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TNF-α Induces Vectorial Secretion of IL-8 in Caco-2 Cells

  • 2010 SSAT Plenary Presentation
  • Published:
Journal of Gastrointestinal Surgery Aims and scope

Abstract

Introduction

Intestinal epithelial cells represent an important component of innate immunity, with sophisticated responses to inflammatory stimuli. The manner in which intestinal epithelial cell polarity affects responses to inflammatory stimuli is largely unknown. We hypothesized that polarized intestinal epithelial cells exhibit a bidirectional inflammatory response dependent upon the location of the stimulus.

Methods

Caco-2 cells were grown on semi-permeable inserts in a dual-compartment culture system and treated with tumor necrosis factor-α (TNF-α; 100 ng/ml) or serum-free media in the apical or basolateral chamber. Interleukin-8 (IL-8) production in each chamber was measured by enzyme-linked immunosorbent assay. To determine receptor specificity, anti-TNF receptor antibodies were added to the apical or basolateral chamber.

Results

Basolateral stimulation with TNF-α resulted in increased apical and basolateral IL-8 production. Apical TNF-α stimulation resulted in increased apical, but not basolateral IL-8 production. Receptor blockade suggested TNF receptor 1 involvement on both apical and basolateral membranes, while TNF receptor 2 was only active on the apical membrane.

Conclusion

Polarized intestinal epithelial cells respond to TNF-α stimulation with focused, directional secretion of the proinflammatory cytokine IL-8. These findings are important because they suggest that intestinal epithelial cells are capable of organizing their response to inflammatory signals and producing inflammatory mediators in a bidirectional, vectorial fashion.

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Authors and Affiliations

  1. Department of Surgery, University of Cincinnati College of Medicine, Mail Location 0558, 231 Albert Sabin Way, Cincinnati, OH, 45267, USA

    Dennis I. Sonnier, Stephanie R. Bailey, Rebecca M. Schuster, Alex B. Lentsch & Timothy A. Pritts

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  1. Dennis I. Sonnier
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  2. Stephanie R. Bailey
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Correspondence to Dennis I. Sonnier.

Additional information

Discussant

Dr. Edward E. Whang (Boston, MA): Good job with the studies and very clear presentation. TNF-stimulated IL-8 secretion in this cell line is a well-known phenomenon. Your identification of directional asymmetry to this process is intriguing. I will ask you to address issues related to validity and biological significance of these findings.

First, you have suggested but not actually shown differential distribution of TNF receptor subtypes. Have you done these studies?

Second, Caco-2 cells are commonly used to model small intestinal epithelium, but they are colon cancer cells, after all. Are you aware whether normal small bowel enterocytes express TNF receptors and whether they respond to TNF by secreting IL-8? How would you validate that the directionality of TNF-stimulated IL-8 secretion you have reported today exists in normal intestine?

Finally, let us assume you were to figure out the detailed mechanisms responsible for this directional phenomenon. What would you do with that information? What would be the potential biological or clinical significance of this information?

Closing Discussant

Dr. Dennis Sonnier: Thank you very much for your comments and insightful questions. I think that looking at this in an ex vivo setting or in other cell lines would be helpful.

In addition, the IBD literature contains data regarding expression of various inflammatory markers in the stool, and they use this to track disease. This suggests that findings similar to ours make occur in the in vivo setting. IBD patients also show considerable responses to anti-TNF receptor therapy. The presence and function of these receptors on the apical membrane in tissue specimens and animal models is something else we plan on looking at as well.

What do we plan to do with this? Transferring this into a mouse model after we work out some of the cellular mechanisms will be going to be very important for studying various causes of intestinal inflammation as well as possible use in assessment of clinical severity of intestinal inflammation after trauma.

Regarding your questions about receptor expression, I agree that we have not demonstrated receptor expression during these studies. We have performed some initial confocal microscopy studies to help determine receptor expression.

Discussant

Dr. Michael Sarr (Rochester, MN): There are several other enterocyte-like cell models, such as RIE and IEC-6 cells. And they are more from younger animals. Have you thought about looking at those cell lines as well?

Closing Discussant

Dr. Dennis Sonnier: Thank you for your comments and questions. We have not yet looked at those specific cell lines, but these and other cell lines should be considered. We used Caco-2 cells in the current experiments because of their known ability to polarize when differentiated on Transwells.

Discussant

Dr. Michael Sarr (Rochester, MN): Why would a cell secrete something into the lumen when it is polarized. We have been struggling with that, looking at the effects of some hormones that are secreted into the lumen that have an effect. Why does this cell line secrete IL-8 into the lumen?

Closing Discussant

Dr. Dennis Sonnier: Dr. Sarr, that is a great question. By way of pure speculation, I think luminal secretion of cytokines may be a way of autocrine or paracrine control of inflammation. Upstream, intestinal epithelial cells can control a response downstream by the mediators that they secrete into the lumen in a way that cannot be achieved by secreting basolaterally into the bloodstream.

Additional effects of IL-8 specifically related to cell restitution or angiogenesis could also be important. In other words, mucosal healing, I think, could also be affected by mediators from cells upstream.

Discussant

Dr. Carlos Chan (Montreal, Canada): I have one quick question about the slide that you showed regarding LPS treatment on the apical side. As far as I understand, the Caco-2 cells do not express Toll-like receptor 4, which is the receptor for the LPS, although you show that CD14 is expressed. So it may not respond. So have you thought of using other cell lines that you have that expresses Toll-like receptor 4?

Closing Discussant

Dr. Dennis Sonnier: Thank you for your comments and questions. Some investigators have demonstrated TLR4 expression in Caco 2 cells, at least under some conditions, but they generally are hyporesponsive to LPS due to lack of MD2 and other proteins related to LPS signaling.

Discussant

Dr. Carlos Chan (Montreal, Canada): Actually, there are some papers that show there is no receptor and some papers show there is a receptor. Have you shown on your study that these cells that you have actually have Toll-like receptor 4?

Closing Discussant

Dr. Dennis Sonnier: I have not demonstrated that myself, but unpublished data from previous residents in our lab indicate that Caco-2 cells do respond to LPS stimulation if MD2 is added to the treatments.

I would like to thank the Society for the privilege of presenting our data.

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Sonnier, D.I., Bailey, S.R., Schuster, R.M. et al. TNF-α Induces Vectorial Secretion of IL-8 in Caco-2 Cells. J Gastrointest Surg 14, 1592–1599 (2010). https://doi.org/10.1007/s11605-010-1321-9

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