外迁移系统中淋巴细胞迁移的评估
Summary
在此协议中，淋巴细胞被放置在迁移系统的顶室中，由多孔膜与底部腔室隔开。化学因子被添加到底部腔室，从而诱导沿化学基质梯度的活性迁移。48小时后，淋巴细胞在两个腔室计数，以定量迁移。
Abstract
在这里，我们提出了一个有效的方法，可以执行与基本的实验室技能和材料，以评估淋巴细胞化学动力学运动在外体迁移系统。组2先天淋巴细胞（ILC2）和CD4+T助剂细胞从脾脏和肺部分离出，对受挑战的BALB/c小鼠。我们比较地确认了CCR4在CD4+T细胞和ILC2上的表达。CCL17 和 CCL22 是 CCR4 的已知配体;因此，使用这种活体迁移方法，我们检查了CCL17– CCL22诱导的CCR4+ 淋巴细胞的运动。为了建立化学梯度，CCL17和CCL22被放置在迁移系统的底部腔室。分离的淋巴细胞然后被添加到顶室，并在48小时期间淋巴细胞通过3μm孔主动迁移到底部腔室的化学素。这是一个有效的系统，以确定淋巴细胞的化学动力学，但可以理解的是，不模仿在体内器官微环境中发现的复杂性。这是通过增加正在研究的器官和淋巴细胞的原位成像来克服的方法的一个限制。相反，这种方法的优点是，入门级技术人员可以以比现场成像更具成本效益的速度执行。当治疗化合物可用于增强迁移时，如肿瘤渗透细胞毒性免疫细胞或抑制迁移，也许在免疫病理学备受关注的自身免疫性疾病中，此方法可用作筛选工具。一般来说，如果感兴趣的化学基因在统计上高于介质控制水平生成化学动力学，则该方法是有效的。在这种情况下，也可以确定给定化合物的抑制/增强程度。
Introduction
这种原始的迁移方法是由斯蒂芬·博伊登于1962年在《实验医学杂志1》上提出的。如果没有博伊登室的发展，我们对化学和化学动力学的了解是不可能实现的。在1977年发现第一个化学药物之前，前体迁移系统被用来了解血清因子，这些血清因子可以阻止巨噬细胞的细胞运动，同时放大中性粒细胞1、2的细胞活力。关于免疫细胞迁移，已经发展了大量的知识，到目前为止，已经发现了47个化学素与19个相应的受体3，4。此外，这些化学药物通路的多种抑制剂/增强剂已发展为治疗目的5，6，7，8。其中许多化合物已经在类似的迁移室中进行了测试，以了解化合物与免疫细胞对给定化学素9的反应之间的直接相互作用。
迁移，或二足动物，进入发炎的组织是一个必要的过程，以健康的炎症反应清除感染10，11。博伊登室、迁移系统或跨井装置一般由由多孔膜1、12隔开的两个腔室组成。底部腔通常装有含有感兴趣的化学素的介质，而白细胞则放置在顶部腔室中。可以根据感兴趣的细胞的大小选择膜中的孔隙大小。对于这个项目，我们选择了3μm多孔膜，因为淋巴细胞的大小是7-20μm，这取决于细胞发育的阶段。这种孔径大小确保这些细胞不会被动地从毛孔中掉落，而是主动迁移以响应化学素梯度。
该协议的主要优点是其成本效益。体内迁移是困难的，因为它需要动物处理和手术方面的广泛培训，并且通常涉及研究人员并不总是可用的高能显微镜。在体内成像之前，可以对被认为能增强或抑制迁移的化合物进行具有成本效益的筛选。由于迁移系统受到严格控制，细胞可以先进行处理，然后添加到转井装置中，反之亦然，可先使用化学抑制剂处理化学药物抑制剂，然后将细胞添加到转井装置中。最后，内皮细胞和/或基底膜蛋白可以在转井实验前1-2天添加到转井插入的底部，以了解这些屏障细胞在化学动力学中的参与程度。同样，这些对系统的操作提供了一种强有力的手段，用于在更复杂的体内研究之前确定有关给定化合物有效性的重要信息。
利用移外室系统是评估淋巴细胞在各种体内和体外条件下的流动性的有效方法12，13，14。在这里，我们描述了一种优化的方法，用于评估外体淋巴细胞对迁移室中化学素的反应。在此示例实验中，CD4+ T 细胞和组 2 先天淋巴细胞 （ILC2） 在 OVA 过敏原暴露后从雄性小鼠和雌性 BALB/c 小鼠中分离。生成一个假设，CCR4+ CD45+系-（LIN-） ILC2 来自过敏原挑战小鼠将比 CCR4 + CD4+ T 帮助细胞更有效地迁移到 CCL17 和 CCL22。CCL17和CCL22是一种化学素，通常由M2（过敏）表型的树突细胞和巨噬细胞等细胞产生，在过敏15、16中。CCL17和CCL22可被视为过敏性炎症的生物标志物，因为它们在气道恶化16、17、18期间很容易在肺部检测到。重要的是，CCR4的表达与未经处理的对照组相比是升高的，从从室内尘虫处理动物中分离出的生物信息数据中所揭示，同样，ILC2来自使用IL-33处理的幼化动物的ILC2（过敏原促进先天细胞因子） 上调节 CCR419，20.此外，根据免疫基因组项目数据库（www.immgen.org）中的ILC2数据，CCR4 mRNA在这些先天免疫细胞中高度表达。迄今为止，关于将ILC2贩运到组织方面鲜为人知，但ILC2和CD4+T细胞很可能使用类似的化学素和受体作为化学和化学动力学，因为它们表达类似的转录因子和受体。因此，我们比较了ILC2和CD4+T淋巴细胞的CCL17与CCL22的响应能力，这些细胞来自男性和女性，OVA挑战动物。
Protocol
Representative Results
Discussion
在这里，我们提出了一个成熟的方法，以评估在外体迁移系统中淋巴细胞引起的化学素迁移。该协议中有几个关键步骤，第一个步骤是验证实验中免疫细胞上正确的化学素受体的表达。在我们的手中，我们选择CCR4是因为文献中强调了CCR4在过敏性炎症中T2帮助T细胞的重要性。Ovalbumin引起的炎症先前被证明至少受到两个CCR4拮抗剂24，25的限制;然而，这是之…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由美国肺脏协会（K.J.W.）、授予T.A.W.和K.J.W.的纪念尤金·肯尼基金、犹他大学为K.J.W.提供的慷慨启动支持以及退伍军人事务部授予T.A.W.（VA I01BX0003635）的资助。T.A.W. 是退伍军人事务部研究职业科学家奖 （IK6 BX003781） 的获得者。作者希望感谢丽莎·丘多梅尔卡女士的编辑协助。作者感谢UNMC流式细胞学核心在收集为本手稿生成的流式细胞学数据方面给予的支持。
Materials
| 0.4% Trypan Blue | Sigma-Aldrich | 15250061 | |
| 1 mL syringe | BD Bioscience | 329424 | U-100 Syringes Micro-Fine 28G 1/2" 1cc |
| 100x Penicillin-Streptomycin, L-Glutamine | Gibco | 10378-016 | Dilute to 1x in RPMI media |
| 15 mL conical tubes | Olympus Plastics | 28-101 | polypropylene tubes |
| 3 um transwell inserts | Genesee Scientific | 25-288 | 24-well plate containing 12 transwell inserts |
| 3x stabilizing fixative | BD Pharmigen | 338036 | Prepare 1x solution according to manufacturers protocol |
| 5 mL polystyrene tubes | STEM Cell Technologies | 38007 | |
| 50 mL conical tubes | Olympus Plastics | 28-106 | polypropylene tubes |
| 8-chamber easy separation magnet | STEM Cell Technologies | 18103 | |
| ACK Lysing Buffer | Life Technologies Corporation | A1049201 | |
| Advanced cell strainer, 40 um | Genesee Scientific | 25-375 | nylon mesh, 40 micron strainers |
| Aluminum Hydroxide, Reagent Grade | Sigma-Aldrich | 239186-25G | 20 mg/mL |
| anti- mouse CCR4; APC-conjugated | Biolegend | 131211 | 0.5 ug/test |
| anti-mouse CD11b | BD Pharmigen | 557396 | 0.5 ug/test |
| anti-mouse CD11c; PE eFluor 610 | Thermo-Fischer Scientific | 61-0114-82 | 0.25 ug/test |
| anti-mouse CD16/32, Fc block | BD Pharmigen | 553141 | 0.5 ug/test |
| anti-mouse CD19; APC-eFluor 780 conjugated | Thermo-Fischer Scientific | 47-0193-82 | 0.5 ug/test |
| anti-mouse CD3; PE Cy 7-conjugated | BD Pharmigen | 552774 | 0.25 ug/test |
| anti-mouse CD45; PE conjugated | BD Pharmigen | 56087 | 0.5 ug/test |
| anti-mouse ICOS (CD278) | BD Pharmigen | 564070 | 0.5 ug/test |
| anti-mouse NK1.1 (CD161); FITC-conjugated | BD Pharmigen | 553164 | 0.25 ug/test |
| anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugated | Biolegend | 145311 | 0.5 ug/test |
| Automated Cell Counter | BIORAD | 1450102 | |
| Automated Dissociator | MACS Miltenyi Biotec | 130-093-235 | |
| Bovine Serum Albumin, Lyophilized Powder | Sigma-Aldrich | A2153-10G | 0.5% in serum-free RPMI |
| Cell Counter Clides | BIORAD | 1450015 | |
| Chicken Egg Ovalbumin, Grade V | Sigma-Aldrich | A5503-10G | 500 ug/mL |
| Collagenase, Type 1, Filtered | Worthington Biochemical Corporation | CLSS-1, purchase as 5 X 50 mg vials (LS004216) | 25 U/mL in RPMI |
| compensation beads | Affymetrix | 01-1111-41 | 1 drop per contol tube |
| Dissociation Tubes | MACS Miltenyi Biotec | 130-096-335 | |
| FACS Buffer | BD Pharmigen | 554657 | 1x PBS + 2% FBS, w/ sodium azide; stored at 4 °C |
| Heat Inactivated-FBS | Genesee Scientific | 25-525H | 10% in complete RPMI & ILC2 Expansion Media |
| mouse CCL17 | GenScript | Z02954-20 | 50 ng/mL |
| mouse CCL22 | GenScript | Z02856-20 | 50 ng/mL |
| mouse CD4+ T cell enrichment kit | STEM Cell Technologies | 19852 | |
| mouse IL-2 | GenScript | Z02764-20 | 20 ng/mL |
| mouse ILC2 enrichment kit | STEM Cell Technologies | 19842 | |
| mouse recombinant IL-33 | STEM Cell Technologies | 78044 | 20 ng/mL |
| RPMI | Life Technologies Corporation | 22400071 | |
| Separation Buffer | STEM Cell Technologies | 20144 | 1 X PBS + 2% FBS; stored at 4C |
| small animal nebulizer and chamber | Data Sciences International | ||
| sterile saline | Baxter | 2F7124; NDC 0338-0048-04 | 0.9% Sodium Chloride |
References
- Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. The Journal of Experimental Medicine. 115, 453-466 (1962).
- Moser, B. Editorial: History of Chemoattractant Research. Frontiers in Immunology. 6, 548 (2015).
- Borroni, E. M., Savino, B., Bonecchi, R., Locati, M. Chemokines sound the alarmin: The role of atypical chemokine in inflammation and cancer. Seminars in Immunology. 38, 63-71 (2018).
- Charo, I. F., Ransohoff, R. M. The many roles of chemokines and chemokine receptors in inflammation. The New England Journal of Medicine. 354 (6), 610-621 (2006).
- Abboud, D., Hanson, J. Chemokine neutralization as an innovative therapeutic strategy for atopic dermatitis. Drug Discovery Today. 22 (4), 702-711 (2017).
- Aldinucci, D., Casagrande, N. Inhibition of the CCL5/CCR5 Axis against the Progression of Gastric Cancer. International Journal of Molecular Sciences. 19 (5), E1477 (2018).
- Chonco, L., et al. Novel DNA Aptamers Against CCL21 Protein: Characterization and Biomedical Applications for Targeted Drug Delivery to T Cell-Rich Zones. Nucleic Acid Therapy. 28 (4), 242-251 (2018).
- Trivedi, P. J., Adams, D. H. Chemokines and Chemokine Receptors as Therapeutic Targets in Inflammatory Bowel Disease; Pitfalls and Promise. Journal of Crohn’s and Colitis. 12 (12), 1508 (2018).
- Pietrosimone, K. M., Bhandari, S., Lemieux, M. G., Knecht, D. A., Lynes, M. A. In vitro assays of chemotaxis as a window into mechanisms of toxicant-induced immunomodulation. Current Protocols in Toxicology. 58 (Unit 18.17), (2013).
- Davis, D. M. How studying the immune system leads us to new medicines. Lancet. 391 (10136), 2205-2206 (2018).
- Culley, F. J., Pennycook, A. M., Tregoning, J. S., Hussell, T., Openshaw, P. J. Differential chemokine expression following respiratory virus infection reflects Th1- or Th2-biased immunopathology. Journal of Virology. 80 (9), 4521-4527 (2006).
- Denney, H., Clench, M. R., Woodroofe, M. N. Cleavage of chemokines CCL2 and CXCL10 by matrix metalloproteinases-2 and -9: implications for chemotaxis. Biochemical and Biophysics Research Communications. 382 (2), 341-347 (2009).
- Burrell, B. E., et al. Lymph Node Stromal Fiber ER-TR7 Modulates CD4+ T Cell Lymph Node Trafficking and Transplant Tolerance. Transplantation. 99 (6), 1119-1125 (2015).
- Warren, K. J., Iwami, D., Harris, D. G., Bromberg, J. S., Burrell, B. E. Laminins affect T cell trafficking and allograft fate. The Journal of Clinical Investigation. 124 (5), 2204-2218 (2014).
- Hirata, H., et al. Th2 cell differentiation from naive CD4(+) T cells is enhanced by autocrine CC chemokines in atopic diseases. Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology. , (2018).
- Lin, R., Choi, Y. H., Zidar, D. A., Walker, J. K. L. beta-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells. American Journal of Respiratory Cell and Molecular Biology. 58 (6), 745-755 (2018).
- Zhang, Y., et al. A new antagonist for CCR4 attenuates allergic lung inflammation in a mouse model of asthma. Science Reports. 7 (1), 15038 (2017).
- Lu, Y., et al. Dynamics of helper CD4 T cells during acute and stable allergic asthma. Mucosal Immunology. 11 (6), 1640-1652 (2018).
- Li, B. W. S., Beerens, D., Brem, M. D., Hendriks, R. W. Characterization of Group 2 Innate Lymphoid Cells in Allergic Airway Inflammation Models in the Mouse. Methods in Molecular Biology. 1559, 169-183 (2017).
- Li, B. W. S., et al. Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation. Frontiers in Immunology. 8, 1684 (2017).
- Warren, K. J., et al. Sex differences in activation of lung-related type-2 innate lymphoid cells in experimental asthma. Annals of Allergy, Asthma, Immunology: Official Publication of the American College of Allergy, Asthma, Immunology. , (2016).
- Warren, K. J., et al. Ovalbumin-sensitized mice have altered airway inflammation to agriculture organic dust. Respiratory Research. 20 (1), 51 (2019).
- Poole, J. A., et al. alphabeta T cells and a mixed Th1/Th17 response are important in organic dust-induced airway disease. Annals of Allergy, Asthma, Immunology: Official Publication of the American College of Allergy, Asthma, Immunology. 109 (4), 266-273 (2012).
- Matsuo, K., et al. A CCR4 antagonist ameliorates atopic dermatitis-like skin lesions induced by dibutyl phthalate and a hydrogel patch containing ovalbumin. Biomedical Pharmacotherapy. 109, 1437-1444 (2019).
- Mikhak, Z., et al. Contribution of CCR4 and CCR8 to antigen-specific T(H)2 cell trafficking in allergic pulmonary inflammation. The Journal of Allergy and Clinical Immunology. 123 (1), 67-73 (2009).
- Monticelli, L. A., et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nature Immunology. 12 (11), 1045-1054 (2011).
- Saenz, S. A., et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. Nature. 464 (7293), 1362-1366 (2010).
- Hoyler, T., et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity. 37 (4), 634-648 (2012).
- Nakajima, H., Shores, E. W., Noguchi, M., Leonard, W. J. The common cytokine receptor gamma chain plays an essential role in regulating lymphoid homeostasis. The Journal of Experimental Medicine. 185 (2), 189-195 (1997).
- Warren, K. J., et al. RSV-specific anti-viral immunity is disrupted by chronic ethanol consumption. Alcohol. , (2016).
- Warren, K. J., Poole, J. A., Sweeter, J. M., DeVasure, J. M., Wyatt, T. A. An association between MMP-9 and impaired T cell migration in ethanol-fed BALB/c mice infected with Respiratory Syncytial Virus-2A. Alcohol. , (2018).
- Molteni, R., et al. A novel device to concurrently assess leukocyte extravasation and interstitial migration within a defined 3D environment. Lab on a Chip. 15 (1), 195-207 (2015).
- Bersini, S., et al. Human in vitro 3D co-culture model to engineer vascularized bone-mimicking tissues combining computational tools and statistical experimental approach. Biomaterials. 76, 157-172 (2016).
- Jeon, J. S., et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proceedings of the National Academy of Sciences of the United States of America. 112 (1), 214-219 (2015).
