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. 2009 Nov;128(3):351-9.
doi: 10.1111/j.1365-2567.2009.03110.x.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

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Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Christel Zufferey et al. Immunology. 2009 Nov.

Abstract

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

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Figures

Figure 1
Figure 1
Schematic representation of the epithelial cell–intraepithelial lymphocytes (IEL) coculture system and formation of transepithelial resistance (TER) by the mouse intestinal crypt-like cell line mlCcl2. (a) Epithelial monolayers were grown on polycarbonate filters for 3 days. At this time, IEL were added to the apical side for subsequent cocultures. Monolayer integrity was assessed by measurement of TER in the absence or presence of IEL. (b) Formation of stable monolayer integrity by mlCcl2 but not by the fibroblast cell line L929 as assessed by measurement of TER. Mean of n = 3 cocultures ± SEM is shown.
Figure 2
Figure 2
Recombinant interferon-γ (IFN-γ) or activated intraepithelial lymphocytes (IEL) reduce the epithelial monolayer integrity. mICcl2 cells were cocultured with IEL in the presence or absence of monoclonal antibodies against CD3/CD28 or treated with recombinant IFN-γ or tumour necrosis factor-α (TNF-α). Transepithelial resistance (TER) was measured after 72 hr of culture with IEL or recombinant cytokines and is expressed as the percentage of TER of mICcl2 in the respective control cell culture medium. Mean values ± SEM of n = 10 to n = 13 cocultures with IEL and n = 5 cocultures with recombinant cytokines from a total of five independent experiments are shown. ***P < 0·001.
Figure 3
Figure 3
Activated T-cell receptor (TCR)-αβ CD8αβ T cells, but not activated TCR-αβ CD8αα intraepithelial lymphocytes (IEL) are responsible for the reduction of transepithelial resistance (TER) in mICcl2. (a) Purified CD8αα and CD8αβ TCR-αβ IEL or splenic CD8 T cells were cocultured with mICcl2 cells in the absence or presence of monoclonal antibodies against CD3/CD28. (b) Purified T-cell subsets derived from P14 TCR transgenic mice (line 318) were cultured with mICcl2 in the presence of their specific peptide gp33 or the irrelevant control peptide adn5, respectively. (a, b) TER was assessed after 72 hr of coculture and is expressed as the percentage of TER of mICcl2 in the respective control cell culture medium. Mean values ± SEM of n = 5 to n = 7 cocultures with IFN-γ, CD3/CD28 or peptides alone, n = 6 to n = 7 cocultures with CD8αβ splenic T cells or CD8αβ IEL, and n = 2 to n = 4 cocultures with CD8αα IEL from a total of three (a) and two (b) independent experiments are shown. ***P < 0·001; NS, non-significant.
Figure 5
Figure 5
Activated T-cell receptor (TCR)-αβ CD8αβ intraepithelial lymphocytes (IEL) increase fluorescein isothiocyanate (FITC)-dextran flux across mICcl2 monolayers in an interferon-γ (IFN-γ)-dependent manner. mICcl2 were cocultured with purified IEL subsets derived from either wild-type (B6) mice (a) or IFN-γ−/− mice (b) in the absence or presence of monoclonal antibodies against CD3/CD28. After 72 hr, FITC-dextran was added to the apical side of the mICcl2 monolayer and paracellular permeability was assessed. FITC-dextran flux is expressed as the fold increase of permeability of mICcl2 in control cell culture medium. Mean values ± SEM of n = 6 to n = 7 cocultures with IFN-γ or CD3/CD28, n = 6 cocultures with CD8αβ IEL, and n = 4 cocultures with CD8αα IEL from a total of three individual experiments are shown. **P < 0·01; ***P < 0·001.
Figure 4
Figure 4
Disruption of epithelial monolayer integrity by activated T-cell receptor (TCR)-αβ CD8αβ strictly and exclusively depends on their secretion of interferon-γ (IFN-γ). (a) mICcl2 cells were cocultured with sorted intraepithelial lymphocyte (IEL) subsets from IFN-γ−/− or wild-type (B6) mice in the presence or absence of monoclonal antibodies against CD3/CD28. Mean values ± SEM of n = 6 cocultures with IFN-γ, n = 6 cocultures with CD8αβ IEL, and n = 4 cocultures with CD8αα IEL from a total of three independent experiments are shown. (b) Experiments were performed as described in (a) but using CMT93 cells (a mouse colon cancer cell line) and unfractionated IEL from wild-type (B6) mice. Bars represent mean values ± SEM of n = 5 cocultures with IFN-γ, n = 4 cocultures with IFN-γ−/− CD8αβ IEL, n = 6 cocultures with B6 IEL, and n = 4 cocultures with CD8αα IEL from a total of two independent experiments. **P < 0·01; ***P < 0·001.

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