The effect of immune cells and their products on the growth and differentiation of small intestinal epithelial cells in vitro

Hutton, Angela Kimberley (1994) The effect of immune cells and their products on the growth and differentiation of small intestinal epithelial cells in vitro. PhD thesis, University of Glasgow.

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Abstract

Crypt hyperplasia in the small intestine is one of the early features of a group of enteropathies which include coeliac disease and it is thought that this phenomenon may be caused by cytokines produced during a DTH response. The work in this thesis was aimed at developing in vitro models to test the hypothesis that cytokines act directly on dividing crypt epithelial cells to produce crypt hyperplasia. The RIE-1 cell line, derived from rat small intestine, appears to represent immature crypt-like cells and thus I considered it to be a suitable model for my studies. Initially, I investigated the effects of T lymphocytes on RIE-1 cell growth. ConA stimulated MEN cells and their supernatants decreased the growth of the RIE-1 cells, and the cytostatic effect of the latter was partially reversed by an anti-IENy monoclonal antibody. Macrophages also had a cytostatic effect on RIE-1 cells, which could be further enhanced by stimulation of the macrophages with IFNy. Inhibiting prostaglandin and NO production had no major effects on the growth inhibition caused by macrophages. EPS stimulated macrophages produced TNFa, and the cytostatic effects of these cells were partly inhibited by antibody against this cytokine. Macrophage supernatants were also cytostatic to RIE-1 cells and, in general, the effects of supernatants from macrophages activated by the different agents correlated with those of macrophages themselves. To examine the role of particular mediators, I next examined the effects of individual cytokines on the growth of RIE-1 cells. lENy, TGFp and TNFa were cytostatic to exponentially growing cultures of RIE-1 cells, but only IFNY was cytostatic to confluent cultures of cells. lEl and TNFa had a proliferative effect on confluent cultures of RIE-1 cells. A number of other cytokines had no effect. iii In an attempt to produce a more accurate model, I co-cultured RIE-1 cells with a fibroblast monolayer. RIE-1 cells in co-culture formed structures several layers thick and a small proportion took on a columnar appearance and had increased numbers of cytoplasmic organelles. However, there were no other obvious features of differentiation and as I was unable to measure the growth of RIE-1 cells quantitatively in this model, I decided it was unsuitable for further use. Finally, I attempted to establish primary cultures of small intestinal epithelial cells. Intestinal epithelial cells isolated from neonatal rats grew in culture and were shown to express digestive enzymes and cytokeratins. EM also showed the presence of columnar epithelial cells with tight junctions and desmosomes and a brush border of microvilh. However, the cultures were contaminated by the presence of non-epithelial cell types making them unsuitable for use in my studies. The overall conclusion of this thesis is that the RIE-1 cell line provides a useful model to study the effects of mediators on the growth of small intestinal epithelial crypt cells in vitro but is unsuitable for differentiation studies. Cytokines derived from T lymphocytes and macrophages did have effects on RIE-1 cells, but these were mainly cytostatic, suggesting that cytokines may not produce crypt hyperplasia via direct action on crypt stem cells in enteropathy as has been hypothesised. If such a stimulatory effect does occur in vivo, I would propose that this occurs via an indirect effect of cytokines on for example mesenchymal cells. One cytokine which may explain this activity is ILl, which is produced by fibroblasts and was stimulatory to RIE-1 cells. My results indicate that it may contribute to hyperplasia by recruiting nondividing crypt epithelial cells back into the cell cycle. Finally, my results underline the difficulty in examining enterocyte growth and iv differentiation in vitro and indicate that there is as yet no perfect model for this important phenomenon.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Allan Mowat
Keywords: Immunology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-73820
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73820

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