Epithelial Sodium Channels in Monocyte‐Macrophage Migration and Regulation by Pro‐inflammatory Cytokines TNFα and IFNγ

Abstract

Migration of monocytes‐macrophages play an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. While Epithelial Na+ Channels (ENaC) are required for normal migratory responses in glial, trophoblast, and vascular smooth muscle cells, their role in monocyte‐macrophage migration signaling is unknown. The Human Protein Atlas data base (http://www.proteinatlas.org/) shows ENaC message in peripheral blood mononuclear cells and tissue resident macrophages in human populations. We used an in vitro model to determine the importance of ENaC in chemotactic migration of monocytes‐macrophages using a standard Boyden chamber assay. Cells were suspended in 0.4% FBS ± amiloride (0.1‐10 µM) or benzamil (0.01‐1 µM) to inhibit ENaC channels, then migrated for 4 hr towards a 10% FBS gradient. In mouse RAW cells, migration responses were inhibited by amiloride (65±6 – 46±4% of control at 0.1 and 10 µM, p=0.0001) and benzamil (55±6 – 55±5% of control at 0.01 and 1.0 µM, p<0.0001). In human THP‐1 cells, migration responses were similarly inhibited by amiloride (73±5 ‐ 40±6% of control at 0.1 and 10 µM, p<0.0001) and benzamil (68±7 – 53±4% of control at 0.01 and 1.0 µM, p<0.0001). Since benzamil and amiloride are selective for ENaC at sub and low micromolar concentrations, respectively, our findings indicate ENaC signaling is required for migration of unstimulated monocyte‐macrophage cells. To determine if pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interferon gamma (INFγ), regulate ENaC expression and function, we used TaqMan quantitative PCR, western blotting and chemotactic migration. Cells were exposed to a standard protocol to activate macrophages: INFγ for 48 hr plus TNFα (10 ng/mL each) during the last 24 hr. Expression of α and βENaC and GAPDH was calculated as fold change using the delta delta CT method (2‐∆∆CT). INFγ48hr + TNFα24hr increased α and βENaC message (9±2 and 5±1, n=4‐6, p=0.01). γENaC was undetectable. Despite increased message, whole cell αENaC protein expression was inhibited 50% by western blotting. The mechanism underlying message stimulation and protein inhibition remains to be determined. A similar effect on message upregulation and migration inhibition also occurred with 18 hr INFγ or TNFα alone (0‐10 ng/mL). Activation with INFγ/TNFα inhibited migration up to 70% and abolished the amiloride (1 µM) sensitive component. In unstimulated cells, amiloride (1 µM) coculture induced a rebound increase in migration following withdrawal, which was abolished by INFγ/TNFα treatment. This finding suggests cytokine induced ENaC loss mediates the migration inhibition. In summary, ENaC mediates monocyte migration, however, monocyte activation by INFγ/TNFα inhibits migration by inhibiting ENaC. Since pro‐inflammatory cytokines initiate monocyte differentiation and polarization to the phagocytic M1 phenotype, future studies will determine if ENaC is required for the phenotypic switch.