Identification and cytokine regulation of gap junction proteins in macrophages and intestinal epithelial cell lines

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In Inflammatory Bowel Disease (IBD), inflammatory ceils, including macrophages, infiltrate the mucosa and are located along epithelial cell basolateral membranes. We have previously presented functional evidence of gap junctional communication between murine macrophages and intestinal epithelial cell (IEC) lines in an in vitro coculture system. Gap junctional channels are formed by the association of two hemichannels (one from each cell). The hemichannel is composed of six structural proteins referred to as connexins. The type of connexin expressed determines the gating and permeation characteristics of the gap junctional channel. AIM: The purpose of this study was to identify the type of connexin(s) expressed by macrophages and IEC and to determine whether inflammatory cytokines could regulate the level of connexin expression in these cells. METHODS: Identification and quantitation of connexins was done by Western Blotting using antibodies to connexin26 (Cx26), Cx32 and Cx43. Cx26 and Cx43 were detectable in lysates of whole cells and isolated plasma membranes, whereas Cx32 could only be detected in membrane lysates. RESULTS: Murine macrophage cell lines P388D1 and J774A. 1 both expressed Cx26, while only J774A.1 expressed Cx43. Cx32 was readily detected in membranes of J774A.1, while P388D1 expressed only very low levels of this connexin. Murine IEC lines Mode-K and CMT93 and the rat IEC line IEC6, expressed Cx26 and C43. Low levels of Cx32 were also detected in Mode-K membranes. One of the most significant findings was that while Cx43 was not detected in unstimulated murine resident peritoneal macrophages (RPM), Cx43 was expressed by thioglycoUateinduced, inflammatory peritoneal macrophages (IPM). This suggested that cytokines may regulate connexin expression. Murine IEC lines Mode-K and CMT93 and macrophage lines P388D1 and J774A.1 were stimulated with IL-1 or interferon gamma (IFN'/) for 18-20 hours prior to sample preparation for Western blotting. IFN~t stimulation resulted in increased expression of Cx43 by macrophage line I774A. 1 (an average of 238% n=4) but did not induce expression of Cx43 by P388D1 or IEC lines. IL-1 induced a 35-176% increase in expression Cx43 by Mode-K (n=3) but not expression by CMT93 or macrophage cell lines. Cx26 expression was unaltered by cytokine stimulation of any cell line. CONCLUSIONS: Macrophages and IEC express multiple connexins allowing for heterocellular communication via channels containing like or different connexins. In IBD, inflammatory cytokines may regulate the expression of these connexins and, by extension, may effect macrophage-IEC gap junctional communication.