Vadim Budagian

Inhibition of keratinocyte apoptosis by IL-15 : a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Rα protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.

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.

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.

Blocking IL-15 Prevents the Induction of Allergen-Specific T Cells and Allergic Inflammation In Vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Rα (sIL-15Rα) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-α, IL-1β, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Rα treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Rα-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4+/CD25+ T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.

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.

Interleukin-15 stimulates macrophages to activate CD4+ T cells: a role in the pathogenesis of rheumatoid arthritis?

Interleukin‐15 (IL‐15) is a proinflammatory cytokine that is overexpressed in rheumatoid arthritis (RA), a disease characterized by activation of monocytes/macrophages (MΦ), and by expansion of autoreactive CD4+ T cells. We hypothesized that IL‐15 plays a major role for this expansion of CD4+ T cells and modulates the phenotype of monocytes/MΦ and their interaction with CD4+ T cells. Here, we show that IL‐15 enhances the proliferation of CD4+ T cells from patients with RA in peripheral blood mononuclear cell cocultures. To further dissect the underlying mechanisms, we employed MΦ from IL‐15−/− or IL‐15 transgenic mice. These were induced to differentiate or were stimulated with IL‐15. Here we show that addition of IL‐15 during differentiation of MΦ (into ‘IL‐15MΦ’) and overexpression of IL‐15 by MΦ from IL‐15tg mice leads to increased levels of major histocompatibility complex class II expression. This resulted in enhanced stimulation of antigen‐specific CD4+ T cells in vitro and was accompanied by reduced messenger RNA expression in MΦ for immunosuppressive SOCS3. The proliferation rates of IL‐15MΦ and IL‐15tgMΦ were high, which was reflected by increased p27Kip1 and reduced p21Waf1 levels. In view of high serum and synovial levels of IL‐15 in patients with RA, our data suggest the possibility that this excess IL‐15 in RA may stimulate monocytes/MΦ to activate the characteristic autoreactive CD4+ T cells in RA.

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

Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, and Institute of Biochemistry, Christian Albrechts University, D-24118 Kiel, Germany; Department of Human Genetics, KU Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB-4), B-3000 Leuven, Belgium; and Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, San Diego, California 92186