Increased production of reactive oxygen species by the vacuolar-type (H⁺)-ATPase inhibitors bafilomycin A1 and concanamycin A in RAW 264 cells

Abstract

-Treatment of the mouse leukemic cell line RAW 264 with bafilomycin A1 or concanamycin A, inhibitors of vacuolar-type (H + )-ATPases (V-ATPases), significantly increased the production of reactive oxygen species (ROS) and decreased cell viability. These effects were significantly suppressed by the presence of N-acetyl cysteine (NAC), an ROS scavenger. si-RNA mediated knockdown of the gene for the c subunit of the V0 domain of V-ATPase also resulted in an increase in ROS production and a decrease in cell viability. These results suggest that decreased cellular V-ATPase activity decreases cell viability by increasing ROS production in RAW 264 cells.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

“…In agreement with increased ROS formation in murine macrophages exposed to the V-ATPase inhibitors bafilomycin A1 or to concanamycin A [31], archazolid enhanced LPS-induced ROS formation in human macrophages, which was sensitive to the NDAPH oxidase inhibitor DPI. Of interest, DPI also blocked archazolid-induced TNFα release implying a requirement of ROS in this respect.…”

“…Previous studies showed that macrophages from different origins and species express V-ATPase and respond to VATPase inhibitors in different ways [13,31,[34][35][36][37][38][39]. We first confirmed V-ATPase expression in human monocyte-derived macrophages [40] by Western blot (not shown) and by using immunofluorescence microscopy where no obvious differences regarding V-ATPase subcellular localization between the macrophage phenotypes were immediately apparent (Fig.…”

“…Pharmacological manipulation of V-ATPase in macrophages by respective inhibitors such as bafilomycin A1 was reported before [13,31,35,37,38,48,50]. Many of these studies focused on osteoclasts [51], macrophage cell lines [31] and macrophages from rodents [37,39,48] and from other animals [52], but only few studies addressed V-ATPase functions in human primary macrophages [40,50], and to the best of our knowledge there are no reports that studied the role of V-ATPase on cytokine/chemokine secretion of different macrophage phenotypes, such as M1 and M2.…”

“…ROS levels were assessed as reported before [31]. Briefly, macrophages were incubated with test compounds or vehicle (0.1% DMSO) for 30 min and stimulated with LPS (100 ng/mL) for 24 hrs or left untreated.…”

“…Many of these studies focused on osteoclasts [51], macrophage cell lines [31] and macrophages from rodents [37,39,48] and from other animals [52], but only few studies addressed V-ATPase functions in human primary macrophages [40,50], and to the best of our knowledge there are no reports that studied the role of V-ATPase on cytokine/chemokine secretion of different macrophage phenotypes, such as M1 and M2. Therefore, one significant novelty of our study is the consideration of distinct human macrophage subtypes, that is, uncommitted M(0), classically-activated M1-like and so-called "resolving" M2-like macrophages that significantly differ in their bioactions [53].…”