Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Abstract

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function

“…Pharmacological inhibition with selective iNOS inhibitors, or genetic ablation (iNOS À/À ) prevented LPS induced epithelial barrier dysfunction and bacterial translocation [29,30]. In addition, activated macrophages secreting NO in epithelial co-cultures inhibited connexion-43 mediated epithelial barrier repair [12]. Collectively, these studies demonstrate that macrophage activation by altered bacterial populations or LPS may profoundly affect NO synthesis, TJ regulation, and bacterial translocation.…”

“…Intestinal macrophages in the duodenum of patients with cirrhosis have an activated phenotype Activated intestinal macrophages and dendritic cells play an important role in intestinal inflammation [10,12]. Immunohistochemistry confirmed a significant increase in CD68 + macrophages ( Fig.…”

“…Enteric pathogens [28] and LPS have been shown to activate iNOS [12]. Endotoxin administered to wild type mice impaired gut barrier function [29], and induced bacterial translocation to mesenteric lymph nodes [30].…”

“…In IBD, however, intestinal macrophages express innate response receptors such as CD14 + , TREM-1, and release pro-inflammatory cytokines [10]. Activated CD14 + macrophages in necrotising enterocolitis produce nitric oxide (NO) that impairs endothelial repair [11,12]. We have shown that macrophages activation in HIV correlated with bacterial translocation and persistent immune activation [13].…”

Modulation of connexin signaling by bacterial pathogens and their toxins

“…Hemichannel activity, however, seems to be consistently upregulated by LPS [45,[57][58][59][60]. A number of mechanisms have been proposed to underlie LPS-induced modifications in GJIC, including nitric oxide signaling [43,54,56] and activation of kinase pathways [55,61]. In fact, some of these pathways may be at the basis of the differential outcome observed for LPS on gap junctions and connexin hemichannels.…”