Apc+/Min colonic epithelial cells express TNF receptors and ICAM-1 when they are co-cultured with large intestine intra-epithelial lymphocytes
Introduction
The identification of the adenomatous polyposis coli (APC) gene as the gene mutated in the germline DNA of patients affected by familial adenomatous polyposis (FAP) was a major event in understanding the pathogenesis of colo-rectal cancers [1], [2]. Mutations in the APC gene are detected in the majority of sporadic colon cancers [3] and are believed to occur early in tumorigenesis [4]. However, recent findings suggesting a role of APC mutation in the development of chromosomal instability may also implicate APC inactivation in the progression of colo-rectal adenoma [5], [6], [7]. The APC protein participates in a variety of cellular functions including proliferation, differentiation, apoptosis, adhesion, migration, and chromosomal segregation that may be critically involved in the tumorigenic process [8]. All of these functions are involved with the heterotypic cellular interactions occurring between intestinal epithelial cells (IECs) and intra-epithelial lymphocytes (IELs) [9], [10]. To our knowledge, the consequences of APC mutation on these crucial interactions within the intestinal epithelium have never been investigated. IEL are located at the basolateral surfaces of IEC and are primarily T cells with potent cytolytic and immunoregulatory capacities involved in sustaining epithelial integrity [9], [10]. In addition, the close interaction between IEC and IEL appears to be crucial for IEL survival [11] and control of their proliferation [12]. These interactions may be of interest in cancer prevention since recent data provided new support for lymphocyte mediated immunosurveillance of epithelial cancers [13], [14]. The present study attempts to determine if APC mutations per se induce changes in the cross-talk between IEC and IEL. We have recently established and characterized conditionally immortalized cell lines from the colon epithelium of C57BL/6J and C57BL/6J-Min/+ mice [15]. C57BL/6J-Min/+ mice (Min, multiple intestinal neoplasia) inherit a chain termination mutation in the Apc gene and develop intestinal tumors in the small and large intestine when they lose or inactivate the second wild-type allele or upon additional transforming mutations [16]. Apc+/+ and Apc+/Min colonocytes were co-cultivated with large-intestine IEL (LI-IEL). ICAM-1 expression in IEC and LFA-1 expression in LI-IEL were investigated since these molecules mediate IEL binding to colon cancer cells [17], which could have critical implications in the interactions between immune and tumor cells [18], [19].
Section snippets
IEC/IEL co-cultures
Apc+/+ and Apc+/Min colon epithelial cells were established in our laboratory, as described previously [15]. Briefly, a heterozygous female “Immortomouse” was mated with a heterozygous male Min/+ mouse or a male C57BL/6J mouse. The transgenic H-2Kb-tsA58 mouse [13] or “Immortomouse” (Charles River Laboratories, Wilmington, MA, USA) has a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) under the control of interferon γ which induces the H-2Kb promoter. The
IEC/LI-IEL co-culture slightly decreased the expression of LFA-1 at the surface of LI-IEL (Fig. 1)
A majority of freshly isolated LI-IEL (more than 80%) were LFA-1 positive. Co-cultures with Apc+/Min or Apc+/+ IEC sustained IEL survival (in the presence of z-VAD-fmk a broad spectrum caspase inhibitor added to the media) as assessed by viability (IP staining, data not shown). At the end of the 24 h co-culture LFA-1 expression was slightly decreased in both types of co-culture.
When co-cultured with LI-IEL, Apc+/Min IEC, but not Apc+/+IEC, expressed high levels of ICAM-1 (Fig. 2)
Both Apc+/Min and Apc+/+ IEC expressed low levels of the LFA-1 ligand ICAM-1 (Fig. 2, left). However, after 24 h
Discussion
The aim of this study was to document if LFA-1/ICAM-1 interactions were affected by the Min mutation in IEC when these cells were in contact with LI-IEL. LFA-1 was present at high levels on LI-IEL but we saw no change in its expression in LI-IEL co-cultivated with Apc+/Min IEC, when compared with co-cultures of Apc+/+ IEC. More interestingly, when co-cultured with LI-IEL, Apc+/Min IEC, but not Apc+/+ IEC, expressed high levels of ICAM-1, the ligand of LFA-1. This induction was specific for the
Acknowledgments
This work was supported by grants from the Association pour la Recherche sur le Cancer (Paris, France). V.F. is a recipient of a fellowship from the Association pour la Recherche sur le Cancer (Paris, France).
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Present address: UMR INRA ENVT 1089, Equipe Aliments et Cancer, 23 Chemin des Capelles, Toulouse 31076, France.