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Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
1992 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 152, no 2, 253-263 p.Article in journal (Refereed) Published
Abstract [en]

A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.

Place, publisher, year, edition, pages
1992. Vol. 152, no 2, 253-263 p.
National Category
Biochemistry and Molecular Biology Immunology
URN: urn:nbn:se:umu:diva-67890DOI: 10.1016/0022-1759(92)90147-LPubMedID: 1500733OAI: diva2:614770
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2013-08-08Bibliographically approved
In thesis
1. Human intraepithelial lymphocytes: a comparative study of phenotype, morphology, and functional properties of intraepithelial lymphocytes in gut and oral mucosa
Open this publication in new window or tab >>Human intraepithelial lymphocytes: a comparative study of phenotype, morphology, and functional properties of intraepithelial lymphocytes in gut and oral mucosa
1995 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human intraepithelial lymphocytes (IEL) constitute a unique cell population situated in the first line of defense of the alimentary tract. Here they are continuously exposed to a massive antigenic load of high complexity. However, different conditions prevail along the alimentary tract. In small intestine food antigens dominate whereas bacterial antigens are abundant in large intestine. The oral cavity is exposed to an enormous variety of antigens from the microflora as well as food constituents. The abundance and selective localization of lymphocytes in the surface epithelium of these challenged tissues implicate important roles for IEL in immune protection.

IEL in normal human jejunum, ileum and colon as well as in normal and chronically inflamed gingiva were studied in situ and after isolation, with regard to phenotype, ultrastructure, cytokine mRNA expression and response to T-cell mitogens. Furthermore, an isolation technique was developed which yielded highly purified, functionally active IEL and enterocytes from the same sample.

Intestinal IEL were situated in the basal part of the epithelium, often in small clusters and in close contact with adjacent lymphocytes and epithelial cells. They had an irregular shape with long processes and some had pseudopodium-like extensions penetrating the basement membrane. This indicates cell co-operation within the epithelium, as well as transmigration of IEL to underlying tissues. Freshly isolated IEL expressed several cytokines (IL-1β, IL-8, IL-2, TNF-α and IFN-γ) and in vitro activation induced expression of IL-2, IL-10, IFN-γ, TNF-α, TNF-β and TGF-β1, suggesting that IEL are involved in cell mediated cytotoxicity and suppressor cell activities.

γδ T cells showed preferential homing to the epithelium both in gingiva and in intestine. They constituted the major lymphocyte population in normal gingiva and on average 30% of IEL at all levels of the intestine. Gingival as well as intestinal γδ IEL showed preferential usage of Vδ1Vγ8, suggesting common reactivity patterns along the alimentary tract. Intestinal γδ IEL and γδ IEL in normal gingiva were CD4-CD8-. In contrast, γδ IEL in chronically inflamed gingiva were predominantly CD8+ and showed induced expression of CD45RO. This indicates that γδ IEL participate in anti-bacterial immune responses in mucosa. Intestinal and gingival γδ IEL displayed ultrastructural features of cytolytic effector cells, e.g. electron-dense cytoplasmic granules and multivesicular bodies. They also expressed cytokines indicative of cell mediated-cytolytic effector functions. γδ IEL from inflamed gingiva expressed IFN-γ, TNF-α, TGF-β1 and IL-6 mRNA while intestinal γδ IEL expressed IL-2, IFN-γ and TNF-α.

Intraepithelial αβ T cells were rare in gingiva while they constituted the major population of intestinal IEL. The phenotype of αβ IEL varied at different levels of the intestine. Thus, CD8+αβ IEL dominated in jejunum while cells with the unusual T-cell phenotype, CD4-CD8- TCR αβ+, constituted a major population of colonic IEL. CD4+ αβ IEL were equally represented, as a minor population, at all three levels of the gut. Intestinal αβ IEL had the same cytokine profile as γδ IEL. Taken together, these data suggest that αβ IEL are involved in immunoregulatory responses to luminal antigens.

IEL with thymocyte-like phenotyped (CD2+TCR/CD3-, CD1+TCR/CD3-, CD1+TCRαβ+ and CD1+TCRγδ+) were present in jejunal epithelium. Furthermore, recombination activating gene-1 (RAG-1) mRNA was expressed in CD2+TCR/CD3- and CD3+/TCR- jejunal IEL. RAG-1 was not expressed in colonic IEL. Thus, the epithelium of small intestine is a site for extrathymic T cell maturation in humans.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 1995. 71 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 50
human intraepithelial lymphocytes, alimentary tract, intestinal epithelium, gut luminal antigens, gingiva, γδ T cells, immunoelectron microscopy, cytokine mRNA, RT-PCR, extrathymic T cell maturation, RAG-1, periodontitis, mucosal immune system, CD4-CD8- αβ T cells, CD8+ γδ T cells
National Category
Immunology in the medical area
urn:nbn:se:umu:diva-79123 (URN)91-7174-997-7 (ISBN)
Public defence
1995-03-10, Major Groove, by 6L, Umeå universitet, Umeå, 09:00
Available from: 2013-08-08 Created: 2013-08-08 Last updated: 2013-08-08Bibliographically approved

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