Summary

通过在小鼠中吸液性淋巴血管阻止淋巴流

Published: May 14, 2020
doi:

Summary

提出了一种通过手术阻断淋巴血管来阻止淋巴流动的方案。

Abstract

淋巴血管通过运输抗原、细胞因子和细胞排干淋巴结(LNs)来保持组织流动性平衡和优化免疫保护至关重要。淋巴流中断是研究淋巴血管功能的重要方法。从杂物足部到流行淋巴结(pLNs)的淋巴血管被明确界定为淋巴排入pLNs的唯一途径。给这些发性淋巴血管进行吸管可以选择性地防止淋巴流向pLAN。此方法允许干扰淋巴流,对排水 pLN 中的淋巴内皮细胞、支淋巴血管以及该区域周围的其他淋巴血管的损害最小。此方法已用于研究淋巴如何影响 LN 中的高内皮淋巴瘤( HEV )和化疗表达,以及淋巴如何在没有功能性淋巴血管的情况经 LN 周围的脂肪组织。随着人们越来越认识到淋巴功能的重要性,这种方法将有更广泛的应用,以进一步解开淋巴血管在调节LN微环境和免疫反应的功能。

Introduction

淋巴系统的空间组织提供结构和功能支持,以有效地去除细胞外液体,将抗原和抗原呈现细胞(APC)运送到排出的LNs。最初的淋巴血管(也称为淋巴毛细血管)由于其不连续的细胞间结,便于从周围的细胞外空间1有效地收集液体、细胞和其他材料, 因此具有高度渗透。最初的淋巴血管合并成收集淋巴血管,这些血管有紧密的细胞间结,连续的地下室膜和淋巴肌肉覆盖。收集淋巴血管负责运输收集的淋巴到排水的LNs,并最终返回淋巴循环2,3。将淋巴推进到排水的LN的收集淋巴血管是发性淋巴血管4,5,6,7。淋巴血管的阻塞可以阻止淋巴流进入LNs,这是研究淋巴流动功能时有用的技术。

先前的研究表明,淋巴流动在运输抗原和药水,以及维持LN平衡方面起着重要作用。众所周知,组织衍生的APCs,通常激活的迁移树突状细胞(DCs),通过发源淋巴血管前往LN激活T细胞8。自由形式的抗原,如微生物或可溶性抗原,被动地与淋巴流动到LN激活LN居民APC的想法在过去十年中得到了接受9,10,11,12。与淋巴一起携带的自由形式抗原在感染后需要几分钟时间才能到达 LN,LN-驻留细胞活化可能在刺激后 20 分钟内发生。这比迁移 DC 的激活要快得多,迁移 DC 进入排空 LN 9 需要 8小时多。除了运输抗原以启动免疫保护外,淋巴还携带细胞因子和DC到LN,以维持其微环境,并支持免疫细胞平衡13,14。此前,通过阻断淋巴血管来阻断淋巴流动表明,淋巴需要维持支持恒流T细胞所需的HV表型,B细胞向LN15、16、17定位。CCL21是一种关键的化学素,它指导直流和T细胞在LN8,18中的定位。阻塞淋巴流会中断 LN 中的 CCL21 表达,并可能中断 LN19中的直流和 T 细胞定位和/或相互作用。因此,阻断淋巴流动可以通过破坏调节 LN 免疫反应的 LN 微环境,直接或间接地消除抗原/直流对排干 LN 的访问。为了更好地研究淋巴流动的功能,提出了一个实验方案(图1),通过将支脚的淋巴血管从脚垫到pLN的吸血,来阻止小鼠的淋巴流动。该方法是今后在健康和患病条件下研究淋巴功能的重要技术。

Protocol

所有动物工作都需要得到机构和政府伦理及动物处理委员会的批准。 这是一个非生存手术。 1. 材料准备 通过将 70 mL 的 100% 乙醇与 30 mL 的无菌水混合,准备 100 mL 的 70% 乙醇。手术前将所有手术工具绝育,并在手术前和手术期间将工具保持为 70% 乙醇,以保持灭菌。 准备注射装置。 切割 ±30 厘米聚乙烯管(直径 0.28 毫米)。将 30 G x 1/2 针(针 A)的尖端?…

Representative Results

淋巴细胞缝合在以前的研究中已经使用15,16,17,19,作为一个重要的工具,研究淋巴流的功能之前,淋巴细胞的分子生物学被更好地理解。阻塞淋巴流中断LN平衡,这导致 HEV 失去最佳淋巴细胞宿主到 LN15、16、 17所需的关键基因表达。<sup…

Discussion

在健康和患病条件下,阻断淋巴流动在操纵抗原输送到LN方面有着广泛的应用。可以使用此方法控制抗原传递的时间,以研究连续淋巴流如何调节排空 LNs 中的免疫反应。这种淋巴流中断方法还可用于研究淋巴如何影响 LN 中的细胞分块、细胞激活、细胞迁移和细胞细胞相互作用。

专门表达人类白喉毒素受体(DTR)的小鼠在淋巴内皮细胞(Flt4-cre-dtr)中已经开发出来;这?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢艾娃·扎迪内扎德对手稿进行校对。这项工作得到了加拿大健康研究所(CIHR,PJT-156035)和加拿大SL创新基金会(32930)以及中国国家自然科学基金会(81901576)的支持。

Materials

0.9% Sodium Chloride Saline Baxter JB1323
100% ethanol Greenfield Global University of Calgary distribution services UN1170.
Depilatory cream Nair Nair Sensitive Formula Hair Removal Crème with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product.
Evans Blue dye Sigma Life Science E2129-10G For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots.
Fluorescein isothiocyanate isomer I (FITC) Sigma Life Science F7250-1G
Forceps Dumont #3 WPI 500337
Forceps Dumont #5 WPI 500233
Injection apparatus Connect one end of polyethylene tubing to 30G × ½ needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G × ½ needle. Insert the blunt end of the 30G × ½ needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 μl.
Insulin syringe Becton Dickinson and Company (BD) 329461
IRIS Forcep straight WPI 15914
IRIS scissors WPI 14218-G
Ketamine Narketan DIN 02374994 The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.
Needles (26Gx3/8) Becton Dickinson and Company (BD) 305110
Needles (30Gx1/2) Becton Dickinson and Company (BD) 305106
Paton Needle Holder ROBOZ RS6403 Straight, Without Lock; Serrated
Phosphate-Buffered Saline (PBS) Sigma Life Science P4417-100TAB
Polyethylene tubing Becton Dickinson and Company (BD) 427401
Surgical tape (1.25cmx9.1m ) Transpore 1527-0
Surgical tape (2.5cmx9.1m ) Transpore 1527-1
Suture Davis and Geck CYANAMID Canada 11/04 0.7 metric monofilament polypropylene
Syringe (1ml) Becton Dickinson and Company (BD) 309659
VANNAS scissors World Precision Instruments (WPI) 14122-G
Xylazine Rompun DIN02169606 The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.
Equipment
Dissecting microscope Olympus Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100
Confocal microscope Leica SP8

References

  1. Pflicke, H., Sixt, M. Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels. The Journal of Experimental Medicine. 206, 2925-2935 (2009).
  2. Schmid-Schonbein, G. W. Microlymphatics and lymph flow. Physiological Reviews. 70, 987-1028 (1990).
  3. Skalak, T. C., Schmid-Schonbein, G. W., Zweifach, B. W. New morphological evidence for a mechanism of lymph formation in skeletal muscle. Microvascular Research. 28, 95-112 (1984).
  4. Johnston, M. G., Hay, J. B., Movat, H. Z. Kinetics of prostaglandin production in various inflammatory lesions, measured in draining lymph. The American Journal of Pathology. 95, 225-238 (1979).
  5. Eisenhoffer, J., Yuan, Z. Y., Johnston, M. G. Evidence that the L-arginine pathway plays a role in the regulation of pumping activity in bovine mesenteric lymphatic vessels. Microvascular Research. 50, 249-259 (1995).
  6. Gasheva, O. Y., Zawieja, D. C., Gashev, A. A. Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct. Journal of Physiology. 575, 821-832 (2006).
  7. Breslin, J. W., et al. Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism. American Journal of Physiology- Heart and Circulatory Physiology. 293, 709-718 (2007).
  8. Randolph, G. J., Angeli, V., Swartz, M. A. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nature Reviews. Immunology. 5, 617-628 (2005).
  9. Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 427, 154-159 (2004).
  10. Gerner, M. Y., Casey, K. A., Kastenmuller, W., Germain, R. N. Dendritic cell and antigen dispersal landscapes regulate T cell immunity. The Journal of Experimental Medicine. 214, 3105-3122 (2017).
  11. Kastenmuller, W., Torabi-Parizi, P., Subramanian, N., Lammermann, T., Germain, R. N. A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread. Cell. 150, 1235-1248 (2012).
  12. Gerner, M. Y., Torabi-Parizi, P., Germain, R. N. Strategically localized dendritic cells promote rapid T cell responses to lymph-borne particulate antigens. Immunity. 42, 172-185 (2015).
  13. Moussion, C., Girard, J. P. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules. Nature. 479, 542-546 (2011).
  14. Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E., Shaw, S. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. The Journal of Experimental Medicine. 192, 1425-1440 (2000).
  15. Mebius, R. E., Breve, J., Duijvestijn, A. M., Kraal, G. The function of high endothelial venules in mouse lymph nodes stimulated by oxazolone. Immunology. 71, 423-427 (1990).
  16. Mebius, R. E., Streeter, P. R., Breve, J., Duijvestijn, A. M., Kraal, G. The influence of afferent lymphatic vessel interruption on vascular addressin expression. Journal of Cell Biology. 115, 85-95 (1991).
  17. Mebius, R. E., et al. Expression of GlyCAM-1, an endothelial ligand for L-selectin, is affected by afferent lymphatic flow. Journal of Immunology. 151, 6769-6776 (1993).
  18. Drayton, D. L., Liao, S., Mounzer, R. H., Ruddle, N. H. Lymphoid organ development: from ontogeny to neogenesis. Nature Immunology. 7, 344-353 (2006).
  19. Tomei, A. A., Siegert, S., Britschgi, M. R., Luther, S. A., Swartz, M. A. Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment. Journal of Immunology. 183, 4273-4283 (2009).
  20. Liao, S., Jones, D., Cheng, G., Padera, T. P. Method for the quantitative measurement of collecting lymphatic vessel contraction in mice. Journal of Biological Methods. 1, 6 (2014).
  21. Lin, Y., et al. Perinodal Adipose Tissue Participates in Immune Protection through a Lymphatic Vessel-Independent Route. Journal of Immunology. 201, 296-305 (2018).
  22. Gardenier, J. C., et al. Diphtheria toxin-mediated ablation of lymphatic endothelial cells results in progressive lymphedema. Journal of Clinical Investigation Insight. 1, 84095 (2016).

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Cite This Article
Lin, Y., Xue, J., Liao, S. Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice. J. Vis. Exp. (159), e61178, doi:10.3791/61178 (2020).

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