Humane Lung dendritiske celler: Rumlig Distribution og Fænotypisk Identifikation i endobronchial Biopsier Brug Immunhistokemi og flowcytometri
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
Lungerne er i løbende kontakt med det ydre miljø og er meget udsat for både harmløse partikler og mikrober med kapacitet til at forårsage sygdom. Derfor er det kritisk for immunsystemet at montere potente immunresponser mod invaderende patogener, men det er lige så vigtigt at opretholde tolerance over for inhalerede antigener, som ikke forårsager sygdom. At tilvejebringe potent immun-overvågning, er åndedrætssystemet foret med et netværk af immunceller, herunder dendritiske celler (DC'er). DC'er er professionelle antigenpræsenterende celler med den unikke evne til at aktivere naive T-celler. I humane lunger, hjemmehørende DC'er støder et antigen og derefter proces og transportere det til lungen-drænende lymfeknuder til præsentation for og aktivering af T-celler 1, 2, 3.
I det humane immunsystem, kan DC'er opdeles i flere delmængder, med Distinct men overlappende funktioner: CD1c + og CD141 + myeloide DCs (MDC'er) og CD123 + plasmacytoide DCs (pdCs) 4, 5. Mens mest detaljerede viden om menneskets DC'er stammer fra undersøgelser i blodet, er det nu klart, at de menneskelige lunger også harbor sjældne populationer af DC delmængder med T-celle stimulerende kapacitet 6, 7, 8, 9. Imidlertid akkumulerende data viser, at immunceller, herunder DC'er, er forskellige i deres frekvens, fænotype og funktion afhængig af deres anatomiske placering 10. Således er det vigtigt at studere immunceller fra det relevante væv for at forstå deres bidrag til lokal immunitet og tolerance. Tilsammen har dette understreger behovet for at undersøge lunge-resident DCs når man behandler lungesygdomme, trods blod DC'er er mere let tilgængelige og tilgængelig hos mennesker.
De første studier, der undersøgte lunge-hjemmehørende DC'er i mennesker anvendte primært morfologi og ekspressionen af enkelte markører, såsom HLA-DR og CD11 c, i vævssnit ved hjælp af immunhistokemi 11, 12, 13. I modsætning hertil nyere undersøgelser har typisk påberåbt flowcytometrisk analyse til at studere forskellige immuncelleundergrupper. Men da det er vanskeligt at finde en enkelt celle-overflademarkør, der entydigt identificerer en specifik DC delmængde, den potentielle begrænsning på studier med kun fire farver flowcytometri er risikoen for herunder cellepopulationer med lignende fænotypiske markører som DC'er. For eksempel er CD11 udtrykt på alle myeloide DC'er og langt de fleste af monocytter. På den anden side, i studier anvende mere avancerede flowcytometri paneler blev ikke-kræft lungevæv fra kirurgiske resektioner af patienter typisk anvendesxref "> 10, 14, 15, 16, selv om det er uklart, om disse sjældne populationer er virkelig repræsentative for udviklingslandene til stede i raske forsøgspersoner. Samlet set er undersøgelser stort set begrænset på grund af det faktum, at kirurgisk fjernet eller hele den menneskelige lungevæv er knappe.
For at overvinde nogle af disse begrænsninger, dette værk beskriver, hvordan du udfører en detaljeret analyse af rumlige fordeling og en fænotypisk identifikation af DC'er i slimhinder endobronchiale biopsier opnået fra raske frivillige, der gennemgår en bronkoskopi. Flere små biopsier kan indsamles fra den enkelte og kan efterfølgende indlejres for sektionering og analyse ved hjælp af immunhistokemi eller enzymatisk fordøjet til avanceret flowcytometrisk analyse. Brug af lungevæv i form af endobronkiale biopsier opnået fra bronchoscopies giver den fordel, som gør det muligt at udføre study på raske frivillige, i modsætning åben kirurgi af lungerne, som af indlysende grunde er begrænset til patienter, der kræver thoraxkirurgi. Endvidere det væv, der er udtaget under en bronkoskopi fra raske frivillige er fysiologisk normal, i modsætning til en ikke-ramte område af lungevævet hos patienter med lungesygdom. På den anden side, biopsierne er små, og antallet af celler hentet frem, selv når pooling flere biopsier, begrænser den type analyser, der kan udføres.
Mens det foreliggende arbejde fokuserer på DC'er, at de beskrevne direkte kan udvides fremgangsmåder indbefatter andre (immun) celler af interesse, der findes i humant mucosalt lungevæv. Endvidere protokollerne er også direkte anvendelse på prøver opnået fra patienter, der lider af lungesygdomme, hvor bronkoskopi er med til at etablere diagnosen, såsom kronisk obstruktiv lungesygdom (KOL), sarcoidose, eller lungecancer.
Protocol
Representative Results
Discussion
This paper describes how to generate a detailed spatial and phenotypical characterization of lung tissue-resident DCs in healthy humans using immunohistochemistry and flow cytometry on endobronchial mucosal biopsies collected during bronchoscopy. In the following paragraphs critical steps in the protocol are discussed in detail.
Critical Steps with the Protocol
Sectioning and immunohistochemistry: It is critical to keep the biopsy blocks at -20 °C when not using t…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank the volunteers who have contributed clinical material to this study. We are also thankful to the staff at the Department of Public Health and Clinical Medicine, Division of Medicine/Respiratory Medicine, University Hospital, Umeå (Norrlands universitetssjukhus) for the collection of all clinical material.
This work was supported by grants to AS-S from the Swedish Research Council, the Swedish Heart-Lung Foundation, the Swedish Foundation for Strategic Research, and the Karolinska Institutet.
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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