CD95-mediated Apoptosis in Vivo Involves Acid Sphingomyelinase (2000)

Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo.We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis. Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo.We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis. acid sphingomyelinase fluorescein isothiocyanate TdT-mediated dUTP-x nick end labeling peripheral blood lymphocyte alanine aminotransferase phytohemagglutinin lymphoproliferative disease generalized lymphoproliferative disease Sphingomyelinases have been implicated in important and diverse cellular functions (1Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1611) Google Scholar, 2Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (729) Google Scholar). Sphingomyelinases are characterized by their optimal pH and are divided accordingly into acid, neutral, and basic sphingomyelinase species (1Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1611) Google Scholar, 2Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (729) Google Scholar). The acid sphingomyelinase (ASM),1 a cellular glycoprotein, has been shown to be located in the acidic lysosomal compartment and contributes to lysosomal sphingomyelin turnover (3Sandhoff K. Klein A. FEBS Lett. 1994; 346: 103-107Crossref PubMed Scopus (43) Google Scholar). Genetic deficiency of ASM results in Niemann-Pick disease that is characterized by an accumulation of sphingomyelin in the cell (4Levade T. Salvayre R. Douste-Blazy L. J. Clin. Chem. Clin. Biochem. 1986; 24: 205-220PubMed Google Scholar). In addition, ASM was recently demonstrated to be secreted upon cellular treatment with inflammatory stimuli (5Schissel S.L. Kessler G.A. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 18250-18259Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The secreted form of ASM seems to be involved in the regulation of lipoprotein composition, and accordingly this form of ASM has been suggested to play a role in atherosclerosis (6Marathe S. Schissel S.L. Yellin M.J. Beatini N. Mintzer R. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 4081-4088Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The secretory ASM is encoded by the same gene as the lysosomal ASM. However, the N-terminal processing and glycosylation pattern of the two proteins are different, and this may direct targeting to different cell compartments (5Schissel S.L. Kessler G.A. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 18250-18259Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 7Schissel S.L. Jiang X. Tweedie-Hardman J. Jeong T. Camejo E.H. Najib J. Rapp J.H. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 2738-2746Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). A similar dichotomy has been demonstrated for ASM in Caenorhabditis elegans with a secretory and lysosomal enzyme encoded by two different genes (8Lin X. Hengartner M.O. Kolesnick R. J. Biol. Chem. 1998; 273: 14374-14379Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In addition to its function in membrane turnover, ASM has been shown to be stimulated by several receptors including the interleukin-1 receptor (9Mathias S. Younes A. Kan C.C. Orlow I. Joseph C. Kolesnick R.N. Science. 1993; 259: 519-522Crossref PubMed Scopus (390) Google Scholar), the tumor necrosis factor receptor (10Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (675) Google Scholar), CD95 (11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar), CD28 (14Boucher L.M. Wiegmann K. Futterer A. Pfeffer K. Machleidt T. Schütze S. Mak T.W. Krönke M. J. Exp. Med. 1995; 181: 2059-2068Crossref PubMed Scopus (190) Google Scholar), CD5 (15Simarro M. Calvo J. Vila J.M. Places L. Padilla O. Alberoloa-Ila J. Vives J. Lozano F. J. Immunol. 1999; 162: 5149-5155PubMed Google Scholar), and the intercellular adhesion molecule (16Ni H.T. Deeths M.J. Li W. Mueller D.L. Mescher M.F. J. Immunol. 1999; 162: 5183-5189PubMed Google Scholar). Further, the enzyme seems to be a primary target of cellular stress, and genetic studies employing ASM knock-out mice or lymphocytes from Niemann-Pick disease type A patients lacking functional ASM have proven that radiation-induced apoptosis of lymphoblasts, splenocytes, or endothelial cells (17Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar) requires ASM. Similarly, ASM plays an indispensable role in the induction of apoptosis in endothelial cells of lipopolysaccharide-challenged mice (18Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (383) Google Scholar). It is unknown, however, whether the enzyme functions in apoptosis under physiological conditions. Such a role has been suggested by several studies that demonstrate activation of ASM upon cellular stimulation via CD95 (11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar) or ligation of the tumor necrosis factor receptor (10Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (675) Google Scholar). Those studies demonstrate a rapid activation of ASM and a release of ceramide upon CD95 or tumor necrosis factor receptor triggering (10Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (675) Google Scholar, 11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar). De-Maria et al. (19De-Maria R. Rippo M.R. Schuchman E.H. Testi R. J. Exp. Med. 1998; 187: 897-902Crossref PubMed Scopus (137) Google Scholar), using ASM-deficient B lymphocytes, have demonstrated a resistance of cells lacking ASM to CD95-mediated apoptosis, strongly suggesting an important role for ASM and ceramide in apoptosis that is induced by CD95. This idea is supported by studies with an inhibitor of ASM, imipramine, which also revealed an inhibition of CD95-induced cell death (20Brenner B. Ferlinz K. Weller M. Grassmé H. Koppenhoefer U. Dichgans J. Sandhoff K. Lang F. Gulbins E. Cell Death Differ. 1998; 5: 29-37Crossref PubMed Scopus (116) Google Scholar). However, the exact function of ceramide in the apoptotic process is still unknown, and studies using high doses or pre-cross-linked anti-CD95 antibodies for stimulation disputed a crucial role for ASM in CD95-triggered death (21Boesen-de Cock J.G. Tepper A.D. de Vries E. van-Blitterswijk W.J. Borst J. J. Biol. Chem. 1998; 273: 7560-7565Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 22Lin T. Genestier L. Pinkowski M.J. Casturo A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). To define the physiological function of ASM in CD95-mediated apoptosis, several in vivo models were employed. In these mouse models we avoided direct non-physiologic manipulation of the CD95/CD95 ligand system. Instead, we attempted to indirectly up-regulate and activate the endogenous CD95/CD95 ligand system, an approach permitting us to test the physiologic requirement for ASM in CD95-mediated cell death. The results show that under physiological in vivoconditions, ASM is required for CD95-mediated apoptosis of peripheral blood lymphocytes or hepatocytes during autoimmune-like disorders. Apoptosis was induced by in vivo injection of anti-CD4 antibodies or phytohemagglutinin, resulting in a CD95/CD95 ligand-mediated death of peripheral blood lymphocytes or hepatocytes, respectively. ASM knock-out mice were resistant to induction of apoptosis in both systems. We have further shown that ASM amplifies thein vivo effect of anti-CD95, which has been injected intravenously into mice. In vitro data on splenocytes or hepatocytes confirm the understanding of ASM as a crucial molecule for CD95 signaling. However, the results also show that the function of ASM can be overcome by a high dose of pre-cross-linked stimulatory anti-CD95. ASM knock-out mice (23Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (412) Google Scholar) or normal control mice were injected intravenously (100-μl total volume) via the retro-orbital venus plexus with anti-CD95 antibody JO2 (PharMingen) at doses of 0.12, 0.16, or 0.2 μg/g; anti-CD4 antibody GK1.5 (PharMingen) at 0.4 μg/g; or phytohemagglutinin (PHA) (Sigma) at 15 μg/g. Control injections were performed with phosphate-buffered saline only. After carefully mincing the livers, the hepatocytes were rested for 60 min in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mmHEPES, pH 7.4, 2 mml-glutamine, 1 mm sodium pyruvate, 100 μm nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μm β-mercaptoethanol. Because we noted a spontaneous death of hepatocytes after incubation times longer than 12 h, all assays were performed within a 10-h period. Splenocytes were purified by a Ficoll gradient and stimulated daily with 10 μg/ml PHA with 4 units/ml interleukin-2. Alternatively, T lymphocytes were depleted by a 45-min incubation with 1 μg/ml each anti-Thy 1.1 and anti-Thy 1.2 (Sigma) at 4 °C followed by a 30-min incubation at 37 °C with 1:10 diluted rabbit complement (Cedarlane). The remaining B lymphocytes were washed and stimulated daily with 100 ng/ml anti-CD40 and 2 μg/ml anti-Ig. All cells were finally treated with anti-CD95 JO2 as indicated. The antibody was either applied under non-cross-linking conditions or after binding the antibody to the plastic plates, i.e. cross-linking conditions. For non-cross-linking conditions, the tissue culture plates were preblocked with 10 mg/ml alcohol-precipitated bovine serum albumin fraction V (Sigma) and gently shaken on an aspherical rotator to prevent binding of the antibody and cells to the plate. For cross-linking conditions, the antibody was coupled to plastic plates for 24 h at 4 °C in phosphate-buffered saline. To determine apoptosis in liver samples after the indicated injection, the mice were killed and the liver immediately transferred into 4% phosphate-buffered saline-buffered formalin, pH 7.0. After a 2-day fixation the tissues were embedded in paraffin, and 6-μm sections were cut, deparaffinized, and digested with proteinase K for 2 min, and endogenous peroxidase was blocked with H2O2. The sections were treated with terminal deoxynucleotidyltransferase in the presence of biotinylated dUTP in terminal deoxynucleotidyltransferase buffer containing cobalt chloride. Staining was developed using the ABC complex and 3-amino-9-ethylcarbazole as a substrate. Counterstaining was done in hematoxylin. To determine apoptosis of ex vivo hepatocytes, the cells were trypsinized to obtain a single-cell suspension and fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 in 0.1% sodium for 2 min, and for min with in the presence of terminal deoxynucleotidyltransferase and a at 37 The was by addition of the and by cells were to determine apoptosis. were performed in Apoptosis of splenocytes or was by to the Apoptosis of cells with from anti-CD4 mice was by of cells as previously (15Simarro M. Calvo J. Vila J.M. Places L. Padilla O. Alberoloa-Ila J. Vives J. Lozano F. J. Immunol. 1999; 162: 5149-5155PubMed Google Scholar). CD95 or were on by incubation of each with 1 μg/ml anti-CD95 anti-CD95 ligand 10 or anti-CD4 followed by the for min at 4 All antibodies were diluted in mm mm 1 mm mm fetal calf serum, and supplemented with 10 mm (Sigma) for CD95 or CD95 After of CD95 or CD95 ligand cells were as with the and a antibody to T All samples were by were in 50 mm pH 7.4, 10 mm 1 mm 10 10 μg/ml each and mg/ml and Triton times for 10 and at for of 50 mm pH 7.4, Triton 1 mm 100 μg/ml each was to the ASM was using a and The were washed times each in buffer and 50 mm sodium pH Triton 1 mm and 10 μg/ml and with in mm sodium pH mm at 37 °C for was and by a in were finally with of and of and in the was by were stimulated with 4 μg/ml anti-CD4 GK1.5 or 50 μg/ml PHA for the indicated The doses were from the in vivo injections on the that the serum is 10% of the of the Cell stimulation was by in mm pH 7.4, 0.1% sodium Triton mm 10 mm each sodium and sodium and 10 μg/ml The were to buffer and and proteins were by transferred to and developed using μg/ml antibody followed by an antibody and in the serum to or 24 h after PHA injection were using the of alanine and to and of with and to and by was ASM knock-out mice were with an of 12 and 2 with 2 cells from a normal The cells were from the and and injected The was to for to To the physiological function of ASM for CD95-triggered we CD95-triggered apoptosis of hepatocytes and peripheral blood lymphocytes upon of the endogenous CD95/CD95 ligand system. In the we an i.e. we apoptosis of hepatocytes by T lymphocytes stimulated by intravenous PHA injection N. K. K. K. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of PHA into mice has been shown to an of CD95 ligand on accumulation of lymphocytes, and apoptosis of hepatocytes via CD95 N. K. K. K. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). apoptosis in this on the of functional CD95 and CD95 ligand or mice are resistant to the of PHA injections and develop N. K. K. K. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Here, we show that injection of 15 PHA into normal mice apoptosis of hepatocytes with an of serum 1 In ASM knock-out mice were resistant to PHA injection and apoptosis in the liver 1 an of the 1 upon PHA The requirement for CD95 in the of apoptosis and is by injection of mice by T. with These were resistant to intravenous PHA apoptosis in intravenous PHA injection requires of ASM. injection of 15 PHA into normal mice apoptosis and the of a as indicated by the in In ASM knock-out or mice are resistant to PHA injection and show of apoptosis or hepatitis. apoptosis was by are shown at two In of by of ASM knock-out mice with from normal mice was by ASM in blood samples The mice overcome resistance to PHA and show apoptosis or an of in the blood serum These that the resistance of hepatocytes in ASM knock-out mice to PHA injection is of an lymphocyte Further, injection of 15 PHA similar of CD95 ligand on peripheral lymphocytes from normal and ASM knock-out mice h after injection CD95 ligand on ASM knock-out lymphocytes was functional as by the to cells a Apoptosis was by and cell of In PHA stimulation of ex vivo peripheral blood lymphocytes from normal or ASM-deficient mice a in the pattern of To an primary stimulation of with PHA in the ASM knock-out mice as for the resistance of mice to CD95-triggered we ASM knock-out mice with from normal mice. The of the was by ASM in peripheral blood lymphocytes The the resistance of ASM knock-out mice to and we apoptosis or and This that the or of ASM in hepatocytes than in T lymphocytes the to In control we have shown that the of CD95 ligand on normal or ASM knock-out mice h after injection of PHA 1 of lymphocytes from normal or ASM knock-out mice with or cells confirm the functional of CD95 ligand on from ASM knock-out mice after PHA injection 1 we in the pattern of in from normal or ASM knock-out mice a activation of from ASM knock-out mice by The was on the that injection of anti-CD4 antibodies or ligation of by in the is to apoptosis in by of CD95 and CD95 ligand A. T. K. Li X. Z. J. Immunol. 1994; 24: PubMed Scopus Google Scholar, C. J. Clin. PubMed Scopus Google Scholar). mice are resistant to apoptosis of that this as an function of the CD95/CD95 ligand A. T. K. Li X. Z. J. Immunol. 1994; 24: PubMed Scopus Google Scholar, C. J. Clin. PubMed Scopus Google Scholar). injected 0.4 of anti-CD4 antibodies into normal control or ASM knock-out mice. GK1.5 injection induced apoptosis of peripheral blood lymphocytes from normal mice whereas apoptosis was in lymphocytes from ASM-deficient mice Apoptosis of with a of and lymphocytes in normal which was in the ASM knock-out mice 2 to the ASM knock-out we apoptosis of or of and lymphocytes in mice the role of the CD95/CD95 ligand in this A and To an primary of ASM knock-out mice to the injected anti-CD4 GK1.5 as a for we the of CD95 on and after injection of the anti-CD4 These studies 2 the same of CD95 on the of normal control and ASM knock-out mice. Further, anti-CD4 GK1.5 an pattern of cellular proteins in from ASM knock-out and normal mice To further confirm the function of the ASM in CD95-triggered apoptosis, we intravenously injected doses of the anti-CD95 JO2 antibody into normal and ASM knock-out mice. of this antibody has been previously shown to result in liver and rapid death of the mice J. R. M. A. T. N. T. S. 1993; PubMed Scopus Google Scholar). In anti-CD95 JO2 induced death a of with an of A and data ASM knock-out mice were resistant to the injection of anti-CD95 to an of the injected antibody dose to 0.2 anti-CD95 JO2 in the death of all ASM knock-out similar to normal mice injected with the same dose of the This that ASM is required for apoptosis via CD95 upon stimulation with doses of anti-CD95 JO2 whereas doses of the antibody the requirement of ASM for CD95-triggered death. This was on hepatocytes and or splenocytes ex with doses of anti-CD95 JO2 required ASM for induction of apoptosis B whereas high doses the for ASM. To confirm that ASM the in apoptosis and to of genes in the ASM knock-out we ASM to the cells during the stimulation induced by CD95. This apoptosis in hepatocytes or splenocytes from ASM knock-out mice B In addition, we by splenocytes from ASM knock-out mice with an of the ASM or control This also a antibody permitting us to cell cells by with antibody The results confirm that of ASM in splenocytes from ASM knock-out mice is to apoptosis the that the CD95 resistance in cells lacking ASM is of of A induced by a high dose of an antibody also can be applied to cells by primary cross-linking the stimulatory whether a manipulation the requirement of ASM for apoptosis. The results in and that primary cross-linking of anti-CD95 antibody the requirement of ASM for CD95 signaling. A similar has been recently by al. T. Genestier L. Pinkowski M.J. Casturo A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) upon induction of CD95-mediated apoptosis in This that ASM functions to the physiologic CD95 However, a is the ASM is for the function of CD95. In the we provide evidence for a pivotal role of ASM in the of CD95-induced death in injection of anti-CD4 antibodies into mice an of the endogenous CD95/CD95 ligand on T lymphocytes resulting in the death of of the ASM the stimulation of lymphocytes by as indicated by assays as as CD95 injection of PHA in of CD95 ligand on T lymphocytes into the liver and apoptosis of The stimulation of T lymphocytes by PHA was by have been previously N. K. K. K. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. K. Li X. Z. J. Immunol. 1994; 24: PubMed Scopus Google Scholar) and have demonstrated to be on the endogenous CD95/CD95 ligand system. These models provide the to test the function of ASM in an in vivo direct manipulation of the CD95/CD95 ligand system. Such an and activation of the endogenous CD95/CD95 ligand us to by the of anti-CD95 antibodies or the of ASM functions in vitro system. in vivo test an of lymphocyte or apoptosis in ASM knock-out a crucial function of ASM for CD95-induced apoptosis. Our in vivo results confirm the of several (11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar, R. Rippo M.R. Schuchman E.H. Testi R. J. Exp. Med. 1998; 187: 897-902Crossref PubMed Scopus (137) Google Scholar) that have an important function for ASM in CD95-triggered apoptosis. studies (11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar) an activation of ASM upon stimulation of CD95 or the tumor necrosis factor receptor and the of the ASM with apoptosis induced by was recently demonstrated that or membrane targeting of the of that ceramide is of the in the apoptotic C. Fuks Z. Kolesnick R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). A using imipramine, a inhibitor of the ASM, supported the idea that ASM is involved in CD95-mediated apoptosis (20Brenner B. Ferlinz K. Weller M. Grassmé H. Koppenhoefer U. Dichgans J. Sandhoff K. Lang F. Gulbins E. Cell Death Differ. 1998; 5: 29-37Crossref PubMed Scopus (116) Google Scholar). a genetic i.e. B lymphocytes from an ASM-deficient Niemann-Pick type A De-Maria et al. (19De-Maria R. Rippo M.R. Schuchman E.H. Testi R. J. Exp. Med. 1998; 187: 897-902Crossref PubMed Scopus (137) Google Scholar) an important role for ASM in CD95 signaling. However, on ex vivo hepatocytes and splenocytes as as the on of anti-CD95 JO2 in mice also that ASM is for CD95-induced death. The requirement of ASM for seems to on the and the of the applied Our provide an for the of Cock et al. (21Boesen-de Cock J.G. Tepper A.D. de Vries E. van-Blitterswijk W.J. Borst J. J. Biol. Chem. 1998; 273: 7560-7565Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and et al. T. Genestier L. Pinkowski M.J. Casturo A. Nicholas S. Mogil R. Paris F. Fuks Z. Schuchman E.H. Kolesnick R.N. Green D.R. J. Biol. Chem. 2000; 275: 8657-8663Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) apoptosis in lymphocytes for ASM. These studies applied high doses of a anti-CD95 or anti-CD95 Our data show that these the requirement of ASM for CD95 and the physiological of ASM for CD95 signaling. an for the function of ASM in receptor that the enzyme membrane by of ceramide Those of the membrane may rapid and the This also the requirement of ASM for CD95-triggered death in in vivo as as in in vitro studies with doses of anti-CD95. However, high or anti-CD95 antibodies may endogenous membrane by ASM. Such a is strongly supported by a from that ASM of CD95 and ASM seems to provide the conditions for via CD95. A role for ASM in a of the receptor in the membrane required for also the that receptors with different functions activate ASM. Those receptors CD95 (11Cifone M.G. De-Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (597) Google Scholar, 12Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 13Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (433) Google Scholar), CD28 (14Boucher L.M. Wiegmann K. Futterer A. Pfeffer K. Machleidt T. Schütze S. Mak T.W. Krönke M. J. Exp. Med. 1995; 181: 2059-2068Crossref PubMed Scopus (190) Google Scholar), CD5 (15Simarro M. Calvo J. Vila J.M. Places L. Padilla O. Alberoloa-Ila J. Vives J. Lozano F. J. Immunol. 1999; 162: 5149-5155PubMed Google Scholar), intercellular adhesion molecule (16Ni H.T. Deeths M.J. Li W. Mueller D.L. Mescher M.F. J. Immunol. 1999; 162: 5183-5189PubMed Google Scholar), and the A further H. Gulbins E. B. Ferlinz K. Sandhoff K. K. Lang F. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that ASM is required for of in However, data the that stimuli activate ASM in different cell compartments resulting in ceramide release ASM a different function of its different in the Our results on ex vivo hepatocytes or splenocytes show that of ASM the to anti-CD95 JO2 by However, the in vivo anti-CD95 JO2 an This that ASM amplifies CD95 in This be by the that a of the liver be or death can be Our data the of ASM knock-out mice develop an or ASM knock-out mice are of mice with a in the by in which also develop or A. D.L. J. 1996; PubMed Scopus Google Scholar). CD95 may have of apoptosis and ASM. data that ASM deficiency results in a of apoptosis in the system. In stimuli during of the be by the of ASM than by stimuli cellular In the evidence for an important function of ASM for CD95-triggered apoptosis in vivo as as in vitro and that ASM is required for apoptosis of different cell under physiological conditions. In the two in vivo which the in or that ASM is involved in the of the apoptotic via CD95. The models that induction of apoptosis in hepatocytes by T lymphocytes or death by CD95 and CD95 lymphocytes requires ASM. Because ASM to be in the CD95 data that the enzyme be a target to apoptotic in We C. for and G. and P. for