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Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of β1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of β1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections. CA - Grassmé

BACKGROUND/AIMS: Major depressive disorder is one of the most common diseases in western countries. The disease is mainly defined by its psychiatric symptoms. However, the disease has also many symptoms outside the central nervous system, in particular cardiovascular symptoms. Recent studies demonstrated that the acid sphingomyelinase/ceramide system plays an important role in the development of major depressive disorder and functions as a target of antidepressants.; METHODS: Here, we investigated (i) whether ceramide accumulates in endothelial cells in the neurogenetic zone of the hippocampus after glucocorticosterone-mediated stress, (ii) whether ceramide is released into the extracellular space of the hippocampus and (iii) whether extracellular ceramide inhibits neuronal proliferation. Ceramide was determined in endothelial cell culture supernatants or extracellular hippocampus extracts by a kinase assay. Endothelial ceramide in the hippocampus was analyzed by confocal microscopy of brain sections stained with Cy3-labelled anti-ceramide antibodies and FITC-Isolectin B4. Neuronal proliferation was measured by incubation of pheochromocytoma neuronal cells with culture supernatants and extracellular hippocampus extracts.; RESULTS: Treatment of cultured endothelial cells with glucocorticosterone induces a release of ceramide into the supernatant. Likewise, treatment of mice with glucocorticosterone triggers a release of ceramide into the extracellular space of the hippocampus. The release of ceramide is inhibited by concomitant treatment with the antidepressant amitriptyline, which also inhibits the activity of the acid sphingomyelinase. Studies employing confocal microscopy revealed that ceramide is formed and accumulates exclusively in endothelial cells in the hippocampus of stressed mice, a process that was again prevented by co-application of amitriptyline. Ceramide released in the culture supernatant or into the extracellular space of the hippocampus reduced proliferation of neurons in vitro.; CONCLUSION: The data suggest a novel model for the pathogenesis of major depressive disorder, i.e. the release of ceramide-enriched microvesicles from endothelial cells that negatively affect neuronal proliferation in the hippocampus, but may also induce cardiovascular disease and other systemic symptoms of patients with major depressive disorder. 2016 The Author(s) Published by S. Karger AG, Basel. OA gold - CA Gulbins

Tuberculosis is one of the most serious infectious diseases worldwide. The initial pulmonal localization of the pathogens often develops into systemic infection with high lethality. We investigated the role of the mammalian neutral sphingomyelinase (Nsm)/ceramide system in systemic infection of mice and murine macrophages with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). Our results demonstrate that BCG infection of RAW cells, a macrophage cell line, results in rapid activation of Nsm but not of acid sphingomyelinase (Asm). Activation of Nsm is associated with a massive release of superoxide. Genetic knock-down of Nsm in RAW cells prevented superoxide production upon BCG infection. Superoxide suppressed autophagy in BCG-infected macrophages in vitro and in vivo: Knock-down of Nsm or inhibition of superoxide restored autophagy in macrophages and increased killing of intracellular bacteria upon BCG infection. Most importantly, autophagy was also massively increased in Nsm-heterozygous mice, protecting these mice from systemic BCG infections, granuloma development, and chronic infections of liver and spleen. These findings indicate that the Nsm/ceramide system plays a role in protecting mice against systemic tuberculosis by preventing superoxide-mediated inhibition of autophagy.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the deadliest and most important infectious diseases worldwide. The sphingomyelinase/ceramide system, which has been shown several times to be a crucial factor in the internalization, processing and killing of diverse pathogens, also modulates the pro-inflammatory response and the state of mycobacteria in macrophages. Both acid and neutral sphingomyelinases are important in this activity. However, studies of the role of sphingomyelinases in TB are still at an early stage.

Background/Aims: Mycobacteria-induced diseases, especially tuberculosis, cause more than 1 million deaths each year, which is higher than any other single bacterial pathogen. Neutral sphingomyelinase 2 (Nsm2) has been implied in many physiological processes and diseases, but the role of Nsm2 in pathogen-host interactions and mycobacterial infections has barely been studied. Methods: We investigated the role of the Nsm2/ceramide system in systemic infection of mice and murine macrophages with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a model for mycobacterial infection. For in vitro assays we isolated bone marrow-derived macrophages from Wildtype mice or Nsm2-heterozygous and investigated the role of Nsm2 for macrophage migration/clustering as well as the involvement of p38 mitogen-activated protein kinases (p38K), c-Jun N-terminal kinase (JNK), β1-integrin and Rac1 activity by Western blot and microscopic studies. For in vivo assays we injected mice intravenously with BCG and analyzed infected tissues for the role of Nsm2-mediated activation of β1-integrin in granuloma formation and bacterial burden. Results: Our results reveal that BCG infection of macrophages results in rapid stimulation of Nsm2. Genetic and pharmacological studies demonstrate that Nsm2 stimulates a signaling cascade via p38K and JNK to an activation of surface β1-integrin and Rac1 that leads to the formation of granuloma-like macrophages clusters in vitro and granuloma in vivo. Heterozygosity of Nsm2 in macrophages or antibody-mediated neutralization of active b1-integrin reduced macrophage clusters in vitro and granuloma formation in vivo. Most importantly, Nsm2 heterozygosity or treatment with neutralizing antibodies against β1-integrin protected mice from systemic BCG infections and chronic infections of the liver and spleen. Conclusion: The findings indicate that the Nsm2/ ceramide system plays an important role in systemic infection of mice with mycobacteria by regulating a signaling cascade via p38K, JNK, b1-integrin and Rac1.

Aims: Staphylococcus aureus plays an important role in sepsis, pneumonia, and wound infections. Acid sphingomyelinase (Asm)-deficient mice are highly susceptible to pulmonary S. aureus infections. Here, we investigated the role of CD44 as a molecule that mediates important aspects of the infection of macrophages with S. aureus. Results: We showed that CD44 activation by S. aureus stimulated Asm via the formation of reactive oxygen species, resulting in ceramide release, clustering of CD44 in ceramide-enriched membrane platforms, CD44/Asm-dependent activation of Rho family GTPases, translocation of phospho-ezrin/radixin/moesin to the plasma-membrane, and a rapid rearrangement of the actin cytoskeleton with cortical actin polymerization. Genetic deficiency of CD44 or Asm abrogated these signaling events and thereby reduced internalization of S. aureus into macrophages by 60–80%. Asm-deficient macrophages also exhibited reduced fusion of phagosomes with lysosomes, which prevented intracellular killi...

Aims: Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils. Results: Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells. Innovation: These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase. Conclusion: These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions. Antioxid. Redox Signal. 22, 1097–1110.

Major depression is one of the most common and severe diseases affecting the world's population. However, the pathogenesis of the disease remains inadequately defined. Previously, a lack of monoaminergic neurotransmitters was the focus of pathophysiological concepts; however, recent concepts focus on a alteration of neurogenesis in the hippocampus. This concept suggests that neurogenesis is decreased in major depression with a rarefication of neuronal networks and a lack of new, immature neurons in the hippocampus, events that may result in the clinical symptoms of major depression. However, molecular targets involved in the pathogenesis of major depression and, in particular, a reduction of neurogenesis, are largely unknown. We have recently discovered that an inhibition of the acid sphingomyelinase/ceramide system mediates the effects of tri- and tetracyclic antidepressants. Moreover, an accumulation of ceramide in the hippocampus results in depression-like symptoms. This suggests the acid sphingomyelinase/ceramide system is very important in the pathogenesis of major depression.

Staphylococcus aureus ( S. aureus ) infections are among the most common and severe infections, garnering notoriety in an era of increasing resistance to antibiotics. It is therefore important to define molecular mechanisms by which this pathogen attacks host cells. Here, we demonstrate that alpha-toxin, one of the major toxins of S. aureus , induces activation of acid sphingomyelinase and concomitant release of ceramide in endothelial cells treated with the toxin. Activation of acid sphingomyelinase by alpha-toxin is mediated via ADAM10. Infection experiments employing alpha-toxin-deficient S. aureus and the corresponding wild-type strain reveal that activation of acid sphingomyelinase in endothelial cells requires alpha-toxin expression by the pathogen. Activation of acid sphingomyelinase is linked to degradation of tight junctions in endothelial cells in vitro , which is blocked by pharmacological inhibition of acid sphingomyelinase. Most importantly, alpha-toxin induces severe degradation of tight junctions in the lung and causes lung edema in vivo , which is prevented by genetic deficiency of acid sphingomyelinase. These data indicate a novel and important role of the acid sphingomyelinase/ceramide system for the endothelial response to toxins and provide a molecular link between alpha-toxin and the degradation of tight junctions. The data also suggest that inhibition of acid sphingomyelinase may provide a novel treatment option to prevent lung edema caused by S. aureus alpha-toxin.

Significance: Tuberculosis (TB), which is caused by Mycobacterium tuberculosis, is one of the most important infections worldwide. The sphingomyelinase/ceramide system, which has been shown to be a crucial factor in internalizing and killing various pathogens, modulates both the proinflammatory response and the state of mycobacteria in macrophages. However, studies about the role of sphingomyelinases in TB are still at an early stage. Recent Advances: Recent studies elucidated several roles of sphingomyelinases in manipulating mycobacterial infections. On the one hand, acid sphingomyelinase (Asm) promotes the fusion of bacteria-containing phagosomes and lysosomes, whereas on the other hand, Asm-derived ceramide induces cell death. Neutral sphingomyelinase (Nsm) enhances the release of reactive oxygen species, which suppress autophagy in infected macrophages in vitro and in vivo, allowing the pathogen to survive within macrophages. These findings indicate that the sphingomyelinase/ceramide system plays an important role in the attack of mycobacteria against the host. Critical Issues: Autophagy is a main strategy of mycobacterial clearance in TB, but the relevant mechanisms are still unknown. Additionally, there are indications that both Asm and Nsm are crucially involved in the formation of granulomas, which are a hallmark and a special structure of TB. However, very few findings have yet been published. Future Directions: Additional studies of the Nsm/ceramide system, which contributes to the resistance or susceptibility, respectively, of the host to mycobacterial infections, will detect currently unknown molecular mechanisms. Because inhibitors of Nsm already exist, targeting Nsm may be a novel approach to developing treatment options for mycobacterial infections. Antioxid. Redox Signal. 28, 935-948.

Background: Cystic fibrosis (CF), the most common autosomal recessive disorder in Western countries, is characterized by chronic pulmonary inflammation, reduced mucociliary clearance, and increased susceptibility to infection. Our studies using Cftr-deficient mice and human CF specimens showed that ceramide accumulates in CF lungs and mediates increased cell death, susceptibility to infections, and inflammation. Methods: We used Cftr-deficient and syngenic wildtype mice as well as Cftr-deficient mice heterozygous for the acid sphingomyelinase. We determined activation and topology of inflammasome components as well as expression of tight junction proteins by confocal microscopy, western blotting and ELISA. Results: We demonstrate an upregulation and membrane recruitment of the adapter protein apoptosis-associated speck-like protein (Asc), a major component of the inflammasome, and caspase 1, an activation of Jun N-terminal kinase as well as an altered distribution and a degradation of the tight junction proteins ZO-1, ZO-2 and Occludin in lungs of CF mice. All of these events are abrogated in CF mice that are heterozygous for the acid sphingomyelinase and, therefore, show normal levels of ceramide in their lungs. These alterations indicate an activation of the inflammasome by ceramide in the lungs of CF mice. Consistent with this notion, we observe a normalization of the increased levels of the cytokines IL-1β and KC/IL-8 in lungs of CF mice upon treatment with caspase 1 inhibitors. Conclusion: Our data suggest a signaling cascade from ceramide via the inflammasome to caspase 1, the release of cytokines and an alteration of tight junction proteins in CF epithelia.

Background: Pulmonary Staphylococcus aureus (S. aureus) infections occur early in a high percentage of cystic fibrosis (CF) patients and it is believed that these infections facilitate further colonization of CF lungs with Pseudomonas aeruginosa (P. aeruginosa). Previous studies demonstrated a marked reduction of sphingosine in tracheal and bronchial epithelial cells in CF compared to wild type mice, while ceramide is massively increased in CF mice. Methods: We investigated the effect of C18-sphingosine and C16-ceramide on S. aureus in vitro. Based on our results we performed pulmonary infections with S. aureus and tested the influence of sphingosine inhalation. Results: In vitro incubation of S. aureus with C18-sphingosine rapidly killed S. aureus, while C16-ceramide did not affect bacterial survival, but abrogated the effect of C18-sphingosine when applied together. The in vivo infection experiments revealed a high susceptibility of CF mice to pulmonary infection with S. aureus. Inhalation of C18-sphingosine rescued CF mice from pulmonary infections with different clinical S. aureus isolates, including a methicillin-resistant S. aureus (MRSA) strain. Conclusions: Our data indicate that the imbalance between ceramide and sphingosine in the CF respiratory tract prevents killing of S. aureus and causes the high susceptibility of CF mice to pulmonary S. aureus infections.

Background: Melatonin has been shown to have antidepressive effects. We tested whether melatonin inhibits the acid sphingomyelinase/ceramide system and mediates its antidepressive effects via inhibition of the acid sphingomyelinase and a reduction of ceramide in the hippocampus. Antidepressants such as amitriptyline and fluoxetine were previously shown to inhibit the acid sphingomyelinase/ceramide system, which mediates neurogenesis and behavioral changes induced by these drugs. Methods: The effect of melatonin on the activity of the acid sphingomyelinase prior to and after treatment with melatonin was determined in cultured neurons and in vivo in the hippocampus of mice by measuring the consumption of [14C] sphingomyelin. Ceramide was measured by DAG kinase assay and fluorescence microscopy of the hippocampus and of cultured neurons. Neurogenesis in the hippocampus was analyzed by in vivo labeling with bromodeoxyuridine. Behavior was assessed in standardized tests. Results: Melatonin treatment inhibited acid sphingomyelinase in vitro in cultured pheochromocytoma cells and in vivo in the hippocampus, which resulted in a reduction of ceramide in vitro and in vivo. The inhibition of the acid sphingomyelinase/ceramide system translated into increased neurogenesis in glucocorticosterone-stressed mice after treatment with melatonin, an effect that is abrogated in acid sphingomyelinase-deficient mice. Likewise, melatonin improved the depressive behavior of stressed mice, a therapeutic effect that was again absent in acid sphingomyelinase-deficient animals. Conclusion: These data indicate that the antidepressive effects of melatonin as well as the induction of neurogenesis triggered by this drug are mediated by an inhibition of the acid sphingomyelinase/ceramide system. This is the first study to identify melatonin as an inhibitor of the acid sphingomyelinase.

Cystic fibrosis is one of the most common autosomal recessive disorders, at least in Western countries. Patients with cystic fibrosis exhibit many symptoms, the most important of which are chronic inflammation and bacterial infections of the lung. Cystic fibrosis is caused by mutations of the cystic fibrosis conductance regulator (CFTR) gene; however, the molecular mechanisms leading to the clinical manifestations of cystic fibrosis are still unclear. Here we discuss recent findings related to the role of sphingolipids, in particular ceramide, in cystic fibrosis and the bacterial infections associated with that disease. Ceramide accumulates in the lungs of cystic fibrosis mice and causes pulmonary inflammation, infection, and cell death, events that are corrected by the genetic deletion or pharmacological inhibition of acid sphingomyelinase; this inhibition normalizes ceramide concentrations in murine models of cystic fibrosis. Initial clinical studies suggest that pharmacological inhibition of acid sphingomyelinase may be a novel strategy for treating patients with cystic fibrosis.

Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhoeae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.

Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr ⁻/⁻Smpd1⁺/⁻ mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis. © 2008 Nature Publishing Group.

Inhaled rhDNase may improve sputum viscosity and mucociliary clearance by cleavage of extracellular DNA derived for instance from dead leukocytes in purulent, highly viscous patient sputum.Here we established a method to quantify rhDNase-mediated DNA fragmentation in sputum using gel electrophoresis. Sputum of Pseudomonas aeruginosa colonized cystic fibrosis (CF) patients with (CF+) or without (CF-) rhDNase treatment or mechanically ventilated non-CF patients receiving rhDNase (non-CF+) or not (non-CF-) was analyzed. DNA measurements from T-lymphocytes served as controls. Absolute DNA content and the relative quantity within eight molecular mass ranges (12000 to 200 bp) was determined by gel electrophoresis and densitometric analysis.Geometric mean sputum DNA concentrations were 0.41 mg/dl for CF- (n=54), 0.78 mg/dl for CF+ (n=60), 0.053 mg/dl for non-CF- (n=41) and 0.049 mg/dl for non-CF+ (n=28). Treatment with rhDNase resulted in fragmentation of DNA that was quantified by separation and densitometric analysis of the DNA on agarose gels. The new analysis method permits analysis of DNA cleavage with high accuracy.This new monitoring method facilitates DNA quantification and in vitro monitoring of rhDNase in sputum.

Cystic fibrosis (CF), the most common autosomal recessive disorder, at least in western countries, is caused by mutations of the cystic fibrosis transmembranous conductance regulator (CFTR) molecule and affects approximately 80,000 patients in Europe and the USA. Most, if not all, CF patients develop a chronic pulmonary infection with Pseudomonas aeruginosa. At present it is unknown why CF patients are highly sensitive to P. aeruginosa infections, and most importantly, no curative treatment for CF is available. P. aeruginosa infection results in an activation of the enzyme acid sphingomyelinase which catalyzes the release of ceramide from sphingomyelin in the cell membrane. Ceramide forms large ceramide-enriched membrane domains that are required for internalization of bacteria, induction of cell death in infected cells and a controlled release of cytokines from infected cells. Ceramide-enriched membrane platforms seem to serve the reorganization of receptors and intracellular signaling molecules involved in the infection of mammalian cells with P. aeruginosa. The significance of the acid sphingomyelinase and ceramide for the infection of mammalian cells with P. aeruginosa was demonstrated on mice genetically deficient for the acid sphingomyelinase. Further studies with N. gonorrhoeae, S. aureus and rhinoviruses indicate that ceramide-enriched membrane domains are also important for the infection of mammalian cells with other bacterial and viral pathogens, suggesting a general role of these membrane domains in infectious biology.

Ceramide generated in the cell membrane has been shown to be central for the induction of apoptosis by death receptors and many stress stimuli such as gamma-irradiation, UV-light or infection with pathogens. Ceramide reorganizes cell membranes and forms large ceramide-enriched membrane domains that serve the spatial and temporal organization of the cellular signalosome upon activation. Thus, ceramide-enriched membrane domains mediate clustering of CD95 and DR5 to facilitate apoptosis, and they are also critically involved in apoptosis after irradiation, UV-light and infection with Pseudomonas aeruginosa. Since ceramide-enriched membrane domains amplify signals, their function is not restricted to the induction of apoptosis and it was shown that ceramide-enriched membrane domains are also involved in internalization of pathogens and the control of cytokine release from infected epithelial cells. Recent studies support the notion that changes of the ceramide metabolism are also critically involved in human diseases, for instance neurological disorders, cancer, infectious diseases and Wilson's disease.

Ceramide has been shown to be critically involved in many aspects of cellular responses to receptor-dependent and -independent stimuli. For instance, ceramide was demonstrated to be a central component of the signaling cascades mediating apoptosis after death receptor stimulation, treatment with chemotherapy or exposure to gamma-irradiation or UV-A light. Further studies indicated the importance of ceramide for the infection of mammalian cells with bacterial, viral and parasitic pathogens. Ceramide is released by the activity of acid, neutral or alkaline sphingomyelinases or de novo synthesized. A concept unifying the diverse biological functions of ceramide indicates that ceramide forms distinct membrane domains, named ceramide-enriched membrane domains or platforms. These domains serve the clustering of receptor molecules, the re-organization of signaling proteins, the exclusion of inhibitory signals and, thus, initiate and greatly amplify a primary signal. In addition, ceramide directly interacts with and stimulates intracellular enzymes that may act together with signals initiated in ceramide-enriched membrane domains to transmit signals into a cell.

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of 51 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C-16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing 1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.

Background/Aims: Major depressive disorder is a common disease with serious morbidity, including increased risk of death from suicide. Major depressive disorder is treated with antidepressants. However, the molecular targets of antidepressants remained ill-defined and require further elucidation. Methods: Mice were treated with corticosterone to induce stress, amitriptyline and the p38-kinase (p38K) inhibitor SB239063 or a combination of these drugs. Phosphorylation of p38K in hippocampal neurons was determined by immunostaining with a phospho-specific antibody, neuronal proliferation using BrdU-labelling and behaviour employing a set of behavioural tests. Results: Corticosterone induced phosphorylation/activation of p38K in the hippocampus in vivo. Antidepressants reversed the effect of corticosterone on p38K activation in wildtype mice, but had no effect in acid sphingomyelinase-deficient animals. Corticosterone also reduced neurogenesis and triggered depression-like behavioural changes, effects that were prevented by pharmacological inhibition of p38K. Conclusion: Stress induces p38K phosphorylation/activation in the hippocampus and thereby reduces neurogenesis and induces depression-like symptoms, events that are prevented by antidepressants via inhibition of the acid sphingomyelinase/ceramide system.

Acid sphingomyelinase and ceramide have previously been shown to play a central role in infections with Neisseria gonorrhoeae, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, and Mycobacterium avium. Recent studies have extended the role of sphingolipids in bacterial infections and have demonstrated that ceramide and sphingosine are central to the defense of lungs against bacterial pathogens. Ceramide accumulates in the airway epithelium of cystic fibrosis and ceramide synthase 2 (CerS2)-deficient mice, which respond to the lack of very long chain (C22-C24-) ceramides with a profound compensatory increase of long chain (mainly C16-) ceramides. In contrast, sphingosine is present in healthy airways and is almost completely absent from diseased or deficient epithelial cells. Both sphingolipids are crucially involved in the high susceptibility to infection of cystic fibrosis and CerS2-deficient mice, as indicated by findings showing that the normalization of ceramide and sphingosine levels rescue these mice from acute infection with P. aeruginosa.

Distinct domains in the cell membrane, termed rafts, emerge as central for the infection of mammalian cells by many pathogens. Rafts consist of sphingolipids and cholesterol that interact strongly, and thus spontaneously separate from other phospholipids in the cell membrane. Recent studies suggest that at least some pathogens activate the acid sphingomyelinase that releases ceramide in membrane rafts. The generation of ceramide transforms small rafts into a signaling unit and results in the fusion of small rafts to large platforms. Membrane rafts and ceramide-enriched membrane platforms have been shown to mediate internalization of bacteria, viruses and parasites into the host cell, to initiate apoptosis of the host cell upon infection and to regulate the release of cytokines from infected mammalian cells. Furthermore, rafts and ceramide have been implicated in the intracellular trafficking of phagosomes and in the budding of viruses from infected cells. The molecular function of rafts and ceramide-enriched membrane platforms seems to be the re-organization of receptor and intracellular signaling molecules in the cell membrane permitting the interaction of the pathogen with the cell. This suggests that rafts and ceramide-enriched membrane platforms function as central structures involved in the infection of mammalian cells by pathogens and as targets for the development of anti-infective drugs.

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassme et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e., within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types.

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.

Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase -/- hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C(16)-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C(16)-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.

Pseudomonas aeruginosa plays a major role in respiratory tract infections or sepsis in patients with cystic fibrosis or upon suppression of the immune system. Several P. aeruginosa strains have been shown to be internalized by human epithelial cells; however, the molecular mechanisms of the invasion process are poorly characterized. Here, we show that the internalization of P. aeruginosa into human epithelial cells results in and requires activation of the Src-like tyrosine kinases p59Fyn and p60Src and the consequent tyrosine phosphorylation of several eukaryotic proteins. The significance of Src-like tyrosine kinase activation is shown by an almost complete blockade of P. aeruginosa internalization, but not adhesion, upon inhibition of Src-like tyrosine kinases. Likewise, inhibition of P. aeruginosa binding to CFTR, which has been shown to block P. aeruginosa internalization, prevents Src and Fyn activation, supporting a pivotal role of Src-like tyrosine kinases for invasion by P. aeruginosa.

Selectins have been shown to be crucial in the rolling process of leukocytes during lymphocyte homing and in the early phase of inflammatory processes. Recently, we and others have shown that binding of L-selectin to its ligands correlates with a rapid induction of several intracellular signaling molecules, in particular, Src-like tyrosine kinases, MAP-kinases, Jun NH2-terminal kinase, the small G-proteins Ras and Rac, and a release of Ca2+in leukocytes. Here, we demonstrate the activation of a novel signaling pathway by L-selectin. Stimulation of Jurkat T-lymphocytes via L-selectin results in an increase of neutral sphingomyelinase activity. This activity correlates with a consumption of cellular sphingomyelin and a release of ceramide. The activation of the neutral sphingomyelinase by L-selectin does not depend on tyrosine kinase activity and, therefore, represents an alternative and novel pathway to stimulate lymphocytes via L-selectin.

The interaction of the CD40 receptor with its ligand has been shown to be crucial for the activation of B-lymphocytes. Here, we provide evidence that the pg39 molecule/CD40 ligand (gp39/CD40L) also functions as a stimulatory molecule for T-lymphocytes. Activation of T-lymphocytes via gp39/CD40L induced a strong activation of Jun-N-terminal kinase (JNK) and p38-K. Activation of these kinases correlates with a stimulation of Rac1 and inhibition of Rac1 prevents gp39/CD40L triggered JNK/p38-K activation. Further, cellular stimulation via the CD40 ligand results in tyrosine phosphorylation of cellular proteins and the activation of p56lck. Inhibition of src-like kinases inhibits Rac1 as well as JNK/p38-K stimulation suggesting a signalling cascade from the gp39/CD40L via p56lck and Rac1 to JNK/p38-K.

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae–induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.

The activation of B-lymphocytes depends critically on the interaction of the CD40 receptor with its ligand. Here, we provide evidence that the CD40 ligand (CD40L) also functions as a direct stimulatory molecule for T-lymphocytes. Activation of T-lymphocytes via CD40L induces tyrosine phosphorylation of cellular proteins including PLC gamma. Tyrosine phosphorylation of PLC gamma correlates with an IP3- and Ca(2+)-release and an activation of PKC. Inhibition of src-like tyrosine kinases by Herbimycin A prevents these activation events suggesting a crucial role of tyrosine phosphorylation in T-lymphocyte activation via CD40L.

Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

Ceramide is released from sphingomyelin primarily by the activity of acid, neutral, or alkaline sphingomyelinases or is synthesized de novo. Several bacteria, viruses, and even parasites infect mammalian cells by exploiting the acid sphingomyelinase or the neutral sphingomyelinase-ceramide system, or both. Sphingomyelinases and ceramide have been shown to be crucially involved in the internalization of pathogens, the induction of apoptosis in infected cells, the intracellular activation of signaling pathways, and the release of cytokines. The diverse functions of ceramide in infections suggest that the sphingomyelinase-ceramide system is a key player in the host response to many pathogens.

Cystic fibrosis (CF), the most common autosomal recessive disorder, at least in western countries, is caused by mutations of CFTR. The disease affects the intestine, the pancreas and the liver, but most important for life quality and expectance of CF patients are alterations in lung functions. The present review describes a novel pathway to explain many aspects of the pathogenesis of CF: ceramide accumulates in bronchial, tracheal and intestinal epithelial cells, and causes pulmonary inflammation, infection susceptibility, peribronchial collagen deposition and cell death, with the consequence of DNA release into the bronchial lumen and concomitant reduction of mucociliary clearance. All of these pathophysiological events are corrected by the normalization of pulmonary ceramide. Phase IIa and IIb clinical studies indicate a beneficial effect of a pharmacological inhibition of acid sphingomyelinase on lung functions in patients with CF.

Neisseria gonorrhoeae enters cultured human mucosal cells following binding of a distinct gonococcal opacity (Opa) outer membrane protein to cell surface proteoglycan receptors. We examined the route of internalization that is activated by Opa-expressing gonococci (strain VP1). Microscopy of infected Chang epithelial cells showed that gonococcal uptake was insensitive to monodansylcadaverine (150 microM), which interferes with clathrin-mediated endocytosis. Similarly, indirect immunofluorescence staining for clathrin in infected cells showed distribution of cellular clathrin unaltered from the distribution in noninfected cells. The microtubule inhibitors colchicine (50 microM) and nocodazole (20 microM) but not the microtubule-stabilizing agent taxol (10 microM) caused a moderate (30 to 50%) reduction in gonococcal entry without affecting bacterial adherence. The most dramatic effects were obtained with the microfilament-disrupting agent cytochalasin D (3 microM), which totally blocked bacterial entry into the cells. Double immunofluorescence staining of gonococci and actin filaments in infected cells demonstrated bacterium-associated accumulations of F-actin as an early signal of bacterial entry. The recruitment of F-actin was transient and disappeared once the bacteria were inside the cells. Cytochalasin D disrupted the actin cytoskeleton architecture but did not prevent the recruitment of F-actin by the bacteria. Adherent, noninvasive gonococcal Opa variants lacked the ability to mobilize F-actin. Recombinant Escherichia coli expressing the gonococcal invasion-promoting Opa of gonococcal strain MS11 (Opa50) adhered to the epithelial cells in an Opa-dependent fashion but was not internalized and did not recruit detectable amounts of F-actin. Coinfection with the E. coli recombinant strain and gonococci resulted in specific entry of the diplococci, despite the presence of large numbers of adherent E. coli cells. Together, our results indicate that Opa-mediated gonococcal entry into Chang cells resembles phagocytosis rather than macropinocytosis reported for Salmonella spp. and sequentially involves gonococcal adherence to the cell surface, Opa-dependent and cytochalasin-insensitive recruitment of F-actin, and cytochalasin D-sensitive bacterial internalization.

We recently demonstrated that the accumulation of ceramide in Cftr-deficient epithelial cells is important for the pathophysiology of CF. However, the role of ceramide in other lung cells, particularly lung macrophages, requires definition. In this study, we report that ceramide is accumulated in Cftr-deficient lung macrophages. Alveolar macrophages contain a vesicle population, which is stained with LysoSensor probes but not by tetramethylrhodamine dextran. These vesicles, presumably secretory lysosomes, exhibit a higher pH in Cftr-deficient macrophages than the corresponding vesicles in lung macrophages isolated from wild-type (WT) mice. Alkalinization of these vesicles in Cftr-deficient macrophages correlates with a failure of the macrophages to respond to infection with various Pseudomonas aeruginosa strains by acutely activating acid sphingomyelinase, releasing ceramide, forming ceramide-enriched membrane platforms that serve to cluster gp91phox, and, most importantly, releasing reactive oxygen species (ROS). In contrast, these events occur rapidly in WT lung macrophages postinfection. Inhibiting ROS in WT macrophages prevents the killing of P. aeruginosa. These findings provide evidence for a novel pH-controlled pathway from acid sphingomyelinase activation via ceramide and clustering of gp91phox to the release of ROS in lung macrophages.

Cystic fibrosis is a hereditary metabolic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and characterized by severe intestinal and pulmonary symptoms, in particular intestinal obstruction, pancreatic insufficiency, chronic pulmonary inflammation, and microbial lung infections. Recent studies have demonstrated an accumulation of ceramide in the lungs of cystic fibrosis patients and in several mouse models. These findings showed that pulmonary ceramide concentrations play an important role in pulmonary inflammation and infection. In this study we investigated whether ceramide concentrations are also altered in the trachea and the intestine of cystic fibrosis mice and whether an accumulation of ceramide in these organs has functional consequences that are typical of cystic fibrosis. Our findings demonstrate a marked accumulation of ceramide in tracheal and intestinal epithelial cells of cystic fibrosis mice. When acid sphingomyelinase activity is inhibited by treating cystic fibrosis mice with amitriptyline or by genetic heterozygosity of acid sphingomyelinase in cystic fibrosis mice, ceramide concentrations in the trachea and the intestine are normalized. Moreover, increased rates of cell death and increased cytokine concentrations in the trachea, the intestine, or both were normalized by the inhibition of acid sphingomyelinase activity and the concomitant normalization of ceramide concentrations. These findings suggest that ceramide plays a crucial role in inflammation and increased rates of cell death in several organs of cystic fibrosis mice.

The molecular pathway of infection of mammalian cells by pathogenic bacteria is still unsolved. The deformation of the cell membrane and especially the role of ceramide molecules during the penetration of the bacteria into the cell is of particular interest for biologists to understand the infection process. By means of modern laser scanning microscopy it is possible to generate spatial images of vital macrophages in spatial relation to bacteria infected the cell. These images show how bacteria diffuse through the membrane. In this study three-dimensional visualizations of microscopic images showing infected mammalian macrophages are presented created by the use of known and adapted 3D reconstruction algorithms which facilitates the analysis of the membrane deformation and of the ceramide aggregations during the cell infection. To depict the topography of the cell surface membrane we used the “Marching Cube” algorithm whereas the use of the “Texture Mapping” algorithm allows us to visualize the dye distribution in a biologically correct manner. Additionally stereoscopic visualization techniques were applied to improve the possibilities of analyzing the images. Here, we show that the production of ceramide and the deformation of the membrane around and underneath the bacterial aggregation is essential for allowing the bacteria to penetrate into the cell.

Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.

Central elements in the infection of mammalian cells with viral, bacterial and parasitic pathogens include the adhesion of the pathogen to surface receptors of the cell, recruitment of additional receptor proteins to the infection-site, a re-organization of the membrane and, in particular, the intracellular signalosome. Internalization of the pathogen results in the formation of a phagosome that is supposed to fuse with lysosomes to form phagolysosomes, which serve the degradation of the pathogen, an event actively prevented by some pathogens. In summary, these changes in the infected cell permit pathogens to trigger apoptosis (for instance of macrophages paralysing the initial immune response), to invade the cell and/or to survive in the cell, but they also serve the mammalian cell to defeat the infection, for instance by activation of transcription factors and the release of cytokines. Distinct membrane domains in the plasma membrane and intracellular vesicles that are mainly composed of sphingolipids and cholesterol or enriched with the sphingolipid ceramide, are critically involved in all of these events occurring during the infection. These membrane structures are therefore very attractive targets for novel drugs to interfere with bacterial, viral and parasitic infections.

The cell membrane contains very small distinct membrane domains enriched of sphingomyelin and cholesterol that are named rafts. We have shown that the formation of ceramide via activation of the acid sphingomyelinase transforms rafts into ceramide-enriched membrane platforms. These platforms are required for infection of mammalian cells with Pseudomonas aeruginosa, Staphylococcus aureus, or Neisseriae gonorrhoeae. In the present study we determined whether the acid sphingomyelinase, ceramide, and ceramide-enriched membrane platforms are also involved in the infection of human cells with pathogenic rhinoviruses. We demonstrate that infection of human epithelial cells with several rhinovirus strains triggers a rapid activation of the acid sphingomyelinase correlating with microtubules- and microfilament-mediated translocation of the enzyme from an intracellular compartment onto the extracellular leaflet of the cell membrane. The activity of the acid sphingomyelinase results in the formation of ceramide in the cell membrane and, finally, large ceramide-enriched membrane platforms. Rhinoviruses colocalize with ceramide-enriched membrane platforms during the infection. The significance of ceramide-enriched membrane platforms for rhinoviral uptake is demonstrated by the finding that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase prevented infection of human epithelial cells by rhinoviruses. The data identify the acid sphingomyelinase and ceramide as key molecules for the infection of human cells with rhinoviruses.

Early events required for induction of apoptosis by CD95 are preassociation of CD95, the formation of the death-inducing signaling complex (DISC) and clustering of CD95 in distinct membrane domains. Here, we identify the molecular ordering of these events and show that the acid sphingomyelinase (ASM) functions upstream of the DISC to mediate CD95 clustering in ceramide-enriched membrane platforms, an event that is required for DISC formation. Experiments in ASM-deficient cells revealed that CD95 ligation, in the absence of ceramide generation, triggers1% of full caspase 8 activation at the receptor. This event, however, is both necessary and sufficient to trigger translocation of ASM onto the outer leaflet of the plasma membrane, ASM activation and ceramide release, but insufficient for apoptosis induction. Ceramide-mediated CD95 clustering then amplifies the primary CD95 signaling and drives the second step of CD95 signaling, that is, formation of the DISC yielding 100% caspase activity and apoptosis. These studies suggest that the most parsimonious interpretation of the molecular ordering of the earliest events in CD95 signaling, at least in some cells, is: CD95 ligation--1% of maximum caspase 8 activation--ASM translocation--ceramide generation--CD95 clustering--DISC formation--100% of maximum caspase 8 activation--apoptosis.

Acid sphingomyelinase (ASM) has been shown to be activated by a variety of receptor molecules and stimuli including CD95, the tumor necrosis factor receptor (TNF-R), CD40, CD28, LFA-1, CD5, during development, irradiation, heat shock, UV light or bacterial and viral infections. The central role of ASM-released ceramide in the response to those stimuli is confirmed by several genetic studies. ASM and ceramide might mediate their biological effects by the activation of several intracellular signaling molecules including cathepsin D, phospholipase A 2 or the kinase suppressor of Ras. In addition, recent fluorescence microscopy studies indicate that distinct, small membrane domains, termed rafts, are modified by ceramide to form larger domains, which serve to cluster receptor molecules. The generation of a high receptor density might be required for initiation of receptor-specific signaling and explain the function of the ASM and ceramide in multiple signaling pathways.

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.

Receptor clustering has been suggested as a crucial mechanism to initiate receptor signaling. Here we show that ceramide in sphingolipid-rich membrane rafts mediates clustering of CD95. Neutralization of surface ceramide or inhibition of its endogenous generation prevented CD95 clustering. Furthermore, application of ceramide at the cell surface triggered clustering of active but not inactive CD95. Apoptosis was inhibited by neutralization of surface ceramide or inhibition of ceramide release in vitro and in vivo. Thus, we conclude that surface ceramide mediates CD95 clustering, which is required for initiation of apoptosis, at least in some cell types.