Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

Abstract

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll-Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-gamma and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-gamma-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-gamma+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial-lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD-even in an inactive state of disease-exert an increased capacity for IFN-gamma induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-gamma-producing CD8+ lymphocytes.

Fig. 1

Influence of intestinal epithelial cells on intracellular IFN-γ and IL-4 expression in CD8⁺ and CD4⁺ lymphocyte populations. After 24 h co-incubation of 2 × 10⁵ peripheral blood mononuclear cells (PBMC) from the whole study group with 2 × 10⁶ Caco-2 cells, samples were prepared, stimulated and stained for intracellular FACS analysis. A significant increase of IFN-γ-producing CD8⁺ lymphocytes (+48%; *P = 0·025; n = 38) was observed by direct short-term co-culture with Caco-2 cells (▪, upper left graph, compared with PBMC monoculture, □ = 100%). A decrease of IL-4⁺/CD8⁺ lymphocytes was also measured in this set (−41%; *P = 0·05, upper right graph). A similar trend was found in Caco-2 transwell co-incubation experiments with an increase of IFN-γ⁺/CD8⁺ (+24%; P > 0·05) and a slight decrease of IL-4⁺/CD8⁺ (−15%; P > 0·05). Intracellular IFN-γ and IL-4 analysed in CD4⁺ lymphocytes showed no shifting after co-culture with Caco-2 cells.

Fig. 2

In contrast to healthy controls, cytotoxic lymphocytes of patients with inactive IBD were strongly inducible for IFN-γ by epithelial cell stimulus. After 24 h co-incubation of 2 × 10⁵ peripheral blood mononuclear cells (PBMC) with 2 × 10⁶ Caco-2 cells, samples were prepared, stimulated and stained for intracellular FACS analysis. Cytotoxic T cell subgroup analysis between healthy controls (□, n = 10) and IBD patients (ulcerative colitis (UC), ▪, n = 16; Crohn's disease (CD), hatched, n = 12) demonstrates differences in basal IFN-γ expression and an increase of IFN-γ-producing lymphocytes by intestinal epithelial cells (iEC) after 24 h co-culture. IFN-γ was strongly inducible by iEC in CD8⁺ lymphocytes in CD and UC patients, whereas controls only poorly responded to the epithelial stimulus. An increase of IFN-γ from 15·5% to 20·1% in transwell samples and a further increase up to 24·0% in direct PBMC/Caco-2 co-cultures (*P = 0·012) with patients suffering from CD were determined. UC patients showed a similarly strong induction of the intracellular IFN-γ signal in CD8⁺/IFN-γ⁺ lymphocytes in transwell and direct co-cultures (**P = 0·0058).

Fig. 3

IFN-γ induction of cytotoxic lymphocytes in ulcerative colitis (UC) patients by co-culture of peripheral blood mononuclear cells (PBMC) with autologous primary colonic crypt cells. After 24 h co-incubation of 2 × 10⁵ PBMC with 2 × 10⁶ primary colonic crypt cells in transwell and direct co-culture, samples were prepared, stimulated and stained for intracellular FACS analysis. A significant increase of IFN-γ⁺/CD8⁺ lymphocytes (+81% mean, *P = 0·014) was measured after direct co-incubation of peripheral mononuclear cells with primary cultures of colonic crypt cells in an autologous co-culture system in the UC patient group (n = 3). No significant induction was observed in the control group (n = 3) or in any transwell preparation.

Fig. 4

Interaction between intestinal epithelial cells (iEC) and CD8⁺ lymphocytes is MHC I-restricted. Interactions between peripheral CD8⁺ cytotoxic T cells and iEC were blocked by anti-MHC I antibody (mouse IgG1κ, clone G46-2.6 anti-human HLA A/B/C; PharMingen) in concentration ranges from 1 to 5 μg/ml. After 24 h of monoculture (▪) and direct co-culture (•) a down-regulation was observed of the intracellular IFN-γ signal in direct co-cultures incubated with anti-MHC I to values below those of basal expression of monoculture samples. In our co-culture system IFN-γ induction may therefore be partially transduced by MHC I molecules. Data represent results of four independent experiments with similar results.