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J. Biol. Chem., Vol. 275, Issue 35, 27316-27323, September 1, 2000

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CD95-mediated Apoptosis in Vivo Involves Acid Sphingomyelinase^*

Susanne Kirschnek, Francois Paris, Michael Weller^§, Heike Grassmé, Klaus Ferlinz, Andrea Riehle, Zvi Fuks, Richard Kolesnick^¶, and Erich Gulbins

From the Department of Physiology, University of Tuebingen, Gmelinstrasse 5 and ^§ Department of Neurology, University of Tuebingen, Hoppe-Seyler-Strasse 3, 72076 Tuebingen, Germany and the ^¶ Laboratory of Signal Transduction and  Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, April 7, 2000, and in revised form, June 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sphingomyelinases have been implicated in important and diverse cellular functions (1, 2). Sphingomyelinases are characterized by their optimal pH and are divided accordingly into acid, neutral, and basic sphingomyelinase species (1, 2). 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 (3). Genetic deficiency of ASM results in Niemann-Pick disease that is characterized by an accumulation of sphingomyelin in the cell (4). In addition, ASM was recently demonstrated to be secreted upon cellular treatment with inflammatory stimuli (5). 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 (6). 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 (5, 7). A similar dichotomy has been demonstrated for ASM in Caenorhabditis elegans with a secretory and lysosomal enzyme encoded by two different genes (8).

In addition to its function in membrane turnover, ASM has been shown to be stimulated by several receptors including the interleukin-1 receptor (9), the tumor necrosis factor receptor (10), CD95 (11-13), CD28 (14), CD5 (15), and the intercellular adhesion molecule (16). 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 (17) requires ASM. Similarly, ASM plays an indispensable role in the induction of apoptosis in endothelial cells of lipopolysaccharide-challenged mice (18).

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 (11-13) or ligation of the tumor necrosis factor receptor (10). Those studies demonstrate a rapid activation of ASM and a release of ceramide upon CD95 or tumor necrosis factor receptor triggering (10-13). De-Maria et al. (19), 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 (20). 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 (21, 22).

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 vivo conditions, 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 the in 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intravenous Injections-- ASM knock-out mice (23) 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.

Hepatocyte and Splenocyte Cultures ex Vivo-- After carefully mincing the livers, the hepatocytes were rested for 60 min in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES, pH 7.4, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM -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.

Apoptosis Assays-- 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 citrate for 2 min, and labeled for 30 min with FITC-dUTP in the presence of terminal deoxynucleotidyltransferase and a sheep alkaline phosphatase-coupled anti-FITC Fab fragment at 37 °C. The signal was converted by addition of the alkaline phosphatase substrate (fast red tablets) and analyzed by light microscopy. At least 300 cells were counted to determine apoptosis. Experiments were performed in triplicate.

Apoptosis of splenocytes or PBLs was determined by FITC-annexin staining according to the manufacturer's instructions (Roche Molecular Biochemicals). Apoptosis of Jurkat cells co-incubated with PBLs from anti-CD4 GK1.5-injected mice was measured by DNA fragmentation of [³H]thymidine-labeled Jurkat cells as described previously (15).

Determination of Surface Antigens by Flow Cytometry-- CD95, CD95 ligand, CD3, or CD4 were measured on Ficoll-purified PBLs by incubation of each with 1 µg/ml anti-CD95 JO2, anti-CD95 ligand Kay 10 (PharMingen), anti-CD3 145-2C11 (PharMingen), or anti-CD4 GK1.5, respectively, followed by the appropriate FITC-labeled secondary Ig for 45 min at 4 °C. All antibodies were diluted in 132 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄, 2% fetal calf serum, and 0.2% NaN₃ supplemented with 10 mM phenanthroline (Sigma) for CD95 or CD95 ligand. After completion of CD95 or CD95 ligand staining, cells were incubated as above with the anti-CD3 145-2C11 and a phycoerythrin-coupled anti-hamster antibody to detect T lymphocytes. All samples were analyzed by flow cytometry.

Acid Sphingomyelinase Activity-- Cells were lysed in ice-cold 50 mM Tris, pH 7.4, 10 mM bacitracin, 1 mM benzamidine, 10 mM Na₃VO₄, 10 µg/ml each aprotinin and leupeptin, 0.1 mg/ml soybean trypsin inhibitor, and 0.2% Triton X-100; sonicated three times for 10 s each; and centrifuged at 600 × g for 5 min. An equal amount of 50 mM Tris, pH 7.4, 3% Nonidet P-40, 1% Triton X-100, 1 mM Na₃VO₄, 100 µg/ml each aprotinin/leupeptin (lysis buffer) was added to the supernatants. ASM was immunoprecipitated using a goat anti-ASM antiserum and protein A/G-coupled-agarose (Santa Cruz Biotechnology, Inc.). The immunoprecipitates were washed three times each in lysis buffer and 50 mM sodium acetate, pH 5.0, 0.2% Triton X-100, 1 mM Na₃VO₄, and 10 µg/ml aprotinin/leupeptin and incubated with [¹⁴C]sphingomyelin (0.5 µCi/sample, 54.5 mCi/mmol, NEN Life Science Products) in 250 mM sodium acetate, pH 5.0, 1.3 mM EDTA, 0.05% Nonidet P-40 (assay buffer) at 37 °C for 30 min. [¹⁴C]Sphingomyelin was dried and solubilized by a 10-min bath sonication in assay buffer. Reactions were finally extracted with 800 µl of CHCl₃/CH₃OH (2:1, v/v) and 250 µl of H₂O, and radioactivity in the upper phase was determined by liquid scintillation counting.

Cellular Tyrosine Phosphorylation-- Cells were stimulated with 4 µg/ml anti-CD4 GK1.5 or 50 µg/ml PHA for the indicated times. The doses were calculated from the in vivo injections on the assumption that the serum volume is approximately 10% of the body weight (~25 g) of the mouse. Cell stimulation was terminated by lysis in 25 mM HEPES, pH 7.4, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 125 mM NaCl, 10 mM each sodium fluoride, Na₃VO₄, and sodium pyrophosphate, and 10 µg/ml aprotinin/leupeptin. The postcentrifugation supernatants were added to 5× SDS sample buffer and 5% -mercaptoethanol, and proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and developed using 0.5 µg/ml monoclonal 4G10 antibody (Upstate Biotechnology Inc.) followed by an alkaline phosphatase-conjugated anti-mouse antibody (Santa Cruz Biotechnology, Inc.) and chemiluminescence (Tropix).

Serum Alanine Aminotransferase-- ALT concentrations in the serum prior to or 24 h after PHA injection were determined using the ALT-catalyzed reaction of alanine and -ketoglutarate to pyruvate and glutamate. Reduction of pyruvate with NADH and H⁺ to lactate and NAD⁺ by lactate dehydrogenase was determined photometrically.

Bone Marrow Transplantation-- ASM knock-out mice were irradiated with an intensity of 12 gray and transplanted 2 days later with 2 × 10⁵ bone marrow cells obtained from a syngenic normal mouse. The bone marrow cells were prepared from the tibia and femur, washed, and injected intravenously. The immune system was allowed to recover for 8 weeks prior to experimentation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ASM Is Required for CD95-mediated Hepatocyte Apoptosis in Vivo-- To investigate the physiological function of ASM for CD95-triggered death, we tested CD95-triggered apoptosis of hepatocytes and peripheral blood lymphocytes upon indirect up-regulation of the endogenous CD95/CD95 ligand system. In the first model, we investigated an autoimmune hepatitis-like syndrome, i.e. we tested apoptosis of hepatocytes by T lymphocytes stimulated by intravenous PHA injection (24). Injection of PHA into mice has been shown to trigger an up-regulation of CD95 ligand expression on CD3⁺ lymphocytes, intrahepatic accumulation of those lymphocytes, and apoptosis of hepatocytes via constitutively expressed CD95 (24). Hepatocyte apoptosis in this model depends on the expression of functional CD95 and CD95 ligand because LPR or GLD mice are resistant to the effects of PHA injections and do not develop hepatitis (24). Here, we show that injection of 15 µg/g PHA into normal mice induces significant apoptosis of hepatocytes (Fig. 1A) correlating with an increase of serum ALT (Fig. 1B). In marked contrast, ASM knock-out mice were completely resistant to PHA injection and displayed neither apoptosis in the liver (Fig. 1A) nor an increase of the ALT levels (Fig. 1B) upon PHA injection. The requirement for CD95 in the development of hepatocyte apoptosis and hepatitis is illustrated by injection of LPR C57/BL6 mice (kindly provided by Dr. T. Möröy) with PHA. These animals were completely resistant to intravenous PHA injection.

View larger version (99K): [in this window] [in a new window]   Fig. 1.   Hepatocyte apoptosis in vivo after intravenous PHA injection requires expression of ASM. Intravenous injection of 15 µg/g PHA into normal mice (+/+, n = 6) induces hepatocyte apoptosis (A) and the development of a hepatitis as indicated by the increase in ALT levels (B). In contrast, ASM knock-out (/) or LPR mice (n = 6 each) are almost completely resistant to PHA injection and do not show significant signs of hepatocyte apoptosis or hepatitis. Hepatocyte apoptosis was determined by TUNEL. Sections are shown at two magnifications. In B, bars indicate mean ± S.D. of six experiments. *, p  0.05 by Student's t test. C-E, transplantation of ASM knock-out mice (n = 5) with bone marrow obtained from normal mice was confirmed by measuring ASM activity in blood samples (C). The mice do not overcome resistance to PHA (E) and do not show hepatocyte apoptosis (D) or an increase of ALT in the blood serum (E). These experiments indicate that the resistance of hepatocytes in ASM knock-out mice to PHA injection is not because of an insufficient lymphocyte stimulation. Further, injection of 15 µg/g PHA induces very similar up-regulation of CD95 ligand on peripheral lymphocytes isolated from normal and ASM knock-out mice 8 h after injection (n = 7 each, F). CD95 ligand on ASM knock-out lymphocytes was functional as evidenced by the ability to kill co-cultured CD95-positive Jurkat cells but not a CD95-resistant Jurkat clone (G). Apoptosis was determined by FITC-annexin staining and fluorescence-activated cell sorter analysis. Bars indicate mean ± S.D. of three independent experiments. In accordance, PHA (50 µg/ml) stimulation of freshly isolated ex vivo peripheral blood lymphocytes from normal or ASM-deficient mice does not reveal a significant difference in the pattern of tyrosine phosphorylation (n = 3, H).

To exclude an insufficient primary stimulation of PBLs with PHA in the ASM knock-out mice as reason for the resistance of those mice to CD95-triggered hepatitis, we transplanted ASM knock-out mice with bone marrow from normal mice. The take of the transplant was confirmed by measuring ASM activity in peripheral blood lymphocytes (Fig. 1C). The transplantation did not alter the resistance of ASM knock-out mice to PHA, and we did not detect significant hepatocyte apoptosis or hepatitis (Fig. 1, D and E). This study indicates that the expression or lack thereof of ASM in hepatocytes rather than in T lymphocytes determines the sensitivity to PHA. In additional control experiments, we have shown that the expression of CD95 ligand on PBLs did not differ between normal or ASM knock-out mice 8 h after injection of PHA (Fig. 1F). Co-culture experiments of lymphocytes from PHA-injected normal or ASM knock-out mice with CD95-sensitive or -resistant Jurkat cells confirm the functional expression of CD95 ligand on PBL from ASM knock-out mice after PHA injection (Fig. 1G). Finally, we detected no differences in the pattern of PHA-triggered tyrosine phosphorylation in freshly isolated PBLs from normal or ASM knock-out mice (Fig. 1H), indicating a sufficient activation of PBLs from ASM knock-out mice by PHA.

ASM Expression Is Necessary for in Vivo Depletion of Activated T Lymphocytes-- The second model was based on the finding that injection of anti-CD4 antibodies or ligation of CD4 by other molecules, in particular the human immunodeficiency virus protein gp120, is known to induce apoptosis in PBLs by up-regulation of CD95 and CD95 ligand expression (25, 26). LPR mice are resistant to anti-CD4-triggered apoptosis of PBLs, indicating that this as an obligate function of the CD95/CD95 ligand system (25, 26). We, therefore, injected 0.4 µg of anti-CD4 antibodies GK1.5/g into normal control or ASM knock-out mice. GK1.5 injection rapidly induced apoptosis of peripheral blood lymphocytes from normal mice whereas significant apoptosis was not detected in lymphocytes from ASM-deficient mice (Fig. 2A). Apoptosis of PBLs correlated with a marked reduction of CD3⁺ and CD4⁺ lymphocytes in normal mice, which was not observed in the ASM knock-out mice (Fig. 2B). Similar to the ASM knock-out mice, we did not detect any apoptosis of PBLs or depletion of CD3⁺- and CD4⁺-positive lymphocytes in LPR mice confirming the role of the CD95/CD95 ligand system in this experimental setting (Fig. 2, A and B). To exclude an insufficient primary response of ASM knock-out mice to the injected anti-CD4 GK1.5 as a reason for resistance, we tested the expression of CD95 on PBLs before and after injection of the anti-CD4 antibodies. These studies (Fig. 2C) reveal the same degree of CD95 up-regulation on the PBLs of normal control and ASM knock-out mice. Further, anti-CD4 GK1.5 (4 µg/ml) induces an almost identical tyrosine phosphorylation pattern of cellular proteins in freshly isolated PBLs from ASM knock-out and normal mice (Fig. 2D).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   ASM is required for CD95-mediated apoptosis of peripheral lymphocytes in vivo. A, intravenous injection of 0.4 µg/g anti-CD4 GK1.5 antibodies results in apoptosis of peripheral blood lymphocytes from normal mice but not from ASM-deficient mice (n = 7 each). The role of CD95 in anti-CD4 GK1.5-triggered death of peripheral lymphocytes is evidenced by the resistance of CD95-deficient LPR mice to this antibody (n = 5). Apoptosis was determined by FITC-annexin staining followed by flow cytometry. Bars indicate mean ± S.D. *, p  0.05, Student's t test. B, normal mice (n = 7, ASM+/+) but not ASM knock-out mice (n = 7, ASM/) reveal a marked reduction of CD3+ as well as CD4+ lymphocytes in the blood. PBLs were purified with Ficoll and stained with 1 µg/ml each anti-CD3 145-2C11 or anti-CD4 GK1.5 followed by FITC-labeled secondary antibodies and flow cytometry. Numbers are the mean ± S.D. C, intravenous injection of 0.4 µg/g anti-CD4 GK1.5 antibodies results in up-regulation of CD95 on peripheral lymphocytes in normal and ASM knock-out mice (n = 7 each). D, in accordance, freshly isolated lymphocytes from both mouse types respond to CD4 triggering with tyrosine phosphorylation of a distinct set of proteins (n = 3). This demonstrates that the applied dose of anti-CD4 stimulates lymphocytes via CD4 regardless of whether they express (+/+) or lack (/) ASM and indicates that the deficiency in apoptosis via CD95 is not because of insufficient primary stimulation through CD4.

ASM Amplifies CD95 Signaling-- To further confirm the function of the ASM in CD95-triggered apoptosis, we intravenously injected increasing doses of the anti-CD95 JO2 antibody into normal and ASM knock-out mice. Injection of this antibody has been previously shown to result in acute liver failure and rapid death of the mice (27). In wild-type mice, anti-CD95 JO2 induced death over a narrow concentration range of 0.03-0.12 µg/g, consistent with an amplification mechanism of action (Fig. 3A and data not shown). ASM knock-out mice were resistant to the injection of anti-CD95 up to 0.12 µg/g (Fig. 3A). Surprisingly, an increase of the injected antibody dose to 0.2 µg/g anti-CD95 JO2 resulted in the death of all ASM knock-out mice, very similar to normal mice injected with the same dose of the antibody. This suggests that ASM is required for apoptosis via CD95 upon stimulation with lower non-saturating doses of anti-CD95 JO2 whereas higher doses of the antibody override the requirement of ASM for CD95-triggered death.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   ASM knock-out mice and isolated hepatocytes or splenocytes ex vivo are relatively resistant to anti-CD95. A, intravenous injection of 0.12 µg/g anti-CD95 JO2 into normal or ASM knock-out mice (n = 7 each) results in rapid death of normal mice whereas death ensues in only 14% of ASM knock-out mice. Increasing the dose of the injected anti-CD95 JO2 antibody to 0.2 µg/g overcomes the resistance of ASM knock-out mice. Anti-CD95 JO2 (8-h incubation each) induces apoptosis of freshly isolated hepatocytes (B) or activated splenocytes (C) from normal mice (open circles) whereas cells from ASM knock-out mice (filled circles) are relatively resistant to application of anti-CD95 JO2. Application of high doses of anti-CD95 JO2 overcomes resistance in ASM-deficient cells. Likewise, addition of pure ASM (107 units/ml, open triangles) to cells lacking ASM or transfection of ASM knock-out splenocytes with an expression vector for ASM restores apoptosis (open squares). Transfection of vector control does not alter resistance to CD95 (filled squares). Artificial cross-linking (D and E) of the antibody by coupling to plastic plates overrides the requirement of ASM for apoptosis, revealing that ASM is not obligatory for apoptosis. Splenocytes were stimulated daily with 10 µg/ml PHA and 2 units/ml interleukin-2 or after T-cell depletion with 100 ng/ml anti-CD40 and 2 µg/ml anti-Ig. The expression of CD95 on the splenocytes after the 2-day stimulation period was confirmed in each single experiment by flow cytometry (data not shown). Bars are the mean ± S.D. of six (hepatocytes) or five (splenocytes) in independent experiments performed in duplicate (*, p  0.05, Student's t test). Apoptosis was determined by TUNEL assay for the hepatocytes and FITC-annexin staining for the lymphocytes.

This hypothesis was validated on freshly isolated hepatocytes and PHA/interleukin-2 or anti-CD40/anti-Ig activated splenocytes ex vivo. Stimulation with lower doses of anti-CD95 JO2 required ASM for efficient induction of apoptosis (Fig. 3, B and C) whereas high doses abolished the need for ASM. To confirm that ASM mediates the defect in apoptosis and to exclude any alterations of other genes in the ASM knock-out cells, we added pure ASM (10⁷ units/ml) to the cells during the stimulation induced by CD95. This maneuver restored apoptosis in hepatocytes or splenocytes from ASM knock-out mice (Fig. 3, B and C). In addition, we transiently transfected by electroporation splenocytes from ASM knock-out mice with an expression vector of the ASM (pJK-asm) or control (pJK). This vector also contains a Myc-tagged single chain antibody permitting us to fluorescence-activate cell sort transfected cells by staining with FITC-labeled anti-Myc 9E10 antibody (1 µg/ml). The results confirm that expression of ASM in splenocytes from ASM knock-out mice is sufficient to restore apoptosis (Fig. 3C), virtually excluding the possibility that the CD95 resistance in cells lacking ASM is because of alterations of other proteins.

A strong stimulus induced by a high dose of an activating antibody also can be applied to cells by primary cross-linking the stimulatory antibody. We, therefore, tested whether such a manipulation alters the requirement of ASM for apoptosis. The results displayed in Fig. 3, D and E, reveal that primary cross-linking of anti-CD95 antibody overrides the requirement of ASM for CD95 signaling. A similar dose-response curve has been recently observed by Lin et al. (22) upon induction of CD95-mediated apoptosis in splenocytes. This suggests that ASM functions to potentiate the physiologic CD95 signal. However, if a strong stimulus is applied, the ASM is dispensable for the biological function of CD95.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we provide evidence for a pivotal role of ASM in the mediation of CD95-induced death in vivo. Intravenous injection of anti-CD4 antibodies into mice induces an up-regulation of the endogenous CD95/CD95 ligand system on T lymphocytes resulting in the death of those cells. Deficiency of the ASM did not affect the stimulation of lymphocytes by CD4 as indicated by assays measuring early tyrosine phosphorylation as well as late CD95 up-regulation. Furthermore, injection of PHA resulted in up-regulation of CD95 ligand on T lymphocytes migrating into the liver and inducing apoptosis of CD95-positive hepatocytes. The stimulation of T lymphocytes by PHA was not affected by PHA. Both systems have been previously described (24, 25) and have demonstrated to be dependent on the endogenous CD95/CD95 ligand system. These models provide the capability to test the function of ASM in an in vivo system without direct manipulation of the CD95/CD95 ligand system. Such an indirect up-regulation and activation of the endogenous CD95/CD95 ligand system permitted us to avoid potential pitfalls created by the use of anti-CD95 antibodies or even the analysis of ASM functions in an in vitro system. Both in vivo test systems reveal an almost complete absence of lymphocyte or hepatocyte apoptosis in ASM knock-out mice, indicating a crucial function of ASM for CD95-induced apoptosis.

Our in vivo results confirm the reports of several groups (11-13, 19) that have established an important function for ASM in CD95-triggered apoptosis. Several studies (11-13) showed an activation of ASM upon stimulation of CD95 or the tumor necrosis factor receptor and correlated the activity of the ASM with apoptosis induced by those receptors. Furthermore, it was recently demonstrated that overexpression or membrane targeting of caspase 8 induces the generation of ceramide, indicating that ceramide is part of the initiator phase in the apoptotic signaling pathway (28). A study using imipramine, a pharmacological inhibitor of the ASM, supported the idea that ASM is involved in CD95-mediated apoptosis (20). Using a genetic approach, i.e. B lymphocytes from an ASM-deficient Niemann-Pick type A patient, De-Maria et al. (19) confirmed an important role for ASM in CD95 signaling. However, our experiments on ex vivo hepatocytes and splenocytes as well as the experiments on increased dosing of anti-CD95 JO2 in mice also suggest that ASM is not absolutely necessary for CD95-induced death. The requirement of ASM for receptor-mediated signaling seems to critically depend on the amount and the aggregation status of the applied agonist stimulus. Our data, therefore, provide an explanation for the findings of Cock et al. (21) and Lin et al. (22) who observed apoptosis in lymphocytes deficient for ASM. These studies applied high doses of a pentamer-forming IgM anti-CD95 or already cross-linked anti-CD95 JO2. Our data show that these maneuvers override the requirement of ASM for efficient CD95 signaling and mask the physiological significance of ASM for CD95 signaling. Thus, an intriguing concept for the function of ASM in receptor signaling suggests that the enzyme modifies membrane fluidity by formation of ceramide microdomains. Those structural alterations of the membrane morphology may then allow rapid and efficient signaling inside the cell. This model also explains the requirement of ASM for CD95-triggered death in our in vivo experiments as well as in in vitro studies with low doses of anti-CD95. However, high concentrations or even aggregated anti-CD95 antibodies may replace endogenous membrane changes triggered by ASM. Such a concept is strongly supported by a recent study from our group indicating that ASM mediates clustering/capping of CD95 and CD40. Thus, ASM seems to provide the conditions necessary for efficient signaling via CD95. A role for ASM in a stimulation-dependent re-assembly of the receptor molecules in the membrane required for signaling might also integrate the finding that many receptors with completely different functions activate ASM. Those receptors include CD95 (11-13), CD28 (14), CD5 (15), intercellular adhesion molecule (16), CD40, CD48, and the B-cell receptor.² A further study (29) demonstrated that ASM is required for internalization of pathogenic bacteria in human epithelial cells.

However, our data certainly do not exclude the possibility that other stress stimuli activate ASM in different cell compartments resulting in ceramide release where ASM causes a completely different biological function because of its different location in the cell.

Our results on ex vivo hepatocytes or splenocytes show that expression of ASM increases the sensitivity to anti-CD95 JO2 by approximately 10-fold. However, the in vivo experiments injecting anti-CD95 JO2 reveal only an approximately 4-fold concentration difference. This suggests that ASM amplifies CD95 signaling in vivo. This amplification phenomenon might be easily explained by the long known finding that a large part of the liver must be destroyed before any clinical symptoms or even death can be observed.

Our data raise the question of why ASM knock-out mice do not develop an LPR or GLD phenotype. First, ASM knock-out mice are reminiscent of other mice with a defect in the signaling machinery triggered by CD95, in particular CrmA transgenic mice, which also do not develop T-cell hyperplasia or autoantibodies (30). Thus, CD95 may have additional effects independent of apoptosis and ASM. Second, our data suggest that ASM deficiency results in a relative but not complete defect of apoptosis in the immune system. In particular, prolonged stimuli acting during development of the immune system might be less affected by the lack of ASM than by acute stress stimuli requiring full cellular activation.

In summary, the present study provides evidence for an important function of ASM for CD95-triggered apoptosis in vivo as well as in vitro and suggests that ASM is required for apoptosis of different cell types under physiological conditions. In particular, the two in vivo models, which closely mimic the situation in autoimmune diseases or viral infections indicate that ASM is crucially involved in the mediation of the apoptotic signal via CD95. The models prove that induction of apoptosis in hepatocytes by autoreactive T lymphocytes or self-induced death by CD95 and CD95 ligand-positive lymphocytes requires ASM. Because ASM does not appear to be redundant in the CD95 signaling pathway, our data suggest that the enzyme might be a preferred target to pharmacologically down-regulate, but not completely inhibit, apoptotic processes in some pathologic situations.

	ACKNOWLEDGEMENTS

We thank Dr. C. Belka for excellent advice and G. von Kürthy and P. Frey for expert technical assistance.

	FOOTNOTES

^* The study was supported in part by Deutsche Forschungsgemeinschaft Grant GU 335/2-3, grants from the Association International Cancer Research, the Interdisciplinary Center for Clinical Research (to E. G.) and the Mildred Scheel Stiftung Grant 1502/5-2 (to M. W. and E. G.), and by National Institutes of Health Grants CA52462 (to Z. F.) and CA42385 (to R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-7071-2972196; Fax: 49-7071-293073; E-mail: erich.gulbins@uni- tuebingen.de.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M002957200

² E. Gulbins, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ASM, acid sphingomyelinase; FITC, fluorescein isothiocyanate; TUNEL, TdT-mediated dUTP-x nick end labeling; PBL, peripheral blood lymphocyte; ALT, alanine aminotransferase; PHA, phytohemagglutinin; LPR, lymphoproliferative disease; GLD, generalized lymphoproliferative disease.

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