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. 1998 Feb;42(2):208–213. doi: 10.1136/gut.42.2.208

Interferon γ induces differential upregulation of α and β chemokine secretion in colonic epithelial cell lines

A Warhurst 1, S Hopkins 1, G Warhurst 1
PMCID: PMC1726988  PMID: 9536945

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 α chemokine interleukin 8 (IL-8), a neutrophil chemoattractant, has been widely studied, but little is known about epithelial secretion of β chemokines, which are preferentially involved in recruiting monocytes. 
Aims—To investigate the profiles of α and β chemokine secretion in colonic cell lines and their differential modulation by interferon γ (IFN-γ), a product of activated T lymphocytes and natural killer cells. 
Methods and results—HT29-19A, a model of the Cl secretory crypt cell, exhibited a parallel secretion of the α chemokines IL-8 and GROα, which could be markedly upregulated by tumour necrosis factor α (TNF-α) and IL-1β. These cells showed no significant expression of the β chemokines RANTES (regulated upon activation T cell expressed and secreted), MIP-1α (macrophage inflammatory protein 1α), and MCP-1 (monocyte chemotactic protein 1) under these conditions, but IFN-γ in combination with TNF-α caused a dose dependent induction of RANTES and MCP-1 secretion. This was accompanied by a marked increase of RANTES mRNA. In contrast, IFN-γ had no significant effect on TNF-α stimulated IL-8 secretion. Caco-2 cells, with features more typical of villus absorptive cells, were relatively poor secretors of α chemokines but secreted high levels of MCP-1 in response to IL-1β. IFN-γ did not influence α or β 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-γ to activate the expression of β chemokines preferentially could facilitate the development of chronic inflammatory infiltrates. 



Keywords: inflammatory bowel disease; RANTES; interferon gamma; chemokine

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Figure 1 .

Figure 1

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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 .

Figure 2

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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 .

Figure 3

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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 .

Figure 4

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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 .

Figure 5

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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 .

Figure 6

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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. 


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