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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Biology

小鼠皮肤和肿瘤淋巴血管白细胞出口的定量

Published: January 07, 2019
doi:
10.3791/58704
, , , , ,
1Department of Cell, Developmental, & Cancer Biology,Oregon Health and Science University, 2Department of Molecular Microbiology & Immunology,Oregon Health and Science University, 3Department of Dermatology,Oregon Health and Science University, 4Knight Cancer Institute,Oregon Health and Science University

Summary

在这里, 我们展示了在体内定量的方法,白细胞出口从天真, 发炎, 恶性小鼠皮肤。我们对两种模型进行了面对面的比较: 透皮 fitc 应用和原位光转换。此外, 我们还论证了光转换在跟踪皮肤肿瘤白细胞出口方面的作用。

Abstract

白细胞从外周组织进入排淋巴结不仅对免疫监测和启动至关重要, 而且有助于外周组织反应的解决。虽然使用了多种方法来量化白细胞从非淋巴, 外周组织, 细胞和分子机制, 控制上下文依赖的出口仍然了解甚少。在这里, 我们描述了使用原位光转换定量分析白细胞出口从小鼠皮肤和肿瘤。光转换允许直接标记白细胞居住在皮肤组织内。虽然皮肤暴露在紫罗兰色的光导致局部炎症反应的特点是白细胞浸润和血管泄漏, 在头部与透皮应用荧光示踪剂的比较, 光转换专门标记迁移树突状细胞群, 同时使骨髓和淋巴出口从皮肤微环境和肿瘤的量化。白细胞出口的机制仍然是我们对肿瘤内白细胞复杂性的理解中缺失的组成部分, 因此本文所述工具的应用将为肿瘤免疫微环境的动态提供独特的见解。在稳定的状态和对治疗的反应。

Introduction

外周组织免疫反应不仅是由白细胞招募到炎症部位, 但也由机制, 调节其随后的保留。因此, 保护性免疫是由累积的细胞和分子机制决定的, 这些机制决定白细胞是进入、停留在内, 还是通过淋巴血管从外周组织中迁移出去。重要的是, 白细胞通过淋巴血管 (称为出口) 退出组织的倾向与它们的专门功能有关。树突状细胞 (dc) 获取迁移行为, 以响应成熟信号, 导致抗原转运和在排水淋巴结 (dln) 中的表现, 这是适应性免疫1所必需的一个过程。清除骨髓细胞, 如巨噬细胞和中性粒细胞, 有助于清除凋亡的碎片, 通过吞噬。在细菌感染期间, 中性粒细胞退出组织, 并最终在 dln 2 和 dss诱导结肠炎模型中发生凋亡, 数据支持巨噬细胞出口是解决局部炎症3所必需的假设。然而, 中性粒细胞和巨噬细胞的退出是否发生在所有炎症环境中尚不清楚。t 淋巴细胞出口的证据,从稳定状态 4,5,6, 7, 感染8, 并发炎4,9,10 ,11,12个外周, 非淋巴组织表明 t 细胞积极循环, 虽然基于组织的信号, 推动这一出口仍然很少了解。几项研究确定了定向迁移以排出淋巴毛细血管和随后的出口所必需的信号, 包括趋化因子 (c-c 母题) 配体 21 (ccl21) 及其受体 ccr7 4,11, 13、趋化因子 (c-x-c 母题) 配体 12 (cxcl12) 及其受体 cxcr42、14和 sphingosine-1-phosphate-磷酸 (s1p)10、15、16。然而, 这些机制并不是在所有情况下都很活跃, 它们是否决定了所有细胞类型的出口仍然是一个悬而未决的问题。重要的是, 要进一步深入了解控制出口的机制及其在疾病中的功能相关性, 需要定量的体内分析方法。

在体内多个动物模型中, 有几种方法被用来量化出口, 包括淋巴血管的直接插管、体内标记白细胞的采用转移、荧光示踪剂的透皮应用,注射标记颗粒, 并在体内光转换17,18。直接插管的外发小鼠淋巴管是困难的, 并限制在小动物的液体量, 可以收集。因此, 插管主要是在大型动物 (如绵羊), 在那里这种手术操作是可行的。这些研究为淋巴 10、19、20中存在淋巴和骨髓细胞提供了直接证据。此外, 卵巢模型显示, 急性和慢性炎症增加淋巴细胞在淋巴中的存在近 100倍 10,21。

通过转移标记和基因操纵的淋巴细胞, 重要地表明 ccr7 是 cd4+ t 细胞从剧烈发炎的皮肤5,11, 而淋巴细胞的预处理所必需的用小分子 s1p 受体激动剂 f其次 720, 仅部分抑制他们的出口10。有趣的是, 从慢性发炎的皮肤转移的淋巴细胞是 ccr7 独立10, 但可能部分需要 cxcr49。然而, 采用转移实验通过注射将体外活化淋巴细胞和标记为组织的非生理数量传递给组织, 从而改变组织的生物力学环境和间质液体压力升高打开最初的淋巴毛细血管, 改变它们的运输特性22。作为替代办法, 在存在或不存在皮肤刺激物 (如邻苯二甲酸二丁酯、dbp) 或感染23、24的情况下, 荧光素异硫氰酸酯 (fitc) 的透皮应用允许跟踪积累示踪剂并迁移到 dln 的吞噬细胞。同样, 荧光标记的肿瘤提供了一种方法来追踪吞噬细胞, 这些细胞已经吞没了肿瘤物质 25。这些方法为指导直流出口 13、14、17、26、27的机制提供了重要的见解, 但无法跟踪非吞噬细胞淋巴细胞和, 解释可以复杂的可溶性 fitc 的游离淋巴引流, 从而标记非迁移, ln 居民 dc。

另外, 体内显微镜是一个强大的工具, 允许在体内实时跟踪与生理相关的白细胞群 28,29.体内显微镜与报告小鼠和抗体联合使用, 在体内进行免疫荧光标记, 揭示了免疫细胞贩运的复杂时空动态, 包括间质迁移30, 在淋巴内皮细胞中的迁移, 在淋巴腔内的通道,以及在 ln 进入 28,31时的迁移。广泛采用的体内成像技术受到费用、设置所需专业知识以及用于量化多种细胞类型的有限吞吐量的限制。尽管如此, 将分析种群动力学组织的定量方法与生命内成像结合起来, 将为淋巴毛细血管的运动和迁移机制提供额外而重要的机械洞察18,31,32岁

因此,体内光转换已成为一种方法, 允许原位标记, 独立于吞噬活性, 并定量生理白细胞出口 (当与流式细胞术结合)没有或存在的挑战。kaede-tg 小鼠本组织地表达从石珊瑚中分离出的一种蛋白质, 这种蛋白质表现出绿色荧光 (kaede 绿色), 直到暴露在紫罗兰色的光线下, 之后它不可逆转地转化为红色荧光 (kaede 红色)33。光转换细胞可以被跟踪, 因为他们从周围组织部位退出和积累在 dln。这和其他类似的光敞篷车模型34,35揭示了重要的生物学, 包括从皮肤的调节 t 细胞的本构出口 36, cxcr4 依赖 b 细胞出口从 peyer 的补丁37在多肽后动员常驻记忆 t 细胞重新挑战38, 从肿瘤微环境中动员广泛的白细胞出口39。在此, 我们对皮肤炎症和感染情况下的光转换与透皮 fitc 应用进行了面对面的比较, 以便将现有数据与光转换方法直接比较。此外, 我们还演示了植入肿瘤的光转换, 并描述了肿瘤微环境中的转化效率和选择性出口。因此, 我们认为, 需要进一步应用这些方法来阐明肿瘤白细胞出口的关键生物学, 这将对解释骨髓内白细胞复杂性、抗肿瘤免疫和对治疗的反应。

Protocol

所有动物协议都已获得俄勒冈州健康 & 科学大学动物护理和使用机构委员会的批准。 1. 小皮纳炎症和 fitc 绘画的归纳 在层流罩中, 使用蒸发异氟烷麻醉 c57bl6 小鼠 (诱导为3-5% 异氟烷, 并保持在1-3% 异氟烷; 氧气流量为 0.5-1.0 lmmin)。通过监测踏板反射的损失、非自愿的运动和呼吸速率的降低, 确保适当的麻醉。 将耳朵扁平, 耳朵的腹侧朝上。将 5% fitc 溶液的 p0μl 溶…

Representative Results

我们首先试图复制发表在文献中的光转换结果, 以评估效率, 并确定相关的炎症在小鼠皮肤。耳朵 pinna 暴露在 100 mw 紫罗兰色光 (405 nm) 3分钟之前所述的 33.暴露后立即从耳皮或宫颈 dln 产生的单个细胞悬浮液显示, 皮肤中所有 cd45+白细胞的转化效率为 78%, 在 dln 中没有观察到转化细胞 (图 1a)。为了评估与光转换相关的炎症反应,…

Discussion

尽管外周、非淋巴组织的白细胞出口对免疫反应的启动和解决至关重要, 但控制出口的分子机制却鲜为人知。这种知识差距在很大程度上是由于可随时提供在体内进行量化的工具。在这里, 我们描述了使用光敞篷小鼠 (kaed-tg) 量化皮肤和肿瘤的白细胞出口, 并提供了一个直接的面对面比较与 fitc 油漆在炎症和感染模型。我们证明, 虽然这两种模型跟踪内源性 dc 种群, fitc 的自由引流和非吞噬细?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢 marcus bosenberg 博士提供 yumm 1.1 和 yumm 1.7 小鼠黑色素瘤线, 并感谢 deborah j. fell 博士提供 b6。cg-tg (cag-g-tdKaede)15Utr 小鼠通过日本 mext 的国家生物资源与 riken brc 达成一致。

Materials

Collagenase D Roche 11088866001
DNase Roche 4536282001
Silver-LED-405B light source with optical fiber and collimtor Prizmatix Ltd V8144
Fluorescein isothiocyanate isomer I Sigma-Aldrich F4274
dibutyl phthalate Sigma-Aldrich 524980
acetone Macron Fine Chemicals 2440-02
29-guage syringes Exel International 26029
Evans Blue Sigma-Aldrich E2129
70 um cell strainers VWR 732-2758
paraformaldehyde Sigma-Aldrich P6148
HBSS Caisson HBL06
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit Invitrogen L34966
Purified Anti-mouse CD16/CD32 Tonbo Biosciences 70-0161-M001
BV605 CD11c (clone N418) Biolegend 117334
PerCP-Cy5.5 MHCII (clone M5/114.15.2) BD Pharmingen 562363
BV421 CD3e (clone 145-2C11) Biolegend 100341
APC CD8a (clone 53-6.7) TonBo Biosciences 20-0081-u100
APC-Cy7 CD45 (clone 30-F11) Biolegend 103116
BV650 CD19 (clone 6D5) Biolegend 115541
PercCP-Cy5.5 Ly6C (clone HK1.4) Biolegend 128011
Alexa Fluor 647 F4/80 (clone BM8) Biolegend 123121
APC-Cy7 Ly6G (clone 1A8) Biolegend 127623
BV711 CD11b (clone M1/70) Biolegend 101241
BV605 CD45 (clone 30-F11) Biolegend 103155
BV711 CD4 (clone RM4-5) BD Biosciences 563726
Bovine serum albumin (Fraction V) Fisher Scientific BP1600-100
Anit-Rat and Anti-Hamster Igk / Negative Control Compensation Particle Set BD Biosciences 552845
Fortessa Flow Cytometer BD Biosciences
FlowJo v10 Software FlowJo
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Tags

Leukocyte EgressLymphatic VesselsMurine SkinTumorsPhotoconversionIn VivoQuantitative AnalysisLeukocyte ResidenceExit DynamicsDisease StatesCutaneous TumorsInflammationKaede Transgenic MouseDibutyl PhthalateVaccinia InfectionLymph NodesCollagenase DDNaseCell Suspension

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Steele, M. M., Churchill, M. J., Breazeale, A. P., Lane, R. S., Nelson, N. A., Lund, A. W. Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors. J. Vis. Exp. (143), e58704, doi:10.3791/58704 (2019).
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