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Human intraepithelial lymphocytes: a comparative study of phenotype, morphology, and functional properties of intraepithelial lymphocytes in gut and oral mucosa
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
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.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 50
Keyword [en]
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
Identifiers
URN: urn:nbn:se:umu:diva-79123ISBN: 91-7174-997-7 (print)OAI: oai:DiVA.org:umu-79123DiVA: diva2:639603
Public defence
1995-03-10, Major Groove, by 6L, Umeå universitet, Umeå, 09:00
Opponent
Supervisors
Available from: 2013-08-08 Created: 2013-08-08 Last updated: 2013-08-08Bibliographically approved
List of papers
1. Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine
Open this publication in new window or tab >>Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine
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.

National Category
Biochemistry and Molecular Biology Immunology
Identifiers
urn:nbn:se:umu:diva-67890 (URN)10.1016/0022-1759(92)90147-L (DOI)1500733 (PubMedID)
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2017-12-06Bibliographically approved
2. Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium
Open this publication in new window or tab >>Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium
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1995 (English)In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 7, no 9, 1473-1487 p.Article in journal (Refereed) Published
Abstract [en]

The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and unusual subpopulations of IEL are present at different levels of human intestine. IEL phenotypes in normal jejunum, ileum and colon were compared using immunoflow cytometry and immunohistochemistry. The expression of mRNA for recombination-activating gene-1 (RAG-1) in IEL from all three levels was compared using reverse-transcription polymerase chain reaction, and the morphology of IEL in situ was determined using immunoelectron microscopy. Surface marker profiles of isolated intestinal epithelial cells at all three levels were also investigated. On average the proportion of TCR gamma delta IEL was comparable in jejunum than ileum and colon and varied in phenotype with gut level. CD4-CD8-TCR alpha beta IEL dominated in colon but were absent in jejunum. CD8+ TCR alpha beta IEL were present at all levels but only in jejunum did they constitute the majority of all IEL. CD4+ TCR alpha beta IEL were present in similar frequencies at all levels of the gut. In general, the majority of IEL had an activated phenotype (CD45RO+, alpha E beta 7+). Furthermore, IEL exhibited phenotypes which are rare in peripheral blood. The thymocyte markers CD1a and CD1c as well as the NK cell marker CD56 were expressed on a fraction of TCR alpha beta and TCR gamma delta IEL. A small population of 'null' cells (CD45+ TCR/CD#-CD20-CD14-CD15- cells) was also present at equal proportions along the gut. Jejunal but not colonic IEL expressed RAG-1 mRNA suggesting that extrathymic T cell maturation occurs in the epithelium of small intestine. RAG-1 was expressed in CD2+TCR/CD3- and CD3+/TCR-IEL. Ultrastructurally, IEL often formed small clusters and intimate contacts with epithelial cells, suggesting cell cooperation within the epithelium. Some IEL had pseudopodium-like extensions penetrating the epithelial basement membrane suggesting transmigration. Epithelial cells in small intestine but not colon expressed heat shock protein 60 and HLA-DR. CD1a, CD1b and CD1c were not expressed on intestinal epithelial cells at any level. The distinct surface marker profiles of IEL and epithelial cells along small and large intestine suggest functional regional specialization and are compatible with the hypothesis that TCR alpha beta IEL participate in immune reactions to lumenal antigens while TCR gamma delta IEL perform surveillance of the epithelium.

National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-67898 (URN)10.1093/intimm/7.9.1473 (DOI)7495755 (PubMedID)
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2017-12-06Bibliographically approved
3. Cytokine profile and ultrastructure of intraepithelial lymphocytes in human gut suggest that they are activated cells with T helper 1 and cytotoxic functions
Open this publication in new window or tab >>Cytokine profile and ultrastructure of intraepithelial lymphocytes in human gut suggest that they are activated cells with T helper 1 and cytotoxic functions
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-79122 (URN)
Available from: 2013-08-08 Created: 2013-08-08 Last updated: 2013-08-08Bibliographically approved
4. T-cell receptor gamma delta-expressing intraepithelial lymphocytes are present in normal and chronically inflamed human gingiva
Open this publication in new window or tab >>T-cell receptor gamma delta-expressing intraepithelial lymphocytes are present in normal and chronically inflamed human gingiva
1993 (English)In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 79, no 1, 38-45 p.Article in journal (Refereed) Published
Abstract [en]

The phenotypic profile of leucocytes in diseased and normal gingival tissue was studied in situ and in isolated gingival mononuclear cell (GMC) preparations. T-cell receptor (TcR)gamma delta + cells showed preferential localization to epithelium, both in normal and inflamed gingiva, and were present in crevicular as well as oral epithelium. In normal gingiva > or = 30% of the isolated leucocytes expressed TcR gamma delta, of which the majority were CD4- CD8-, and expressed CD45RA. The proportion of TcR gamma delta + cells in GMC from periodontitis tissue varied between 2 and 32%. In contrast to normal gingiva the majority of TcR gamma delta + cells in diseased tissue were CD8+ and expressed CD45RO. Thus expression of the CD8 antigen on gingival TcR gamma delta + cells is probably a consequence of immune activation. Numerous Langerhans' cells and keratinocytes expressing the major histocompatibility complex (MHC) class I-like antigen, CD1, were present within normal and inflamed gingival epithelium in close proximity to the TcR gamma delta + cells. Most CD1a+ cells were scattered within oral epithelium. CD1c+ cells were localized close to the basal layer of crevicular epithelium. No CD1b+ cells were found. TcR alpha beta + cells, CD4+ and B cells were restricted to lamina propria of periodontitis lesions. The presence of intraepithelial TcR gamma delta + cells in normal gingiva suggests that they constitute the 'first line of defence' against the potentially harmful microflora in the oral cavity. Induction of CD8 and CD45RO antigens on TcR gamma delta + cells in periodontitis tissue indicate that they play a significant role in the disease. CD1 molecules on Langerhans' cells and keratinocytes may be the restriction elements for the CD8+ TcR gamma delta + cells.

National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-67893 (URN)7685315 (PubMedID)
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2017-12-06Bibliographically approved
5. Cytokine profile and ultrastructure of intraepithelial gamma delta T cells in chronically inflamed human gingiva suggest a cytotoxic effector function
Open this publication in new window or tab >>Cytokine profile and ultrastructure of intraepithelial gamma delta T cells in chronically inflamed human gingiva suggest a cytotoxic effector function
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1994 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 153, no 5, 2302-2312 p.Article in journal (Refereed) Published
Abstract [en]

We have shown that gamma delta T cells in human gingiva have an intraepithelial location and, that in the chronic inflammatory disease periodontitis, the expression of CD45RO and CD8 or CD4 is induced on gamma delta T cells. To study the role of gamma delta T cells in local antibacterial responses, we determined the cytokine profiles of isolated human gingival cells. Different T cell subpopulations, isolated by positive selection with mAb-coated magnetic beads and macrophages, as well as epithelial cells, were analyzed for expression of mRNA for 15 cytokines by reverse transcriptase-PCR. The ultrastructure of gingival gamma delta T cells was also studied. The gamma delta T cells expressed mRNA for IFN-gamma, TNF-alpha, TGF-beta 1, and IL-6. Expression of IFN-gamma was a consequence of inflammation. CD4+ gamma delta T cells expressed IFN-gamma only, whereas CD8+ gamma delta T cells expressed all four cytokines. CD8+ cells expressing IFN-gamma, TNF-alpha, and IL-6 in combination suggest a cytotoxic effector function. Gingival gamma delta T cells contained cytoplasmic electron-dense membrane-bound granules and multivesicular bodies that are ultrastructural characteristics of cytotoxic cells. Epithelial cells from inflamed gingiva expressed HLA-DR, CD1a, CD1c, and heat shock protein 60 on the cell surface. They also expressed mRNA for IL-1 beta, IL-6, IL-8, TNF-alpha, and TGF-beta 1. Thus, epithelial cells may function as accessory cells in immune activation and, at the same time, be target cells for CD8+ gamma delta T cells reactive with CD1 Ag or heat shock protein. These results suggest that gamma delta T cells constitute a first line of defense in gingiva, preventing entrance of pathogens by cytotoxicity against infected and stressed epithelial cells, and by control of epithelial cell growth through secretion of regulatory cytokines.

National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-67897 (URN)8051426 (PubMedID)
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2017-12-06Bibliographically approved

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