CD154 inhibits tumor-induced apoptosis in dendritic cells and tumor growth.

We have recently demonstrated that murine and human tumors induce apoptosis of dendritic cells (DC). Here, we evaluated the effect of CD40 ligation on the survival of tumor-associated DC and tumor growth. Retroviral transduction of MC38 colon carcinoma cells with the CD154 gene resulted in inhibition of tumor growth. This effect was abrogated in IL-12 knockout mice. Immunohistochemical analysis revealed an increase in CD11c+ (N418) and CD8+ but not NLDC-145+ cells in CD154-transfected tumors in wild-type mice. This increase was less pronounced in IL-12-deficient mice. In vitro, overexpression of CD154 on tumor cells significantly decreased the level of tumor-induced DC apoptosis. Surprisingly, the CD154-induced protection of DC from tumor-induced apoptosis was IL-12 independent in vitro, suggesting an IL-12-dependent and an IL-12-independent mechanism of CD154-induced anti-tumor immunity. Thus, our data suggest a new strategy to improve immunotherapy of cancer by protecting DC from tumor-induced apoptosis.     

Spontaneous programmed cell death of peripheral blood mononuclear cells from HIV-infected persons is decreased with interleukin-15

Interleukin 15 (IL-15) is an important regulatory cytokine in cellular immunity. In vitro replacement of IL-15 has been shown to enhance immunity in Human immunodeficiency virus type 1 (HIV-1) infected lymphocytes. We evaluated the effect of IL-15 on the survival of peripheral blood mononuclear cells of HIV patients by examining in vitro lymphocyte apoptosis, and correlated the process with Bcl-2 and Fas gene regulation. Peripheral blood mononuclear cells (PBMC) from 21 HIV-infected adults and 24 HIV-seronegative healthy individuals were isolated and cultured to determine the effect of escalating doses of IL-15 (0, 1, 10, 100, 1000 ng/mL) on apoptosis. Lymphocyte proliferation assay with (3H) TdR was measured and Bcl-2 and Fas gene regulation was observed. The results were as follows: 1) IL-15 reduced culture induced lymphocyte apoptosis in HIV patients in a dose dependent manner, and reached a plateau level at a concentration of 100 ng/ml; 2) IL-15 significantly reduced the level of apoptosis after 3 days (14%) and 5 days (15%) of culture in HIV patients, while no difference was observed in HIV (-) donors; 3) The percentage of viable cells among the total number of lymphocytes was significantly enhanced by 25% in HIV patients with IL-15; 4) Bcl-2 expression was decreased in HIV patients (53.9 +/- 12.3%) compared to HIV (-) donors (93.0 +/- 3.7%), and IL-15 increased Bcl-2 expression by 21.2 +/- 5.2% in HIV patients; 5) Fas expression was increased in HIV patients (70.2 +/- 4.6%) compared to HIV (-) donors (32.4 +/- 4.3%), and IL-15 increased Fas expression by 8.4 +/- 1.2% in HIV (-) donors. Our findings indicate that IL-15 may influence immunologic abnormalities in HIV infection, particularly its ability to prevent apoptosis of lymphocytes by suppressing the down-modulation of Bcl-2. This may provide an experimental basis for IL-15 immunotherapy.     

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.     

The generation of human dendritic and NK cells from hemopoietic progenitors induced by interleukin-15.

Interleukin-15 (IL-15) is a pleiotropic cytokine that induces the generation and differentiation of lymphoid cells and shares many biological activities with IL-2. We have shown here the development of dendritic cells (DC) from human CD34+ hemopoietic precursor cells cultured for 2-4 weeks with IL-15 alone. DC generated with IL-15 have typical morphological, immunocytochemical, phenotypic, and functional characteristics of mature DC. Dual flow cytometry analysis performed weekly demonstrated increasing co-expression of CD1a or CD83 with HLA-DR, CD80, CD86, IL-2R alpha, beta, and gamma. Two populations of cells were distinguished among CD34+ progeny. Small and medium-size cells were mainly natural killer (NK) cells (72.6-85.2% CD56+) and low numbers of DC (9.1-21.3% CD1a+). Large cells were mostly DC (75.4-95.4% CD1a+). Isolated CD34+ cells did not express IL-2R subunits but after 2-3 days in culture with IL-15, they were found to express IL-2Rgamma. Induced expression of IL-2Rgamma on CD34+ cells may explain the primary mechanism of IL-15-regulated differentiation of hemopoietic precursor cells. Thus, our data suggest that IL-15 stimulates CD34+ cells to differentiate into NK and DC and may represent a new growth and survival factor for lymphoid DC.     

白细胞介素12诱导T细胞凋亡对Bcl-2表达和信号分子的影响

目的:了解和探讨白细胞介素12(IL-12)作用于T细胞,活化T细胞表面的IL-12受体β,诱导T细胞凋亡对Bcl-2表达和信号转导的作用。方法:用AnnexinV流式细胞仪检测细胞凋亡率和半定量PCR的方法测定在不同抑制剂作用下对Bcl-2信号转导表达的影响。结果:IL-12在高浓度下诱导人的白血病T细胞株TIB-152和人的淋巴瘤T细胞株HTB-176及正常人T细胞的凋亡。在IL-12处理6h后Bcl-2的表达明显升高,表达的高峰值在24h,Bcl-2的升高不能抑制和阻碍T细胞的凋亡;IL-12诱导上调T细胞Bcl-2mRNA表达。HA100能促进T细胞的凋亡,PKC是IL-12诱导T细胞凋亡信号转导的负性调节因子;AG490不抑制IL-12上调的Bcl-2mRNA表达作用,AG490阻断的Jak2通路不参与IL-12诱导T细胞mRNA表达的信号转导及其调节;HA1004不影响T细胞表达mRNA,PKA通路不影响IL-12诱导T细胞表达Bcl-2mRNA的信号传递。结论:Bcl-2是凋亡介导分子之一,但是Bcl-2不能阻断Fas-FasL介导的T细胞凋亡。     

Activation, survival and apoptosis of CD45RO+and CD45RO−T cells of human immunodeficiency virus-infected individuals: effects of interleukin-15 and comparison with interleukin-2

HIV infection is associated with increased representation of T cells bearing an activated, memory (CD45RO+) phenotype. Although administration of antiretroviral agents and interleukin-2 (IL-2) augment depleted CD4+ T-cell numbers, such therapies have been preferentially beneficial for CD45RO+ T cells. Interleukin-15 (IL-15) exhibits many biological activities in common with IL-2, including promoting T-cell survival and proliferation. The present study found that these two cytokines differed in their ability to induce proliferation, enhance survival, and control apoptosis of CD45RO+ and CD45RO- T-cell populations of human immunodeficiency- (HIV) infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 in activating and stimulating proliferation of CD4+CD45RO+, CD8+CD45RO+ and CD8+CD45RO- cells, but failed to be more effective than IL-2 in reducing apoptosis. Poor activation of CD4+CD45RO- cells by IL-15 and to IL-2 appeared to be attributable to low expression of the beta receptor chain utilized by both cytokines. However, IL-15 was more effective than IL-2 in enhancing survival of the CD4+CD45RO- population, suggesting a greater protective effect of IL-15 for naive CD4+ T cells, which are preferentially lost in HIV-infected individuals.     

Bcl-xL is associated with the anti-apoptotic effect of IL-15 on the survival of CD56(dim) natural killer cells

Human NK cells can be distinguished into CD56(bright) and CD56(dim) subsets based on cell surface CD56 density. It has been shown that IL-2 and IL-15 have opposing effects on life and death of CD8(+) T cells. However, the roles of IL-2 and IL-15 in regulating these two NK cell subsets remain elusive. In this study, we comparatively analyzed the effects of IL-2 and IL-15 on two NK cell subsets. IL-15 improved the proliferation and activation of CD56(dim) NK cells in long-term cord blood mononuclear cell culture, but IL-2 only maintained the survival of CD56(bright) NK cells. The percentage of CD56(+)Annexin V(+) NK cells cultured with IL-15 was lower than that with IL-2; moreover, most of Annexin V(+) NK cells were primarily in the CD56(dim) NK cells. IL-15 cultured NK cells expressed higher level of Bcl-xL than IL-2 cultured cells. Furthermore, IL-15 more strongly upregulated CD25 expression and better maintained the expression of IL-15Ralpha than IL-2. These results suggest that CD56(dim) NK cells undergo apoptosis when cultured with IL-2, but IL-15 inhibits their apoptosis and Bcl-xL is associated with the anti-apoptotic effect of IL-15. So IL-15 played a crucial role in sustaining long-lasting functions of CD56(dim) NK cells.     

Distinct mechanisms for rescue from apoptosis in Ramos human B cells by signaling through CD40 and interleukin-4 receptor: role for inhibition of an early response gene,Berg36

The role of interleukin-4 (IL-4) and CD40 signaling in negative regulation of apoptosis in human Ramos B cells induced in response to different agents was investigated. CD40 ligation protected cells from apoptosis induced by calcium ionophore through an initial, rapid and apparently Bcl-2-independent mechanism, associated with up-regulation of Bcl-x_L. However, rescue from apoptosis induced by inhibition of macromolecular synthesis required several hours of prior stimulation with CD40 ligand/antibody and was accompanied by up-regulation of Bcl-2. In contrast, IL-4 did not up-regulate Bcl-2 or Bcl-x_L and did not inhibit apoptosis induced by inhibitors of macromolecular synthesis. However, IL-4 did protect Ramos cells from apoptosis induced by calcium ionophore and this effect was accompanied by inhibition of ionophore-induced expression of an immediate early gene encoding a 36-kDa zinc-finger protein, Berg36. Anti-sense blockade of Berg36 expression partially inhibited ionophore-induced apoptosis to an extent commensurate with the level of IL-4 protection, implicating Berg36 function as a requirement for apoptosis induced through calcium signaling and as a target for IL-4 through which this cytokine inhibits apoptosis in Ramos B cells. These distinct mechanisms for rescue from apoptosis by CD40 and IL-4 may help explain the co-operative roles of these T cell-derived signals for B cell survival.     

IFN-α primes T- and NK-cells for IL-15-mediated signaling and cytotoxicity

Recently it has become clear that interferon (IFN)-α, a type I interferon produced rapidly in response to infection, not only plays a key role in innate immunity, but also promotes adaptive immune responses by influencing the production or function of other cytokines. During infections IFN-α fosters the production of IL-15, which plays a pivotal role in the development, survival and function of NK cells and recruitment and activation of T cells. Since these two cytokines exert overlapping functions during infections, this investigation was undertaken to study the priming effect of IFN-α on the effect of IL-15 on human T and NK cells. We show that IFN-α induces an increased expression of IL-15Rα in human activated peripheral T cells, and in CD8⁺ and CD4⁺ T-cell lines. Functionally, the IFN-α-enhanced IL-15Rα expression resulted in an enhanced IL-15-mediated phosphorylation of STAT5 and STAT3 followed by a further increase in IL-15Rα expression. Moreover, IFN-α significantly increased the IL-15-induced cytotoxic activity of freshly isolated T and NK cells. Taken together, our data show that IFN-α boosts signaling and functional effects of IL-15, at least in part by fostering the increased IL-15R expression, thus add new facet to the emerging role of IFN-α as an important primer of adaptive immune responses.     

Differential Role for IL-2 and IL-15 in the Inhibition of Apoptosis in Short Term Activated Human Lymphocytes

Interleukin (IL)-15 is a newly described cytokine with properties similar to IL-2. Even though it does not share sequence homology with IL-2, both cytokines bind to the same receptor with the noted exception of a cytokine specific α-chain. In this study the authors compared IL-2 and IL-15 to determine their ability to rescue short term activated lymphocytes (phytohaemagglutinin stimulation of peripheral blood mononuclear cells for 6 days, followed by expansion in medium containing IL-2 for 2 days) from apoptotic cell death. The authors found that both IL-2 and IL-15 can inhibit induction of apoptosis in this experimental model with similar time and dose kinetics. On mRNA or protein levels induction of pro- and anti-apoptotic gene products like fasL, bcl-2, or bax with minor effects on fas/Apo-1 or bcl-x_L was observed under culture conditions with both IL-2 and IL-15. Next, it was found that phytohaemagglutinin (PHA) blasts were less responsive (in terms of cellular proliferation and prevention from apoptosis) to IL-2 if signals through the α-chain were blocked, with no effect on β-chain specific monoclonal antibodies (MoAb). By contrast, IL-15 was less effective in induction of cellular proliferation and prevention of apoptosis if IL-2R β-chain specific MoAb were added to cell cultures. Testing intracellular signalling induced by IL-2 or IL-15, the authors found identical changes in tyrosine phosphorylation patterns in PHA blasts cultured in medium or under IL-2 or IL-15 stimulation. By contrast, they found consistent differences if PHA stimulated peripheral blood mononuclear cells (PBMC) were expanded in medium containing IL-15 (instead of IL-2). These IL-15 expanded PHA blasts showed a significantly increased percentage of apoptosis after growth factor withdrawal. Furthermore, IL-2 was more efficient than IL-15 in rescuing IL-15 expanded PHA blasts from apoptosis. In IL-15 expanded PHA blasts expression of IL-2R α-chain was lower than that in IL-2 expanded PHA blasts. A model presenting a differential role for IL-2 and IL-15 in inhibition of apoptosis in vivo is discussed.     

Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL-12 and NF-kappa B. In this paper, we demonstrate that transfection of DC with IL-10-specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA was accompanied by increased CD40 expression and IL-12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL-10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL-10 siRNA-treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL-10 siRNA-transfected DC enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL-10 siRNA-transfected antigen-presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.     

Expression of IL-1Rrp2 by human myelomonocytic cells is unique to DCs and facilitates DC maturation by IL-1F8 and IL-1F9

We report for the first time that expression of the novel IL-1 cytokine receptor IL-1Rrp2 (IL-1R6) is unique to DCs within the human myelomonocytic lineage. IL-1Rrp2 was expressed by monocyte-derived dendritic cells (MDDCs) which was dose-dependently increased by IL-4 and correlated with increased numbers of differentiated MDDCs. Human plasmacytoid DCs also express IL-1Rrp2 but the receptor is not expressed by either myeloid DC type 1 (mDC1) or mDC2 cells. We also show that IL-1F8 or IL-1F9 cytokines, which signal through IL-1Rrp2, induce maturation of MDDCs, as measured by increased expression of HLA-DR and CD83 and decreased expression of CD1a. Furthermore, IL-1F8 stimulated increased CD40 and CD80 expression and IL-18 and IL-12 p70 production by MDDCs, which induced proliferation of IFN-γ-producing CD3(+) lymphocytes (indicative of inflammatory Th1 subsets). IL-1F8 and IL-1F2 were equipotent in their ability to stimulate IL-18 secretion from MDDCs but IL-1F8 was not as potent as IL-1F2 in stimulating secretion of IL-12p70 from MDDCs or inducing lymphocyte proliferation Therefore, IL-1Rrp2 expression by some DC subsets may have an important function in the human immune response in vivo via its role in differentiation of inflammatory Th1 lymphocytes.     

Enhanced survival and potent expansion of the natural killer cell population of HIV-infected individuals by exogenous interleukin-15.

The CD56+CD16+ natural killer (NK) cell population plays a crucial role in eliminating virus-infected cells and is diminished in HIV-infected individuals. This study examined the effects of exogenous interleukin (IL)-15 on proliferation and survival of CD56+ and CD16+ cells of HIV-infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 as a growth factor for CD56+ cells, as well as for CD16+ cells and also CD4+ and CD8+ T cells. Analysis of cell survival in etoposide-treated cultures indicated that IL-15 was also more potent than IL-2 as a survival factor for CD56+ cells by virtue of its greater ability to up-regulate bcl-2 expression. Although IL-15-induced proliferation of CD56+ cells was accompanied by increased apoptosis, IL-15 was more effective than IL-2 in increasing the representation of viable CD56+ cells in the peripheral blood mononuclear cell population, but less effective in increasing T cell representation. The immunotherapeutic potential of IL-15 appears superior to IL-2 in regard to expanding NK cell populations in HIV-infected individuals, but needs to be weighed against poorer increases in T cell populations.     

Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.     

IL-24基因修饰的树突状细胞促进CIK细胞对肺癌细胞的杀伤作用

目的 研究IL-24基因修饰的树突状细胞(DC)与同源细胞因子诱导的杀伤细胞(cyto-kine-induced killer,CIK)共培养后对A549肺癌细胞的杀伤作用及其机制.方法 从健康人外周血单个核细胞中常规诱导DC、CIK细胞,同时抽提重组腺病毒质粒pAdEasy-1-pTrack-CMV-IL-24,Pac I酶切线性化后脂质体转染QBl-293A细胞,收获Ad-IL-24重组病毒.将IL-24基因通过重组病毒导入已负载肿瘤抗原的DC,获得细胞称为DC-IL-24.RT-PCR和ELISA法检测DC中IL-24基因的表达,FCM和ELISA法检测DC表型及分泌细胞因子能力的变化,将DC和CIK细胞混合培养,溶血试验检测CIK细胞产生穿孔素的能力,FCM法检测共培养的DC-CIK细胞对A549肺癌细胞细胞毒活性的变化.结果 获得了高滴度的重组病毒Ad-IL-24并成功将IL-24基因导入DC,在倒置荧光显微镜下可观察到荧光,IL-24可上调Dc表而CD80、CD83、HLA-DR、CD40、CXCR4分子的表达,转染后Dc分泌IL-12、TNF-α和IL-24的能力显著增强,DC-IL-24更能促进CIK细胞产生穿孔素,与同源CIK细胞共培养后对A549肺癌细胞的细胞毒活件明显增强.结论 IL-24基因修饰的DC能增强自体CIK细胞产生特异性抗肿瘤免疫,其机制与IL-24能促进DC表型成熟,分泌Th1型细胞因子,维持DC活化状态,进而促进CIK细胞活化密切相关.     

Preactivation with IL-12, IL-15, and IL-18 Induces CD25 and a Functional High-Affinity IL-2 Receptor on Human Cytokine-Induced Memory-like Natural Killer Cells

Natural killer (NK) cells are effector lymphocytes that are under clinical investigation for the adoptive immunotherapy of hematologic malignancies, especially acute myeloid leukemia. Recent work in mice has identified innate memory-like properties of NK cells. Human NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief preactivation with IL-12, IL-15, and IL-18, which later exhibit enhanced functionality upon restimulation. However, the optimal cytokine receptors and signals for maintenance of enhanced function and homeostasis after preactivation remain unclear. Here, we show that IL-12, IL-15, and IL-18 preactivation induces a rapid and prolonged expression of CD25, resulting in a functional high-affinity IL-2 receptor (IL-2Rαβγ) that confers responsiveness to picomolar concentrations of IL-2. The expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2Rαβγ. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to costimulate IFN-γ production by preactivated NK cells, an effect that was CD25 dependent. Picomolar concentrations of IL-2 also enhanced NK cell proliferation and cytotoxicity via the IL-2Rαβγ. Further, after adoptive transfer into immunodeficient NOD-SCID-γ_c^−/− mice, human cytokine–preactivated NK cells expand preferentially in response to exogenous IL-2. Collectively, these data demonstrate that human CIML NK cells respond to IL-2 via IL-2Rαβγ with enhanced survival and functionality, and they provide additional rationale for immunotherapeutic strategies that include brief cytokine preactivation before adoptive NK cell transfer, followed by low-dose IL-2 therapy.     

Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Ralpha chain

Interleukin-15 (IL-15) is a cytokine that possesses interesting, potential therapeutic properties. However, based on several parameters including activation of neutrophils, it is also recognized as a proinflammatory cytokine. The mechanisms by which IL-15 activates human neutrophil functions are not fully understood. Although these cells express a functional IL-15 receptor (IL-15R) composed of IL-15Ralpha, IL-2/15Rbeta (CD122), and gamma(c) (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15Ralpha, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15Ralpha, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2Ralpha (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15Ralpha. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15Ralpha chain plays a key role in mediating this response, at least by interacting with Syk kinase.     

Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206⁺ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206⁺ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.