Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells

Several studies have shown that exposure to cigarette smoke and/or house dust mite (HDM) can lead to increased airway inflammation in susceptible individuals. The underlying mechanisms, however, are not defined. To investigate the interaction between cigarette smoke and HDM allergen on mediator release from primary cultures of human bronchial epithelial cells. Confluent human bronchial epithelial cell cultures were exposed to cigarette smoke in the absence or presence of HDM allergen and investigated for the release of IL-8, IL-1beta, and sICAM-1. Damage to the epithelial cells themselves was assessed by release of 51Cr. On separate occasions, we investigated the effect of PTL11028, a highly potent and selective Der p1 inhibitor, on HDM allergen-induced release of IL-8, following activation of HDM allergen by incubation with cysteine. The effect of cigarette smoke exposure on the stability of these released mediators in prepared solutions in the absence/presence of reduced glutathione was also studied. Both HDM allergens and short-term (20 min) cigarette smoke exposure led to a significantly increased release of IL-8, IL-1beta and sICAM-1 from the epithelial cell cultures. Longer exposure (1-6 h) to cigarette smoke led to a dramatic decrease in the amount of these mediators detected in the culture medium. Whilst incubation of epithelial cultures with HDM allergen did not cause any significant change in the release of 51Cr from pre-loaded cells, cigarette smoke on its own led to a marked, exposure and incubation-time dependent increase in the release of 51Cr. Incubation with HDM allergen led to a significant, dose and time-dependent increase in the release of IL-8, which was further enhanced when the allergen extract was pre-activated with cysteine. This effect was completely abrogated by PTL11028, a novel Der p1 inhibitor. Prepared solutions of various concentrations of IL-8, IL-1beta and sICAM-1 exposed to cigarette smoke demonstrated a dramatic exposure time-dependent decrease in the detectable amount of these mediators, an effect which was abrogated by GSH. HDM-induced airway inflammation may include Der p-mediated release of inflammatory mediators from epithelial cells. Additionally, short-term cigarette smoke exposure may induce airway inflammation by release of inflammatory mediators from these cells, an effect which may be potentiated by Der p allergens. Longer term cigarette smoke exposure may cause damage to epithelial cells and changes in the structure of inflammatory mediators.     

沉默NLRP3炎性小体表达降低香烟烟雾提取物导致的人肺动脉内皮细胞凋亡

目的探讨NLPR3炎性小体(含NLR家族pyrin结构域蛋白3)对香烟烟雾提取物(CSE)诱导人肺动脉内皮细胞(HPAECs)凋亡的影响。方法用NLRP3 siRNA和NC siRNA(阴性对照)分别转染HPAECs后(分别为siNLRP3组、siNC组),再以10%CSE刺激12 h,应用annexin V/PI流式法检测这两组细胞在CSE刺激前后的凋亡水平。再将HPAECs分为对照(control)组、CSE组(加入10%CSE液)、NAC(乙酰半胱氨酸,N-acetylcysteine)+CSE组(以10 mmol/L NAC预处理1h后再以10%CSE培养液处理)及NAC组(以10 mmol/L NAC单独处理),均培养12 h后以DCFH-DA光探针标记法观察细胞内活性氧(reactive oxygen species,ROS),Western blot法测定NLRP3、caspase-1蛋白表达。结果 siNC组在CSE刺激后,HPAECs存活率下降,凋亡率较CSE刺激前明显增加(P<0.05),SiNLRP3组在CSE刺激后,细胞凋亡率较siNC组同等CSE刺激后有...     

Cigarette smoke-induced pulmonary inflammation, but not airway remodelling, is attenuated in chemokine receptor 5-deficient mice

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory response of the airways and lungs to noxious particles and gases, mostly cigarette smoke (CS). Pathological changes characteristic of COPD include airway wall thickening, peribronchial fibrosis, peribronchial lymphoid follicles and destruction of lung parenchyma (emphysema). The recruitment of inflammatory cells into the lung in response to CS is thought to play an important role in the development of COPD. OBJECTIVE: Our aim was to study the contribution of chemokine receptor 5 (CCR5) to the pathogenesis of COPD and specifically whether the development of airway remodelling is a direct result of airway inflammation or rather occurs through an independent mechanism. METHODS: In this study, C57BL/6 wild-type mice and CCR5-deficient mice were subjected to sub-acute (4 weeks) and chronic (24 weeks) CS exposure. RESULTS: Both sub-acute and chronic CS exposure significantly increased CCR5 mRNA expression and protein levels of CCR5 ligands [macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and regulated upon activation, normal T expressed and secreted (RANTES)], and induced the recruitment of neutrophils, macrophages, dendritic cells, and lymphocytes to the bronchoalveolar lavage (BAL) of wild-type mice. Chronic CS exposure also increased the number and extent of peribronchial lymphoid follicles. In CCR5 knockout (KO) mice, these CS-induced increases in CCR5 ligands, inflammatory cells in BAL and peribronchial lymphoid follicles were all significantly attenuated compared with wild-type animals. Importantly, chronic CS exposure induced airspace enlargement in wild-type mice, while CCR5 KO mice were partially protected against the development of emphysema. However, CCR5 deficiency did not affect CS-induced airway wall remodelling, because chronic CS exposure induced a similar increase in airway wall thickness, smooth muscle mass and peribronchial deposition of collagen and fibronectin in both wild-type and CCR5 KO mice. CONCLUSION: Our data suggest that CCR5 contributes to pulmonary inflammation and to the development of emphysema in response to CS. CCR5 is, however, not implicated in CS-induced airway wall remodelling, suggesting that the mechanisms that lead to airway inflammation are distinct to those responsible for airway remodelling.     

Hypoxia modulates phenotype,inflammatory response,and leishmanial infection of human dendritic cells

Bosseto MC, Palma PVB, Covas DT, Giorgio S. Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells. APMIS 2010; 118: 108–14. Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA‐DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL‐12p70, the bioactive interleukin‐12 (IL‐12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro‐inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.     

血红素加氧酶-1抑制香烟烟雾提取物致体外人肺A549细胞黏液高分泌

目的探讨血红素加氧酶-1(HO-1)对香烟烟雾提取物(CSE)所致人气道黏液高分泌的影响。方法用CSE刺激A549细胞,复制黏液高分泌细胞模型。分为对照组、CSE组、氯化高铁血红素(Hemin)组和锌原卟啉(ZnPPIX)组,观察黏蛋白(MUC)5AC、表皮生长因子受体(EGFR)、磷酸化(p)-EGFR、HO-1及双功能氧化酶1(Duox1)的表达。用MTT法测定细胞活性;RT-PCR法检测HO-1、Duox1、EGFR及MUC5AC的mRNA;Western blot法检测p-EGFR、EGFR、Duox1和HO-1蛋白;ELISA法检测细胞裂解液中MUC5AC蛋白表达。结果CSE组MUC5AC的mRNA吸光度积分相对值和蛋白水平分别为0.660±0.044和(157±3)μg/mg,较对照组0.412±0.043和(105±8)μg/mg明显升高(P<0.05),p-EGFR蛋白水平、EGFR、HO-1及Duox1的mRNA和蛋白水平也较对照组明显增高。与CSE组相比,Hemin组HO-1mRNA及蛋白进一步增高,而p-EGFR蛋白水平、Duox1、EGFR及MUC5AC的mRNA和蛋白水平明显回降。与...     

香烟烟雾水溶物对人外周血淋巴细胞SCE的影响

SCE频率分析是研究环境诱变剂和致癌剂引起人类遗传物质损伤的有效手段。本文以人外周血淋巴细胞姐妹染色单体交换(SCE)为指标,研究了香烟烟雾水溶物对体外培养的人外周血淋巴细胞SCE的影响。结果表明香烟烟雾水溶物引起人的外周血淋巴细胞SCE数的增加,证明香烟烟雾水溶物具有诱发突变作用。     

Bhas 42 cell transformation activity of cigarette smoke condensate is modulated by selenium and arsenic

Cigarette smoking remains a major health risk worldwide. Development of newer tobacco products requires the use of quantitative toxicological assays. Recently, v‐Ha‐ras transfected BALB/c3T3 (Bhas 42) cell transformation assay was established that simulates the two‐stage animal tumorigenesis model and measures tumor initiating and promoting activities of chemicals. The present study was performed to assess the feasibility of using this Bhas 42 cell transformation assay to determine the initiation and promotion activities of cigarette smoke condensate (CSC) and its water soluble fraction. Further, the modulating effects of selenium and arsenic on cigarette smoke‐induced cell transformation were investigated. Dimethyl sulfoxide (DMSO) and water extracts of CSC (CSC‐D and CSC‐W, respectively) were tested at concentrations of 2.5–40 µg mL⁻¹ in the initiation or promotion assay formats. Initiation protocol of the Bhas 42 assay showed a 3.5‐fold increase in transformed foci at 40 µg mL⁻¹ of CSC‐D but not CSC‐W. The promotion phase of the assay yielded a robust dose response with CSC‐D (2.5–40 µg mL⁻¹) and CSC‐W (20–40 µg mL⁻¹). Preincubation of cells with selenium (100 nM) significantly reduced CSC‐induced increase in cell transformation in initiation assay. Co‐treatment of cells with a sub‐toxic dose of arsenic significantly enhanced cell transformation activity of CSC‐D in promotion assay. The results suggest a presence of both water soluble and insoluble tumor promoters in CSC, a role of oxidative stress in CSC‐induced cell transformation, and usefulness of Bhas 42 cell transformation assay in comparing tobacco product toxicities and in studying the mechanisms of tobacco carcinogenesis. Environ. Mol. Mutagen. 57:220–228, 2016. © 2016 Wiley Periodicals, Inc.     

T cells stimulated in vitro have a suppressive function but do not contain only regulatory T cells

The generation of regulatory T cells (Tregs) in vitro represents an attractive possibility to set up cellular therapies that could prevent and cure autoimmune disorders. Different methods have been proposed to generate Tregs in vitro and to evaluate their phenotype and function. Moreover, the overlap between generation of activated and regulatory cells could often be underestimated. We showed that in vitro treatment of CD4+ CD25- lymphocytes with different stimuli leads to a good expression of CD25 and forkhead box P3 (FoxP3) on most cells, but to a full Treg phenotype (including CD127 negativity) in only a minor percentage of cells, ranging from 17.38% of cells treated with phytohaemagglutinin (PHA) to 50.91% of cells treated with T cell receptor (TCR) stimulation in association with transforming growth factor (TGF)-beta. Some suppressive activity was demonstrated for T cells activated with all the different stimuli. However, while suppression mediated by TCR/TGF-beta treated T cells was associated with an inhibition of both interleukin (IL)-2 and interferon (IFN)-gamma in the co-culture supernatant, the suppression observed for PHA-activated cells occurred in the presence of large amounts of these cytokines. In conclusion, also taking into account other recent publications, caution should be taken in interpretation of data in the field of regulatory T cells.     

辛伐他汀对香烟烟雾提取物诱导的人脐静脉内皮细胞表达sEPCR和mEPCR的影响

目的: 研究辛伐他汀对香烟烟雾提取物(CSE)诱导的人脐静脉内皮细胞(HUVECs) 表达可溶性内皮细胞蛋白C受体(sEPCR)和膜联内皮细胞蛋白C受体(mEPCR)的干预作用。方法: 体外培养的4~6代HUVECs 随机分为对照组、5%CSE组、不同浓度辛伐他汀组及辛伐他汀干预组,辛伐他汀组分别加入50、100、200 μmol/L辛伐他汀液孵育24 h,辛伐他汀干预组先以50、100、200 μmol/L辛伐他汀预处理细胞2 h,再与5%CSE孵育24 h。收集各组细胞及上清液,ELISA法检测上清液中sEPCR蛋白含量,实时定量PCR法检测各组细胞中mEPCR mRNA的表达。结果: (1)5%CSE组sEPCR蛋白含量高于对照组,mEPCR mRNA表达低于对照组,差异均有统计学意义(均P<0.05);(2)100 μmol/L与200 μmol/L辛伐他汀组sEPCR蛋白含量均高于对照组,低于5%CSE组,其mEPCR mRNA表达均低于对照组,高于5%CSE组,差异均有统计学意义(均P<0.05);(3)各辛伐他汀干预组sEPCR蛋白含量均低于5%CSE组,但高于对照组及相应浓度的辛伐他汀组;相反,各辛伐他汀干预组mEPCR mRNA表达均高于5%CSE组,低于对照组及相应浓度的辛伐他汀组,差异均有统计学意义(均P<0.05)。结论: 在体外,辛伐他汀通过上调HUVECs mEPCR mRNA的表达,降低sEPCR的分泌,对CSE介导的内皮细胞凝血功能障碍可能具有一定的改善作用。     

Phenotypic comparison of extrathymic human bone-marrow-derived T cells with thymic-selected T cells recovered from different tissues

We have previously described extrathymic generation of human T cells from purified stem cells in the bone marrow of athymic immune deficient mice. This system provides a pure population of extrathymic human T cells that is devoid of contamination by peripheral expansion of thymic-selected T cells. In the current studies, we phenotypically compared the extrathymic human T cells (Ex-T) to T cells from human peripheral blood leukocytes (PBL), umbilical cord blood (CB), bone marrow (BM), and postnatal thymus. There were few CD4(+)/CD8(+) double positive (DP) cells in PBL, CB, BM, and Ex-T, in comparison with over 85% DP cells in thymus. More CD8(+) and CD4(dim) cells were observed in Ex-T than in the thymic-selected cells. Ex-T and T cells in thymus and peripheral tissues differed in their CD8 isoforms. There were more TCRgamma/delta T cells in PBL, CB, BM, and Ex-T than in thymus. Similar to the bright CD3(+) T cells in thymus, T cells in PBL, CB, and BM were CD3 bright and expressed the adhesion molecules CD44 and L-selectin (CD62L), while intermediate CD3 T cells in thymus lacked CD44 and L-selectin. However, the majority of Ex-T only expressed CD44 but not L-selectin. In summary, thymic- and extrathymic-derived T cells are phenotypically different. The identification of extrathymically derived T cells in humans will allow us to begin to understand their role in the early contribution to immune recovery posttransplantation and their possible involvement in autoimmunity and other disease states.     

香烟烟雾暴露对肺气肿小鼠CD4+IL-17+辅助性T细胞的影响

目的 探讨香烟烟雾暴露对肺气肿小鼠肺实质、外周血中CD4+IL-17+辅助性T细胞(Th17)的影响。方法 将40只雄性BALB/c小鼠按随机数字表法分为4组：对照12周组(C12)、对照24周组(C24)、烟雾暴露12周组(S12)、烟雾暴露24周组(S24),每组10只。香烟烟雾暴露法建立小鼠肺气肿模型。HE染色观察小鼠肺气肿的改变,计算平均内衬间隔(Lm)和肺泡破坏指数(DI);酶联免疫吸附法检测小鼠支气管肺泡灌洗液中IL-17、干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α水平及肺实质中IFN-γ和TNF-α水平;流式细胞术检测小鼠肺实质、外周血中CD4+ IL-17+T(Th17)细胞占CD4+T细胞的百分比;免疫荧光PCR法检测小鼠肺实质、外周血中RORγt和IL-17的mRNA表达,并分析这些指标的相互关系。结果 (1)S12组和S24组Lm为(39.19±3.51) μm和(46.87±7.16) μm,DI为39.13±1.57和45.16±3.13,均明显高于C12组和C24组(32.60±3.21) μm和(32.38±3.73) μm及28.23±1.62和28.86±2.07,以S24组增高更为明显,差异均有统计学意义(均P＜0.05)。(2) S12组和S24组BALF中IL-17、IFN-γ及TNF-α水平[(119.72±10.72) ng/L和(296.40±14.00) ng/L、(129.7±22.2) ng/L和(251.1±62.4) ng/L,(17.35±1.60) ng/L和(36.35±1.43) ng/L]较C12组和C24组[(52.06±4.70) ng/L和(51.89±6.82) ng/L、(85.8±26.8) ng/L和(88.9±11.5) ng/L,(6.41±0.90) ng/L和(5.85±0.92) ng/L]明显增高;在肺实质中,S12组和S24组IFN-γ及TNF-α水平[(1124.3±174.4) ng/L和(1342.7±206.1) ng/L,(77.2±13.7) ng/L和(101.7±19.0) ng/L]亦较C12组和C24组[(946.2±81.9) ng/L和(1027.2±188.3) ng/L,(62.1±16.1) ng/L 和(64.4±15.1) ng/L]明显增高;且以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(3)S12组和S24组肺实质中Th17细胞比例[(3.27±1.12)％和(7.19±2.24)％]明显高于C12组和C24组[(1.80±0.75)％和(1.99±0.59)％];S12组和S24组外周血中Th17细胞比例[(1.96±0.61)％和(3.82±1.26)％]亦明显高于C12组和C24组[(0.90±0.37)％和(0.97±0.32)％];且以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(4) S12组和S24组肺实质中RORγt和IL-17 mRNA表达量（1.73±0.35和4.52±1.15,4.00±0.87和83.43±21.01）较C12组和C24组（0.83±0.21和0.90±0.36,0.80±0.22和0.64±0.31）明显增高,以S24组增高更为明显,差异均有统计学意义(均P＜0.05);外周血中,S12组和S24组RORγt和IL-17 mRNA表达量(1.48±0.32和2.75±0.79,201.25±5.49和416.22±99.64)亦较C12组和C24组(0.59±0.25和0.75±0.36,24.90±5.49和22.28±4.24)明显增高,以S24组增高更为明显,差异均有统计学意义（均P＜0.05）。(5)烟雾暴露组中,外周血和肺实质的Th17细胞比例均与Lm、DI值显著正相关(r值分别为0.767、0.772;0.722、0.706,均P＜0.05)。结论 Th17细胞在香烟暴露肺部炎症、肺气肿小鼠外周血及肺内表达增高,并伴活性的增强,且随烟雾暴露时间延长而增强,提示香烟暴露肺气肿小鼠Th17细胞上调可能是导致小鼠肺内及全身炎症反应放大的原因之一。     

CD4+Foxp3+调节性T细胞表达升高提示免疫应答而非移植耐受

目的 探讨诱导CD4+Foxp3+调节性T细胞(regulatory T cells,Tregs)表达的机制及其在移植耐受中可能的作用.方法 构建新生移植耐受小鼠动物模型,分析移植耐受与同种异体反应性T细胞、Tregs表达及嵌合体的关系;运用过继转移实验、EGFP转基因小鼠,研究移植排斥过程中Tregs表达及抗原特异性.结果 新生耐受小鼠形成嵌合体,同种异体反应性T细胞被克隆清除,但Tregs表达与初始小鼠相同;同种异体反应性T细胞识别抗原诱导免疫反应,Tregs在移植排斥反应中表达升高,且不仅同种异体反应性Tregs表达升高,非同种异体反应性Tregs表达也升高.结论 Tregs在移植排斥中非特异性诱导产生,可能为一种负反馈免疫调控机制.     

DC-SIGN, but not sDC-SIGN, can modulate IL-2 production from PMA- and anti-CD3-stimulated primary human CD4 T cells

Dendritic cell (DC)-specific intercellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN) is expressed on the surface of DCs and specialized macrophages and can support T cell proliferation. Antibody-mediated co-ligation of CD3 and ICAM-3, the ligand for both DC-SIGN and leukocyte function-associated antigen-1, leads to T cell activation. Therefore, we tested to see whether DC-SIGN or a splice variant of dendritic cell-specific intercellular cell adhesion molecule-3-grabbing non-integrin (sDC-SIGN) can co-stimulate primary human T cells. The sDC-SIGN lacking the transmembrane domain encoded by exon 3 localizes to the cytoplasm of cells and is not secreted. Both B7 and DC-SIGN co-stimulated phorbol myristate acetate-stimulated CD4+ cells as compared with controls. However, unlike B7, both DC-SIGN and sDC-SIGN failed to co-stimulate CD4+ T cells treated with sub-optimal amounts of anti-CD3 (2 microg ml(-1)) as defined by a lack of CD69 and CD25 up-regulation, cell division and cytokine secretion. Instead, DC-SIGN, and not sDC-SIGN, induced a small but consistent down-regulation of IL-2 production by these CD4+ T cells. In contrast, DC-SIGN in the presence of 30 mug ml(-1) of anti-CD3 modestly up-regulated cytokine production as compared with control. These results suggest that DC-SIGN can differentially modulate T cell stimulation.     

Nicotine modulates molecules of the innate immune response in epithelial cells and macrophages during infection with M. tuberculosis

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.     

Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis

BACKGROUND: Apart from the long-used corticosteroids, topical calcineurin inhibitors (tacrolimus, pimecrolimus) represent novel therapeutic options for the treatment of atopic dermatitis. OBJECTIVE: This study was designed to investigate the pathophysiological target cells and mode of action of pimecrolimus in atopic skin. METHODS: Twenty-two patients were randomly assigned to treatment with pimecrolimus cream 1%, matching vehicle cream, or beta-methasone-17-valerate (BMV) cream 0.1% in a randomized, double-blind, vehicle-controlled, parallel group clinical trial. Treatment was given twice daily for 3 weeks. Cryostat sections of skin biopsies were taken before as well as at selected time points after initiation of therapy. For certain experiments, healthy volunteers were topically treated with the creams described twice a day on 5 consecutive days. Epidermal sheets were prepared from suction blisters. For in vitro experiments, untreated, healthy human skin was obtained from patients undergoing plastic surgery. The effect of pimecrolimus and BMV on Langerhans cells (LCs), inflammatory dendritic epidermal cells, and T cells was investigated by using immunofluorescence and flow-cytometry techniques. RESULTS: While topical BMV treatment depleted LCs in healthy and atopic skin, pimecrolimus did not affect their number. Correlating with the disappearance of inflammatory cells, we observed a depletion of inflammatory dendritic epidermal cells and T cells on pimecrolimus and BMV treatment. Furthermore, we show that pimecrolimus depletes T cells by the induction of apoptosis. CONCLUSION: In summary, our data show that pimecrolimus reduces pathological T cells in atopic dermatitis skin via apoptosis, whereas LCs remain unaffected.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

低氧增加人皮肤微血管内皮细胞系迁移并促进凋亡

目的研究低氧环境对人皮肤微血管内皮细胞(HDMEC)迁移、凋亡及相关因子HIF-1α、VEGF、i NOS基因及蛋白表达的影响。方法低氧培养人微血管内皮细胞,分为常氧组(对照组)和低氧组(Co Cl2模拟化学低氧)。CCK-8细胞生存实验确定合适处理浓度(200μmol/L Co Cl2),划痕实验检测细胞迁移能力,流式细胞技术观察细胞凋亡;用RT-PCR和Western blot技术检测HIF-1α、VEGF、i NOS mRNA和蛋白表达。结果与常氧组比较,低氧组细胞迁移能力增加(P0.05),凋亡增多(P0.05),均呈时间依赖性。低氧组HIF-1α、HIF-1β、VEGF、i NOS mRNA表达较常氧组比较均增加(P0.05),HIF-1α、VEGF、i NOS蛋白表达较常氧组比较均增加(P0.05),具有一定时间依赖性。结论低氧能增强人皮肤微血管内皮细胞迁移能力,并促进其细胞凋亡,其可能机制与HIF-1α、VEGF、i NOS蛋白表达升高有关。     

New cohorts of naive T cells exacerbate ongoing allergy but can be suppressed by regulatory T cells

Although as pretreatment oral tolerance is a potent means to achieve systemic suppression, its application in ongoing disease is controversial. Here we propose that availability of naive T cells may critically determine whether immunological tolerance is achieved during ongoing antigenic reactivity. Infusion of naive antigen-specific T cells into mice directly prior to eliciting a secondary Th2 response induces these naive cells to actively engage in the antigenic response despite presence of established memory. Naive antigen-specific T-cells divided faster, produced more interleukin (IL)-2, IL-4 and IL-5 and enhanced immunoglobulin E (IgE) release during a secondary Th2 response, compared with naive T cells that were infused prior to a primary response. Despite such contribution by new cohorts of naive T cells co-infusion of mucosal Tr together with naive T cells could suppress enhanced IgE release during a secondary Th2 response. We conclude that naive T cells contribute to a secondary Th2 response and although they can still be suppressed in the presence of sufficient numbers of mucosal Tr, they may interfere with potential therapeutic application of mucosal tolerance.     

Gene‐environment interaction between the MMP9 C–1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease

The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well‐established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene‐environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C–1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C–1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C–1562T genetic variant and cigarette smoking was assessed using a case‐control model. The response of the C–1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C–1562T variant demonstrated a dose‐dependent and allele‐specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9–1562T allele and cigarette smoke in COPD, emphasising gene‐environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447–454, 2016. © 2016 Wiley Periodicals, Inc.