Biomedical subjects

Erich Gulbins

Publications and source records attributed to Erich Gulbins.

At least 19 recordsLinked to original sources

PubMed2006

Suicidal erythrocyte death in sepsis.

Sequelae of sepsis include anemia which presumably results from accelerated clearance of erythrocytes from circulating blood. The underlying mechanisms, however, remained hitherto elusive. Most recent studies disclosed that increased cytosolic Ca2+ activity and ceramide both trigger suicidal erythrocyte death (i.e., eryptosis), which is characterized by lipid scrambling of the cell membrane leading to phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing erythrocytes may adhere to vascular walls or may be engulfed by macrophages equipped with phosphatidylserine receptors. To explore whether sepsis leads to eryptosis, erythrocytes from healthy volunteers were exposed to plasma of patients suffering from sepsis, or to supernatants from sepsis producing pathogens. Then, phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Challenge of erythrocytes with plasma from the patients but not with plasma from healthy individuals triggered annexin V binding. The effect of patient plasma on erythrocyte annexin V binding was paralleled by formation of ceramide and a significant increase of cytosolic Ca2+ activity. Exposure of erythrocytes to supernatant of pathogens similarly induced eryptosis, an effect correlating with sphingomyelinase activity. The present observations disclose a novel pathophysiological mechanism leading to anemia and derangement of microcirculation during sepsis. Exposure to plasma from septic patients triggers phosphatidylserine exposure leading to adherence to the vascular wall and clearance from circulating blood.

Adult↗

PubMed2006

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.

Animals↗

PubMed2006

Membrane rafts in host-pathogen interactions.

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.

Animals↗

PubMed2006

Rhinoviral infections activate p38MAP-kinases via membrane rafts and RhoA.

Rhinoviral infections belong to the most frequent human infections characterized by common cold, chronic bronchitis, exacerbations of asthma, otitis media and sinusitis. Here, we define molecular mechanisms that mediate infections of human epithelial cells with human rhinovirus strain 14 (RV14). We demonstrate that RV14 activates p38-MAPKinase (p38-K) in a biphasic time course. Early stimulation of p38-K by RV14 was observed a few minutes after initiation of the infection, while the late increase of p38-K activity occurred 7-12 hrs upon infection. The stimulation of p38-K was mediated by the small G-protein RhoA,which was activated by RV14. Transfection of a genetic construct preventing RhoA activation blocked RV14-induced p38-K activation. Further, integrity of cholesterol and sphingolipid-enriched membrane domains was required for RV14-mediated p38-K activation, which was inhibited by destruction of membrane rafts. The data indicate that RV employs a signaling cascade from membrane rafts via the small G-protein RhoA to p38-K to infect human cells.

Blotting, Western↗

PubMed2006

Regulation of pulmonary Pseudomonas aeruginosa infection by the transcriptional repressor Gfi1.

Pulmonary Pseudomonas aeruginosa infections are characterized by the release of proinflammatory mediators, focal induction of apoptosis in respiratory epithelial cells and internalization of the bacteria. Here, we demonstrate that the transcriptional regulator Gfi1 is critically involved in the regulation of proinflammatory cytokine release and the induction of apoptosis in respiratory epithelial cells and macrophages upon P. aeruginosa infection. Gfi1-deficient mice responded to a pulmonary P. aeruginosa infection with uncontrolled pulmonary release of interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha, sepsis and death, which were delayed by injection of IL-1- and TNF-alpha-neutralizing antibodies. The uncontrolled release of cytokines seems to be caused by a failure of Gfi1-deficient respiratory epithelial cells in large to small bronchi and macrophages to respond to P. aeruginosa infection with an induction of apoptosis. Pharmacological inhibition of apoptosis in wild-type mice by intravenous injection of the broad-spectrum caspase inhibitor zVAD-cmk mimicked the phenotype of Gfi1-deficient mice and resulted in a profound sensitization of mice to P. aeruginosa, an increased release of cytokines, sepsis and death of the animals. Thus, Gfi1 controls apoptosis of respiratory epithelial cells and macrophages upon infection with P. aeruginosa. Inhibition of apoptosis by Gfi1 deficiency or caspase blockers sensitizes mice to P. aeruginosa infections, suggesting that apoptosis functions as a novel defence mechanisms in the regulation of the local innate immune response.

Animals↗

PubMed2006

Phospholipase A2 functions in Pseudomonas aeruginosa-induced apoptosis.

Pseudomonas aeruginosa, a gram-negative, facultative pathogen, causes severe and often even lethal infections in immunocompromised patients, as well as cystic fibrosis patients. We show here that a variety of P. aeruginosa strains activate phospholipase A2 (PLA2), cultured epithelial cells, and fibroblasts, resulting in increased intracellular and extracellular arachidonic acid release. The use of different PLA2 inhibitors revealed that P. aeruginosa-induced arachidonic acid release is mediated by activation of cytosolic PLA2 (cPLA2), whereas iPLA2 or sPLA2 do not seem to be involved in the response to P. aeruginosa. Likewise, the cPLA2-specific inhibitors MAFP and AACOCF3 prevented apoptosis of cultured epithelial cells upon P. aeruginosa infection, whereas inhibitors specific for iPLA2 or sPLA2 were without effect. The physiological significance of these findings is indicated by an inhibition of apoptosis in tracheal epithelial cells upon in vivo infection with P. aeruginosa. The data indicate that arachidonic acid generation by activation of cPLA2 during P. aeruginosa infection plays an important role in the induction of host cell death.

Animals↗

PubMed2006

Physiological and pathophysiological aspects of ceramide.

Activation of cells by receptor- and nonreceptor-mediated stimuli not only requires a change in the activity of signaling proteins but also requires a reorganization of the topology of the signalosom in the cell. The cell membrane contains distinct domains, rafts that serve the spatial organization of signaling molecules in the cell. Many receptors or stress stimuli transform rafts by the generation of ceramide. These stimuli activate the acid sphingomyelinase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. Surface acid sphingomyelinase generates ceramide that serves to fuse small rafts and to form large ceramide-enriched membrane platforms. These platforms cluster receptor molecules, recruit intracellular signaling molecules to aggregated receptors, and seem to exclude inhibitory signaling factors. Thus ceramide-enriched membrane platforms do not seem to be part of a specific signaling pathway but may facilitate and amplify the specific signaling elicited by the cognate stimulus. This general function may enable these membrane domains to be critically involved in the induction of apoptosis by death receptors and stress stimuli, bacterial and viral infections of mammalian cells, and the regulation of cardiovascular functions.

Animals↗

PubMed2006

Ion channels and cell volume in regulation of cell proliferation and apoptotic cell death.

Cell proliferation must be accompanied by increase of cell volume and apoptosis is typically paralleled by cell shrinkage. Moreover, profound osmotic cell shrinkage may trigger apoptosis. In isotonic environment cell volume changes require the respective alterations of transport across the cell membrane. Cell proliferation is typically paralleled by activation of K(+) channels, which is required for the maintenance of the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. The Ca(2+) entry leads to oscillations of cytosolic Ca(2+) activity which is followed by activation of Ca(2+) dependent transcription factors and by depolymerization of the actin filament network. The latter disinhibits the Na(+) H(+) exchanger and Na(+) , K(+) , 2Cl(-)cotransport thus leading to cell swelling. At some point transient activation of Cl(-) channels is required leading to transient decrease of cell volume. Apoptosis is typically paralleled by sustained activation of Cl(-) channels leading to Cl(-) , HCO-(3) and osmolyte exit. The subsequent cell shrinkage and cytosolic acidification are not counter-regulated by activation of the Na(+) /H(+) exchanger, which is inhibited and eventually degraded during apoptosis. At a later stage K(+) exit through K(+) channels decreases intracellular K(+) concentration and facilitates cell shrinkage. Sustained or excessive increase of Ca(+) triggers apoptotic cell death, typically paralleled by cell shrinkage due to activation of Ca(2+) sensitive K(+) channels. Cellular K(+) loss and cell shrinkage are supportive but not required for the induction of apoptosis. On the other hand, several studies point to a critical role of K(+) -channel inhibition in the initiation of apoptosis. Thus, alterations of K(+) channel and Ca(2+) channel activities may participate in the triggering of both, cell proliferation and apoptosis. The impact of those channels depends on magnitude and temporal organization of channel activation and on the activity of further signaling mechanisms. Accordingly, the same ion channel blockers may interfere with both, cell proliferation and apoptosis depending on cell type, regulatory environment and condition of the cell.

Animals↗

PubMed2005

Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells.

Recent studies have indicated that lipid rafts (LRs) in the cell membrane are clustered in response to different stimuli to form signaling platforms for transmembrane transduction. It remains unknown whether this LR clustering participates in redox signaling in endothelial cells. The present study tested a hypothesis that clustering of LRs on the membrane of coronary endothelial cells produces aggregation and activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, thereby forming a redox signaling platform. By confocal microscopic analysis of agonist-stimulated rafts patch formation, we found that several death receptor ligands or apoptotic factors, including tumor necrosis factor alpha, Fas ligand, or endostatin, stimulated the clustering and trafficking of individual LRs on the plasma membrane of coronary endothelial cells. Interestingly, double labeling of a membrane-bound NADPH oxidase subunit, gp91phox, and LRs showed that gp91phox colocalized within the LR patches when endothelial cells were stimulated by Fas ligand. In isolated LR fractions from Fas-stimulated endothelial cells, gp91phox, p47phox (a crucial cytosolic regulatory subunit of NADPH oxidase), and Rac GTPase were markedly increased and blocked by nystatin, a compound that disrupts LRs. These clustered LRs contained high NADPH oxidase activity, which increased in response to Fas stimulation. Functionally, Fas ligand-induced inhibition of endothelium-dependent vasorelaxation was reduced if LRs were disrupted or NADPH oxidase was inhibited. These results suggest that LR clustering occurs in coronary endothelial cells. The formation of redox signaling platforms on the cell membrane mediates transmembrane signaling of death receptors, resulting in endothelial dysfunction.

Animals↗

PubMed2005

Ceramide-enriched membrane domains.

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.

Animals↗

PubMed2005

Rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms.

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.

Actin Cytoskeleton↗

PubMed2005

TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing store-operated Ca2+ influx.

Tumor necrosis factor alpha (TNF-alpha) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF-alpha alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca(2+)](i)) signal in Jurkat T cells after just 30 min exposure to TNF-alpha, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca(2+)](i) was also observed in human CD4+ T lymphocytes. The change in Ca(2+) signal was related to a decrease in the plasma membrane Ca(2+) influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca(2+) influx. The role of TNF-alpha-induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF-alpha led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca(2+) response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca(2+) responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF-alpha-induced inhibition of the Ca(2+) influx in an ASM-deficient murine T cell line (OT-II(+/+)ASM(-/-)). This potent ability of TNF-alpha to rapidly modulate the TCR Ca(2+) signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.

Antibodies, Monoclonal↗

PubMed2005

Annexin II is a novel receptor for Pseudomonas aeruginosa.

Infections with Pseudomonas aeruginosa (P. aeruginosa) are critical in ventilated and poly-traumatized patients. Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P. aeruginosa is urgently required. Here, we aimed to identify novel receptors that are involved in internalization of P. aeruginosa into mammalian epithelial cells. Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P. aeruginosa. The significance of the interaction of annexin II with P. aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P. aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P. aeruginosa into human epithelial cells.

Amino Acid Sequence↗

PubMed2005

A novel potassium channel in lymphocyte mitochondria.

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance.

Blotting, Western↗

PubMed2004

Reactive oxygen species limit neutrophil life span by activating death receptor signaling.

Neutrophils are abundant, short-lived leukocytes, and their death by apoptosis is central to hemostasis and the resolution of inflammation, yet the trigger for their entry into apoptosis is unknown. We show here that death receptor signaling, including CD95 death-inducing signaling complex (DISC) formation and caspase 8 activation, occurred early in neutrophil apoptosis. However, death receptor ligation was not required for apoptosis, suggesting a novel mechanism for caspase 8 activation. We detected ceramide generation and clustering of CD95 in lipid rafts early in neutrophil apoptosis, and neutrophil apoptosis and ceramide generation were both significantly inhibited in acid sphingomyelinase knockout (ASM(-/-)) mice compared to wild-type littermates. Further studies revealed that ceramide generation, CD95 clustering, and neutrophil apoptosis were dependent on reactive oxygen species (ROSs) and were preceded by a fall in reduced glutathione levels. We propose that accumulation of ROSs, as a consequence of altered redox status, initiates ligand-independent death receptor signaling via activation of ASM and clustering of preformed DISC components in lipid rafts and is therefore a primary factor limiting neutrophil life span.

Animals↗

PubMed2004

Ceramide, membrane rafts and infections.

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.

Animals↗

PubMed2004

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide.

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.

Animals↗

PubMed2003

Raft ceramide in molecular medicine.

Ceramide, generated by the action of acid sphingomyelinase (ASM), has emerged as a biochemical mediator of stimuli as diverse as ionizing radiation, chemotherapy, UVA light, heat, CD95, reperfusion injury, as well as infection with some pathogenic bacteria and viruses. ASM activity is also crucial for developmental programmed cell death of oocytes by apoptosis. Recently, we proposed a comprehensive model that might explain these diverse functions of ceramide: Upon contacting the relevant stimuli, ASM translocates into and generates ceramide within distinct plasma membrane sphingolipid-enriched microdomains termed rafts. Ceramide, which manifests a unique biophysical property, the capability to self-associate through hydrogen bonding, provides the driving force that results in the coalescence of microscopic rafts into large-membrane macrodomains. These structures serve as platforms for protein concentration and oligomerization, transmitting signals across the plasma membrane. Preliminary data suggest that manipulation of ceramide metabolism and/or the function of ceramide-enriched membrane platforms may present novel therapeutic opportunities for the treatment of cancer, degenerative disorders, pathogenic infections or cardiovascular diseases.

Animals↗