaccharide (LPS)-induced Signaling

Ceramide and ceramide-activated enzymes have been implicated in responses to bacterial lipopolysaccharide (LPS) and the proinflammatory cytokines tumor necrosis factor-α (TNF) and interleukin-1β (IL-1). Although TNF and IL-1 cause elevation of cellular ceramide, which is thought to act as a second messenger, LPS has been proposed to signal by virtue of structural similarity to ceramide. We have investigated the relationship between ceramide and LPS by comparing the effects of a cell-permeable ceramide analog (C2-ceramide) and LPS on murine macrophage cell lines and by measuring ceramide levels in macrophages exposed to LPS. We found that while both C2-ceramide and LPS activated c-Jun N-terminal kinase (JNK), only LPS also activated extracellular signal-regulated kinases (ERKs). C2-ceramide was also unable to activate NF-κB, a transcription factor important for LPS-induced gene expression. Upon measurement of cellular ceramide in macrophage lines, we observed a small but rapid rise in ceramide, similar to that seen upon IL-1 or TNF treatment, suggesting LPS induces an increase in ceramide rather than interacting directly with ceramide-responsive enzymes. We found that C2-ceramide activated JNK and induced growth arrest in macrophages cell lines from both normal mice (Lps n) and mice genetically unresponsive to LPS (Lps d), whereas onlyLps n macrophages made these responses to LPS. Surprisingly, LPS treatment of Lps d macrophages induced a rise in ceramide similar to that observed in LPS-responsive cells. These results indicate that the wild type Lps allele is not required for LPS-induced ceramide generation and suggest that ceramide elevation alone is insufficent stimulus for most responses to LPS. Ceramide and ceramide-activated enzymes have been implicated in responses to bacterial lipopolysaccharide (LPS) and the proinflammatory cytokines tumor necrosis factor-α (TNF) and interleukin-1β (IL-1). Although TNF and IL-1 cause elevation of cellular ceramide, which is thought to act as a second messenger, LPS has been proposed to signal by virtue of structural similarity to ceramide. We have investigated the relationship between ceramide and LPS by comparing the effects of a cell-permeable ceramide analog (C2-ceramide) and LPS on murine macrophage cell lines and by measuring ceramide levels in macrophages exposed to LPS. We found that while both C2-ceramide and LPS activated c-Jun N-terminal kinase (JNK), only LPS also activated extracellular signal-regulated kinases (ERKs). C2-ceramide was also unable to activate NF-κB, a transcription factor important for LPS-induced gene expression. Upon measurement of cellular ceramide in macrophage lines, we observed a small but rapid rise in ceramide, similar to that seen upon IL-1 or TNF treatment, suggesting LPS induces an increase in ceramide rather than interacting directly with ceramide-responsive enzymes. We found that C2-ceramide activated JNK and induced growth arrest in macrophages cell lines from both normal mice (Lps n) and mice genetically unresponsive to LPS (Lps d), whereas onlyLps n macrophages made these responses to LPS. Surprisingly, LPS treatment of Lps d macrophages induced a rise in ceramide similar to that observed in LPS-responsive cells. These results indicate that the wild type Lps allele is not required for LPS-induced ceramide generation and suggest that ceramide elevation alone is insufficent stimulus for most responses to LPS. The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; JNK, c-Jun N-terminal kinase; BMMO, bone marrow macrophages; SRBC, sheep red blood cells; ERK, extracellular signal-regulated kinases; GPI, glycosylphosphatidylinositol; MAPK, mitogen-activated protein kinases; IL, interleukin; TNF, tumor necrosis factor; CAPK, ceramide-activated protein kinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; BrdUrd, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; GST, glutathioneS-transferase; dH-C2-ceramide, dihydro-C2-ceramide; SMase, sphingomyelinases; ASMase, acid sphingomyelinases; KSR, kinase suppressor of activated Ras; MEKK, MAPK kinase kinase. is a classic and common initiator of inflammation. Macrophages exposed to LPS undergo a differentiation program that involves the up-regulation of genes whose products enhance the ability of macrophages to invade tissue, destroy bacteria, attract other immune system cells, and coordinate their responses. A localized proinflammatory response to LPS promotes host defense against bacterial infection, but if this response becomes systemic, as can occur during bacterial sepsis, it can result in endotoxic shock, which is often fatal. The principal high affinity receptor for LPS on myeloid cells is CD14, a glycosylphosphatidylinositol (GPI)-linked protein, which recognizes LPS in a complex with LPS-binding protein. Binding of LPS activates a variety of well characterized signaling pathways, possibly through an as yet unidentified coreceptor (1Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar, 2DeFranco A.L. Crowley M.T. Finn A. Hambleton J. Weinstein S.L. Levin J. Pollack M. Yokichi T. Nakano M. Endotoxin and Sepsis: Molecular Mechanisms of Pathogenesis, Host Resistance, and Therapy. Wiley-Liss, Inc., New York1997Google Scholar, 3Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar). Lipid A, the conserved core structure of LPS, retains the essential biological and signaling properties of intact LPS (4Morrison D.K. Kaplan D.R. Rapp U. Roberts R.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8855-8859Crossref PubMed Scopus (279) Google Scholar). It has been suggested that the lipid A portion of LPS shares some structural similarity with the cellular lipid ceramide (5Joseph C.K. Wright S.D. Bornmann W.G. Randolph J.T. Kumar E.R. Bittman R. Liu J. Kolesnick R.N. J. Biol. Chem. 1994; 269: 17606-17610Abstract Full Text PDF PubMed Google Scholar) and that LPS may act by mimicking ceramide (6Wright S.D. Kolesnick R.N. Immunol. Today. 1995; 16: 297-302Abstract Full Text PDF PubMed Scopus (88) Google Scholar). Ceramide can be generated by cleavage of membrane sphingomyelin by either acid or neutral sphingomyelinases (7Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar, 8Speigel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (474) Google Scholar), which remain to be fully characterized, or by de novo ceramide synthesis. Increases in cellular ceramide have been reported in many cell types in response to a variety of stimuli. These include the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as environmental stresses, such as UV light, differentiating agents, like vitamin D3, and other immunomodulatory signals, including Fas and CD28 (9Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Abstract Full Text PDF PubMed Scopus (915) Google Scholar, 10Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar). Micro-organisms have also been shown to stimulate increased cellular ceramide production as follows: binding of P-fimbriated Escherichia coli (11Hedlund M. Svensson M. Nilsson A. Duan R.-D. Svanborg C. J. Exp. Med. 1996; 183: 1037-1044Crossref PubMed Scopus (138) Google Scholar) or internalization ofNeisseria gonorrhoeae by nonphagocytic cells reportedly increases ceramide (12Grassme H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Cell. 1997; 91: 605-615Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Membrane-permeable ceramide analogs have been used to investigate the function of cellular ceramide, and exposure of mammalian cells to micromolar concentrations of these analogs has profound effects on gene expression, cell growth, and cell survival (10Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar). One of the earliest signaling events following LPS treatment of macrophages is tyrosine phosphorylation and activation of mitogen-activated protein kinases (MAPKs), including members of the ERK family, as well as the stress-activated MAPKs, JNK and p38 (13Weinstein S.L. Sanghera J.S. Lemke K. DeFranco A.L. Pelech S.L. J. Biol. Chem. 1992; 267: 14955-14962Abstract Full Text PDF PubMed Google Scholar, 14Weinstein S.L. June C.H. DeFranco A.L. J. Immunol. 1993; 151: 3829-3838PubMed Google Scholar, 15Hambleton J. Weinstein S. Lem L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778Crossref PubMed Scopus (414) Google Scholar, 16Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2413) Google Scholar). Ceramide analogs have likewise been shown to activate ERK and JNK, and more recently p38, although the subset of MAPKs reported to be activated varies and may be cell line-dependent (17Cifone M.G. Roncaioli P. De Maria R. Camarda G. 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Activated MAPKs phosphorylate and regulate multiple transcription factors; among these AP-1/c-Jun and ATF2 have both been shown to be activated by either LPS or ceramide analog treatment (21Welsh N. J. Biol. Chem. 1996; 271: 8307-8312Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 25Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar, 26Sawai H. Okazaki T. Yamamoto H. Okano H. Takeda Y. Tashima M. Sawada H. Okuma M. Ishikura H. Umehara H. Domae N. J. Biol. Chem. 1995; 270: 27326-27331Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The NF-κB/Rel family of transcription factors is also activated by LPS and is critical for induced expression of many proinflammatory genes (3Sweet M.J. Hume D.A. J. 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The coordinate activation of NF-κB and other transcription factors by LPS results in the expression of genes encoding adhesion molecules, enzymes involved in the production of oxygen and nitrogen radicals, and inflammatory cytokines, notably TNF, IL-1, and IL-6 (1Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar). These changes in gene expression reflect differentiation to an activated phenotype and are accompanied by growth arrest in bone marrow-derived macrophages (30Vadiveloo P.K. Vairo G. Novak U. Royston A.K. Whitty G. Filonzi E.L. Cragoe Jr., E.J. Hamilton J.A. Oncogene. 1996; 13: 599-608PubMed Google Scholar), as well as in the murine macrophage line RAW 264.7 (31Paul A. Bryant C. Lawson M.F. Chilvers E.R. Plevin R. Br. J. Pharmacol. 1997; 120: 1439-1444Crossref PubMed Scopus (28) Google Scholar). In some macrophage cell lines and in fibroblasts, ceramide analogs have also been shown to induce expression of cytokines and cell adhesion molecules (32Barber S.A. Perera P.-Y. Vogel S.N. J. Immunol. 1995; 155: 2303-2305PubMed Google Scholar, 33Barber S.A. Detore G. McNally R. Vogel S.N. Infect. Immun. 1996; 64: 3397-3400Crossref PubMed Google Scholar, 34Laulederkind S.J.F. Bielawska A. Raghow R. Hannun Y.A. Ballou L.R. J. Exp. Med. 1995; 182: 599-604Crossref PubMed Scopus (75) Google Scholar, 35Modur V. Zimmerman G.A. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1996; 271: 13094-13102Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and ceramide treatment causes growth arrest in many eukaryotic cell types, including yeast (36Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Yu. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 37Dbaibo G.S. Pushkareva M.Y. Jayadev S. Schwarz J.K. Horowitz J.M. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1347-1351Crossref PubMed Scopus (204) Google Scholar, 38Fishbein J.D. Dobrowsky R.T. Bielawska A. Garrett S. Hannun Y.A. J. Biol. Chem. 1993; 268: 9255-9261Abstract Full Text PDF PubMed Google Scholar). Direct evidence for a link between LPS and ceramide was initially provided by the observation that LPS up-regulated a 97-kDa serine/threonine protein kinase activity, thought to correspond to ceramide-activated protein kinase (CAPK) (5Joseph C.K. Wright S.D. Bornmann W.G. Randolph J.T. Kumar E.R. Bittman R. Liu J. Kolesnick R.N. J. Biol. Chem. 1994; 269: 17606-17610Abstract Full Text PDF PubMed Google Scholar). This led to the proposal that LPS may act as a structural mimic of ceramide (6Wright S.D. Kolesnick R.N. Immunol. Today. 1995; 16: 297-302Abstract Full Text PDF PubMed Scopus (88) Google Scholar). A report that CD14 and LPS-binding protein can transfer LPS into phospholipid bilayers supported the idea that LPS might interact directly with ceramide-responsive enzymes at the plasma membrane (39Wurfel M.M. Wright S.D. J. Immunol. 1997; 158: 3925-3934PubMed Google Scholar). The molecular mimickry hypothesis was further strengthened by observations that bacterial sphingomyelinase or synthetic ceramide analogs induced expression of an array of LPS-inducible mRNAs in macrophages from LPS-responsive (Lps n) mice but not in those from C3H/HeJ (Lps d) mice, which are genetically hyporesponsive to LPS (32Barber S.A. Perera P.-Y. Vogel S.N. J. Immunol. 1995; 155: 2303-2305PubMed Google Scholar). Additionally, the intracellular trafficking of both fluorescently labeled LPS and labeled ceramide analogs is reportedly altered in Lps d macrophages (40Thieblemont N. Wright S.D. J. Exp. Med. 1997; 185: 2095-2100Crossref PubMed Scopus (56) Google Scholar), although LPS binding and internalization occur with normal kinetics in the LPS-unresponsive macrophages (41Kitchens R.L. Munford R.S. J. Immunol. 1998; 160: 1920-1928PubMed Google Scholar). In contrast, other recent studies suggest that ceramide analogs and LPS, while having some overlapping effects, induced different patterns of gene expression (33Barber S.A. Detore G. McNally R. Vogel S.N. Infect. Immun. 1996; 64: 3397-3400Crossref PubMed Google Scholar) and differed in their ability to prime myeloid cells for superoxide production (42Nakabo Y. Pabst M.J. Immunology. 1997; 90: 477-482Crossref PubMed Scopus (15) Google Scholar). In an attempt to clarify the role of ceramide in LPS-induced signaling we have assayed the effects of LPS and the ceramide analog C2 on many of the above signaling molecules in murine macrophage lines. We found that both C2-ceramide and LPS activated JNK, but only LPS activated the ERK MAPKs. Similarly, NF-κB was not activated by C2-ceramide over a range of concentrations, in contrast to rapid activation by LPS treatment, arguing against the molecular mimickry hypothesis of LPS action. Measurement of cellular ceramide in LPS-treated macrophages revealed a rapid increase in ceramide levels, comparable to that induced by IL-1 or TNF. Both C2-ceramide and LPS induced growth arrest in a concentration-dependent manner in the RAW 264.7 macrophage line, suggesting that LPS-induced cellular ceramide could contribute to this response. In an Lps d macrophage line, responses to LPS were deficient, but C2-ceramide responses, namely JNK activation and growth arrest, were intact. Elevation of cellular ceramide was also observed in Lps dmacrophages treated with LPS, indicating that normal Lpsfunction is not required for this effect and that cellular ceramide increases alone cannot elicit many downstream LPS responses. Murine macrophage cell lines were expanded and maintained in Dulbecco's modified Eagle's medium with glutathione supplemented with 5–10% fetal bovine serum (containing <0.06 units/ml endotoxin) at 37° C in 5% CO2 for a maximum of 8 weeks of passage. The RAW 264.7 cell line was obtained from the American Type Culture Collection (Rockville, MD). The Lps n and Lps dVN-11 retrovirally transformed lines were the kind gift of Dr. Paula Ricciardi-Castagnoli (CNR, Milan, Italy). The latter macrophage lines originated from infection of primary thymic cultures derived from Balb/c and C3H/HeJ mice, respectively. Bone marrow macrophages (BMMO) from C57Bl/6 mice were isolated and cultured as described previously (43Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L.J. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (409) Google Scholar). Cells were stimulated as noted in figure legends and text with wild type LPS from E. coli K-235 (purified by phenol extraction and gel filtration; <1% protein), diphosphoryl lipid A from E. coli F-583, or paclitaxel from Taxus yannanensisobtained from Sigma, ReLPS from Salmonella minnesota (List Biological Laboratories, Campbell, CA); C2-ceramide (N-acetyl-d-erythro-sphingosine) and dihydro-C2-ceramide (dihydro-N-acetyl-d-erythro-sphingosine) obtained from Calbiochem or Biomol (Plymouth Meeting, PA); and recombinant mouse IL-1β or recombinant human or mouse TNF-α obtained from Genzyme Diagnostics (Cambridge, MA). Cell viability was determined by trypan blue exclusion. Cells were plated 18–24 h prior to stimulation in tissue culture dishes of desired size to give ∼80% confluency on the day of the experiment. Cells were stimulated in culture medium and then washed twice with cold phosphate-buffered saline (PBS) solution. Whole-cell extracts were prepared by direct addition of 0.3–1.0 ml of cold lysis buffer to the dish. Lysis buffer consisted of 20 mm Tris (pH 7.9), 137 mm NaCl, 5 mm EDTA, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 10 mm NaF and was supplemented immediately prior to use with 1 mm phenylmethylsulfonyl fluoride or PefablocTM (Boehringer Mannheim), 1 mm Na3VO4, 1 mmaprotinin, and 1 mm leupeptin. Following high speed centrifugation to remove debris, the protein concentration of extracts was determined using the BCA (Pierce) or Bio-Rad (Bio-Rad) protein assay. Whole-cell extracts containing 30–50 μg of total protein were separated by electrophoresis in 10% SDS-PAGE gels for IκBα immunoblotting or in 12% SDS-PAGE gels (acrylamide/bis-acylamide ratio of 120:1) for ERK1/ERK2 and JNK immunoblots (44Hambleton J. McMahon M. DeFranco A. J. Exp. Med. 1995; 182: 147-154Crossref PubMed Scopus (104) Google Scholar). The gels were transferred to nitrocellulose, blocked with 4% dried non-fat milk in Tris-buffered saline with 0.5% Tween 20 (TBST), and incubated with specific antibody overnight at 4° C. After extensive washing in TBST, a horseradish peroxidase-coupled sheep (Boehringer Mannheim) or donkey (Amersham Pharmacia Biotech) anti-rabbit IgG antibody was added to the blots for 1 h. After further washes the peroxidase activity was revealed using Renaissance™ chemiluminescence reagents (NEN Life Science Products). The primary antibodies used for immunoblotting were raised against IκBα- (sc-203, -371, -847) or ERK1-derived (sc-94) immunogenic peptides (Santa Cruz Biotechnology, Santa Cruz, CA) or against JNK1 (PharMingen, San Diego, CA). Whole-cell extracts containing 200–500 μg of total protein were diluted to a 1-ml volume with lysis buffer and immunoprecipitated with 10 μl of specific antibody to JNK1 (sc-474) or ERK2 (sc-154) (Santa Cruz Biotechnology) overnight at 4° C and then for 1–2 h with 15 μl of protein A-Sepharose (Zymed Laboratories Inc., San Francisco, CA). The resulting immunoprecipitate was washed extensively with lysis buffer and once with kinase assay buffer (25 mmHepes (pH 7.6), 20 mm MgCl2, 2 mmdithiothreitol, 20 mm β-glycerol phosphate, 1 mm Na3VO4). The kinase reaction was performed in kinase assay buffer with 20 μm unlabeled ATP and 10 μCi of [33P] or [γ-32P]ATP (Amersham Pharmacia Biotech or NEN Life Science Products) and 1 mg/ml substrate in a 30-μl volume. A glutathione S-transferase (GST) c-Jun (amino acids 1–79) fusion protein, prepared in our laboratory as described previously (15Hambleton J. Weinstein S. Lem L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778Crossref PubMed Scopus (414) Google Scholar), provided the substrate for JNK kinase assays; GST-Elk1 (New England Biolabs) was used as substrate for ERK2 activity assays. Kinase reactions were performed at 30° C for 15 min and terminated by addition of Laemmli gel-loading buffer. The kinase reactions were loaded on SDS-PAGE gels and following electrophoresis were either dried down and exposed or in some cases were transferred for immunoblotting (see above) to verify equivalent kinase precipitation and loading across samples. A κB oligonucleotide probe (described in Ref. 44Hambleton J. McMahon M. DeFranco A. J. Exp. Med. 1995; 182: 147-154Crossref PubMed Scopus (104) Google Scholar) was filled in with the Klenow fragment of DNA polymerase and labeled with [γ-33P]ATP using T4 polynucleotide kinase (New England Biolabs). Whole-cell extracts were prepared as above, but the salt concentration was increased to 400 mm to ensure complete extraction of nuclear proteins. Specific binding of extract proteins to the κB probe was assessed by incubation for 15 min at room temperature in a solution containing 7.5 mm Hepes (pH 8.0), 35 mm NaCl, 1.5 mm MgCl2, 0.05 mm EDTA, 1 mm dithiothreitol, and 7.5% glycerol, and 0.5 μg of poly(dI·dC) (Boehinger Mannheim), followed by electrophoretic separation in a 5% polyacrylamide gel. Ceramide was labeled in vivo by incubation of cells in [3H]palmitate (10 μCi/well) in 6-well plates for 24–48 h prior to stimulation, using a method similar to that described by Liu and Anderson (45Liu P. Anderson R.G.W. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Cells were stimulated with LPS or IL-1 in triplicate or quadruplicate wells in the same culture medium containing [3H]palmitate at 37° C. Unstimulated control wells were included on these plates and were labeled and processed simultaneously with stimulated samples. Following stimulation, cells were washed once with cold PBS, and flash-frozen in a dry-ice/ethanol bath. Water was added to each well, and cells were removed by scraping and transferred to glass tubes. Wells were rinsed with methanol/hydrochloric acid (40:1), and cellular lipids were extracted in chloroform and NaCl (1 n), and the chloroform phase was dried under nitrogen. The resulting labeled lipid mixture was resuspended in chloroform with 5% methanol and 30 μg of cold C18-ceramide from bovine brain as carrier. Samples were separated by thin layer chromatography (TLC) on silica gel plates (19-channel LK6D; Whatman) in a solvent of toluene/methanol (85:15) along with lipid standards for phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and C16- and/or C18-ceramide. The ceramide band was visualized by iodine vapor staining and autoradiography, and the corresponding area of the plate was recovered, scintillation fluid (Cytoscint, ICN) was added, and samples were counted in a β-scintillation counter. Alternatively, ceramide content of RAW 264.7 cells was assessed by diacylglycerol kinase labeling in vitro according to the method of Preiss et al. (46Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar) with modifications. RAW 264.7 macrophages were seeded in 12- or 6-well tissue culture plates (Corning) 18–24 h prior to stimulation. Following stimulation, total cellular lipids were extracted and dried as described above. Extracted total cellular lipids and ceramide (Avanti Polar Lipids) and diolein standards (Sigma) were resuspended in a solution containing 5 mm cardiolipin and subjected to phosphorylation by diacylglycerol kinase (Calbiochem) in a reaction containing 50 mm imidazole, 50 mm NaCl, 12.5 mmMgCl2, 2 mm dithiothreitol, 1 mmEGTA, 1 mm ATP, 10 μmdiethylenetriaminepentaacetic acid; (Sigma), and 50 μCi of [32P] or [γ-33P]ATP. Labeled phosphatidic acid and ceramide phosphate were extracted, dried under nitrogen, resuspended in chloroform with 5% methanol, and separated by TLC in a chloroform/methanol/acetic acid (65:15:5) mixture. Labeled ceramide phosphate signals were quantitated by PhosphorImager analysis (Molecular Dynamics). Phosphorylation of ceramide was linear over the concentration range tested (100–1000 pmol of bovine brain ceramide). Cellular ceramide levels in unstimulated cells were on the order of 100 pmol/105 cells, as judged by signals obtained with ceramide standards. Flow cytometry was used to determine the cell surface expression of various markers on murine macrophage lines and bone marrow-derived macrophages. Cells (∼106/sample) were removed from tissue culture plastic by gentle pipetting following treatment with 6 mm EDTA/PBS, washed, and resuspended in 100 μl of staining buffer (PBS, 2% fetal bovine serum, 0.02% azide) with rat anti-mouse FcγR blocking antibody (0.25 μg), and 10 min later 5 μg of specific fluorescein isothiocyanate (FITC)-conjugated antibody was added. FITC-conjugated antibodies (PharMingen) used for staining were rat anti-mouse antibodies to Mac-1, CD14, B7.2, I-Ab, I-Ad, B220, CD3, and anti-rat IgG (for FcγR staining) and a hamster anti-mouse antibody to B7.1. After 30 min staining with antibody on ice, the cells were washed twice and then resuspended in staining buffer with 1 μg/ml propidium iodide. FITC staining was determined on a logarithmic scale, after exclusion of propidium iodide positive (dead) cells. Flow cytometry was also used to quantify growth arrest in macrophages, much as described by Page and DeFranco (47Page D.M. DeFranco A.L. Mol. Cell. Biol. 1990; 10: 3003-3012Crossref PubMed Scopus (46) Google Scholar). Cells were divided into 6-well plates (∼2 × 105/well), and 16 h later were treated with C2-ceramide or LPS. Treatment continued for 24 h prior to cell harvesting, and 5-bromo-2-deoxyuridine (BrdUrd) (15 μm) was added during the final 8 h of culture. Cells were removed by EDTA treatment, washed in PBS, and fixed in 70% ice-cold ethanol. Fixed cells were permeabilized with 2.5m HCl containing 0.5% Triton X-100, washed in PBS plus 0.5% Tween, and labeled with FITC-conjugated anti-BrdUrd antibody (20 μl/106 cells) (Becton Dickinson, San Jose, CA). Following extensive washing, BrdUrd-positive and -negative fractions were assessed by flow cytometry in staining buffer with 50 μg/ml propidium iodide. In separate experiments, cell death following 1–6 h treatment with C2-ceramide or dihydro-C2-ceramide was assessed by flow cytometric analysis of propidium iodide (1 μg/ml) uptake by nonpermeabilized cells. Sheep red blood cells (SRBC) (Accurate Chemical and Scientific Corp., Westbury, NY) were washed in PBS and resuspended in a 5% solution (∼109/ml). This solution was incubated with Na251CrO4 (300 μCi/ml) for 2–3 h, and then washed with PBS to remove unabsorbed 51Cr label. SRBC were then opsonized with anti-SRBC IgG 30 min at room temperature, washed in PBS, and resuspended in RPMI at a dilution of 1:300. The 51Cr-labeled opsonized SRBC (0.5 ml/well) were added to confluent macrophages growing in 24-well plates that had been chilled on ice. Plated were centrifuged at 150 × g for 5 min to settle the 51Cr-labeled IgG-coated SRBC on the adherent macrophages. Phagocytosis was initiated by adding warm media to the wells. After a 30-min incubation at 37° C, phagocytosis was terminated by removing the plates to ice and washing with cold PBS. Uningested labeled SRBC were lysed by addition of water (750 μl/well) for 1 min. After a further wash in PBS, adherent macrophages with ingested 51Cr-labeled SRBC were lysed in 500 μl of 1% SDS and transferred to tubes for quantitation of radioactivity in a CliniGamma counter (LKB-Wallac, Finland). Percent phagocytosis was calculated as the mean cpm of 3–4 wells treated as above/mean total cpm of replicate wells treated as described above but without hypotonic lysis of uningested 51Cr-SRBC, i.e.ingested/bound SRBC × 100%. Macrophages growing in 24-well plates (∼106/well) in 1 ml of medium were stimulated with LPS or C2-ceramide, as indicated. Supernatants were ha