Biomedical subjects

Elena Bulanova

Publications and source records attributed to Elena Bulanova.

14 recordsLinked to original sources

PubMed2006

IL-15/IL-15 receptor biology: a guided tour through an expanding universe.

The cytokine interleukin-15 (IL-15) has a key role in promoting survival, proliferation and activation of natural killer (NK) and CD8+ T cells. Despite its functional similarities to IL-2, IL-15 affects a wider range of target cell populations and utilizes different mechanisms of signaling. Here, we review recent advances in the IL-15-mediated signaling, and in the functional properties on cells besides T lymphocytes and NK cells. These are discussed in the context of their potential clinical and therapeutic relevance.

Animals↗

PubMed2006

The interleukin-15/interleukin-15 receptor system as a model for juxtacrine and reverse signaling.

Interleukin-15 (IL-15) is a pleiotropic cytokine of the 4 alpha-helix bundle family, which binds to a receptor complex that displays common elements with the IL-2 receptor and a unique high-affinity alpha chain. This review focuses on juxtacrine and reverse signaling levels in the IL-15/IL-15R system. Specifically, we discuss how agonistic stimulation of membrane-bound IL-15 induces phosphorylation of members of the MAP kinase family and of focal adhesion kinase (FAK), thereby upregulating processes including cytokine secretion, cell adhesion and migration. In addition, we explore IL-15 trans-presentation and intracellular signaling, and define promising molecular targets for future pharmacological intervention in infectious diseases and immunological disorders. These frontiers in IL-15/IL-15Ralpha research serve as highly instructive examples for key concepts, unsolved problems and therapeutic opportunities in juxtacrine and reverse signaling in general.

Animals↗

PubMed2005

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 alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha 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-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, 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-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.

Animals↗

PubMed2005

A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L.

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

Adenine Nucleotides↗

PubMed2005

TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment.

Mast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon beta, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell-deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.

Animals↗

PubMed2005

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 P2X(7), 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 Ca(2+) 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-alpha. 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 P2X(7) 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 P2X(7) 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 P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

Adenosine Triphosphate↗

PubMed2005

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.

Animals↗

PubMed2005

Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum.

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.

ADAM Proteins↗

PubMed2004

Reverse signaling through membrane-bound interleukin-15.

The results from this study implicate membrane-anchored interleukin (IL)-15 constitutively expressed on the cell surface of PC-3 human prostate carcinoma cells and interferon-gamma-activated human monocytes in reverse signaling upon stimulation with soluble IL-15 receptor-alpha or anti-IL-15 antibodies, mediating the outside-to-inside signal transduction that involves the activation of members of the MAPK family (ERK and p38) and focal adhesion kinase. The presence of membrane-bound IL-15 was not dependent on the expression of the trimeric IL-15 receptor complex by these cells and resisted treatment with acidic buffer or trypsin. Reverse signaling through membrane-bound IL-15 considerably increased the production of several pro-inflammatory cytokines by monocytes, such as IL-6, IL-8, and tumor necrosis factor-alpha, thereby indicating the relevance of this process to the complex immunomodulatory function of these cells. Furthermore, stimulation of transmembrane IL-15 also enhanced the transcription of IL-6 and IL-8 in the PC-3 cell line and promoted migration of PC-3 cells as well as LNCaP human prostate carcinoma cells stably expressing IL-15 on the cell surface. Thus, IL-15 can exist as a biologically active transmembrane molecule that possesses dual ligand-receptor qualities with a potential to induce bidirectional signaling. This fact highlights a new level of complexity in the biology of IL-15 and offers novel important insights into our understanding of the cellular responses modulated by this pleiotropic cytokine.

Cell Line, Tumor↗

PubMed2004

Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17).

This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.

ADAM Proteins↗

PubMed2003

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-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha 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-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha 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-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

Alternative Splicing↗

PubMed2003

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 Th1 immune responses by employing two delayed-type hypersensitivity (DTH) models. IL-15-deficient (IL-15(-/-)) mice or mice treated with soluble IL-15Ralpha 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) from IL-15(+/+), IL-15(-/-) or IL-15Ralpha(-/-) 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-15Ralpha 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-15Ralpha 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 therapeutic modulation of clinically relevant DTH reactions.

Animals↗

PubMed2002

Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate multiple downstream signaling events in a human T-lymphoblastoid cell line.

Adenosine Triphosphate↗

PubMed2002

Novel B cell population producing functional IgG in the absence of membrane IgM expression.

Surface expression of IgM is a characteristic feature of the development of most B cells. Only pre-B cells bearing functional IgM heavy chains mu chains) are selected for clonal expansion and differentiation. Cells lacking mu chains are normally eliminated. muMT mice carrying a deletion of the first exon coding for the transmembrane domain of the immunoglobulin mu chain gene were described as mice deficient for mature B cells, plasma cells and immunoglobulins in serum. In this study, we describe in muMT/BALB/c mice the presence of a novel B cell population, producing IgG, IgA and IgE in the absence of IgM membrane expression. Moreover, this small population of B cells is able to recognize antigens and to differentiate into plasma cells. These "non-conventional" mu(- / -) B cells produce functional immunoglobulins after immunization, undergo germinal center reactions, and maintain B cell memory. Our findings support the concept, that a small percentage of mu -non-expressing pre-B cells can escape elimination, switch to downstream immunoglobulin heavy chains and respond to antigens. It remains an open question how the reactivity of these B cells is regulated and in which extent such B cells play a role in physiological and pathological processes such as autoantibody production and autoimmunity.

Animals↗