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Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
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1996 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 157, no 5, 1926-34 p.Article in journal (Refereed) Published
Abstract [en]

The functional properties of intraepithelial lymphocytes (IEL) in normal human jejunum, ileum, and colon were investigated. Cytokine mRNA expression in IEL and enterocytes was determined by reverse transcriptase-PCR and IFN-gamma+ IEL by immunohistochemistry. Polyclonal activators were used to study proliferation and IFN-gamma secretion of IEL, and an anti-CD3-mediated redirected cytotoxicity assay was used to determine the lytic potential of IEL. Freshly isolated IEL at all three gut levels expressed mRNA for IL-1 beta, IL-2, IL-8, IFN-gamma, and TNF-alpha. Approximately 10% of IEL produced IFN-gamma, suggesting that IEL are immunologically active in vivo, performing similar functions along the intestine. IEL could be stimulated further in vitro to express IL-10, TNF-beta, and TGF-beta 1, while no Th2-type cytokines were induced, suggesting suppressive and cytolytic functions for IEL. All three jejunal IEL subpopulations (CD4-CD8-TCR-gamma delta+, CD4+TCR-alpha beta+, CD8+TCR-alpha beta+) expressed the same four cytokines, IL-2, IL-8, IFN-gamma, and TNF-alpha, indicating that CD4+TCR-alpha beta+ IEL are Th1 cells and that TCR-gamma delta+ IEL and CD8+TCR-alpha beta+ IEL include cytotoxic effector cells. Indeed, freshly isolated jejunal IEL displayed cytolytic activity. IEL were induced to proliferation by anti-CD3/TCR complex mAbs and leukoagglutinin, but not by Con A. There was no correlation between the magnitude of the proliferative response and the amounts of secreted IFN-gamma. Enterocytes expressed IL-1 beta and IL-8, and sometimes TNF-alpha. Although jejunal enterocytes express HLA-DR and hsp60, Ag presentation by these cells may induce anergy since their cytokine profile is different from that of classical APCs.

Place, publisher, year, edition, pages
1996. Vol. 157, no 5, 1926-34 p.
National Category
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-67900PubMedID: 8757311OAI: oai:DiVA.org:umu-67900DiVA: diva2:614794
Available from: 2013-04-07 Created: 2013-04-07 Last updated: 2017-12-06
In thesis
1. Specific and nonspecific immune mechanisms in human gut: a comparative study of normal and ulcerative colitis intestine
Open this publication in new window or tab >>Specific and nonspecific immune mechanisms in human gut: a comparative study of normal and ulcerative colitis intestine
1996 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The intestine, with its large mucosal surface area, digests and absorbs food nutrients and maintains a beneficial microbial flora in the colon. Local protective immune responses against intestinal pathogens ensure the survival of the individual. These immune reactions are both specific and non-specific in nature. The intestinal epithelium is single-layered and constantly renewed with differentiating epithelial cells moving from the crypt to the luminal surface. Intraepithelial lymphocytes (IEL) are interspersed between the epithelial cells. Ulcerative colitis (UC) is a life-threatening chronic inflammation affecting colon.

In this study three molecules belonging to the carcinoembryonic antigen (CEA) family, namely CEA proper, nonspecific cross-reacting antigen 50/90 (NCA 50/90) and biliary glycoprotein (BGP), were found to be specifically localized to the apical surface of colonic epithelial cells. Full expression of these molecules occurs when the cells reach the upper 1/3 of the crypts and are maintained on the mature cells at the luminal surface. Ultrastructurally, CEA, NCA50/90 and BGP are localized to microvesicles and microfilaments of the fuzzy coat/glycocalyx as well as to the microvilli of the epithelial cells. Their unique localization and documented bacterial binding capacities suggest that they have a role in innate immunity.

Functional analysis of IEL in normal jejunum, ileum and colon revealed that IEL are in vivo activated T-lymphocytes expressing mRNA for the cytokines IL-1β, IL-2, IL-8, IFNγ and TNFα and that jejunal IEL have T-cell receptor (TCR)/CD3 dependent cytolytic capacity. As many as 10% of IEL actively produce IFNγ. CD4+TCRαβ+IEL, CD8+TCRαβ+IEL and CD4-CD8-TCRαβ+IEL all had a TH1/cytotoxic cytokine profile. IEL could be further stimulated in vitro to express IL-10, TNFβ and TGFβ1, to proliferate and to secrete IFNγ. Thus, active protection and/or regulation of the epithelium via cell-mediated immune reactions are prominent in the gut.

UC colon was characterized by a marked lymphocyte infiltration in the lamina propria, 10-50 times the normal level. Most lymphocytes were present in follicle-like cell aggregates containing both T- and B-cells. An unexpected finding was that γδT-cells constituted about 15% of the cells in the aggregates. Such cells are only found intraepithelially in normal gut. γδT-cells of both the intestinal- (TCR-Vδ1/Vγ8) and blood type (TCR-Vδ2/Vγ9) were seen. T-cells in UC colon were activated but nonproliferating and had a down-regulated TCR/CD3 complex. RT-PCR and quantitative immunohistochemistry for cytokine mRNA (n=11) and protein respectively, revealed that the T-cells in UC colon did not produce IL-2, in marked contrast to T-cells in normal colon and to ileal T-cells from UC patients. This was a selective defect since TNFα, IFNγ, IL-1β, IL-8 and TGFβ1 were similarly expressed in normal colon. No TH2 cytokines were seen. Lamina propria leukocytes in UC colon expressed IL-6, a cytokine not found in normal colon. The epithelial cells of UC colon were activated expressing MHC class II antigens, heat shock proteins and the co-stimulatory molecule B7.1/CD80.

Our study demonstrates that UC is an immunological disease. The immunopathological picture seen in UC colon probably reflects an inappropriate down-regulation of local immune responses perhaps due to a selective loss of the key cytokine IL-2 in a situation of extreme antigenic stress.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 1996. 95 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 485
Keyword
human intestine, ulcerative colitis, lymphoid aggregates, intraepithelial lymphocytes, lamina propria lymphocytes, γδT-cells, cytokines, IL-2, carcinoembryonic antigen, biliary glycoprotein, epithelial cells, glycocalyx
National Category
Cancer and Oncology Gastroenterology and Hepatology
Identifiers
urn:nbn:se:umu:diva-73387 (URN)91-7191-235-5 (ISBN)
Public defence
1996-11-08, Astrid Fagraeussalen (A103), Norrlands universitetssjukhus, Umeå, 10:00
Opponent
Supervisors
Available from: 2013-06-20 Created: 2013-06-20 Last updated: 2013-06-20Bibliographically approved
2. Human intestinal T lymphocytes: a comparative analysis of phenotype and function in normal and inflamed mucosa
Open this publication in new window or tab >>Human intestinal T lymphocytes: a comparative analysis of phenotype and function in normal and inflamed mucosa
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The epithelial lining of the gut must allow immediate contact with beneficial components as nutrients and normal microflora. At the same time it runs the constant risk of attack from pathogenic microbes and noxious agents. Only a singel layer of epithelial cells separates the body's largest lymphocyte population from foreign components in the gut lumen. I have addressed two fundamental questions: 1) How does the immune system in the gut specifically protect against infectious agents and other harmful substances and at the same time avoid overreaction against antigens in food and commensals? and 2) What causes the immune protection breakdown in inflammatory bowel disease? To this end, the phenotype, distribution and functions of T lymphocytes, key players in adaptive immunity, were compared to T lymphocytes in the chronically inflamed intestine of patients with ulcerative colitis (UC) and Crohn's disease (CD). Intestinal T lymphocytes are present both within the epithelium, intraepithelial lymphocytes (IEL), and in the underlying lamina propria (LP). Even though there are many T lymphocytes in normal intestine, there is a highly significant increase in inflamed intestine. They seem to be involved in the pathogenesis of both UC and CD. In UC, T lymphocytes populate the basal lymphoid aggregates, which occupy up to 45% of LP.

Phenotype was analysed in situ by immunomorphometry and immunoelecton microscopy, and by immunoflow cytometry of isolated IEL and lamina propria lymphocytes (LPL). Cytokine production was monitored as the frequencies of cells expressing the proteins in situ and as mRNA expression in purified T lymphocytes using quantitative RT-PCR. Cytotoxicity was measured in functional assays using purified IEL and LPL as effector cells and as expression of cytotoxic effector molecules. The responsiveness to polyclonal T cell activators was assayed as proliferation and secretion of cytokines in vitro.

Major findings were: (1) Interleukin-2 (IL-2), the Th1 cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), the down-regulatory cytokine transforming growth factor-β1 (TGF-β1) and the chemokine IL-8 are normally produced by IEL and LPL in both small and large intestine. Activation in vitro enhanced IFN-γ and TGF-β1 production and induced IL-10. These data indicate ongoing controlled cell mediated immune activity and readiness for both pro-inflammatory and immunosuppressive responses. (2) IEL and LPL of both small and large intestine exhibited a capacity to kill via the Fas/FasL pathway in a T cell antigen receptor (TCR)/CD3 independent manner. FasL expressing cells were present both intraepithelially and in LP and were most abundant in LP of colon, where they constitute 30% of the immune cells. These results suggest that local immune activity in the intestinal mucosa is controlled by activation induced cell death, AICD. Small intestinal mucosa harbours a small population of CD8+ αβ+T lymphocytes with cytolytic machinery that can be triggered through the TCR/CD3 complex. These T cells use the perforin/granzyme exocytosis pathway for killing. Such cells are not detected in colon. Most probably these cytotoxic T lymphocytes are "caught in the act" of eliminating virus infected epithelial cells. (3) Inflammation in colon leads to activation of γδ T cells. In UC, Vδ1+ γδ T cells, which normally reside within the epithelium, were numerous in LP. They constituted as much as 15% of the cells in the lymphoid aggregates and showed TCR-γδ internalisation and surface down-regulation, signs of receptor mediated activation. Thus, γδ T cells appear to be recruited from the epithelium in response to nominal antigens present in LP. (4) The cytokine profile of LP T cells was markedly distorted in UC patients and regulatory T cells were induced. IL-10 production was increased and expression levels correlated with disease activity while TGF-β1 levels were unchanged. CD4+ T cells were the major source of IL-10. Moreover, cells with the regulatory/suppressor phenotype CD4+CD28-TCR-αβ+ were frequent, particularly in the aggregates. IL-2 production was shut off and production of IFN-γ and TNF-α was decreased. However, we found no evidence for a corresponding induction of the Th2 cytokines. (5) There was a parallel accumulation of FasL expressing cells with retained capacity to kill via the Fas/FasL pathway and apoptosis resistant bcl-2 expressing lymphocytes in UC. Inflammation in ileum of CD patients led to an enhanced cytotoxicity. The frequency of perforin expressing LPL was increased and the capacity of LPL to execute TCR/CD3 mediated cytotoxicity via the perforin exocytosis pathway was elevated.

In summary, it appears that intestinal T lymphocytes are constantly involved in cell mediated immune activities and that cytotoxic memory CD8+ αβ T cells accumulate in the small intestinal mucosa. IEL and LPL seem capable of down-regulatory actions in the context of proinflammatory cytokines and the local homeostasis of immune reactions to luminal components seems to be regulated by IL-2 dependent AICD. The cytokine profile in UC suggests a generalised activation of regulatory T cells along the intestine. Inflammation in colon, but not in ileum, point to the importance of colonic microflora for precipitating the proinflammatory actions of IL-10.

 

 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2002. 70 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 769
Keyword
human intestinal mucosa, intraepithelial lymphocyte, lamina propria lymphocyte, γδ T cell, regulatory T cell, cytotoxicity, cytokine, basal lymphoid aggregate, ulcerative colitis, Crohn's disease
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-79076 (URN)91-7305-165-9 (ISBN)
Public defence
2002-01-19, Astrid Fagraeussalen (A103), Norrlands universitetssjukhus, Umeå, 10:00
Opponent
Available from: 2013-08-06 Created: 2013-08-06 Last updated: 2013-08-06Bibliographically approved

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