Interferon gamma induces differential upregulation of alpha and beta chemokine secretion in colonic epithelial cell lines

Abstract

Background: Production of chemoattractant factors by the intestinal epithelium may contribute to mucosal infiltration by inflammatory cells in inflammatory bowel disease. Secretion of the alpha chemokine interleukin 8 (IL-8), a neutrophil chemoattractant, has been widely studied, but little is known about epithelial secretion of beta chemokines, which are preferentially involved in recruiting monocytes.

Aims: To investigate the profiles of alpha and beta chemokine secretion in colonic cell lines and their differential modulation by interferon gamma (IFN-gamma), a product of activated T lymphocytes and natural killer cells.

Methods and results: HT29-19A, a model of the CT secretory crypt cell, exhibited a parallel secretion of the alpha chemokines IL-8 and GRO alpha, which could be markedly upregulated by tumour necrosis factor alpha (TNF-alpha) and IL-1 beta. These cells showed no significant expression of the beta chemokines RANTES (regulated upon activation T cell expressed and secreted), MIP-1 alpha (macrophage inflammatory protein 1 alpha), and MCP-1 (monocyte chemotactic protein 1) under these conditions, but IFN-gamma in combination with TNF-alpha caused a dose dependent induction of RANTES and MCP-1 secretion. This was accompanied by a marked increase of RANTES mRNA. In contrast, IFN-gamma had no significant effect on TNF-alpha stimulated IL-8 secretion. Caco-2 cells, with features more typical of villus absorptive cells, were relatively poor secretors of alpha chemokines but secreted high levels of MCP-1 in response to IL-1 beta. IFN-gamma did not influence alpha or beta chemokine secretion in these cells.

Conclusions: These studies suggest that intestinal epithelial cells may produce chemokines capable of attracting both neutrophils and monocytes. The ability of IFN-gamma to activate the expression of beta chemokines preferentially could facilitate the development of chronic inflammatory infiltrates.

Figure 1

Secretion of α and β chemokines in (A) HT29-19A and (B) Caco-2 human colonic cell lines. Cultures were incubated for 24 hours with control media alone or in the presence of TNF-α (2 ng/ml in HT29-19A and 50 ng/ml in Caco-2), IL-1 (100 IU/ml), LPS (1 µg/ml), or LTA (1 µg/ml). Values are mean (SEM) of 3-6 observations in each group. *p<0.05; **p<0.01.

Figure 2

Dose dependent stimulation of α chemokine secretion by IL-1 in HT29-19A and Caco-2 cells. Chemokine levels were measured 24 hours after addition of IL-1 to confluent cultures. Values are mean (SEM) for 3-8 observations in each group.

Figure 3

Influence of IFN-γ on basal and TNF-α stimulated secretion of α and β chemokines in HT29-19A cells. Chemokines were measured 24 hours after incubation with control media or 50 ng/ml TNF-α in the presence or absence of 50 IU/ml IFN-γ. Results are mean (SEM) of four observations in each group.

Figure 4

Synergistic effect of TNF-α and IFN-γ on RANTES mRNA expression in the HT29-19A cell line. Cells were incubated with TNF-α alone (50 ng/ml), IFN-γ alone (50 IU/ml), or TNF-α + IFN-γ for 24 hours. The expression of RANTES specific product (243 bp) is shown after RT-PCR analysis of varying amounts (3-50 ng) of total RNA isolated from each treatment group. RT-PCR for actin using a single concentration (12 ng) confirmed the integrity of the RNA in each group.

Figure 5

Dose dependence of RANTES secretion in response to combinations of TNF-α and IFN-γ in the HT29-19A cell line. Cells were incubated with varying concentrations of TNF-α, either alone or in the presence of 10 or 50 U/ml IFN-γ. Results are mean (SEM) of four experiments. (Inset: IFN-γ dose response for RANTES secretion in the presence of 50 ng/ml TNF-α showing mean (SEM) for 6-8 observations in each group.)

Figure 6

Effect of TNF-α and IFN-γ on vectorial secretion of chemokines from polarised HT29-19A monolayers. Cells were grown to confluency on Transwell filter supports and the integrity of the resulting monolayer checked by monitoring development of transepithelial resistance. TNF-α (50 ng/ml), IFN-γ (50 IU/ml), or TNF-α + IFN-γ was added to the basolateral reservoir. The concentration of IL-8, MCP-1, and RANTES in the medium was measured in both apical and basolateral reservoirs after 24 hours. Values are mean of four monolayers in each group except IFN-γ alone on MCP-1 secretion where n=2.