Summary

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

Published: January 20, 2017
doi:

Summary

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

Abstract

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Introduction

肺是在与外部环境的连续接触,并且高度暴露于既无害颗粒和微生物有能力引起的疾病。因此,它是免疫系统装入针对入侵病原体有效的免疫反应的关键,但要保持耐受吸入抗原不引起疾病是同样重要的。为了提供有效的免疫监视,呼吸系统衬有免疫细胞,包括树突细胞(DC)的网络。 DC是专业的抗原呈递细胞与激活幼稚T细胞的独特能力。在人的肺部,驻留的DC遇到抗原,然后进程并将其输送到肺引流淋巴结以供呈现给和活化的T细胞1,2,3的。

在人类的免疫系统,DCS可分为几个子集,具有次方CT但重叠的功能:CD1C +和CD141 +骨髓DC(的MDC)和CD123 +浆的DC(PDC上)4,5。而在人DCs最详细的知识从血液中的研究茎,现在明显的是,人体肺部也怀有DC亚群与T细胞刺激能力6,7,8,9的罕见群体。然而,积累的数据表明,免疫细胞,包括树突状,不同在它们的频率,表型,和功能取决于它们的解剖位置10。因此,为了研究免疫细胞从相关组织,以了解它们对局部免疫和容忍的贡献是重要的。总之,这强调需要解决肺部疾病时,研究肺居民的DC,尽管血液DC是更容易使用和访问在人类。

在人类研究肺驻留的DC的首批研究采用免疫组织化学11,12,13主要依靠形态和单标记物,如HLA-DR和CD11c的表达,在组织切片。与此相反,最近的研究通常是在流式细胞依靠分析来研究不同的免疫细胞亚群。然而,由于很难找到唯一地标识特定的DC子集的单个细胞表面标记物,研究的潜在的限制施加仅四色流式细胞仪是包含具有类似表型标记如树突细胞群的风险。例如,CD11c的是在所有的骨髓DC和广大单核细胞表达。另一方面,在研究中采用更先进的流式细胞仪面板,从患者的外科手术切除非癌肺组织中通常使用外部参照“> 10,14,15,16,虽然目前还不清楚这些稀有人群是否真正代表的DC存在于正常人。总体而言,研究在很大程度上是由于一个事实,即手术切除或整个人类的肺组织是稀缺的限制。

为了克服这些限制,这项工作介绍了如何执行空间分布及区议会在从谁接受支气管镜检查健康志愿者获得支气管粘膜活检表型鉴定的详细分析。几个小活检可以从每个单独的收集和随后可以嵌入用于切片和免疫组织化学或酶促消化先进流式细胞分析分析。在从支气管镜检查获得的支气管活检的形式使用的肺组织赋予使得能够执行ST的优点UDY上健康的志愿者,不像肺部开放手术,对于明显的原因,被限制为那些需要开胸手术的患者。此外,从健康志愿者支气管期间被取样的组织是生理上正常的,而相比之下,肺组织的非患部患者肺部疾病。另一方面,在活检小和检索单元的汇集几个活检即使当数,限制了可进行分析的类型。

虽然目前的工作重点是对DC,描述可以直接扩展的方法,包括其他(免疫)利益驻留在人体黏膜肺组织细胞。此外,该协议也直接适用于从肺部疾病,其中支气管镜检查是确立诊断,如慢性阻塞性肺疾病(COPD),结节病,或者肺癌的一部分的患者获得的样品。

Protocol

注:此研究于默奥,瑞典批准了区域伦理审查委员会。 1.支气管镜从人类受试者取样活检支气管获得所有参与者的知情同意。 治疗口服咪达唑仑(4-8毫克)和静脉注射格隆(0.2-04毫克)的支气管镜检查前30分钟科目。应用表面麻醉用喉和支气管利多卡因。让我们用〜3个毫升利多卡因4%的受漱口并申请3毫升舌根并经由一个注射器喉喉。优化8-10剂量利多卡因喷表…

Representative Results

研究表征人呼吸道组织驻留的免疫细胞,包括树突状,是有限的,这主要是由于这一事实,即外科手术切除或整个人肺组织是稀少。这里,从健康志愿者和开发的协议的支气管活检(EBB)获得的肺组织使用免疫组织化学研究中的组织中的免疫细胞或流式细胞术的微创方法进行了概述。 健康志愿者进行支气管镜检查,如前所述<s…

Discussion

本文介绍了如何利用免疫组织化学生成健康人肺组织居民DC的详细的空间和表型鉴定和支气管镜检查期间收集支气管粘膜活检流式细胞仪。在以下各段中的协议的关键步骤中详细讨论。

关键步骤议定书

切片和免疫组化:这是至关重要的,当不使用它们(步骤2.5),以保持活检块在-20℃。温馨块有时会变得柔软,不会一节为好。此外,保持块在-20℃将保留抗?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

笔者想感谢谁临床资料已经促成了这一研究志愿者。我们也感谢工作人员在公共卫生与临床医学,医学/呼吸内科的科,大学医院,于默奥(Norrlands universitetssjukhus)部的所有临床资料的收集。

这项工作得到了补助金从瑞典研究理事会,瑞典心肺基金会,瑞典战略研究基金会,并卡罗琳斯卡医学院AS-S支持。

Materials

Bronchoscopy
Bronchoscope BF1T160 Olympus BF1T160
Light source  Olympus Exera CV-160
Fenestrated forceps Olympus FB21C Used to take biopsies
Bite Block Conmed 1429 20x27mm
Glucose 25%  500mL intravenous
Glycopyrronium bromide 0.2mg/mL Intravenous. Prevents mucus/saliva secretion
Mixt. Midazolam 1mg/mL p.o Can be used for extra relaxation
Lidocaine, 40mg/mL Mouth and throat administration / Gargled
Lidocaine 100mg/ml spray Administered to back of throat
Lidocaine 20mg/ml spray Administered via bronchoscope to airways
Name of Reagent/ Equipment Company Catalog Number Comments/Description
GMA processing and embedding
Glass vials 5mL
Acetone Sigma-Aldrich 32201-1L
Molecular sieves, 4A Alfa Aesar 88120 3-4mm diameter pellets
Phenylmethylsulfonyl fluoride Sigma-Aldrich P-7626 0.035g/100ml acetone
Iodoacetamide Sigma-Aldrich I-6125 0.37g/100ml acetone
Polythene-flat  TAAB embedding capsules TAAB laboratories C094 x500 8mm diameter, polythene, flat-bottom capsules
Capsule holder TAAB laboratories C054 Holds 25 8mm capsules
JB-4 GMA embedding kit Polysciences 00226 Contains JB-4 Solution A (0026A-800), JB-4 solution B (0026B-3.8), benzoyl peroxide (02618-12)
Methyl benzoate Sigma-Aldrich 27614-1L
Silica gel with humidity indicator Scharlau GE0043 2.5-6mm 
Name of Reagent/ Equipment Company Catalog Number Comments/Description
GMA sectioning
Glass microscope slides ThermoFisher Scientific 10143562CEF Cut edges, frosted end
Poly-L-Lysine solution Sigma-Aldrich P8920-500mL 1:10 for working solution
Sheet glass strips for ultramicrotomy Alkar
Tween 20 Sigma-Aldrich P2287 Wash solution (0.1% Tween20)
LKB 7800B Knifemaker LKB
Capsule splitter TAAB laboratories C065
Carbon steel single edge blades TAAB laboratories B054
Vice
Ammonia, 25% VWR 1133.1000 2mL in 1L, 1:500 (0.05%)
Microtome Leica Leica RM 2165
Light source Leica Leica CLS 150 XE
Microscope with swing arm stand Leica Leica MZ6
Name of Reagent/ Equipment Company Catalog Number Comments/Description
GMA Immunohistochemistry
Diamond tipped pen Histolab 5218
Hydrogen peroxide 30% solution AnalaR Normapur 23619.264
Sodium azide Sigma-Aldrich S8032
Tris Roche 10708976001
Sodium chloride VWR chemicals 27810.295
Bovine serum albumin Millipore 82-045-2 Probumin BSA diagnostic grade
Dulbecco's modified eagle medium (DMEM) Sigma-Aldrich D5546
Anti-human CD45 antibody BioLegend 304002 Mouse monoclonal, clone HI30, isotype IgG1k. Working concentration of 500 ng/ml
Anti-human CD1a antibody AbD Serotech MCA80GA Mouse monoclonal, clone NA1/34-HLK, isotype IgG2a. Working concentration of 10 µg/ml
Mouse monoclonal IgG1 isotype control Abcam ab27479
Mouse monoclonal IgG2a isotype control Dako X094301-2
Vectastain ABC Elite standard kit Vector Labs PK-6100
AEC (3-amino-9-ethylcarbazole) peroxidase substrate kite Vector Labs SK-4200
Mayers haematoxylin HistoLab 01820
Permanent Aqueous Mounting Medium AbD Serotech BUF058C
Drying oven
DPX permanent mounting solution  VWR 360292F
Light microscope Leica Leica DMLB
Microscope camera Leica Leica DFC 320
Analysis software Leica Leica Qwin V3
Name of Reagent/ Equipment Company Catalog Number Comments/Description
Enzymatic digestion
Hank's Balanced Salt Solution (HBSS) Sigma-Aldrich 55021C
Dithiothreitol (DTT) Sigma-Aldrich DTT-RO
Collagenase II Sigma-Aldrich C6885
DNase Sigma-Aldrich 10104159001 ROCHE
RPMI 1640 Sigma-Aldrich R8758
Forceps
Platform rocker Grant instruments PMR-30
50 mL conical tubes Falcon 14-432-22
40 µm cell strainer Falcon 352340
Name of Reagent/ Equipment Company Catalog Number Comments/Description
Flow cytometry
Phosphate Buffered Saline (PBS)
LIVE/DEAD Aqua fixable dead cell stain kit Life Technologies L34957
CD45 BD 555485
CD3 BD 557757
CD20 BD 335829
CD56 Biolegend 318332
CD66abce Miltenyi 130-101-132
HLA-DR BD 555813
CD14 BD 557831
CD16 Biolegend 302026
CD11c BD 560369
CD1c Miltenyi 130-098-009
CD141 Miltenyi 130-090-514
CD103 Biolegend 350212
Paraformaldehyde Sigma-Aldrich F8775
LSR II Flow cytometer BD Flow cytometer
FlowJo FlowJo Software for analysis

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Baharom, F., Rankin, G., Scholz, S., Pourazar, J., Ahlm, C., Blomberg, A., Smed-Sörensen, A. Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry. J. Vis. Exp. (119), e55222, doi:10.3791/55222 (2017).

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