IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol

Identification of individual response-signal pathway induced by UVA-irradiation is necessary for understanding photo-biological and -pathological mechanisms with respect to the prevention of UV-irradiated skin damage and aging. Here, we investigated the role of D-alpha-tocopherol in the regulation of IL-8 production and AP-1 binding activity in UVA-irradiated human keratinocytes. UVA dramatically upregulated IL-8 mRNA expression and protein secretion and enhanced the AP-1-DNA binding activity. These effects of UVA irradiation were effectively reduced by D-alpha-tocopherol in a dose-dependent manner. The human keratinocytes expressed various NAD(P)H oxidase components, gp91phox homologues Nox1, and p22phox, p47phox, p67phox, as well as NOXO1, suggesting that cellular stress induced by UVA included the activation of non-phagocytic NADPH oxidase system, leading to AP-1 transactivation and IL-8 expression. D-alpha-tocopherol significantly inhibited the NADPH oxidase activity and the formation of malondialdehyde-thiobarbituric acid under UVA exposure. These results demonstrated that D-alpha-tocopherol may be able to prevent the IL-8 upregulation and the increase in AP-1 activation induced by UVA irradiation through down-modulating cellular oxidative stress.     

IL-8-mediated angiogenic responses of endothelial cells to lipid antigen activation of iNKT cells depend on EGFR transactivation

iNKT cells are a unique T cell subset, which is CD1d-restricted and specific for glycolipid antigens. In advanced atherosclerotic plaques, focal collections of inflammatory cells correlate with areas of intraplaque neovascularization. We reported recently that iNKT cells might facilitate intraplaque neovascularization by enhancing EC migration and sprouting in an IL-8-dependent manner. This study investigated the participating effector mechanisms. In ECs, CM, derived from antigen-stimulated human iNKT cells (CM+), induced up-regulation of IL-8R CXCR2 and the phosphorylation of EGFR and of multiple intracellular signaling effectors, including FAK, Src, Erk, Jnk, p38-MAPK, and STAT1 and -3. We found that a cascade of events, which were IL-8-dependent and involved EGFR activation, was responsible for signaling through FAK and Src kinases and necessary for acquisition of angiogenic morphology, migration in a two-dimensional wound assay, and sprout outgrowth in a three-dimensional model of angiogenesis in vitro. The data support that IL-8-dependent activation of angiogenic behavior in ECs, in response to activated iNKT, involves CXCR2, transactivation of EGFR, and subsequent FAK/Src signaling. We found too that activated iNKT increased VEGFR2 expression in ECs. Functional studies confirmed that EGF is the motogenic-enhancing factor in CM+ and is necessary, together with an exogenous source of VEGF, for iNKT-promoted sprout formation. EGFR inhibition may represent a novel therapeutic modality aimed at plaque stabilization through control of neovascularization within developing atherosclerotic plaques.     

Interleukin 1 (IL-1) and tumour necrosis factor synergise in the induction of IL-1 synthesis by human vascular endothelial cells

We have demonstrated that recombinant interleukin 1 (IL-1) induces IL-1 alpha and IL-1 beta production by human vascular endothelial cells (EC) in vitro. The effect of IL-1 on EC was dose-dependent and not due to contamination by endotoxin or secondary to the production of tumour necrosis factor (TNF). Thymocyte co-mitogenesis was shown to be due to IL-1 by treating EC supernatants with neutralising antibodies specific for IL-1 alpha and IL-1 beta. Recombinant TNF alpha synergised with IL-1 in the induction of IL-1 secretion by EC. Synergy was particularly striking at concentrations of IL-1 and TNF which, when used alone, had no effect - 2 U/ml IL-1 with 10 U/ml TNF. Thus we provide more evidence that EC play an important role in the perpetuation or possibly even initiation of chronic inflammation by amplifying cytokine production initiated by small numbers of infiltrating leucocytes.     

Proteasome inhibition suppresses cisplatin-dependent ERCC-1 mRNA expression in human ovarian tumor cells

One of the major problems with ovarian cancer treatment is the clinical development of resistance to cisplatin. Considerable efforts have been directed toward the identification of biological and pharmacological agents that would reverse drug resistance to cisplatin. ALLnL is an inhibitor of the proteasome that can inhibit the ubiquitin-proteasome pathway, which plays an essential role in many processes in cell life. We have recently shown that ALLnL, at concentrations that do not appear harmful, has significantly enhanced DNA platination and decreased DNA repair of cisplatin-DNA adducts in human A2780/CP70 ovarian carcinoma cells. These activities of proteasome inhibition were also associated with substantially increased cisplatin toxicity in these cells. In this communication, we demonstrate that treatment of A2780/CP70 cells with ALLnL blocks cisplatin-induced ERCC-1 mRNA expression in a concentration- and time-dependent manner, as measured by Northern blot analysis. In addition, we showed that the cisplatin-dependent increase in steady-state levels of ERCC-1 mRNA was prevented by pretreatment with lactacystin, a potent and specific inhibitor of proteasome. These results suggest that the effect of proteasome inhibition on cisplatin cytotoxicity, DNA platination, and DNA repair of cisplatin adducts in ovarian cancer cells may be through down-regulating ERCC-1 expression.     

Bleomycin induces IL-8 and ICAM-1 expression in microvascular pulmonary endothelial cells

To investigate the pathomechanisms of bleomycin-induced early inflammation of lung parenchyma which is known to result in pulmonary fibrosis, we examined the in vitro effect of bleomycin (BLM) on primary human pulmonary microvascular endothelial cells (HMVEC-L).

After incubation of microvascular endothelial cells with BLM we detected an induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) by immunoblotting. Further, after BLM-exposure an increased concentration of interleukin-8 (IL-8) in culture supernatant and an increased expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on the cell surface have been observed. Real-time PCR revealed up-regulated mRNA expression levels of both, IL-8 and ICAM-1 after treatment with BLM. Finally, pre-treatment with a selective p38 MAPK-inhibitor, SB 203580, potently reduced the BLM-induced up-regulation of IL-8 expression but did not show any effect on expression of ICAM-1.

These results demonstrate that BLM induces the expression of pro-inflammatory molecules in the pulmonary microvascular endothelium, which thereby may actively contribute to the development of early inflammation and later fibrosis of the lung. Furthermore, investigating the effect of an inhibitor of p38 MAPK the data indicate the involvement of p38 MAPK-dependent as well as p38 MAPK-independent mechanisms in the effects of BLM on the pulmonary microvasculature.     

CpG oligodeoxynucleotides induce IL-8 expression in CD34+ cells via mitogen-activated protein kinase-dependent and NF-kappaB-independent pathways

To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-kappaB, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-kappaB (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative IkappaB alpha plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-kappaB-independent pathways.     

Lysophosphatidic acid induces lymphangiogenesis and IL-8 production in vitro in human lymphatic endothelial cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.     

Effect of protein kinase inhibitors on IL-8/NAP-1 release from human umbilical vein endothelial cells

Several protein kinase inhibitors (PKIs) were investigated for their effects on IL-1β, TNFα and PMA-induced IL-8 production from human umbilical vein endothelial cells (HUVEC). IL-1β (ED₅₀ 0.07 ng/ml), TNFα (ED₅₀ 100 ng/ml) and PMA (ED₅₀ 20 ng/ml) induced IL-8 production that could be detected as early as 2 h following stimulation. Staurosporine, a potent but non-specific inhibitor of protein kinases, inhibited PMA-induced (IC₅₀ 2 nM) but not IL-1β or TNFα (IC50>200 nM) induced IL-8 production. Neither the cAMP-dependent PKI, KT5720, nor the tyrosine PKIs, genistein, tyrphostin (1–100 μM) or lavendustin A (0.0001–1 μM), inhibited IL-8 production elicited by IL-1β. However, the macrolide protein kinase inhibitor geldanamycin (IC₅₀=30 nM), but not the closely related analog herbimycin A (5–500 nM), inhibited IL-8 production by 60%. Northern blot analysis of IL-8 mRNA revealed that staurosporin suppressed mRNA increase following stimulation by PMA but not by IL-1. It is proposed that a novel protein kinase susceptible to geldanamycin inhibition may be involved in IL-1-mediated signal transduction.

     

Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production

During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24 h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.     

HIF-1α介导低氧诱导的内皮细胞Brm表达上调的机制研究

目的:本研究拟探讨低氧诱导因子1α(HIF-1α)在低氧诱导的血管内皮细胞Brm表达上调中的作用及机制,为低氧性肺动脉高压的防治提供理论依据。方法:在内皮细胞中过表达或siRNA干扰HIF-1α的表达,并给予1%低氧处理24 h后,运用萤光素酶报告基因检测方法检测Brm启动子的转录活性,运用RT-qPCR检测Brm的mRNA表达水平,运用Western blot检测Brm的蛋白表达水平。运用ChIP技术检测低氧条件下HIF-1α与Brm启动子区域的结合变化情况。结果:过表达HIF-1α可显著上调Brm的启动子活性、mRNA及蛋白表达,而siRNA干扰HIF-1α可显著抑制Brm的启动子活性、mRNA及蛋白表达,ChIP实验揭示低氧可显著增强HIF-1α与Brm启动子的结合。结论:低氧上调内皮细胞中Brm的表达依赖HIF-1α的转录调控作用。     

Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins

The capacity of endothelial cells to produce and release cytokines (IL-6, IL-8 and G-CSF) in response to exposure to Staphylococcus aureus strains or staphylococcal exotoxins (alpha-toxin, enterotoxin A and TSST-1) was investigated. An endothelial cell culture model of human umbilical vein endothelial cells (HUVEC) was used. Five out of ten clinical isolates of S. aureus were found to induce cytokine production and release from endothelial cells. Four of the five isolates that induce cytokine release produced enterotoxin A, B, C, D and/or TSST-1, compared with two of those that did not induce release. Purified staphylococcal exotoxins (1 pg/ml-1 microg/ml) did not act as primary stimuli and induced no detectable cytokine secretion. When endothelial cells were prestimulated with IL-1beta or TNF alpha at a concentration of 1 ng/ml for 2 h, IL-1beta served as a potent primary stimulus for IL-6, IL-8 and G-CSF production, whereas TNF alpha did not induce any significant cytokine release during the subsequent 24 h. A further increase in IL-6 and G-CSF release, but not of IL-8, was observed when IL-1beta prestimulated cells were exposed to alpha-toxin or TSST-1. However, to potentiate cytokine production (IL-6 and IL-8) by SEA, both IL-1beta and the toxin had to be present simultaneously. Our data show that S. aureus, but not staphylococcal exotoxins, have the capacity to act as primary stimuli of endothelial cells and induce production and release of cytokines. IL-1beta may prime HUVEC to release IL-6, IL-8 and G-CSF prior to subsequent stimulation with staphylococcal exotoxins.     

补体旁路激活对内皮细胞表达IL-8的影响

目的:探讨补体旁路活化途径直接促进内皮细胞表达IL-8,以及核转录因子kappa B(NF-κB)对该效应的调控作用。方法:应用酵母多糖活化人血清(ZAHS)直接作用于体外培养的人脐静脉内皮细胞(HUVECs)单层,检测如下指标:①放射免疫(RIA)检测IL-8的表达水平,并用原位杂交(ISH)检测胞浆中mRNA水平;②凝胶电泳迁移率(EMSA)方法测定NF-κB的活化。 结果:① 0.14 mL ZAHS能够诱导内皮细胞表达IL-8,4 h开始即有明显上升,ZAHS作用6 h,内皮细胞胞浆中IL-8 mRNA含量明显增加;②ZAHS能够激活NF-κB,30 min时即有活化,60 min明显增加,120 min达到高峰。NF-κB抑制剂咯啶二硫氨基甲酸脂(PDTC) 20 μmol/L预处理HUVECs 单层2 h,可以明显降低IL-8的表达(P＜0.05)。结论:补体旁路活化直接在转录水平诱导HUVECs表达IL-8,NF-κB参与调控该过程。     

IL-4 primes human endothelial cells for secondary responses to histamine

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E(2) (PGE(2)) release. IL-4 alone did not enhance PAF synthesis or PGE(2) release; however, pretreatment with IL-4 primed for PAF synthesis and PGE(2) release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), and IL-1beta did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE(2) release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.