抽象
In the present study, the hypothesis that dendritic cells (DCs), key players in immunity and tolerance, might be involved in the immunopathology of idiopathic pulmonary arterial hypertension (IPAH) was tested.
The phenotype and localisation of DCs were characterised by immunohistochemistry and double-labelling immunofluorescence in lung samples from controls, human IPAH patients and an experimental pulmonary hypertension model (monocrotaline-exposed rats).
与对照组相比,形态学分析表明,在IPAH和OX-62阳性DC中的肌肉肺动脉中的肌肉肺动脉中的树突状细胞特异性细胞间粘附分子抓取数量增加的数量增加。在人类样品中,平均值±SEM.直流符号阳性细胞数·动脉-1100-300μm直径为1.4±0.4的对照versus26.。4.±2.7 in IPAH. In rats, the number of OX-62-positive cells·artery-1of 50–150 μm diameter was 0.5±0.2 in controls, and 0.7±0.5, 3.1±0.5 and 8.4±0.6 at day 7, 14 and 28 after monocrotaline exposure, respectively. Human complex lesions of muscular pulmonary arteries showed transmural DC infiltration. Phenotyping revealed an immature DC profile in human and experimental pulmonary hypertension.
结果支持该概念,即未成熟的树突细胞积聚在改造的肺血管中,因此可以参与肺动脉高压的免疫病理学。
Inflammatory mechanisms play a role in pulmonary arterial hypertension (PAH)1。实际上,严重的PAH可能使系统炎症状况的过程复杂化,并且通过皮质类固醇和免疫抑制剂的治疗已经显示出改善患者的结缔组织疾病,如系统性红斑狼疮2那3.。此外,靶向内皮细胞的病原自身抗体能够诱导血管内皮细胞凋亡,并可启动PAH的发育4.那5.。已显示特发性Pah(IPAH)患者的副本具有循环的自身抗体(抗核,抗纤维细胞抗纤维细胞抗体)6.那7.as well as elevated circulating levels of the pro-inflammatory cytokines interleukin (IL)-1 and IL-68.。Lung histology of patients displaying severe IPAH frequently reveals inflammatory cell infiltrates corresponding to macrophages and lymphocytes in the range of plexiform lesions with local expression of chemokines such as RANTES (regulated on activation, normal T-cell expressed and secreted) and fractalkine9.那10。来自大鼠偏菌碱诱导的肺动脉高压的实验数据进一步支持肺血管炎症和重塑之间的联系。
Among the different pathways related to the inflammatory process, the role of dendritic cells (DCs) has been increasingly recognised, not only as simple antigen-presenting cells responsible for the initiation of the inflammatory response, but, especially, as a key modulator of the whole process, thus raising its importance in many human disorders, including allergy11那autoimmunity12那tumour immunology13.and allograft rejection14.。Numerous data clearly demonstrate that pulmonary DCs are at least involved in the pathogenesis of a whole spectrum of highly prevalent respiratory conditions, and that for some of those (such as asthma), DCs have been proven to be essential in the development of the disease15.。In mice, pulmonary DCs have been shown to be the key cells in the pathogenesis of asthma16.and there is circumstantial evidence from animal models that pulmonary DCs might contribute to the development of chronic obstructive pulmonary disease in smokers17.。此外,肺部DC的数量在结节病中增加18.and diffuse panbronchiolitis19.。DCs中区分的能力to other cell phenotypes, including endothelial cells20那expands their potential role in vascular disorders. In the present study, data supporting the involvement of DCs in human and experimental pulmonary hypertension are reported for the first time.
方法
主题and sample processing
人类得到了PAH肺标本time of lung transplantation in eight patients displaying severe IPAH. Right-heart catheterisation demonstrated severe pre-capillary pulmonary hypertension in all cases. Control pulmonary specimens consisted of eight lung biopsies performed during surgery for pneumothoraces or at a distance from localised pulmonary lesions. Controls had no evidence of pulmonary vascular disease. PAH and control lungs were distended by infusion of ornithyl carbamyl transferase compound (OCT; VWR International, West Chester, PA, USA) diluted in PBS (1:5) into the main bronchi or by needle injection into the parenchyma to preserve lung morphology. Specimens for histology were collected from lung periphery, then snap-frozen inISOpentane on dry ice and stored at -80°C until further analysis was required. All patients and controls studied were part of the French Network on Pulmonary Hypertension, a programme approved by the institutional Ethics Committee, and gave written informed consent.
动物模型
Adult male Wistar rats (8–10 weeks old) were euthanised by overdose of sodium pentobarbital 7, 14 and 28 days after a single subcutaneous injection of saline (n = 10) or 60 mg·kg-1龙玺(Sigma-Aldrich, St Louis, MO, USA; n = 30). Lungs from controls (saline) and monocrotaline-exposed rats were distended by infusion of OCT:PBS (1:1) into the trachea, then snap-frozen. Monocrotaline-exposed rats displayed severe pulmonary hypertension at day 28, while mild haemodynamic and histological alterations occurred at day 14. Saline-exposed rats and rats evaluated 7 days after monocrotaline exposure had normal pulmonary haemodynamic values and pulmonary vascular histology. Animal experiments were approved by the administrative panel on animal care at Centre de Chirurgie Expérimentale Marie Lannelongue, Le Plessis-Robinson, France.
免疫组织化学
免疫组织化学was performed on 7-μm sections of frozen lung tissue. After routine preparation and peroxydase inhibition, human samples were processed with the following antibodies: monoclonal antibodies against dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN; Pharmingen clone DCN46; BD Pharmingen, San Diego, CA, USA), CD1a (Pharmingen clone HI149; Pharmingen) and CD83 (Immunotech clone HB15A; Immunotech, Mississauga, ON, Canada) diluted in PBS containing 1% calf foetal serum and 5% human AB serum. Rat DCs were stained with the monoclonal anti-CD103 antibody (Pharmingen clone OX-62; Pharmingen) diluted in PBS containing 2% normal rat serum. After overnight incubation at 4°C, human lung sections were labelled with biotinylated anti-mouse immunoglobulins and peroxidase-labelled streptavidin (Biogenex, San Ramon, CA, USA). Rat OX-62 labelling was revealed with the labelled streptavidin-biotin (LSAB)2 kit for use on rat specimens (Dako A/S, Glostrup, Denmark). Staining was completed after incubation with substrate-chromogen (3-amino-9-ethylcarbazole) solution (Dako A/S). Slides were counterstained with Mayer′s hematoxylin (Réactifs RAL, Martillac, France) and mounted with aqueous medium (Glycergel; Dako A/S). Controls used for these antibodies included omission of the primary antibody and substitution of the primary antibody by isotype control. Positive control for CD1a and CD83 was obtained by staining hilar lymph nodes of explanted lungs (data not shown).
免疫荧光标记
For double immunofluorescence, antihuman DC-SIGN (Pharmingen clone DCN46; Pharmingen) and OX-62 (Pharmingen) antibodies were labelled by biotinylated anti-mouse immunoglobulins and streptavidin (Biogenex or Dako A/S), and AlexaFluor 594 or 488 conjugate (Molecular Probes, Paisley, UK) for 1 h. The tissues were subsequently incubated overnight either with fluorescein isothiocyanate conjugated, anti-smooth muscle α-actin (Sigma Aldrich clone 1A4, Sigma Aldrich), antihuman CD68 (Dako clone KP1, Dako A/S), anti-rat RTIB (HMC class II, Serotec clone OX6; Serotec, Oxford, UK), rhodamine-conjugated vimentin (Santa Cruz sc-6260, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), or Alexa Fluor 488 conjugated anti-rat CD68 (Serotec clone ED1; Serotec) and anti-rat CD4 (Serotec clone W3/25; Serotec). The slides were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA).
免疫组织化学电池量化
使用尼康80i显微镜进行免疫组化细胞定量,其中尼康80I显微镜与形态学分析软件NSI元素(尼康,东京,日本)进行。通过在每个样品的三个载玻片上计数表达细胞的DC-符号(人DC)或OX-62(大鼠DC)计算平均肺动脉DCS的平均数量。对于定量,分别考虑了人和大鼠肺的横截面直径为100-300或50-150μm的肺肌动脉,分别考虑。
统计分析
All values are expressed as mean±SEM.。In human samples, statistical significance was evaluated with a two-tailed unpaired t-test for comparisons between control and PAH groups. ANOVA using repeated measures and the Fisher projected least significant difference (PLSD) post test was performed on rat results (4 groups: control, day 7, 14 and 28 after monocrotaline exposure). A p-value <0.05 was considered significant.
结果
Detection of dentritic cells in pulmonary arteries from IPAH patients
在来自iPAH患者的所有人肺样本中,在肺脉管系统的过滤组织中鉴定出呈现网络状组织的DC标志表达的DCS(图1⇓)。Affected vessels in PAH lungs displayed DC infiltration into the adventitial and the medial layer. Transmural infiltration by DCs was frequently observed in plexiform lesions. Morphometric analysis revealed a dramatic increase in the number of DC-SIGN-positive cells in muscular pulmonary arteries: the mean number of DC-SIGN-positive cells·artery-1100-300μm直径为1.4±0.4的对照versus26.。4.±2.7 in IPAH (p<0.001, two-tailed unpaired t-test; fig. 2⇓)。免疫组织化学表明,这些细胞是CD1A-和CD83阴性的,对应于未成熟的DC(数据未显示)。用CD68,α-肌动蛋白和Vimentin的免疫荧光双标记排除了与巨噬细胞或成纤维细胞的可能交叉反应(图1⇓)。对照肺的肺动脉显示散射的过度直流染色。在较小的程度上,从iPAH患者和对照中检测到血浆加湿肺实质,小叶肺癌和肺的内脏胸膜肺癌中检测到DC标志阳性细胞。在Visules或毛细血管中未发现DC标志阳性细胞。
实验性肺动脉高压肺动脉中牙牙细胞的检测
The recruitment of DCs in monocrotaline-induced pulmonary hypertension was studied in Wistar rats (fig. 3⇓)。通过CD103克隆OX-62检测到DC,其已被证明是大鼠DCS的重要标志物21。An increase in the number of DCs was first observed 14 days after monocrotaline exposure. By 28 days after monocrotaline injection, pulmonary hypertensive rats had a marked recruitment of OX-62-positive DCs in the adventitial layer of muscular pulmonary arteries, as well as in the vessel wall of pulmonary veins (fig. 2⇓)。OX-62阳性细胞的平均数量·动脉-1of 50–150 μm diameter was 0.5±0.2 in controls, and 0.7±0.5, 3.1±0.5 and 8.4±0.6 at day 7, 14 and 28 after monocrotaline exposure, respectively (p<0.001, ANOVA; fig. 2⇓)。DC计数随着肺动脉内侧改造的程度而增加,对照容器中较低。免疫荧光双标记显示招募的DCS的未成熟图案:CD4-阴性;Ox-62阳性;CD68-负;主要是主要的组织相容性复合体II类阴性表型(图3⇓)。OX-62-positive scattered DCs were present in bronchial-associated lymphatic tissue, as well as in the bronchial wall of PAH rats and controls.
讨论
The present study reports the first evidence that DCs may contribute to the immunopathology of human and experimental pulmonary hypertension. The results suggest that in human and experimental pulmonary hypertension, immature DCs accumulate in remodelled pulmonary vessels and hence could be involved in the evolution of the pulmonary vascular lesions. The present study has shown that mean numbers of arterial DCs increase during the development of vasculopathy in monocrotaline-exposed rats. The arterial DC accumulation precedes pulmonary arterial thickening and haemodynamic alteration and is constantly present in remodelled vessels, indicating that DC influx is not merely a consequence of increased pulmonary arterial pressure. Functional testing of DC recruitment, such asin vivo在动物模型中迁移,尚未在本研究中进行。因此,肺动脉高血压病变中的直流积聚可能是招生而且局部增殖的结果,从肺血管壁或甚至从渗透巨噬细胞的转化引起的渗透率下降。
据讨论了炎症过程在PAH自然历史中的作用,因为几种研究人员已描述了人类PAH中的重新模糊血管的炎症浸润。此外,已经描述了血液,肺部牙科和受影响的肺动脉中细胞因子的增加和表达。在结缔组织紊乱中增加PAH的患病率,并且在某种形式下,其成功治疗免疫抑制治疗,支持炎症途径在疾病的发展和调节中的概念。因此,更好地理解炎症与肺血管重塑之间的关系具有重要意义。
Inflammatory mechanisms and DCs could play a role in the early stages of human PAH development, as demonstrated in the rat inflammatory model of monocrotaline-induced pulmonary hypertension. All human PAH samples in the present study came from patients suffering from IPAH. This study design was chosen in order to avoid associated inflammatory conditions which can be encountered in PAH related to connective tissue diseases, autoimmune thyroiditis or HIV infection. The goal was to confront an exaggerated inflammatory animal model of pulmonary hypertension (monocrotaline-induced pulmonary hypertension) to “pure” IPAH and to describe common inflammatory mechanisms. Indeed, such mechanisms have not yet been well defined in human IPAH.
如前所述,人肺DCS缺乏CD83表达22那corresponding to immature DCs23.。此外,CD1a,存在于表皮和血液衍生的DC上的特征细胞表面分子,持续在血管外肺DC上不存在22。CD1a was occasionally seen in bronchial epithelium (data not shown), as confirmed by Demedtset al。24.。这种观察到的缺乏成熟的DC可以解释:在加工外/自抗原之后,DC随后迁移到淋巴组织,实现成熟。这里,DCS引发抗原特异性T细胞的激活。因此,大鼠肺DC呈现不成熟的CD4-阴性OX-62阳性表型25.。纤维纤维包含单核细胞/巨噬细胞CD68阳性谱系的循环单核细胞亚核细胞,其可以在组织损伤部位(α-肌动蛋白和vimentin生产,分化为肌纤维细胞的分析)中表现出成纤维细胞性质26.那27.,这被证明参与在动物模型中发生的缺氧诱导的PAH中的动脉重塑26.。The current authors checked, by immunofluorescent double-labelling, that human DC-SIGN- and rat OX-62-positive cells did not express vimentin or α-actin fibrocytic markers. Hence, it can be concluded that a DC subpopulation distinct from fibroblast/fibrocyte lineages accumulates in human and rat remodelled pulmonary arteries.
在炎症细胞的不同谱系中,DC与血管障碍有关。在全身动脉中,血管相关的DC在主要血液动力学应力下的动脉区域密集地积聚通过turbulent flow conditions28.。Interestingly, branching points of pulmonary arteries with altered flow conditions are predisposed to the development of plexiform lesions in human PAH29.。值得注意的是,目前的作者观察到DCS最常观察到DC的透射渗透在红色病变中。伴随着血液动力学变化,PAH中氧化应激的发生可以充当DC吸引的“危险信号”。实际上,已经表明,氧化应激的标志物不断存在于PAH中的内皮和内侧层中的内皮和内侧层3.0。
DC-SIGN is a C-type lectin which is highly expressed on the surface of immature DCs3.1并且已被证明允许单核细胞衍生的DC识别各种微生物,包括病毒,寄生虫和细菌3.2。DC-SIGN can also bind adhesion molecules on the surface of naive T-cells and endothelium suggesting its involvement in T-cell activation and DC trafficking3.1。值得注意的是,最近发现通过接受内皮样分化来突出DCS子集参与DCS在肿瘤血管生成中的累积ex vivo和组装新生动物in vivo20。
Novel therapeutic strategies may target pulmonary artery remodelling and its inflammatory component in PAH. Schermulyet al.3.3.have reported that the platelet-derived growth factor receptor antagonist STI571 (imatinib mesylate) reverses pulmonary vascular remodelling in two different animal models of pulmonary hypertension, including monocrotaline-exposed rats. Moreover, preliminary case reports provide evidence in support of imatinib as an antiremodelling agent in severe human IPAH3.4.-3.6.。Although the curative properties of imatinib in experimental and human PAH are supposed to rely onin vitro- 对肺动脉平滑肌细胞的抗癫痫抗抗溶剂,抗牵种和促进效应3.3.,最近的出版物突出了伊马替尼的直流调节效果in vitroandin vivo3.7.-4.0。Indeed, Mohtyet al.3.9.已经表明,对慢性髓性白血病(CML)患者对伊马替尼的疾病反应伴随着血浆骨质特性DCS功能的恢复in vivo。These findings provide evidence that imatinib is capable of restoring some DC-related immune functions in CML. In addition, Smyth4.0说明iMatinib可以激活名为“产生干扰素的杀手DCS”的单元格类型,被认为具有DC和天然杀伤单元的特征。此外,最近的报告表明,伊马替尼在癌症的小鼠模型中激活了产生干扰素的杀手DC,解释了这种药物在一些抗增殖作用的胃肠道基质肿瘤中的治疗效果4.1。因此,IPAH中的伊马替尼效应可以至少部分地链接到DC功能的调制。
In conclusion, the possible contribution of dendritic cells to the immunopathology of both human and experimental pulmonary hypertension is of major interest. Further phenotyping of the immature dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-positive subsets in lungs of human pulmonary arterial hypertension should help to better characterise dendritic cells in pulmonary arterial hypertension. The possible involvement of immature dendritic cells in a well-established animal model of inflammatory pulmonary hypertension will aid the analysis of the effects of specific therapeutic interventions to modulate the role of these cellsin vivo。Finally, evaluation of the cross-talk between immature dendritic cells and endothelial and smooth muscle cells should be evaluatedin vivoandin vitro。These data will allow the better understanding of the links between immune cells, abnormal angiogenesis and pulmonary artery remodelling in pulmonary hypertension.
树枝状细胞特异性细胞间粘附分子抓住非抗体素(DC-符号) - 阳性树突状细胞(DCS)a)围绕正常肺动脉和b)和c)位于肺动脉高压的复杂病变内(PAH)内部) 肺。d)DC-符号阳性细胞(红色)不同于巨噬粒CD68阳性群(绿色)。e)DCS(红色)与α-肌动蛋白阳性细胞(绿色)分开,不包括具有平滑肌细胞和/或肌纤维细胞的可能交叉反应。f)DCS(绿色)未表达波形蛋白(红色),间充质标志物,消除了与纤维细胞/纤维细胞元素的任何混淆,可参与纤维化PAH病变。秤条=100μm,除b)外,秤条=200μm。
人和大鼠肺动脉中树突细胞的定量。a)在特发性肺动脉高压(IPAH)患者中,观察到肺动脉中的树突细胞特异性细胞间粘附分子抓住非integrin(DC-Sign)阳性细胞的增加。b)单甲胺醇暴露的大鼠表现出直径为50-150μm直径为50-150μm的肺动脉的平均数量的显着增加。#:p<0.001, two-tailed unpaired t-test;¶:p<0.001, ANOVA.
OX-62-positive rat dendritic cells (DCs) were detected only a) in bronchus-associated lymphatic tissue of control lungs, while b) presenting a network-like organisation in the adventitia of affected arteries in monocrotaline-exposed rats. Recruited OX-62-positive DCs (red) were distinct from the c) medial smooth muscle cells (green), and also from d) macrophagic populations (green), e) rat monocytic/lymphocytic cells and DC CD4-positive subsets (green). f) OX-62 (red) and major histocompatibility complex class II (green) were occasionally coexpressed (yellow staining), highlighting different maturation stages or different DC subpopulations. a) Scale bar = 200 μm, b) scale bar = 100 μm, c–f) scale bars = 25 μm.
脚注
社论评论见435页。
目前的出版物仅反映了提交人的意见和欧洲共同体的意见,绝不会对任何可能由其中所载信息进行的任何使用责任。
- ReceivedJuly 19, 2006.
- 公认November 13, 2006.
- ©ers Journals Ltd