Elena Bulanova

Ceramide mediates caspase-independent programmed cell death

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.—Thon, L., Möhlig, H., Mathieu, S., Lange, R., Bulanova, E., Winoto‐Morbach, S., Schütze, S., Bulfone‐Paus, S., Adam, D. Ceramide mediates caspase‐independent programmed cell death. FASEB J. 19, 1945–1956 (2005)

Extracellular ATP Induces Cytokine Expression and Apoptosis through P2X7 Receptor in Murine Mast Cells

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X7, and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca2+ influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-α. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4′-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X7 receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X7 receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X7-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

IL-15 constrains mast cell–dependent antibacterial defenses by suppressing chymase activities

Sepsis remains a global clinical problem. By using the mouse cecal ligation and puncture model of sepsis, here we identify an important aspect of mast cell (MC)-dependent, innate immune defenses against Gram-negative bacteria by demonstrating that MC protease activity is regulated by interleukin-15 (IL-15). Mouse MCs express both constitutive and lipopolysaccharide-inducible IL-15 and store it intracellularly. Deletion of Il15 in mice markedly increases chymase activities, leading to greater MC bactericidal responses, increased processing and activation of neutrophil-recruiting chemokines, and significantly higher survival rates of mice after septic peritonitis. By showing that intracellular IL-15 acts as a specific negative transcriptional regulator of a mouse MC chymase (mast cell protease-2), we provide evidence that defined MC protease activity is transcriptionally regulated by an intracellularly retained cytokine. Our results identify an unexpected breach in MC-dependent innate immune defenses against sepsis and suggest that inhibiting intracellular IL-15 in MCs may improve survival from sepsis.

Dendritic cell-derived IL-15 controls the induction of CD8 T cell immune responses

The development and the differentiation of CD8+ T cells are dependent on IL‐15. Here, we have studied the source and mechanism of how IL‐15 modulates CD8+ T cell‐mediated Th1immune responses by employing two delayed‐type hypersensitivity (DTH) models. IL‐15‐deficient (IL‐15–/–) mice or mice treated with soluble IL‐15Rα as an IL‐15 antagonist showed significantly reduced CD8+ T cell‐dependent DTH responses, while activation of CD4+ T cell and B cell functions remained unaffected. Injection of antigen‐labeled dendritic cells (DC) fromIL‐15+/+, IL‐15–/– or IL‐15Rα–/– mice revealed that DC‐derived IL‐15 is an absolute requirement for the initiation of DTH response. The re‐establishment of the interaction of IL‐15 with the IL‐15Rα by incubating IL‐15–/– DC with IL‐15 completely restored the capacity to prime T cells for DTH induction in vivo. Moreover, IL‐15 also enhanced secretion of pro‐inflammatory cytokines by DC and triggered in vitro CD8+ T cell proliferation and IL‐2 release. Taken together, the data suggest that an autocrine IL‐15/IL‐15Rα signaling loop in DC is essential for inducing CD8+‐dependent Th1 immune responses in mice. Therefore, targeted manipulation of this loop promises to be an effective, novel strategy for therapeuticmodulation of clinically relevant DTH reactions.

The IL-15Rα Chain Signals Through Association with Syk in Human B Cells

The α-chain of the IL-15R (IL-15Rα) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Rα-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Rα and IL-2Rγ chains, but lacks the IL-2Rβ chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Rα chain. Upon association, the activated Syk kinase phosphorylates the IL-15Rα chain as well as phospholipase Cγ, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Rα and phospholipase Cγ phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Rα (Tyr227) abrogates both the IL-15Rα/Syk association and IL-15Rα phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Rα chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a ‘promiscuous liaison’ between both receptors that enables interleukin (IL)‐15 to transactivate the signaling pathway of a tyrosine kinase. IL‐15 protects murine L929 fibroblasts from tumor necrosis factor α (TNFα)‐induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl‐overexpressing fibroblasts are TNFα‐resistant. IL‐15Rα and Axl colocalize on the cell membrane and co‐immunoprecipitate even in the absence of IL‐15, whereby the extracellular part of Axl proved to be essential for Axl/IL‐15Rα interaction. Most strikingly, IL‐15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL‐15Rα, and activation of the phosphatidylinositol 3‐kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild‐type but not Axl−/− or IL‐15Rα−/− mice. Thus, IL‐15‐induced protection from TNFα‐mediated cell death involves a hitherto unknown IL‐15 receptor complex, consisting of IL‐15Rα and Axl RTK, and requires their reciprocal activation initiated by ligand‐induced IL‐15Rα.

Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

ABSTRACT Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

P2 receptor-mediated signaling in mast cell biology

Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

How a Cytokine Is Chaperoned through the Secretory Pathway by Complexing with Its Own Receptor: Lessons from Interleukin-15 (IL-15)/IL-15 Receptor α

ABSTRACT While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.

Mast cells express novel functional IL-15 receptor alpha isoforms.

Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15Rα chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15Rα gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15RαΔ4, IL-15RαΔ3,4, IL-15RαΔ3,4,5). After transient transfection in COS-7 cells, all IL-15Rα isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15Rα isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15Rα isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15Rα isoforms, which can explain the selective regulatory effects of IL-15 on these cells.