Effects of bacterial products on enterocyte-macrophage interactions in vitro.

We describe a coculture model of a human intestinal epithelial cell line and human peripheral blood monocytes in which monocytes differentiate into cells with features of resident intestinal macrophages. Caco-2 cells are grown on the lower surface of a semipermeable filter with pore size of 3 µm (Transwells) until they differentiate into enterocytes. Peripheral-blood monocytes are added and the co-culture incubated for two days. Monocytes migrate through the pores of the membrane, come into direct contact with the basolateral surfaces of the epithelial cell monolayer, and develop characteristics of resident intestinal macrophages including downregulation of CD14 expression and reduced pro-inflammatory cytokine responses (IL-8, TNF and IL-1ß) to bacterial products. The apical application of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) resulted in an increased number of integrated monocytes, but abrogated the downregulation of CD14 expression and the diminished cytokine responses. MDP also reduced tight-junctional integrity, whilst LPS had no effect. These data indicate that LPS and MDP have significant pathophysiological effects on enterocyte-monocyte interactions, and confirm other studies that demonstrate that enterocytes and their products influence monocyte differentiation. This model may be useful in providing insights into the interaction between monocytes, epithelial cells and intestinal bacteria in health and disease.

Receptor mediated targeting of M-cells.

The intestinal epithelium is a complex system of highly specialised cells that provide digestive and absorptive functions as well as innate and adaptive immunity. Induction of an adaptive immune response in the intestine can occur through the interaction of antigen with M-cells that overlay the lymphoid aggregates of the intestine (Peyer's patches). This study demonstrated that specific common microbial pathogen-associated molecular patterns are recognised by pattern recognition receptors on the surface of the M-cells and this interaction initiates transcytosis through the M-cell of particulate antigen from the intestinal milieu to underlying antigen presenting cells within the Peyer's patch. The study has found that among the pattern recognition molecules that have a role in recognising bacterial components, the apical expression of alpha5beta1 integrin was important for the transcytotic function of M-cells. A proportion of intestinal enterocytes transform to an M-cell morphology in vitro, when cultured with Peyer's patch cells and our studies have demonstrated that CD4+ cells are integral for the development of M-cells in vitro.

Validation and quantitation of an in vitro M-cell model.

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyer's patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyer's patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyer's patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.