接触超敏反应作为过敏性接触性皮炎的小鼠模型
1Department of Clinical Immunology,Wroclaw Medical University, 2Department of Medical Physiology, Chair of Biomedical Sciences, Institute of Physiotherapy, Faculty of Health Sciences,Jagiellonian University Medical College, 3Chair of Biomedical Sciences, Faculty of Health Sciences,Jagiellonian University Medical College
Summary
接触性超敏反应（CHS）是过敏性接触性皮炎（ACD）的小鼠实验模型。CHS基于通过涂上胸部和腹部的剃须皮肤对反应性半抗原的致敏，随后用稀释的半抗原进行耳朵皮肤激发，引起肿胀反应，以各种方式评估。
Abstract
接触超敏反应（CHS）是过敏性接触性皮炎（ACD）的实验模型，可以在小鼠中研究。本研究旨在提出一种客观的实验室方法，该方法可能有助于研究小鼠的CHS反应，该方法可以通过各种测试进行测量和定量。为了诱导CHS，在第“0”天，小鼠通过用丙酮 – 乙醇混合物中的半抗原2，4，6-三硝基氯苯（TNCB）进行腹部皮肤涂漆，使小鼠在先前剃须的斑点上致敏，而阴性对照小鼠则用载体单独 – 丙酮 – 乙醇混合物进行假敏化。在第“4天”，在诱发CHS（激发）之前用千分尺测量基线耳朵厚度，方法是在测试组和对照组中用稀释的TNCB涂上两只耳朵。24小时后，用千分尺测量耳朵肿胀。CHS是T细胞介导的免疫反应的一个例子，它导致发炎组织肿胀，在皮肤挑战后24小时达到峰值，同时出现半抗原。耳水肿的增加与耳朵重量增加、髓过氧化物酶 （MPO） 活性、耳提取物中的促炎细胞因子浓度、组织学检查中水肿真皮增厚增加以及耳血管通透性相关。与对照小鼠相比，测试组血清中TNP特异性IgG1抗体的浓度也有所增加。此外，CHS可以与先前用TNCB致敏的供体获得的CHS效应细胞成功转移。将CHS效应细胞静脉注射到幼稚的受体小鼠中，随后用相同的稀释半抗原挑战。24小时后用千分尺测量耳朵肿胀。
Introduction
过敏性接触性皮炎（ACD）是工业化国家常见的皮肤炎症性疾病，由暴露于称为半抗原的低分子量化学物质引起的IV型超敏反应引起。在人类中引起接触致敏的物质包括重金属离子（铬、镍、铁、钴）、松节油、香料、染料和化妆品中存在的防腐剂（例如，对苯二胺）、一些药物（例如新霉素、苯佐卡因）、β内酰胺类抗生素（即青霉素）、植物产生的化学物质（十五甲苯甲酚，毒藤中存在的物质）以及照相工业中使用的对苯二酚1，2.ACD的病因剂非常高，因为仅在工业中使用了100，000多种化学品，并且每年合成2，000种新的化学品。迄今为止，已经鉴定出3，700多个分子可能是接触性半抗原/过敏原3。接触超敏反应（CHS）是ACD的实验模型，可以在小鼠，豚鼠和大鼠中研究，并且可以通过局部皮肤应用溶解在有机溶剂中的活性化学半抗原4，5，6来诱导。本研究旨在描述一种客观的实验室方法，该方法可能有助于研究小鼠中的CHS反应，该方法可以通过各种测试进行测量和定量。
CHS由致敏（诱导）和效应器（激发）阶段组成。在动物模型中，半抗原首先与体内的蛋白质共价结合以产生新抗原。在致敏阶段，活化的角质形成细胞通过产生促炎细胞因子 – 肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）7来促进皮肤树突状细胞（sDC）的迁移和成熟。表皮朗格汉斯细胞（LC）在CHS诱导和效应器阶段8期间存在抗原。在致敏过程中暴露于半抗原的LC促进了调节细胞和效应细胞的诱导9。来自几项研究的越来越多的证据表明，CHS反应可以由CD4 + MHC II类限制性Th1细胞介导，局部释放干扰素γ（IFN-γ）以采用特征性炎症浸润，CD8 + MHC I类限制性Tc1淋巴细胞，其也可以释放IFN-γ但主要介导对角质形成细胞的细胞毒性损伤，现在还有白细胞介导素17（IL-17）产生Th17细胞10，11.
已经开发了采用各种物种12，13，14 和半抗原的几种不同的CHS模型（不同半抗原，溶剂和施用时间的详细比较总结在 表1中）。小鼠是一种常用的实验室物种，在研究CHS方面具有一些优势。与其他物种相比，小鼠中有更多的菌株，敲除（KO）和转基因动物，这使它们成为非常有吸引力的动物15。此外，CHS模型需要许多动物，而老鼠在这里更经济。动物模型并非在所有方面都模仿ACD;特别是，它们表现出结痂和脱屑，这在人类中并不常见16，17。慢性病的特征难以重现，主要是因为所描述的模型没有假设半抗原在很长一段时间内的应用。然而，这里已经证实，ACD的许多重要方面都被复制了。还表明，与人类一样，这些特征与局部过敏反应有关。本方案中概述的半抗原，其溶剂及其应用的选择取决于这样一个事实，即结果已被众多 体外 测试证实，并且在实验室中对其进行了多年的测试和修改，直到建立当前版本为止。小鼠模型允许分析参与ACD开发的细胞亚群或细胞因子，并且对于新疗法的临床前评估至关重要。
Protocol
本文中介绍的所有实验均根据克拉科夫第一届当地动物试验伦理委员会的指导方针进行。所描述的所有程序都是根据当地的建议进行的，特别是关于使用氯胺酮/甲苯噻嗪作为麻醉剂，使用耳朵的两侧涂抹该物质/半抗原，切断耳朵，并通过去除眼球来收集血液。本研究使用BALB/c（单倍型H-2d）、CBA/J（H-2k）和C57BL/6（H-2b）雄性和雌性小鼠，6-12周龄（见 材料表）?…
Representative Results
对于CHS诱导， 通过 皮肤涂漆（腹部）用150μL5%TNCB或假用载体敏化动物。在第“4天”，在先前与TNCB接触的小鼠（测试组）和对照组小鼠（假敏化）中，用10μL0.4%TNCB接触绘画（激发）诱导两只耳朵的耳朵肿胀反应。提供的数据表明，与同样受到挑战的假敏小鼠相比，对TNCB敏感并4天后受到挑战的小鼠的耳朵肿胀显着增加（图3， 表2，测试与对照组）。耳部?…
Discussion
CHS 通过 半抗原诱导，半抗原与皮肤中的自身蛋白抗原结合，产生新抗原。CHS由循环抗原特异性CHS效应T细胞的局部血管外募集介导，这导致受攻击组织肿胀，在次级皮肤暴露于同一半抗原6后24小时达到峰值。组织的肿胀主要是由白细胞浸润和白细胞依赖性纤维蛋白沉积24引起的。这些变化可以用一个千分尺测量半抗原致敏和激发小鼠与假敏和激发小鼠的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了亚必胜区补助金的支持。波兰弗罗茨瓦夫医科大学的A020.22.060，以及科学和高等教育部N N401 545940至MS和IP2012 0443 72至MMS的赠款。
Materials
| 70% ethanol | Merck KGaA, Darmstadt, Germany | 65350-M | for surface disinfection |
| 96-well flat-bottom plates, polypropylene | Greiner Bio-One GmbH, Kremsmunster, Austria | 655101 | for MPO and Evans blue measurement – plates should be made of polypropylene, that has a lower binding capacity so proteins or DNA will not bind |
| Acetone (ACS reagent, ≥99.5%) | Merck KGaA, Darmstadt, Germany | 179124 | |
| Aluminum foil | Merck KGaA, Darmstadt, Germany | Z185140 | |
| Analytical balance | Sartorius Weighing Technology GmbH, Goettingen, Germany | PRACTUM224-1s, 29105177 | |
| Automated tissue processor | MediMeas Instruments, Sarsehri, Haryana, India | MTP-E-212 | automatically prepare tissue samples by fixing, dehydrating, clearing, and infiltrating them with paraffin |
| BD Vacutainer SST II Advance (tube with gel for obtaining serum) | Becton Dickinson (BD), Franklin Lakes, NJ, USA | BD 366882 | |
| Bicinchoninic acid kit for protein determination | Merck KGaA, Darmstadt, Germany | BCA1-1KT | |
| Biotin Rat Anti-Mouse IgG1 | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 553441 | |
| BSA (bovine serum albumine) | Merck KGaA, Darmstadt, Germany | A9418 | protein assays & analysis, 2 mg/mL |
| Cell strainer, pore size 70 μm | BIOLOGIX, China | 15-1070 | suitable for 50 mL tubes |
| Coverslip | VWR, Radnor, Pennsylvania, United States | 631-1583 | 24 mm, but it possible to use different size |
| Disposable pipettes capacity: 5 mL, 10 mL, 25 mL | Merck KGaA, Darmstadt, Germany | Z740301, Z740302, Z740303 | |
| DPBS (Dulbecco′s phosphate buffered saline) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 14190144 | no calcium, no magnesium, mammalian cell culture |
| DPX Mountant for histology | Merck KGaA, Darmstadt, Germany | 6522 | mounting media for H-E, might be used some other e.g, Canada balsam |
| Dumont 5 tweezers – straight | Animalab, Poznan, Poland | 11251-10FST | surgical instruments for procedures on mice (should be steriled) |
| Dumont 7 tweezers – bent | Animalab, Poznan, Poland | 11272-50FST | surgical instruments for procedures on mice (should be steriled) |
| Eosin Y solution, alcoholic | Merck KGaA, Darmstadt, Germany | HT110116 | |
| Eppendorf Safe-Lock Tubes 1.5 mL | Eppenforf, Germany | 3,01,20,086 | polypropylene |
| Eppendorf Safe-Lock Tubes, 2.0 mL | Eppenforf, Germany | 3,01,20,094 | polypropylene, round bottom (the homogenization beads can easily move) |
| Ethanol 100% (absolute alkohol) | Merck KGaA, Darmstadt, Germany | 1.07017 | |
| Ethanol 96% | Merck KGaA, Darmstadt, Germany | 1.59010 | |
| Evans blue | Merck KGaA, Darmstadt, Germany | E2129 | |
| FBS (fetal bovine serum) | ThermoFisher Scientific, Waltham, Massachusetts, USA | A3160802 | |
| Formalin solution, neutral buffered, 10% | Merck KGaA, Darmstadt, Germany | HT501128 | |
| Formamide 99.5% (GC) | Merck KGaA, Darmstadt, Germany | F7503 | |
| Freezer -20 °C | Bosch, Germany | GSN54AW30 | |
| Fridge +4 °C / freezer -20 °C | Bosch, Germany | KGV36V10 | mammalian Cell Culture, qualified, Brazil, 10 x 50 mL |
| Glass microskope slides, SuperFrost Plus | VWR, Radnor, Pennsylvania, United States | 631-0108, 631-0446, 631-0447, 631-0448, 631-0449 | Slides that eliminates the need to apply adhesive materials or protein coatings, to preventing any tissue sections loss during staining. |
| Graph Pad Prism | GraphPad Software Inc. | v. 9.4.0 | |
| Grey soap | Pollena Ostrzeszów, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland | Bialy jelen soap bar | grey Soap Bar Natural Hypoallergenic. Generally available product |
| H2SO4 (sulfuric acid) 1 mol/l (1 M) | Merck KGaA, Darmstadt, Germany | 1.60313 | |
| Harris hematoxylin solution | Merck KGaA, Darmstadt, Germany | HHS16 | |
| Hemocytometer | VWR, Avantor, U.S.A | 612-5719 | manual counting chamber is recommend, which is more accurate |
| Hexadecyltrimethylammonium bromide | Merck KGaA, Darmstadt, Germany | H5882 | |
| Homogenizer | QIAGEN Hilden, Germany | Tissue Lyser LT, SN 23.1001/05234 | homogenizer with stainless steel beads (diameter 5 mm) for 2 mL centrifuge tubes |
| Horseradish peroxidase streptavidin (HRP streptavidin) | Vector Laboratories, Inc., Burlingame, CA, USA | SA-5004-1 | |
| Hydrogen peroxide solution (H202) | Merck KGaA, Darmstadt, Germany | H1009 | |
| Incubator Heracell 150i | Thermo Electron LED Gmbh, Germany | 41071068 | 37 oC in the atmosphere of 5 % CO2, and 65 0C for deparaffinization the sections for histology |
| Ketamine 100 mg/mL, solution for injection | Biowet Pulawy Sp. z o.o., Pulawy, Poland | cat.# not avaliable | |
| KH2PO4 (potassium dihydrogen phosphate) 99.995% anhydrous basis | Merck KGaA, Darmstadt, Germany | 1.05108 | |
| Laboratory Centrifuge | Heraeus Megafuge 1.0R, Thermo Scientific, Germany | B00013899 | speed to 300 x g, with cooling to 4 0C |
| Laboratory Centrifuge | Heraeus Fresco 21, Thermo Scientific, Germany | 75002425 | speed to 3,000 x g, with cooling to 4 0C |
| Mask (FFP2) | VWR, Radnor, Pennsylvania, United States | 111-0917 | for working with ortho-dianisine dihydrochloride |
| Mice | Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland | CBA/J, C57BL/6 | |
| Micrometer | Mitutoyo, Tokyo, Japan | 193-111 | digit Outside Micrometer, Ratchet Stop, 0-25mm Range, 0.001mm Graduation, +/-0.002mm Accuracy, https://shop.mitutoyo.pl/web/mitutoyo/pl_PL/all/all/Mikrometr%20analogowy%20/PR/193-111/index.xhtml |
| microplate, 96 well, microlon, high binding (for ELISA test) | Greiner Bio-One GmbH, Kremsmunster, Austria | 655061 | with maxi-sorp binding surfaces for reliable and reproducible results in colormetric assays |
| Microscope with objectives | Leica Microsystems CMS GmbH, Germany | DM1000, 294011-082007 | histology presented in the paper was performed under ThermoFisher Scientific EVOS M5000 Imaging System, with objectives: FL 20X LWDPH, 0.40NA/3.1WD and FL 40X LWDPH 0.65NA/1.79WD |
| Myeloperoxidase from human leukocytes (MPO standard) | Merck KGaA, Darmstadt, Germany | M6908 | |
| Na2HPO4 x 7 H2O (sodium phosphate dibasic heptahydrate) | Merck KGaA, Darmstadt, Germany | S9390 | |
| Olive-oil | Merck KGaA, Darmstadt, Germany | 75343 | pure, natural |
| OptEIA Mouse IFN-γ ELISA Set | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 555138 | |
| Ortho-dianisine dihydrochloride | Merck KGaA, Darmstadt, Germany | D3252 | |
| Paraffin wax | Merck KGaA, Darmstadt, Germany | 76242 | beads, waxy solid |
| PBS (phosphate buffered saline) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 20012027 | pH 7.2, mammalian cell culture |
| ph meter | Elmetron, Poland | CP-401 | |
| Pipettes, variable volume with tips | Merck KGaA, Darmstadt, Germany | EP3123000900-1EA | 3-pack, Option 1, 0.5-10 uL/10-100 uL/100-1000 uL, includes epT.I.P.S. |
| Razor blade | VWR, Radnor, Pennsylvania, United States | PERS94-0462 | scraper and cutter blades, single edge, aluminium spine, 100 blades per box, individually wrapped |
| Rotary microtome | MRC Laboratory-Instruments, Essex, CM20 2HU UK | HIS-202A | |
| Scissors – straight, sharp / sharp | Animalab, Poznan, Poland | 14060-10FST | Surgical instruments for procedures on mice (should be steriled) |
| Screw cap (open top) | Merck KGaA, Darmstadt, Germany | 27056 | black polypropylene hole cap, for use with 22 mL vial with 20-400 thread |
| Spectrophotometer | BioTek Instruments, U.S.A | 201446 | universal microplate spectrophotometer: λ range: 200 – 999 nm, absorbance measurement range: 0.000 – 4.000 Abs |
| Staining dish 20 slides with rack | Merck KGaA, Darmstadt, Germany | S6141 | e.g. 20 slide staining dishes complete with covers, slide rack and handle |
| Sterile Disposable Biopsy Punch 6mm | Integra LifeSciences, Princeton, NJ, USA | 33-36 | |
| Surgical scissors | Animalab, Poznan, Poland | 52138-46 | surgical instruments for procedures on mice (should be steriled) |
| Tissue processing cassettes | Merck KGaA, Darmstadt, Germany | Z672122 | tissue processing/ embedding cassettes with lid |
| TMB Substrate Reagent Set | Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA | 555214 | |
| TNCB (2,4,6-trinitrochlorobenzene) | Tokyo Chemical Industry CO., LTD, Japan | C0307 | |
| TNP-BSA (bovine serum albumin conjugated with 2,4,6-trinitrophenyl) | Biosearch Technologies LGC, Petaluma, CA, USA | T-5050 | |
| T-PER (tissue protein extration reagent) | ThermoFisher Scientific, Waltham, Massachusetts, USA | 78510 | |
| Tubes 15 mL sterile | Merck KGaA, Darmstadt, Germany | CLS430055 (Corning) | polypropylene, conical bottom |
| Tubes 50 mL, sterile | Merck KGaA, Darmstadt, Germany | CLS430290 (Corning) | polypropylene, conical bottom |
| Tween 20 | Merck KGaA, Darmstadt, Germany | P1379 | |
| Vials, screw top, clear glass (vial only) 22 mL | Merck KGaA, Darmstadt, Germany | 27173 | for the preparation of hapten, screwed on so that it does not evaporate |
| Water bath | AJL Electronic, Poland | LW102 | |
| Wax (paraffin) dispenser | VWR, Radnor, Pennsylvania, United States | 114-8737 | |
| Xylazine (xylapan 20 mg/mL) solution for injection | Vetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Poland | cat.# not avaliable | |
| Xylene (histological grade) | Merck KGaA, Darmstadt, Germany | 534056 |
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Cite This Article
Zemelka-Wiacek, M., Majewska-Szczepanik, M., Gajdanowicz, P., Szczepanik, M. Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis. J. Vis. Exp. (187), e64329, doi:10.3791/64329 (2022).