Abstract
Dendritic cells (DCs) migrate constitutively from the intestine via the lymph to the mesenteric lymph nodes. These migrating intestinal lymph DCs (ilDCs) carry antigens acquired in the intestine and play important roles in both the initiation of immune responses and the maintenance of oral tolerance. The ilDC population is made up of at least three functionally different DC subsets. Like many DC populations, ilDCs are exquisitely sensitive to their environment, changing their phenotype and maturing in response to the procedures associated with their extraction from solid tissues. We have developed and refined a method for collecting and purifying these DC subsets from rats, without inducing them to mature. This method involves two separate surgical procedures, separated by at least 6 weeks. Initially, mesenteric lymph nodes are removed. After the animals have fully recovered we cannulate the thoracic duct and collect the ilDCs on ice, minutes after they have left the lymph vessel. The DCs are then enriched using magnetic beads and purified by flow cytometric sorting. We describe this method here, including our recent refinements to limit the use of the restraining “Bollman” cage.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jiang A, Bloom O, Ono S, et al. Disruption of E-cadherin-mediated adhesion induces a functionally distinct pathway of dendritic cell maturation. Immunity (2007); 27:610–24.
Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature (2003); 425:516–21.
Worbs T, Bode U, Yan S, et al. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J Exp Med (2006); 519–27.
Itano AA, McSorley SJ, Reinhardt RL, et al. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity (2003); 19:47–57.
Milling SW, Yrlid U, Jenkins C, Richards CM, Williams NA, MacPherson G. Regulation of intestinal immunity: effects of the oral adjuvant Escherichia coli heat-labile enterotoxin on migrating dendritic cells. Eur J Immunol (2007); 37:87–99.
Pugh CW, MacPherson GG, Steer HW. Characterization of nonlymphoid cells derived from rat peripheral lymph. J Exp Med (1983); 157:1758–79.
Yoffey JM, Courtice C. Lymphatics, lymph and the lymphomyeloid complex. London, New York: Academic Press; 1970; 548.
Smith IB, McIntosh GH, and Morris B. The traffic of cells through tissues: a study of peripheral lymph in sheep. J Anat (1970); 107:87–100.
Bollman JL, Cain JC, Grindley JH. Techniques for the collection of lymph from the liver, small intestine or thoracic duct of the rat. J Lab Clin Med (1948); 33:1349.
Milling SWF, Jenkins C, MacPherson G. Collection of lymph-borne dendritic cells in the rat. Nature Protocols (2006); 1:1–8.
Zhou Q, Renard JP, Le Friec G, et al. Generation of fertile cloned rats by regulating oocyte activation. Science (2003); 302:1179.
Ionac M, Laskay T, Labahn D, Geisslinger G, Solbach W. Improved technique for cannulation of the murine thoracic duct: a valuable tool for the dissection of immune responses. J Immunol Methods (1997); 202:35–40.
Pham TH, Okada T, Matloubian M, Lo CG, Cyster JG. S1P1 receptor signaling overrides retention mediated by G alpha i-coupled receptors to promote T cell egress. Immunity (2008); 28:122–33.
Massberg S, Schaerli P, Knezevic-Maramica I, et al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell (2007); 131:994–1008.
Rhodes JM. Isolation of large mononuclear Ia-positive veiled cells from the mouse thoracic duct. J Immunol Methods (1985); 85:383–92.
Rhodes JM, Agger R. Comparison of membrane antigens of mouse dendritic cell types. Immunol Lett (1987); 16:107–12.
Rhodes JM, Balfour BM, Blom J, Agger R. Comparison of antigen uptake by peritoneal macrophages and veiled cells from the thoracic duct using isotope-, FITC-, or gold-labelled antigen. Immunology (1989); 68:403–9.
Turnbull EL, Yrlid U, Jenkins CD, Macpherson GG. Intestinal Dendritic Cell Subsets: Differential Effects of Systemic TLR4 Stimulation on Migratory Fate and Activation In Vivo. J Immunol (2005); 174:1374–84.
Yrlid U, Milling SWF, Miller JL, Cartland S, Jenkins CD, Macpherson GG. Regulation of intestinal dendritic cell migration and activation by plasmacytoid dendritic cells, TNF-a and Type 1 IFNs after feeding a TLR7/8 ligand. J Immunol (2006); 176:5205–12.
Huang FP, Platt N, Wykes M, et al. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med (2000); 191:435–44.
Cousins L, Graham M, Tooze R, et al. Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene. Gastroenterology (2006); 131:1475–85.
Tilney NL. Patterns of lymphatic drainage in the adult laboratory rat. J Anat (1971); 109:369–83.
Acknowledgements
The authors would like to thank Chris Jenkins for helping to develop these methods. The work was supported by BBSRC Grant BB/D012643/1.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Milling, S., MacPherson, G. (2010). Isolation of Rat Intestinal Lymph DC. In: Naik, S. (eds) Dendritic Cell Protocols. Methods in Molecular Biology, vol 595. Humana Press. https://doi.org/10.1007/978-1-60761-421-0_19
Download citation
DOI: https://doi.org/10.1007/978-1-60761-421-0_19
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-420-3
Online ISBN: 978-1-60761-421-0
eBook Packages: Springer Protocols