Novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ARDS model: a translational study.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-ß1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase.

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.

Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

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.

Acid sphingomyelinase serum activity predicts mortality in intensive care unit patients after systemic inflammation: a prospective cohort study.

INTRODUCTION: Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation. METHODS: 40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT before), during (PCT peak) and after (PCT low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed. RESULTS: ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT before was not different, but further increased at PCT peak in non-survivors and was significantly higher at PCT low compared to survivors. Survivors exhibited decreased ASM at PCT peak and PCT low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT low. CONCLUSIONS: In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.

Cell fate decisions regulated by K63 ubiquitination of tumor necrosis factor receptor 1.

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.

TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells.

BACKGROUND: The cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, its efficacy is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Programmed necrosis is an alternative, molecularly distinct mode of programmed cell death that is elicited by TRAIL under conditions when the classical apoptosis machinery fails or is actively inhibited. The potential of TRAIL-induced programmed necrosis in tumor therapy is, however, almost completely uncharacterized. We therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin. METHODS: Cell death/viability was measured by flow cytometry/determination of intracellular ATP levels/crystal violet staining. Cell surface expression of TRAIL receptors was detected by flow cytometry, expression of proteins by Western blot. Ceramide levels were quantified by high-performance thin layer chromatography and densitometric analysis, clonogenic survival of cells was determined by crystal violet staining or by soft agarose cloning. RESULTS: TRAIL-induced programmed necrosis killed eight out of 14 tumor cell lines. Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested. TRAIL synergized with chemotherapeutics in killing tumor cell lines by programmed necrosis, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. Susceptibility/resistance of the investigated tumor cell lines to programmed necrosis seems to primarily depend on expression of the pro-necrotic kinase RIPK3 rather than the related kinase RIPK1 or cell surface expression of TRAIL receptors. Furthermore, interference with production of the lipid ceramide protected all tested tumor cell lines. CONCLUSIONS: Our study provides evidence that TRAIL-induced programmed necrosis represents a feasible approach for the elimination of tumor cells, and that this treatment may represent a promising new option for the future development of combination therapies. Our data also suggest that RIPK3 expression may serve as a potential predictive marker for the sensitivity of tumor cells to programmed necrosis and extend the previously established role of ceramide as a key mediator of death receptor-induced programmed necrosis (and thus as a potential target for future therapies) also to the tumor cell lines examined here.

18:1/18:1-Dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome.

BACKGROUND: 18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro, secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. MATERIALS AND METHODS: Respiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2-6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. RESULTS: Within 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-ß1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. CONCLUSIONS: We conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.

Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma show alterations of genes encoding the NADPH oxidase complex and impaired reactive oxygen species synthesis capacity.

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<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.

Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine.

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-ß1) 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.

IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model.

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.

Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury.

D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-ß1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes.

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.

The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase.

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.

CXCL4-induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1.

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.

Intracellular IL-15 controls mast cell survival.

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.

Selective NF-kappaB inhibition, but not dexamethasone, decreases acute lung injury in a newborn piglet airway inflammation model.

Acute respiratory failure in neonates (e.g. ARDS, meconium aspiration pneumonitis, pneumonia) is characterized by an excessive inflammatory response, governing the migration of polymorpho-nuclear leukocytes (PMNLs) into lung tissue and causing consecutive impairment of gas exchange and lung function. Critical to this inflammatory response is the activation of nuclear factor-kappaB (NF-kappaB) that is required for transcription of the genes for many pro-inflammatory mediators. We asked whether the inhibition of NF-kappaB activity using either a selective inhibitor (IKK-NBD peptide) or dexamethasone would be more effective in decreasing NF-kappaB activity and chemokine expression in pulmonary cells. Changes in lung function were repeatedly assessed for 24h following induction of acute respiratory failure and therapeutic intervention. We conducted a randomized, controlled, prospective animal study with mechanically ventilated newborn piglets which underwent repeated airway lavage (20+/-2 [SEM]) to remove surfactant and to induce lung inflammation. Admixed to 100 mg kg(-1) surfactant, piglets then received either IKK-NBD peptide (S+IKK), a selective inhibitor of NF-kappaB activation, its control peptide without intrinsic activity, dexamethasone (S+Dexa), its solvent aqua, or an air bolus only (all groups n=8). After 24h of mechanical ventilation, the following differences were measured: PaO(2)/FiO(2) (S+IKK 230+/-9 mm Hg vs. S+Dexa 188+/-14, p<0.05); ventilation efficiency index (0.18+/-0.01 [3800/(PIP-PEEP)(*)f(*)PaCO(2)] vs. 0.14+/-0.01, p<0.05); extravascular lung water (24+/-1 ml kg(-1) vs. 29+/-2, p<0.05); PMNL in BAL fluid (112+/-21 cells microl(-1) vs. 208+/-34, p<0.05), IL-8 (351+/-117 pg ml(-1) vs. 491+/-144, p=ns) and leukotriene B(4) (23+/-7 pg ml(-1) vs. 71+/-11, p<0.01) in BAL fluid. NF-kappaB activity in the nucleus of pulmonary cells differed by 32+/-5% vs. 55+/-3, p<0.001. Differences between these two intervention groups were more pronounced in the second half of the observation period (hours 12-24). At 24h of mechanical ventilation, inhibition of NF-kappaB activity by IKK-NBD peptide admixed to surfactant as a carrier caused improved gas exchange, lung function and reduced pulmonary inflammation, as evidenced by reduction in PMNL migration into lung tissue due to reduced nuclear NF-kappaB activity. We conclude that IKK-NBD admixture to surfactant in acute neonatal respiratory failure is superior to dexamethasone administration within the first 24h.

Evidence for an association of prion protein and sphingolipid-mediated signaling.

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.

Improved pulmonary function by acid sphingomyelinase inhibition in a newborn piglet lavage model.

RATIONALE: In acute inflammatory lung disease in newborn infants, exogenous surfactant only transiently improves lung function. We hypothesized that the transient nature of this protection is in part explained by elevated acid sphingomyelinase (a-SMase) activity that may inactivate surfactant and promote proinflammatory responses. OBJECTIVES: We investigated the intermediate-term effects (>12 h) of a-SMase inhibition in a neonatal piglet model of repeated airway lavage by the intratracheal use of the a-SMase inhibitor imipramine, together with exogenous surfactant as a carrier substance. METHODS: After surfactant washout and induction of pulmonary inflammation, lung function was monitored over 24 hours of mechanical ventilation and followed by ex vivo analyses. In addition, we studied the effect of lipopolysaccharide inhalation in a-SMase-deficient mice at 48 hours. MEASUREMENTS AND MAIN RESULTS: Surfactant washout increased both pulmonary a-SMase activity and ceramide content; this was attenuated by surfactant and prevented in the surfactant plus imipramine group. Compared with surfactant alone, Pa(O(2)), dynamic compliance, and extravascular lung water were improved in the final 12 hours in the surfactant plus imipramine group. At 24 hours, lavage fluid leukocyte counts and IL-8 concentrations decreased, and physical surfactant film properties improved. In the mouse model at 48 hours, a-SMase-deficient mice showed reduced pulmonary ceramide levels and attenuated leukocyte influx into the alveolar space. CONCLUSIONS: We conclude that stabilization of exogenous surfactant by adding imipramine to create a "fortified surfactant preparation" improves lung function in a clinically relevant piglet model, and that this effect can be attributed to the inhibition of a-SMase as evidenced in the mouse model.

Surfactant "fortification" by topical inhibition of nuclear factor-kappaB activity in a newborn piglet lavage model.

OBJECTIVE: In acute respiratory distress syndrome of term newborn infants, surfactant replacement may be effective because endogenous surfactant is decreased and structurally changed. Inflammation is central to acute respiratory distress syndrome, and hence, attenuation of proinflammatory transcription factor nuclear factor (NF)-[kappa]B activation in the lung might prevent secondary loss of surfactant function. In this study, we tested the hypothesis that the topical use of a NF-[kappa]B inhibitor (I[kappa]B kinase-NF-[kappa]B essential modulator binding domain [IKK-NBD] peptide), together with surfactant as a carrier substance, improves surfactant function by attenuation of pulmonary inflammation during 24 hrs of mechanical ventilation in a neonatal piglet model of acute respiratory distress syndrome by repeated airway lavage. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory of a university children's hospital. SUBJECTS: A total of 24 anesthetized, mechanically ventilated newborn piglets. INTERVENTIONS: After 20 +/- 6 (mean +/- sd) lavages to induce lung failure and inflammation, a porcine surfactant (100 mg/kg) with (S+IKK) or without (S) 1.25 mg of IKK-NBD peptide, or an air bolus (control) was administered into the airways. Lung function was monitored throughout 24 hrs of mechanical ventilation and completed by ex vivo analyses. MEASUREMENTS AND MAIN RESULTS: Pao2 (S+IKK, 125 +/- 16 mm Hg; S, 105 +/- 33; control, 61 +/- 20), ventilation efficiency index, functional residual capacity, compliance of the respiratory system, and extravascular lung water (S+IKK, 24 +/- 2 mL/kg; S, 30 +/- 7; control, 34 +/- 8) were all significantly improved in S+IKK piglets after 24 hrs. Decreased leukocyte concentrations in bronchoalveolar lavage (S+IKK, 152 +/- 94 cells/microL; S, 202 +/- 100; control, 276 +/- 57) were observed together with reduced acid sphingomyelinase activity, lowered ceramide concentrations, improved surfactant function (minimum surface tension: S+IKK, 10.8 +/- 6.1 mN/m; S, 13.2 +/- 3.9; control, 20.9 +/- 8.5), and decreased NF-[kappa]B activation in lung tissue. CONCLUSION: Supplementation of exogenous surfactant with a NF-[kappa]B inhibitor to create a "fortified" surfactant improves gas exchange, lung function, and pulmonary edema during 24 hrs of mechanical ventilation, without a secondary functional relapse. Inhibition of NF-[kappa]B suppressed acid sphingomyelinase activity and ceramide generation, indicating a novel proinflammatory link of NF-[kappa]B.