Search

// Mario Stephan 1, * , Barbel Edelmann 2, * , Supandi Winoto-Morbach 1 , Ottmar Janssen 1 , Uwe Bertsch 1 , Cristiana Perrotta 3 , Stefan Schutze 1, * , Jurgen Fritsch 1, * 1 Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany 2 Department of Hematology and Oncology, University Hospital Magdeburg, Magdeburg, Germany 3 Department of Biomedical and Clinical Sciences “Luigi Sacco” (DIBIC), Universita degli Studi di Milano, Milano, Italy * These authors have contributed equally to this work Correspondence to: Jurgen Fritsch, email: Juergen.Fritsch@uksh.de Keywords: acid sphingomyelinase, ceramide, CD95 ligand, internalization, CD95-receptosomes Received: November 16, 2016 Accepted: January 24, 2017 Published: February 16, 2017 ABSTRACT Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner. Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD. Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation. In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding.

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.

Human monocytes respond to a variety of stimuli with a complex spectrum of activities ranging from acute defense mechanisms to cell differentiation or cytokine release. However, the individual intracellular signaling pathways related to these functions are not well understood. CXC chemokine ligand 4 (CXCL4) represents a broad activator of monocytes, which induces acute as well as delayed activities in these cells including cell differentiation, survival, or the release of ROS, and cytokines. Here, we report for the first time that CXCL4-treated monocytes significantly upregulate sphingosine kinase 1 (SphK1) mRNA and that CXCL4 induces SphK1 enzyme activity as well as its translocation to the cell membrane. Furthermore, we could show that pharmacological inhibition of SphK results in reversal of CXCL4-induced monocyte survival, cytokine expression, and release of oxygen radicals, which was confirmed by the use of SphK1-specific siRNA. CXCL4-mediated rescue from apoptosis, which is accompanied by inhibition of caspases, is controlled by SphK1 and its downstream element Erk. Taken together, these data assign SphK1 as a central regulator of acute and delayed monocyte activation and suggest SphK1 as a potential therapeutic target to suppress pro-inflammatory responses induced by CXCL4.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1.

The regulation of mast cell activities and survival is a central issue in inflammatory immune responses. Here, we have investigated the role of mouse interleukin-15, a pro-inflammatory and pleiotropic cytokine, in the control of mast cell survival and homeostasis. We report that aged IL-15-/- mice show a reduced number of peritoneal mast cells compared to WT mice. Furthermore, IL-15 deficiency in bone marrow derived mouse mast cells (BMMCs) results in increased susceptibility to apoptosis mediated by growth factor deprivation and A-SMase-treatment. IL-15-/- BMMCs show a constitutive stronger mRNA and protein expression as well as enzymatic activity of the members of the mitochondrial apoptotic pathways including acidic lysosomal aspartate protease cathepsin D (CTSD), endogenous acid sphingomyelinase (A-SMase), caspase-3 and -7 compared to wild type (WT) BMMCs. Furthermore, IL-15-/- BMMCs constitutively generate more A-SMase-derived ceramide than WT controls and display a decreased expression of pro-survival sphingosin-1-phosphate (SPP) both in cytosol and membrane cell fractions. Furthermore, pre-treatment of mast cells with imipramine or pepstatin A, inhibitors of the intracellular acid sphingomyelinase and cathepsin D pathways respectively, increases survival in IL-15-/- BMMCs. These findings suggest that intracellular IL-15 is a key regulator of pathways controlling primary mouse mast cell homeostasis.

The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI DeltaTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI DeltaTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI DeltaTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis.

In aged skin, decreased levels of stratum corneum ceramides have been described. Epidermal ceramides are generated by sphingomyelin hydrolysis or synthesis from sphingosin and fatty acids and are degraded by ceramidase. We recently showed that epidermal acid sphingomyelinase (A-SMase) generates ceramides with structural function in the stratum corneum lipid bilayers, which provide for the permeability barrier function of the skin. Here, we examined the activities of epidermal A-SMase, ceramide synthase, and ceramidase in chronologically aged versus young hairless mouse skin. We found reduced A-SMase and ceramide synthase activities in the epidermis of aged mice. However, studies on enzyme localization revealed unchanged, ongoing high A-SMase activity in the outer epidermis, which correlated with reported normal barrier function found in aged skin under basal conditions. Reduced A-SMase and ceramide synthase activity was noted in the inner epidermis, correlating with reduced capacity for permeability barrier repair in aging. Ceramidase activity was not age dependent. In summary, we found reduced activities of ceramide-generating SMase and ceramide synthase in the inner epidermis of aged skin, explaining its reduced capacity in barrier repair. In contrast, A-SMase activity in the outer epidermis was unchanged, indicating that this enzyme is crucially involved in basal permeability barrier homeostasis.

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.

The adhesion of immunomagnetic particles to surfaces of target cells based on ligand–receptor interactions was evaluated by counting the number of magnetically labeled cells using magnetophoresis and analyzed by the Langmuir adsorption theory. As few as 200 tumor necrosis factor (TNF) receptor molecules per cell could be detected with streptavidin-coated magnetic particles coupled to biotinylated TNF as ligand.

Airway epithelial NF-κB is a key regulator of host defence in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (aSMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defence as well as in pathologic processes of acute lung injury. Little is known about the regulatory mechanisms of pulmonary sphingolipid metabolism in bacterial infections of the lung. The objective of this study was to evaluate the influence of NF-κB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation. In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS we investigated TNF-α, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to aSMase activity and ceramide and S1-P lung tissue concentrations. Airway epithelial NF-κB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of aSMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-κB translocation. This confirms the critical role of airway epithelial NF-κB pathway for the inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defence mechanisms.

Tumor necrosis factor-α (TNF-α) is known as a highly pleiotropic cytokine. Stimulation of TNF-receptor 1 (TNF-R1) by TNF-α elicits the transduction of intracellular signals that on the one side promote cell death by apoptosis. However, TNF-R1 may also transduce non-apoptotic signals that lead to inflammatory responses through the activation of the transcription factor nuclear factor-κB (NF-κB) or to cell proliferation through activation of the mitogen-activated protein kinase (MAPK) cascade. A clue to the understanding of these contradictory biological phenomena may arise from recent findings which reveal a regulatory role of receptor endocytosis and intracellular receptor trafficking in selective transmission of signals, which either promote apoptosis or rather cell survival. Although internalization of cell surface receptors has traditionally been regarded as a means to shut down signaling via receptor degradation, there is now good evidence for an active role of many internalized surface receptors in the continuation of signal transmission along the endocytic pathway. Thus endocytosis may control the quality, intensity, duration, and spatial distribution of signaling events. TNF-induced apoptotic signals lead to an enhanced generation of ceramide by the enzyme sphingomyelin phosphodiesterase 1 (SMPD1, also known as acid sphingomyelinase [A-SMase]). Since TNF-triggered activation of A-SMase is linked to the death domain of TNF-receptor 1 (TNF-R1) and since the death domain adapter proteins FADD and caspase-8 are recruited during internalization of TNF-R1 to endosomes (TNF-receptosomes), we examined the possibility that A-SMase could be activated by caspase-8 within this compartment. Since we observed TNF-induced proteolytic processing and activation of pro-A-SMase that depended on the presence of caspase-8, we propose that activation of A-SMase within TNF-receptosomes requires activation of caspase-8 and probably further downstream proteases. Thus the fusion of internalized TNF-receptosomes with trans-Golgi vesicles containing the proform of A-SMase should be recognized as a novel mechanism to transduce death signals along the endocytic route.

The physiological function of the cellular prion protein (PrP(c)) is unclear. PrP(c) associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP(c) and sphingolipid-associated signaling pathways. Using PrP(c)-wt and PrP(c)-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP(c)-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP(c)-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P(3)-receptor expression was higher in PrP(c)-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P(3) receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP(c) is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP(c) plays a role in neuroprotection.

We have identified the aspartic protease cathepsin D as a novel intracellular target protein for the lipid second messenger ceramide. Ceramide specifically binds to and induces CTSD proteolytic activity. A-SMase deficient cells derived from Niemann-Pick patients show decreased CTSD activity that was reconstituted by transfection with A-SMase cDNA. Ceramide accumulation in cells derived from A-ceramidase defective Farber patients correlates with enhanced CTSD activity. These findings suggest that A-SMase-derived ceramide targets endolysosomal CTSD.

Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.

We previously demonstrated that the aspartate protease cathepsin D is activated by ceramide derived from acid sphingomyelinase. Increased expression of cathepsin D in the skin has been reported in wound healing, psoriasis and skin tumors. We explored specific functions of cathepsin D during epidermal differentiation. Protein expression and enzymatic activity of cathepsin D increased in differentiated keratinocytes in both stratified organotypic cultures and in mouse skin during epidermal barrier repair. Treatment of cultured keratinocytes with exogenous cathepsin D increased the activity of transglutaminase 1, known to cross-link the cornified envelope proteins involucrin and loricrin during epidermal differentiation. Inhibition of cathepsin D by pepstatin A suppressed the activity of transglutaminase 1. Cathepsin D-deficient mice revealed reduced transglutaminase 1 activity and reduced protein levels of the cornified envelope proteins involucrin and loricrin. Also, amount and distribution of cornified envelope proteins involucrin, loricrin, filaggrin, and of the keratins K1 and K5 were significantly altered in cathepsin D-deficient mice. Stratum corneum morphology in cathepsin D-deficient mice was impaired, with increased numbers of corneocyte layers and faint staining of the cornified envelope only, which is similar to the human skin disease lamellar ichthyosis. Our findings suggest a functional link between cathepsin D activation, transglutaminase 1 activity and protein expression of cornified envelope proteins during epidermal differentiation.

Acidic noncaspase proteases-like cathepsins have been introduced as novel mediators of apoptosis. A clear role for these proteases and the acidic endolysosomal compartment in apoptotic signalling is not yet defined. To understand the role and significance of noncaspases in promoting and mediating cell death, it is important to determine whether an intersection of these proteases and the caspase pathway exists. We recently identified the endolysosomal aspartate protease cathepsin D (CTSD) as a target for the proapoptotic lipid ceramide. Here, we show that tumor necrosis factor (TNF)-induced CTSD activation depends on functional acid sphingomyelinase (A-SMase) expression. Ectopic expression of CTSD in CTSD-deficient fibroblasts results in an enhanced TNF-mediated apoptotic response. Intracellular colocalization of CTSD with the proapoptotic bcl-2 protein family member Bid in HeLa cells, and the ability of CTSD to cleave directly Bid in vitro as well as the lack of Bid activation in cathepsin-deficient fibroblasts indicate that Bid represents a direct downstream target of CTSD. Costaining of CTSD and Bid with Rab5 suggests that the endosomal compartments are the common 'meeting point'. Caspase-9 and -3 activation also was in part dependent on A-SMase and CTSD expression as revealed in the respective deficiency models. Our results link as novel endosomal intermediates the A-SMase and the acid aspartate protease CTSD to the mitochondrial apoptotic TNF pathway.

The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p

A defective permeability barrier leads to the penetration of environmental allergens into the skin and initiates immunological reactions and inflammation crucially involved in the pathogenesis of atopic dermatitis (AD). Decreased stratum corneum ceramide content may cause the defect in permeability barrier function consistently found in AD. Acid and neutral sphingomyelinase (A- and N-SMase) generate ceramides with structural and signal transduction functions in epidermal proliferation and differentiation. We determined epidermal SMase activities, DNA synthesis, involucrin, loricrin, filaggrin, and keratin expression in lesional and non-lesional skin of AD patients. We found decreased epidermal A-SMase activity in lesional and non-lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function. N-SMase activity was reduced in non-lesional skin and more significantly reduced in lesional skin, correlating with impaired expression of cornified envelope proteins and keratins, important for skin barrier function. Changes in involucrin, loricrin, filaggrin, keratin K 5 (basal) and K 16 (proliferation associated) were noticed in non-lesional and lesional skin, whereas changes in K 10 (suprabasal), K 6 (proliferation associated), and K 17 (inflammation associated) were found only in lesional skin. In summary, reduction in SMase-generating ceramides and impaired differentiation are involved in the defective barrier function found in AD.

Because successful human islet transplantation requires large quantities of viable islets that must be separated from the highly immunogenic exocrine tissue and because handpicking is too time-consuming and laborious to be clinically relevant, a new approach for solving this problem has been established in rat models. It is based on the principle that magnetic microspheres (MMSs) coupled to lectins with binding specificity for the exocrine tissue portion are trapped in an electromagnetic field, thus providing effluent islets of a high degree of purity. In this study our aim was to adapt this principle to human islet preparations. In this context our prime interest was focused on a lectin suitable for human pancreatic tissue. Of 19 different lectins tested, only 1, Wisteria floribunda agglutinin (WFA), is suitable, as shown by immunofluorescence, MMS-lectin binding, and magnetic separation.

In a recently identified novel TNF-induced apoptotic signaling pathway the generation of ceramide by acid sphingomyelinase (A-SMase) and the activation of the aspartic protease cathepsin D (CTSD) occur in the same endosomal compartment [1]. CTSD subsequently mediates cleavage of Bid leading to activation of caspase-9 and -3. Since activation of A-SMase is linked to the death domain of TNF-receptor 1 (TNF-R1) and since the death domain adapter proteins FADD and caspase-8 are recruited during internalization of TNF-R1 in endosomes (TNF-receptosomes) [2], we addressed the question, whether A-SMase can be activated directly by caspase-8 within this compartment. We here show by confocal laser scan microscopy and in immunomagnetically isolated TNF-receptosome preparations that the active form of caspase-8 colocalizes with A-SMase within TNF-receptosomes. Activation of caspase-8 correlates with cleavage of a 70/72 kDa pro-A-SMase molecule, paralleled by enhanced A-SMase activity, activation of CTSD as well as Bid-cleavage in isolated TNF-receptosomes. The functional link between caspase-8 activity and A-SMase stimulation is revealed by the lack of TNF-induced A-SMase activation in caspase-8 deficient Jurkat cells, which can be restored in vivo after retransfection of caspase-8 as well as in vitro by the addition of exogenous caspase-8 to lysates from caspase-8 deficient cells.