Abstract
Dendritic cells are prime antigen presenting cells for stimulation of T cell immune responses. These cells are present in trace amounts in normal tissue. At sites of disease the increased frequency of these cells interacting with T cells may provide the basis for the release of pro-inflammatory mediators and contribute to localised cell and tissue damage. Studies on dendritic cells in the colon lamina propria of inflammatory bowel disease (IBD) mice have been limited due to the difficulties encountered in the isolation and purification of sufficient numbers of these cells. This is the first detailed, reproducible method provided in the literature for the isolation of colon lamina propria dendritic cells from mice with colitis, yielding optimum purity of cells and sufficient numbers to advance the study of dendritic cell function in the colons of mice. The most frequently used identification marker of murine DC is the CD11c surface antigen. We have adapted, combined, and improved procedures developed for the isolation of other cell types, to develop an efficient procedure for the isolation of dendritic cells from colon tissue. This protocol describes a step-by-step method for optimising the purity and recovery of lamina propria CD11c+ dendritic cells from mice colons.
Keywords: Collagenase digestion, Dendritic cell enrichment, Dendritic cell purification, Inflammatory bowel disease mice, Lamina propria dendritic cells
Full Text
The Full Text of this article is available as a PDF (350.5 KB).
Glossary
- DC
dendritic cells
- PBS
Dulbecco’s phosphate buffered saline
- FACS
fluorescent activated cell sorting
- IBD
inflammatory bowel disease
- LPMC
lamina propria mononuclear cells
- MACS
magnetic activated cell sorting
References
- Banchereau J., Steinman R.M. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
- Cebra J.J., Gearhart P.J., Kamat R., Robertson S.M., Tseng J. The secretory immunoglobulin A response in the gut. Biochem. Soc. Trans. (proc) 1977;5:1565–1569. doi: 10.1042/bst0051565. [DOI] [PubMed] [Google Scholar]
- Davies M.D., Parrott D.M. Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine. Gut. 1981;22:481–488. doi: 10.1136/gut.22.6.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dillon S.B., MacDonald T.T. Functional properties of lymphocytes isolated from murine small intestinal epithelium. Immunology. 1984;52:501–509. [PMC free article] [PubMed] [Google Scholar]
- Drakes M.L., Lu L., Subbotin V.M., Thomson A.W. In vivo administration of flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver. J. Immunol. 1997;159:4268–4278. [PubMed] [Google Scholar]
- Finkelman F.D., Lees A., Birnbaum R., Gause W.C., Morris S.C. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J. Immunol. 1996;157:1406–1414. [PubMed] [Google Scholar]
- Hart D.N. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood. 1997;90:3245–3287. [PubMed] [Google Scholar]
- Krajina T., Leithauser F., Moller P., Trobonjaca Z., Reimann J. Colonic lamina propria dendritic cells in mice with CD4+ T cell-induced colitis. Eur. J. Immunol. 2003;33:1073–1083. doi: 10.1002/eji.200323518. [DOI] [PubMed] [Google Scholar]
- Leithauser F., Trobonjaca Z., Moller P., Reimann J. Clustering of colonic lamina propria CD4(+) T cells to subepithelial dendritic cell aggregates precedes the development of colitis in a murine adoptive transfer model. Lab. Invest. 2001;81:1339–1349. doi: 10.1038/labinvest.3780348. [DOI] [PubMed] [Google Scholar]
- Liu Y.J. Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity. Cell. 2001;106:259–262. doi: 10.1016/s0092-8674(01)00456-1. [DOI] [PubMed] [Google Scholar]
- Morrissey P.J., Charrier K., Braddy S., Liggitt D., Watson J.D. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 1993;178:237–244. doi: 10.1084/jem.178.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Powrie F., Leach M.W., Mauze S., Caddle L.B., Coffman R.L. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int. Immunol. 1993;5:1461–1471. doi: 10.1093/intimm/5.11.1461. [DOI] [PubMed] [Google Scholar]
- Pillarisetty V.G., Shah A.B., Miller G., Bleier J.I., DeMatteo R.P. Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition. J. Immunol. 2004;172:1009–1017. doi: 10.4049/jimmunol.172.2.1009. [DOI] [PubMed] [Google Scholar]
- Rescigno M., Rotta G., Valzasina B., Ricciardi-Castagnoli P. Dendritic cells shuttle microbes across gut epithelial monolayers. Immunobiology. 2001;204:572–581. doi: 10.1078/0171-2985-00094. [DOI] [PubMed] [Google Scholar]
- Spencer D.M., Veldman G.M., Banerjee S., Willis J., Levine A.D. Distinct inflammatory mechanisms mediate early versus late colitis in mice. Gastroenterology. 2002;122:94–105. doi: 10.1053/gast.2002.30308. [DOI] [PubMed] [Google Scholar]
- Stagg A.J., Hart A.L., Knight S.C., Kamm M.A. The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria. Gut. 2003;52:1522–1529. doi: 10.1136/gut.52.10.1522. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tseng S. Expression of immunoglobulin isotypes by lymphoid cells of mouse intestinal lamina propria. Cell. Immunol. 1982;73:324–336. doi: 10.1016/0008-8749(82)90459-2. [DOI] [PubMed] [Google Scholar]
- Heijden P.J., Stok W. Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine. J. Immunol. Methods. 1987;103:161–167. doi: 10.1016/0022-1759(87)90285-7. [DOI] [PubMed] [Google Scholar]