Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X(7) receptor subtype (P2X(7)R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1beta. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X(7)R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X(7)R by extracellular ATP causes IL-1beta release from the vesicle lumen. Previous studies demonstrated that high extracellular K(+) inhibits IL-1beta processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1beta loading.     

Extracellular signal-regulated protein kinase signaling pathway negatively regulates the phenotypic and functional maturation of monocyte-derived human dendritic cells

Dendritic cells (DC) are highly specialized antigen-presenting cells that on activation by inflammatory stimuli (eg, tumor necrosis factor alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) or infectious agents (eg, lipopolysaccharide [LPS]), mature and migrate into lymphoid organs. During maturation, DC acquire the capacity to prime and polarize resting naive T lymphocytes. Maturation of monocyte-derived DC (MDDC) is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. This study found that in the presence of the mitogen-activated protein kinase kinase 1-extracellular signal-regulated kinase (ERK) inhibitors PD98059 or U0126, TNF-alpha- and LPS-induced phenotypic and functional maturation is enhanced. ERK pathway inhibitors increased expression of major histocompatibility complex and costimulatory molecules; loss of mannose-receptor-mediated endocytic activity; nuclear factor-kappaB DNA-binding activity; release of IL-12 p40; and allogeneic T-cell proliferation induced by LPS or TNF-alpha. Moreover, PD98059 and U0126 enhanced LPS-triggered production of IL-12 p70. In agreement with the effect of ERK inhibitors, maturation of MDDC was delayed in the presence of serum, an effect that was reversed by U0126. These results indicate that the ERK and p38 MAPK signaling pathways differentially regulate maturation of MDDC and suggest that their relative levels of activation might modulate the initial commitment of naive T-helper (Th) cells toward Th1 or Th2 subsets. The findings also suggest that maturation of MDDC might be pharmacologically modified by altering the relative levels of activation of both intracellular signaling routes.     

Constitutive expression of IL-12R beta 2 on human multiple myeloma cells delineates a novel therapeutic target

The interleukin-12 (IL-12) receptor (R) B2 gene acts as tumor suppressor in human acute and chronic B-cell leukemias/lymphomas and IL-12rb2–deficient mice develop spontaneously localized plasmacytomas. With this background, we investigated the role of IL-12Rβ2 in multiple myeloma (MM) pathogenesis. Here we show the following: (1) IL-12Rβ2 was expressed in primary MM cells but down-regulated compared with normal polyclonal plasmablastic cells and plasma cells (PCs). IL-6 dampened IL-12Rβ2 expression on polyclonal plasmablastic cells and MM cells. (2) IL-12 reduced the proangiogenic activity of primary MM cells in vitro and decreased significantly (P = .001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of proangiogenic genes and up-regulated expression of the antiangiogenic genes IFN-, IFN-, platelet factor-4, and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the proangiogenic genes CCL11, vascular endothelial-cadherin, CD13, and AKT and to up-regulation of an IFN-–related antiangiogenic pathway. Thus, IL-12Rβ2 directly restrains MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.     

PTHrP inhibits BMP-6 expression through the PKA signaling pathway in breast cancer cells

Background  

PTHrP, a mediator of humoral hypercalcemia of malignancy, is considered as a potential activator to induce breast cancer cells metastasizing to bone. However, recent clinical evidences and basal research results prove that PTHrP expression in primary tumors indicates good prognosis. BMP-6, as a member of TGF-β superfamily, is closely correlated with tumor differentiation and skeletal metastasis.     

Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets

Adenosine diphosphate (ADP), an important platelet agonist, acts through 2 G-protein-coupled receptors (GPCRs), P2Y(1) and P2Y(12), which signal through Gq and Gi, respectively. There is increasing evidence for cross-talk between signaling pathways downstream of GPCRs and here we demonstrate cross-talk between these 2 ADP receptors in human platelets. We show that P2Y(12) contributes to platelet signaling by potentiating the P2Y(1)-induced calcium response. This potentiation is mediated by 2 mechanisms: inhibition of adenylate cyclase and activation of phosphatidylinositol 3 (PI 3)-kinase. Furthermore, the Src family kinase inhibitor PP1 selectively potentiates the contribution to the calcium response by P2Y(12), although inhibition of adenylate cyclase by P2Y(12) is unaffected. Using PP1 in combination with the inhibitor of PI 3-kinase LY294002, we show that Src negatively regulates the PI 3-kinase-mediated component of the P2Y(12) calcium response. Finally, we were able to show that Src kinase is activated through P2Y(1) but not P2Y(12). Taken together, we present evidence for a complex signaling interplay between P2Y(1) and P2Y(12), where P2Y(12) is able to positively regulate P2Y(1) action and P2Y(1) negatively regulates this action of P2Y(12). It is likely that this interplay between receptors plays an important role in maintaining the delicate balance between platelet activation and inhibition during normal hemostasis.     

Both the ADP receptors P2Y1 and P2Y12, play a role in controlling shape change in human platelets

Two types of ADP receptors, P2Y(1) and P2Y(12) are involved in platelet aggregation. The P2X(1) receptor is also present but its role, in terms of platelet function, is not yet defined. The aim of this study was to establish if the ADP receptors, P2Y(1,) P2Y(12) and P2X(1) play a role in controlling platelet shape change (PSC) in human platelets. PSC is an early phase of platelet activation that precedes aggregation. Using a high-resolution channelyzer, PSC was assessed by measuring the median platelet volume (MPV). The P2Y(1) receptor antagonist MRS 2179 (1.06 - 10.25 micro mol/l) blocked ADP-induced PSC (by 100%). The median IC(50) was 3.16 micro mol/l. MRS 2179 also significantly (P = 0.01) inhibited PSC induced by the combination of ADP + serotonin (5HT). The P2Y(12) receptor antagonist AR-C69931MX significantly inhibited (at 10s, P = 0.009; 15 s, P = 0.001 and 30 s, P = 0.015) ADP-induced PSC. The P2X(1) receptor antagonist TNP-ATP had no significant effect on ADP- or ADP + 5HT-induced PSC. We conclude that the IC(50) of a P2Y(1)-blocker can be derived because of the high-resolution and reproducibility of the channelyzer technique. In addition to the P2Y(1) purinoceptor, the P2Y(12)receptor appears to be involved in ADP-induced PSC since this process was significantly inhibited by AR-C69931MX. The channelyzer technique may be more reliable than optical aggregometry to assess PSC.     

Expression of Tyk2 in dendritic cells is required for IL-12, IL-23, and IFN-gamma production and the induction of Th1 cell differentiation

It is well documented that dendritic cells (DCs), representative antigen-presenting cells, are important sources of Th1-promoting cytokines and are actively involved in the regulation of T-helper-cell differentiation. However, the intracellular event that regulates this process is still largely unknown. In this study, we examined the role of Tyk2, a JAK kinase that is involved in the signaling pathway under IL-12 and IL-23, in DC functions. While the differentiation and maturation of DCs was normal in Tyk2-deficient (Tyk2(-/-)) mice, IL-12-induced Stat4 phosphorylation was diminished in Tyk2(-/-) DCs. IL-12-induced IFN-gamma production was also significantly diminished in Tyk2(-/-) DCs to levels similar to those in Stat4(-/-) DCs. Interestingly, Tyk2(-/-) DCs were defective in IL-12 and IL-23 production upon stimulation with CpG ODN. Furthermore, Tyk2(-/-) DCs were impaired in their ability to induce Th1-cell differentiation but not Th2-cell differentiation. Taken together, these results indicate that the expression of Tyk2 in DCs is crucial for the production of Th1-promoting cytokines such as IL-12 and IFN-gamma from DCs and thereby for the induction of antigen-specific Th1-cell differentiation.     

不同条件下尼古丁对致敏大鼠树突状细胞分泌白介素10及白介素12的影响

目的研究不同实验条件尼古丁对致敏大鼠树突状细胞（DC）及白介素10（IL-10）、白介素12（IL-12）表达的影响,从细胞水平探讨尼古丁加重哮喘的免疫学机制。方法制备致敏大鼠模型,培养骨髓来源的DC,接种于6孔培养板中,随机分为对照组及实验组,进行以下研究。①尼古丁作用的浓度依赖性研究：分别用50、100、200、400mg/L的尼古丁孵育细胞,72h后收集各组细胞的上清液;②尼古丁作用的时间依赖性研究：用400mg/L的尼古丁与细胞分别孵育0、4、8、12、24、72h,收集各组细胞的上清液。采用酶联免疫吸附双抗体夹心分析法测定各组细胞上清IL-10和IL-12的浓度。结果①DC与不同浓度尼古丁孵育72h后,200、400mg/L尼古丁组IL-10的表达较对照组均明显减少,差异有统计学意义（P〈0.01）;400mg/L尼古丁组IL-12较对照组均明显减少,差异有统计学意义（P〈0.01）;②DC与400mg/L的尼古丁分别孵育4、8、12、24、72h,其中12、24、72h组IL-10蛋白均较对照组明显减少,差异有统计学意义（P〈0.01）,12h组IL-12与对照组比较明显升高,72h组IL-12与对照组比较明显降低,差异有统计学意义（P〈0.01）。结论一定浓度的尼古丁可以抑制致敏大鼠DCIL-10及IL-12的表达,并且呈时间依赖性,这可能是吸烟加重和改变哮喘的气道炎症反应的免疫学机制之一。     

不同条件下尼古丁对致敏大鼠树突状细胞分泌白介素10及白介素12的影响

目的 研究不同实验条件尼古丁对致敏大鼠树突状细胞(DC)及白介素10(IL-10)、白介素12(IL-12)表达的影响,从细胞水平探讨尼古丁加重哮喘的免疫学机制.方法 制备致敏大鼠模型,培养骨髓来源的DC,接种于6孔培养板中,随机分为对照组及实验组,进行以下研究.①尼古丁作用的浓度依赖性研究:分别用50、100、200...     

Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs

Dendritic cells (DCs) are known to induce the growth and function of natural killer (NK) cells. Here, we address the capacity of DCs to interact with NK cells in human lymphoid organs and identify the role of specific DC-derived cytokines. We demonstrate that DCs colocalize with NK cells in the T cell areas of lymph nodes. In culture, DCs from either blood or spleen primarily stimulate the CD56(bright)CD16- NK cell subset, which is enriched in secondary lymphoid tissues. Blocking of IL-12 abolished DC-induced IFN-gamma secretion by NK cells, whereas membrane-bound IL-15 on DCs was essential for NK cell proliferation and survival. Maturation by CD40 ligation promoted the highest IL-15 surface presentation on DCs and led to the strongest NK cell proliferation induced by DCs. These results identify secondary lymphoid organs as a potential DC/NK cell interaction site and identify the distinct roles for DC-derived IL-12 and IL-15 in NK cell activation.     

MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells

IL-12 exerts several regulatory effects on natural killer (NK) cells by activating IL-12 signaling. IL-12 signaling is tightly auto-regulated to control its onset and termination, with prolonged IL-12 treatment resulting in IL-12 hyporesponsiveness. However, the mechanisms underlying IL-12 auto-regulation are still unclear. In this study we report that prolonged IL-12 treatment significantly up-regulates microRNAs (miRNAs), including miR-132, -212, and -200a in primary human NK cells. This up-regulation correlates temporally with gradually decreasing STAT4 levels and decreasing IFN-γ expression, after an initial increase within the first 16 hours of IL-12 treatment. The IL-12 hyporesponsiveness is dependent on IL-12 concentration, and associated up-regulation of miR-132, -212, and -200a. Furthermore, IL-12-hyporesponsive cells regain responsiveness of IFN-γ production 24 hours after IL-12 removal, which correlates with decreases in miR-132, -212, and -200a levels. Overexpression of miR-132, -212, and -200a by transfection into NK cells mimics IL-12 priming, inducing IL-12 hyporesponsiveness, whereas transfection of miR-132, -212, and -200a inhibitors largely abolishes IL-12 induction of IL-12 hyporesponsiveness. These data suggest that miR-132, -212, and -200a up-regulation during prolonged IL-12 treatment, negatively regulates the IL-12 signaling pathway by reducing STAT4 expression in primary human NK cells.     

Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation

Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A2A adenosine receptors and HMEC-1 preferentially express A2B receptors. Neither cells expressed A1 or A3 receptors. The nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Cotransfection of each type of adenosine receptors with a luciferase reporter in HMEC-1 showed that A2B receptors, but not A1, A2A, or A3, activated IL-8 and VEGF promoters. These effects were mimicked by constitutively active alphaG(q), alphaG12, and alphaG13, but not alphaG(s) or alphaG(i1-3). Furthermore, stimulation of phospholipase C indicated coupling of A2B receptors to G(q) proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes to functional heterogeneity of human endothelial cells. A2B receptors, predominantly expressed in human microvascular cells, modulate expression of angiogenic factors via coupling to G(q), and possibly via G12/13.     

Recombinant HBsAg inhibits LPS-induced COX-2 expression and IL-18 production by interfering with the NFkappaB pathway in a human monocytic cell line, THP-1

BACKGROUND/AIMS: Hepatitis B virus suppresses the human immune-system and HBsAg inhibits the induction of cytokines by LPS in human macrophages, but the mechanisms involved remain unclear. COX-2 and its product, PGE2, play a role in hepatitis B and IL-18 has also been shown to inhibit HBV infection in vivo. We investigated whether rHBsAg affects induction of COX-2 and IL-18 by LPS and, if so, which signal pathways are involved in a human monocytic cell line, THP-1. METHODS: Cell culture, Western blotting for COX-2, ERK and IKB-alpha, immunofluorescence for HBsAg and NFkappaB protein and ELISA for PGE2, IL-18 and IL-12 were performed. RESULTS: rHBsAg inhibits LPS-induced COX-2 expression in a time- and dose-dependent manner by blocking the ERK and NFkappaB pathways. LPS-induced IL-18 production was also down-regulated by rHBsAg by interfering mainly with the NFkappaB pathway. PGE2 reversed the inhibition of LPS-induced IL-18 production by rHBsAg. rHBsAg was also found to inhibit the induction of IL-12 by LPS in THP-1 cells. CONCLUSIONS: These results showed a novel anti-inflammatory property of rHBsAg which involves inhibition of COX-2 and suggested that hepatitis B virus may regulate IFN-gamma production by inhibiting IL-18 and IL-12 production.