Human bronchial intraepithelial T cells produce interferon-gamma and stimulate epithelial cells

Abstract

Intraepithelial lymphocytes (IELs) can be identified among epithelial cells in systemic mucosal tissues. Although intestinal IELs play a crucial role in mucosal immunity, their bronchial counterparts have not been well studied. The purpose of this study was to determine the immunological functions of human bronchial IELs, which interact directly with epithelial cells, unlike lamina propria lymphocytes (LPLs). We isolated successfully bronchial IELs and LPLs using a magnetic cell separation system from the T cell suspensions extracted from bronchial specimens far from the tumours of resected lungs. Human bronchial IELs showed an apparent type 1 cytokine profile and proliferated more actively in response to CD2 signalling than did bronchial LPLs. CD8(+) IELs were identified as the most significant sources of interferon (IFN)-gamma. Human bronchial epithelial cells constitutively produced the T cell growth factors interleukin (IL)-7 and IL-15, and levels of those factors increased when cells were stimulated by IFN-gamma. Bronchial epithelial cells expressed cell surface proteins CD58 and E-cadherin, possibly enabling adhesion to IELs. In summary, human bronchial IELs have immunological functions distinct from bronchial LPLs and may interact with epithelial cells to maintain mucosal homeostasis.

Fig. 1

(a) Subsegmental bronchial wall section prior to removal of the epithelial layer. There are no indications of epithelial damage, inflammation and tumour invasion. (b) Subsegmental bronchial wall section after removal of epithelial layer. The basement membrane and lamina propria are maintained, and very few epithelial cells remain. Bronchi in (a) and (b) were obtained from one donor. Bars indicate 100 µm.

Fig. 2

CD103 expression in isolated human bronchial intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Freshly purified T cells were stained directly by fluorescein isothiocyanate (FITC)-labelled anti-human CD103 monoclonal antibody and expressions were determined by flow cytometry. The percentage of CD103⁺ cells increased in bronchial IELs compared with LPLs.

Fig. 3

Cytokine-secreting cells in human bronchial intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) examined by enzyme-linked immunospot assay. The number of spot-forming cells (SFCs) (open circles) of interferon (IFN)-γ (a), interleukin (IL)-4 (b), IL-2 (c), IL-10 (d) per 10⁴ of IELs and LPLs is shown. (e) The ratio of the number of IFN-γ SFCs to the number of IL-4 SFCs. Solid circles and bars indicate mean values ± standard error of the mean. Lines connect IEL and LPL values from the same donor. The number of IFN-γ-producing cells in bronchial IELs was significantly greater than in LPLs (a), and the IFN-γ SFCs/IL-4 SFCs ratio in IELs was also significantly greater than that in LPLs (e). *P < 0·05.

Fig. 4

Cytokine-secreting cells of human bronchial intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) purified to the CD4⁺ or CD8⁺ compartment. Shown are the number of spot-forming cells (SFCs) (open circles) of interferon (IFN)-γ (a), interleukin (IL)-4 (b), IL-2 (c), IL-10 and (d) per 10⁴ of CD4⁺ or CD8⁺ IELs and CD4⁺ or CD8⁺ LPLs, and the ratio of IFN-γ SFCs to IL-4 SFCs (e). Solid circles and bars indicate values ± standard error of the mean. Lines connect IEL and LPL values from the same donor. Larger numbers of CD8⁺ IELs produced IFN-γ compared with CD4⁺ IELs (a), and the ratio of IFN-γ SFCs to IL-4 SFCs was greater in CD8⁺ IELs than in CD4⁺ IELs (e). *P < 0·05.

Fig. 5

Interleukin (IL)-7 (a) and IL-15 (b) production from bronchial epithelial cell line/BEAS-2B cells treated with various concentrations of interferon (IFN)-γ, IL-4 or tumour necrosis factor (TNF)-α for 24 h; 0 indicates untreated cells assayed in the same manner. Shown is IL-7 (c) and IL-15 (d) production from BEAS-2B cells grown for 1, 6, 12 or 24 h without cytokine, with IFN-γ or with TNF-α. IFN-γ stimulates IL-7 and IL-15 production. *P < 0·05, different from untreated cells; **P < 0·05, different from untreated cells incubated for the same period.

Fig. 6

Interleukin (IL)-7 (a) and IL-15 (b) production from primary human bronchial epithelial cells (HBECs) grown in the presence of interferon (IFN)-γ IL-4 and tumour necrosis factor (TNF)-α for 24 h; 0 indicates untreated cells assayed in the same manner. IFN-γ stimulates production of both IL-7 and IL-15 in primary HBECs.

Fig. 7

Proliferation of human bronchial intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) by stimulation with monoclonal antibodies (mAbs) to T cell surface molecules. Stimulation index = [³H]-thymidine incorporation stimulated by mAb/[³H]-thymidine incorporation stimulated by control immunoglobulin G₁. IEL proliferation was increased by CD2 or CD3 stimulation, and additional CD28 stimulation increased proliferation further. Increased proliferation resulting from CD2 stimulation of IELs was significantly greater than that seen in LPLs and peripheral blood memory T cells (PB CD45⁺ T cells). **P < 0·05.

Fig. 8

Immunohistochemistry of a human bronchial specimen using mouse immunoglobulin G₁ (a), mouse anti-human CD58 monoclonal antibody (mAb) (b) and mouse anti-human E-cadherin mAb (c). Human bronchial epithelium was CD58- and E-cadherin-positive. Bars indicate 100 µm. CD58 and E-cadherin were also detected on bronchial epithelial cell line/BEAS-2B and primary human bronchial epithelial cells (HBECs) HBECs by Western blotting (d).