Gap junctional communication between murine macrophages and intestinal epithelial cell lines

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.