IL-13对成纤维细胞IL-13受体和IL-4受体表达的影响

目的研究IL-13对人肺成纤维细胞株HFL-1和人肝星状细胞株LX-2 IL-13受体和IL-4受体表达的调节作用。方法 RT-PCR法检测HFL-1细胞株和LX-2细胞株IL-13Rα1、IL-4R和IL-13Rα2 mRNA的表达;凝胶电泳定量软件Image Tool2.0对RT-PCR电泳条带进行光密度分析;ELISA法检测HFL-1细胞株和LX-2细胞株分泌可溶型IL-13Rα2以及检测细胞裂解液总IL-13Rα1、IL-4R和IL-13Rα2含量。结果 IL-13(5～100 ng/ml)对HFL-1细胞株和LX-2细胞株表达IL-13Rα1和IL-4R无影响;IL-13为5、10、20 ng/ml时能诱导HFL-1细胞株表达IL-13Rα2并呈现剂量依赖,当IL-13为50 ng/ml时,对HFL-1细胞株IL-13Rα2表达的诱导作用明显减弱,IL-13 100 ng/ml组没有检测到HFL-1细胞株IL-13Rα2的表达;LX-2细胞株IL-13Rα2表达缺失且IL-13不能诱导LX-2细胞株表达IL-13Rα2。结论 IL-13不能上调人肺成纤维细胞株HFL-1和人肝星状细胞株LX-2表达功能型受体IL-13Rα1和IL-4R,表明IL-13不能通过上调IL-13Rα1和IL-4R表达量来放大自身作用;一定浓度的IL-13能诱导人肺成纤维细胞株HFL-1表达抑制型受体IL-13Rα2,表明IL-13的自身负调控。     

Interleukin-1 down-regulates gene and surface expression of interleukin-1 receptor type I by destabilizing its mRNA whereas interleukin-2 increases its expression.

The interleukin-1 receptor type I (IL-1RtI) plays an important role in the biological effects of IL-1, but regulation of its surface and gene expression remains unknown. We found that occupancy of 2-15% of the IL-1 surface receptor results in dramatic down-regulation of IL-1RtI both at the mRNA and cell surface level in murine D10S cells, a subline of T-helper type 2 cells. At these low occupancy levels (3 x 10(-12) to 3 x 10(-13) M), the reduction in IL-1RtI surface expression appears at 24 hr and continues to 48 and 72 hr. At the mRNA level, low occupancy of the IL-1R results in decreased IL-1RtI mRNA stability; steady state half-life of the IL-1RtI mRNA is reduced from 6 to 1 hr after exposure to 3 x 10(-12) M IL-1. This down-regulation of IL-1RtI by IL-1 is blocked by cycloheximide, suggesting de novo protein synthesis is necessary for decreased RNA stability. Low concentrations of human IL-1 beta, murine and rabbit IL-1 alpha or beta similarly down-regulated IL-1RtI, whereas low concentrations of human IL-1 alpha failed to reduce the receptor surface expression, despite inducing a full proliferative response. We also observed that the effect of IL-1 on this down-regulation was not through protein kinase C (PKC), since PMA rapidly increased IL-1RtI mRNA levels within 30 min and persisted for 24 hr. IL-2 up-regulated IL-1RtI in D10S cells at both mRNA and protein levels. These results demonstrate that low occupancy of IL-1 receptors induces down-regulation of IL-1RtI surface as well as mRNA expression. The regulation of IL-1RtI gene expression may be one of the mechanisms by which IL-1-mediated events are controlled.     

Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.     

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.     

Seminal plasma and prostaglandin E2 up-regulate fibroblast growth factor 2 expression in endometrial adenocarcinoma cells via E-series prostanoid-2 receptor-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase pathway

BACKGROUND: Prostaglandin E(2) (PGE(2)) has been shown to modulate angiogenesis and tumour progression via the E-series prostanoid-2 (EP2) receptor. Endometrial adenocarcinomas may be exposed to endogenous PGE(2) and exogenous PGE(2), present at high concentration in seminal plasma. METHODS: This study investigated fibroblast growth factor 2 (FGF2) mRNA expression and cell signalling in response to seminal plasma or PGE(2), using an endometrial adenocarcinoma (Ishikawa) cell line stably expressing the EP2 receptor (EP2 sense cells) and endometrial adenocarcinoma explants. RESULTS: Seminal plasma and PGE(2) induced a significant up-regulation of FGF2 expression in EP2 sense but not parental untransfected Ishikawa (wild-type) cells (P < 0.05). These effects were inhibited by co-treatment with EP2 receptor antagonist or inhibitors of protein kinase A, c-Src, epidermal growth factor receptor (EGFR) kinase or extracellular signal-regulated kinase (ERK) signalling. The treatment of EP2 sense cells with seminal plasma induced cAMP accumulation and phosphorylation of c-Src, EGFR kinase and ERK via the EP2 receptor. Finally, seminal plasma and PGE(2) significantly increased FGF2 mRNA expression in endometrial adenocarcinoma tissue explants via the EP2 receptor (P < 0.05). CONCLUSIONS: Seminal plasma and PGE(2) can similarly activate FGF2 expression and EP2 receptor signalling in endometrial adenocarcinoma cells. These data highlight the potential for seminal plasma exposure to facilitate tumorigenesis-angiogenesis in endometrial adenocarcinomas in vivo.     

Regulation of human alveolar macrophage- and blood monocyte-derived interleukin-8 by prostaglandin E2 and dexamethasone.

Mononuclear phagocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophages produce a vast array of regulatory and chemotactic cytokines. Interleukin-8 (IL-8), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammatory events. In this investigation, we describe the effects of prostaglandin E2 (PGE2) and dexamethasone (Dex) on IL-8 mRNA and protein expression from lipopolysaccharide (LPS)-treated human peripheral blood monocytes (PBM) and alveolar macrophages (AM). We demonstrate the dose-dependent suppression of IL-8 from LPS-stimulated PBM by PGE2. Treatment of stimulated PBM with 10(-6) M PGE2 resulted in maximal inhibition, causing 60% suppression of both IL-8 mRNA and extracellular protein levels. In contrast, PGE2 (10(-6) to 10(-8) M) did not significantly alter IL-8 mRNA or protein expression from LPS-treated AM. Treatment of LPS-stimulated PBM and AM with Dex (10(-6) to 10(-8) M) resulted in 75% decline in IL-8 mRNA and extracellular protein from either cell population. Pretreatment of PBM with PGE2 or Dex 1 or 2 h before LPS stimulation caused a significant suppression of steady-state IL-8 mRNA levels; however, administration of either of these modulators 1 or 2 h after LPS stimulation failed to have an inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 and the increase of intracellular cAMP inhibit the expression of interleukin 2 receptors in human T cells

We have analyzed the effect mediated by prostaglandin E2 (PGE2) and different reagents that increase intracellular cAMP on the expression of the p55 subunit (CD25) of interleukin 2 receptors (IL 2R), on the levels of CD25-specific mRNA and on the expression of high affinity IL 2R. In purified T cells, activated either by an anti-CD3 monoclonal antibody or phytohemagglutinin, the addition of PGE2 (10(-6) M), forskolin (5 X 10(-5) M), cholera toxin (0.2 microgram/ml) or dibutyryl cAMP (dBcAMP) (10(-4) M) decreased the cell surface expression of IL 2R by reducing (40%-78% inhibition) the proportions of CD25+ cells as well as the expression of high affinity IL 2R, detectable after 24 h. Furthermore, it was observed that PGE2 reduced the concentration of IL 2R-specific mRNA after a 6-h period of activation, indicating that its regulatory activity takes place at a pretranslational level. The addition of exogenous recombinant IL 2 only partially reversed the inhibition, thus suggesting that PGE2 and increased intracellular concentration of cAMP directly interfered with CD25 expression and that their effect could not be merely attributed to a lack of IL 2-dependent positive feedback. Cells cultured under the same conditions in the presence of phorbol myristate acetate, that activates protein kinase C, were refractory to the cAMP-mediated regulation. Finally, we demonstrate that both PGE2 and dBcAMP inhibit the generation of inositol metabolites after T cell activation, thus indicating that these reagents interfere with early signal transduction mechanisms which precede the synthesis of IL 2R.     

Interleukin-1 induces protein tyrosine phosphorylation in T cells.

Interleukin (IL)-1 alpha activates multiple signal transmission pathways in the T helper type 2 cell line, D10A, and these pathways are linked to two separate IL-1 receptors (IL-1R). In the present report we show that IL-1 induces the activation of tyrosine kinase in these cells, leading to tyrosine phosphorylation of a subset of proteins of 38, 75, 97 and 115 kDa. This type of phosphorylation is prevented by a monoclonal antibody directed against the 80-kDa IL-1R and by tyrphostins which are specific inhibitors of tyrosine kinases. In addition, this inhibitor blocks IL-1-and IL-2-induced proliferation in D10A cells as well as the c-myc and c-myb proto-oncogene mRNA expression in response to IL-1. Interestingly, the inhibitor of cAMP-dependent kinase, H-8, only blocks IL-1-induced c-myb, but not c-myc mRNA expression. Altogether, our results demonstrate that the activation of a tyrosine kinase(s) is an early and major event that happens after IL-1/IL-1R interaction, leading to an increase in intracellular cAMP which results in c-myb and IL-5 mRNA expression. Independent of cAMP, by tyrosine phosphorylation of specific substrates IL-1 also induces c-myc and IL-6 mRNA expression and cellular proliferation.     

Inducible cyclooxygenase-2 gene expression in the human thyroid epithelial cell line Nthy-ori3-1

OBJECTIVE: To investigate whether the genes encoding Cyclooxygenase-1 and -2 are expressed in thyroid epithelial cells, in vitro. MATERIALS AND METHODS: COX-1/-2 gene expression was examined in the thyroid epithelial cell line Nthy-ori3-1 using semi-quantitative RT-PCR and Western blot analysis. ELISAs were employed to assess whole cell COX-enzyme activity, PGE2 and IL-6 formation. RESULTS: In response to IL-1beta and TNF-alpha combined cells of the thyroid epithelial cell line Nthy-ori3-1 secreted marked amounts of PGE2 in a time-dependent fashion. This is attributed to increased levels of COX-2 specific mRNA, increased amounts of COX-2 protein and COX enzyme activity in the absence of detectable COX-1 protein. The inhibition of the induced COX enzyme activity by the selective COX-2 inhibitor NS-398 demonstrated the presence of COX-2 pharmacologically. The expression of the COX-2 gene was also accompanied by a marked induction of IL-6 formation, a well described inflammatory response of thyroid epithelial cells. CONCLUSIONS: Our observation presents first evidence that COX-2 gene expression is inducible in thyroid epithelial cells, in vitro, upon stimulation with the pro-inflammatory cytokines IL-1beta and TNF-alpha. This finding may indicate that thyroid epithelial cells could play an inflammatory controlling role perhaps during auto-immune thyroid diseases.     

Prostaglandin E2 is a potent regulator of interleukin-12- and interleukin-18-induced natural killer cell interferon-gamma synthesis

Synthesis of interferon (IFN)-gamma by natural killer (NK) cells is an important pro-inflammatory event with interleukin (IL)-12 and IL-18 playing major inductive roles. However, other temporal events are likely to regulate such processes and as prostaglandin E2 (PGE2) is ubiquitous during inflammation this study tested the hypothesis that PGE2 was capable of directly modulating cytokine-induced NK cell IFN-gamma synthesis in the absence of other immune cells. Using homogeneous NK cell lines to establish direct effects, PGE2 (0.1-1 micro m) was found to suppress NK cell IFN-gamma synthesis and antagonized the potent synergistic IFN-gamma-inducing effects of IL-12 and IL-18. The actions of PGE2 were mimicked by synthetic PGE2 analogues including misoprostol and butaprost. The selective EP2 receptor agonist butaprost, but not the EP1/EP3 agonist sulprostone, suppressed IFN-gamma synthesis and exclusively competed with PGE2 for receptor binding on NK cells. Further analysis showed that PGE2 did not modulate IL-12 receptor mRNA expression and the effects of PGE2 could be mimicked by the phosphodiesterase inhibitor 3-iosobutyl-1-methylxanthine. The absence of demonstrable receptor modulation coupled with the observed suppression of IFN-gamma synthesis by both EP2 receptor-selective agonists and IBMX suggest that PGE2 acts directly on NK cells via EP2 receptors with its downstream effects on cAMP metabolism. This conclusion is further supported by findings that PGE2 and its analogues consistently elevated levels of cAMP in NK cells. The ability of PGE2 to antagonize the potent inductive signal provided by the combination of IL-12 and IL-18 supports the concept that PGE2 may play an important role in limiting innate inflammatory processes in vivo through direct suppression of NK cell IFN-gamma synthesis.     

Synergy of interleukin 1 (IL-1) with arachidonic acid and A23187 in stimulating PGE synthesis in human fibroblasts: IL-1 stimulates fibroblast cyclooxygenase

The stimulation of prostaglandin E (PGE) synthesis by interleukin 1 (IL-1) has important physiologic effects in many target tissues. The mechanism(s) whereby IL-1 stimulates PGE synthesis is not well understood. Two alternative mechanisms have been postulated: hydrolysis of membrane phospholipid with release of arachidonic acid substrate and induction of cyclooxygenase activity. We examined these mechanisms in IL-1 stimulation of human dermal fibroblast PGE synthesis. IL-1 failed to induce release of previously incorporated radiolabeled arachidonic acid from fibroblasts under conditions where there was a 30-fold or greater stimulation of PGE synthesis. The calcium ionophore, A23187, gave much less stimulation of PGE synthesis while inducing 30-100% release of incorporated [14C]arachidonic acid. There was marked synergy between IL-1 and agents which increased availability of arachidonic acid. For example, the combination of IL-1 and A23187, used at concentrations where neither agent alone stimulated PGE synthesis, increased PGE synthesis from 3.1 to 22.5 ng/ml; similar synergy was seen between IL-1 and exogenous arachidonic acid. To examine a possible effect of IL-1 on cyclooxygenase synthesis, fibroblast cyclooxygenase was measured by radioimmunoassay. Following treatment with IL-1, fibroblasts demonstrated a two- to threefold increase in content of immunoreactive cyclooxygenase. These results suggest that IL-1 increases fibroblast PGE synthesis by a mechanism(s) other than making available increased membrane arachidonic acid, and that at least part of its effect may be mediated by induction of cyclooxygenase. Furthermore, the effect of IL-1 on fibroblast PGE synthesis is markedly potentiated by agents which increase available substrate.     

EP1 and EP4 Receptors Mediate Exocytosis Evoked by Prostaglandin E2 in Guinea-Pig Antral Mucous Cells

Effects of prostaglandin E(2) (PGE(2)) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE(2) elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca(2+) and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 microM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30 % of that evoked by 1 microM PGE(2)). Stimulation with an EP1 agonist (17-P-T-PGE(2), 1 microM), which activates Ca(2+) signals, increased the frequency of exocytotic events to a lesser extent (5 % of that evoked by 1 microM PGE(2)), while addition of an EP1 antagonist (ONO-8713, 10 microM) decreased the frequency of exocytotic events (approximately 40 % of that evoked by 1 microM PGE(2)). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca(2+) signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE(2) stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE(2)-stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. Experimental Physiology (2001) 86.4, 451-460.     

Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour

We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.