Proinflammatory cytokine synthesis by mucosal fibroblasts from mouse colitis is enhanced by interferon-gamma-mediated up-regulation of CD40 signalling

Abstract

Gut mesenchymal fibroblasts form complex phenotypical and functional populations. They participate actively in homeostatic maintenance of the extracellular matrix, epithelial barrier function, repair mechanisms and leucocyte migration. In inflammation, they become activated, change matrix expression and synthesize proinflammatory mediators. Subpopulations of mucosal fibroblasts express CD40 and the aim of this study was to define its role in their proinflammatory function. Stable primary fibroblast lines derived from normal mouse colon and inflamed colon from CD4(+) CD45RB(high)-transplanted SCID mice were used as models to explore the role of mucosal fibroblast CD40 in the inflammatory process. Phenotype correlated with in situ fibroblast phenotype in the tissues of origin. Lines from both sources co-expressed CD40 and Thy1.2 independently of alpha-smooth muscle actin. A subpopulation of CD40(+) fibroblasts from normal colon expressed CD40 at high levels and expression was enhanced by interferon (IFN)-gamma treatment, whereas all CD40(+) fibroblasts from colitis expressed at low levels and expression was unaffected by IFN-gamma treatment. Despite lower-level expression of CD40 by cells from colitis, they secreted constitutively interleukin (IL)-6 and C-C chemokine (CCL)2. Ligation of CD40 enhanced secretion of these mediators and induced secretion of CCL3. CD40 in cells from colitis was more responsive to ligation than CD40 on cells from normal tissue and this sensitivity was amplified selectively by the action of IFN-gamma. We conclude that the inflammatory milieu in colitis induces long-lasting changes in phenotype and proinflammatory function in colonic fibroblasts. In particular, proinflammatory signalling from fibroblast CD40 is amplified synergistically by the Th1 effector T cell cytokine, IFN-gamma.

Fig. 1

In situ CD40 expression by fibroblasts in normal and inflamed mouse colon. Frozen sections of non-transplanted SCID mouse colon (a), Balb/c mouse colon (b) and inflamed, T cell-transplanted SCID mouse colon (c) were stained for CD40 (rat anti-mouse CD40; donkey anti-rat biotin; streptavidin–Texas red) and type I collagen [rabbit anti-mouse type I collagen; goat anti-rabbit fluorescein isothiocyanate (FITC)]. Frequent double-stained (yellow, arrowheads) CD40⁺ putative fibroblasts are visible in the lamina propria of non-inflamed colons (a,b), but all type I collagen⁺ cells in inflamed colon (c) are CD40^–.

Fig. 2

Phenotype of mucosal fibroblast cell lines. Representative lines from normal colon growing in chamber slides were stained for α-smooth muscle actin (SMA) (red) and either Thy1 [(a) CD 90 or (b) CD40].

Fig. 3

CD40 expression by subpopulations of fibroblast lines from normal and inflamed mouse colon. Normal cells lines CFN3 (a), CFN4 (c) and inflamed lines CFI5 (b), CFI6 (d) were stained with hamster anti-mouse CD40 fluorescein isothiocyanate (FITC) [filled histogram, untreated; open histograms, treated with 200 U/ml interferon (IFN)-γ for 24 h], isotype control (dotted line), unstained control (dashed line).

Fig. 4

Forward- and side-scatter characteristics of fibroblast lines from normal and inflamed mouse colon. Normal [ (a) CFN3] and inflamed [ (b) CFI5] cell lines were removed at confluence from plates and analysed by flow cytometry for forward- and side-scatter characteristics. Gates 1 and 2 were set for major definable populations of normal cells and also applied to inflamed cells for analysis.

Fig. 5

Subpopulation analysis of CD40 expression on fibroblast lines from normal and inflamed mouse colon. Confluent cells were removed from plates and analysed by flow cytometry for CD40 expression using hamster anti-mouse CD40 fluorescein isothiocyanate (FITC) (filled histogram), isotype control (open histogram) or unstained (dotted line histogram). Major subpopulations are indicated by arrows. Results are representative of three separate experiments.

Fig. 6

Detailed CD40 expression by gated subpopulations of fibroblast lines from normal and inflamed mouse colon. Normal cells (a,b) and inflamed cells (c,d) were stained with hamster anti-mouse CD40 fluorescein isothiocyanate (FITC) (filled histogram), isotype control (open histogram) or unstained (dotted line histogram) and gated populations defined in Fig. 1[gate 1 (a,c), gate 2 (b,d)] were examined. Representative of three separate experiments.

Fig. 7

Effect of interferon (IFN)-γ treatment on CD40 expression by gated subpopulations of fibroblast lines from normal and inflamed mouse colon. Cells were grown to confluence and stimulated for 24 h with 0, 100 and 200 U/ml IFN-γ, released into suspension culture and stained for CD40 expression with hamster anti-mouse CD40 fluorescein isothiocyanate (FITC) (filled histogram, untreated; open histograms, treated with 100 or 200 U/ml IFN-γ for 24 h), isotype control (dashed line), or unstained (dotted line). Gated populations, as defined in Fig. 1, were examined for normal cells [gate 1 (a), gate 2 (b)] and inflamed cells [gate 1 (c), gate 2 (d)]. Results are representative of three separate experiments.

Fig. 8

Secretion of proinflammatory cytokines and chemokines by fibroblast lines from normal and inflamed mouse colon and effects of CD40 ligation, with and without pretreatment with interferon (IFN)-γ. Cells were grown to confluence and stimulated with (+, solid bars) or without (–, open bars) IFN-γ (200 U/ml) and with or without sCD40L (0; 0·1, 1·0 and 10 μg/ml) for 24 h. Secretion of interleukin (IL)-6, CCL2, 3 and 5 was measured simultaneously in cell supernatants by multiplex enzyme-linked immunosorbent assay. (a) Interleukin (IL)-6 synthesis by normal and inflamed fibroblasts; (b) CCL5 synthesis by normal and inflamed fibroblasts; (c) CCL2 synthesis by normal and inflamed fibroblasts; (d) CCL3 synthesis by normal and inflamed fibroblasts. Bars represent means ± s.d. of duplicate values from three separate experiments. *P < 0·05, **P < 0·01, ***P < 0·001; n.s., not significantly different.