Elsevier

Experimental Cell Research

Volume 312, Issue 19, 15 November 2006, Pages 3909-3919
Experimental Cell Research

Research Article
Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha

https://doi.org/10.1016/j.yexcr.2006.08.018Get rights and content

Abstract

Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-α antibody and etanercept, strongly suggesting that TNF-α secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-κB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.

Introduction

The mucosal surface of the gastrointestinal tract is lined by a monolayer of intestinal epithelial cells joined together at their apical poles by tight junctions, thus forming a barrier that separates the luminal contents from the effector immune cells underneath. It has become increasingly clear that the intense immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells [1]. Intestinal epithelial cells are understood to be regulated by immune cells beneath the intestinal epithelial monolayer via soluble factors. Previous studies have suggested that intestinal intraepithelial lymphocytes (IELs) are effector cells capable of secreting cytokines in response to stimulation [2]. These cytokines secreted by IELs include tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL)-1β, IL-2, IL-4 and IL-5, and it has been suggested that IFN-γ and TNF-α can act directly on intestinal epithelial cells to mediate changes in the epithelial permeability and the capacity for electrogenic ion transport [3], [4], [5], [6], [7]. As well as IELs, macrophages and dendritic cells (DC) also affect the intestinal epithelial cells via such soluble factors as cytokines. Any abnormal activation of such immune cells as macrophages overproduces the inflammatory cytokines that cause destructive cell damage to the intestinal epithelial monolayers and subsequent mucosal inflammation; for example, inflammatory bowel disease (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) [8]. However, the occurrence of an abnormal balance via inflammatory cytokines remains unknown.

The analysis of intestinal inflammation has been mainly performed by using animal models. Several experimental models of colitis induced either by the administration of dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) have mostly been used for the analysis of intestinal inflammation [9], [10], [11], [12], [13], [14]. It has also recently been reported that the effect of several cytokines on knockout or transgenic mice such as the IL-2 knockout mouse [15], IL-10 knockout mouse [16], [17], IL-7 transgenic mouse [18] and IL-15 transgenic mouse [19] showed itself as colitis. These mutant mice have also been utilized as the experimental models for colitis to examine or search for anti-inflammatory drugs or food factors [20]. However, details of the regulatory mechanism of these drugs or food factors have not been revealed, especially at the cellular level.

We therefore constructed an in vitro artificial model of intestinal inflammation by using a coculture between human intestinal epithelial-like cells and macrophages, especially activated macrophage-like cells. This coculture system was used to examine the interaction between different types of cells via soluble factors. Previous studies using a coculture system have reported the interaction between neurons and astrocytes [21], endothelial cells and astrocytes, which has been developed as a blood–brain barrier model [22], and intestinal epithelial cells and immune cells [23]. We have previously reported the interaction between Caco-2 cells as a model of human intestinal epithelial cells and PC12 cells as a model of neuronal cells [24], [25]. We have also reported preliminary results on the interaction between intestinal epithelial cells and immune cells [26].

We further characterize in more detail in the present study the cell-to-cell interaction between intestinal epithelial cells and activated macrophages. We also examine whether the in vitro coculture system could be utilized for an assay to search for drugs or food substances, which could prevent intestinal inflammation. The human colonic adenocarcinoma Caco-2 cell line was used as a model for IEC, and the human acute monocytic leukemia THP-1 cell line was differentiated to activated macrophages by a PMA treatment and used as a model for activated macrophages.

Section snippets

Materials

The Caco-2 cell line was obtained from American Type Culture Collection (Rockville, MD, USA), and THP-1 cells were purchased from Health Science Research Resources Bank (Osaka, Japan). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceuticals (Tokyo, Japan), and fetal calf serum (FCS) was from Sigma (St. Louis, MO, USA). Non-essential amino acids were purchased from Cosmobio (Tokyo, Japan). Recombinant human TNF-α, recombinant human IFN-γ, recombinant human IL-1β and

Induced damage to the human intestinal epithelial-like Caco-2 cell monolayers by coculturing with the human macrophage-like THP-1 cells

A schematic diagram representing the in vitro coculture system used in the present study is shown in Fig. 1. The two different types of cell used with this system were not in contact with each other, enabling crosstalk between the two types of cell via the soluble factors secreted from each cell line to be observed.

The intestinal epithelial-like Caco-2 cell monolayers were cultured on a semipermeable membrane for 14 days, and the human monocytic THP-1 cells were cultured in 12-well plates and

Discussion

We examined in this study the effect of activated macrophages on the intestinal epithelial Caco-2 cell monolayers by constructing an in vitro coculture model and using the cultured cell lines. We revealed that coculturing with human macrophage-like THP-1 cells induced damage, involving increased LDH release and decreased TER value, to human intestinal epithelial-like Caco-2 cell monolayers. The increase in LDH released from the Caco-2 cell monolayers was completely blocked, and the decrease in

Acknowledgments

This work was partially supported by Grant-in Aid for Young Scientists (B)(17780100) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also partially supported by the research grants from the Skylark Food Science Institute and from Kampou Science Foundation.

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