Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast

The P2X7 receptor (P2X7R), an ATP-gated ion channel, has been implicated in the process of cell-to-cell fusion into multinucleated macrophages (MA), but its contribution to MA fusion driven by physiological/pathological stimuli is not clearly established. Based on several lines of evidence, we demonstrate that P2X7R is critical for the induction of multinucleated MA by the inflammatory cytokine GM-CSF: 1) pharmacological inhibition of P2X7R with oxidized ATP (oATP), KN-62, and the selective antagonist A740003 abrogated GM-CSF action on rat alveolar MA and murine peritoneal MA; 2) a murine J774 P2X7 low MA clone, selected for defective P2X7R function, was unresponsive; 3) MA from mice lacking P2X7R failed to respond to GM-CSF, in contrast to wild-type. GM-CSF also stimulated ATP-induced membrane permeabilization in J774 P2X7 high MA and rat alveolar MA, an effect absent in the P2X7 low MA clone and inhibited by the P2X7 blockers oATP and KN-62. Notably, the stimulatory effects of GM-CSF on pore formation and MA fusion were both inhibited by blocking functional Pannexin-1 (Panx-1), and GM-CSF failed to stimulate MA fusion in cells from Panx-1 knockout mice. We provide further evidence that extracellular ATP release from peritoneal MA is dependent on P2X7 but not on Panx-1 expression and that its metabolism to adenosine mediates P2X7-dependent MA fusion. These data demonstrate that both P2X7 and Panx-1 are required for GM-CSF promotion of MA fusion but likely act independently through different signaling pathway(s).

Section: Discussionmentioning

confidence: 99%

The P2X7 Receptor and Pannexin-1 Are Both Required for the Promotion of Multinucleated Macrophages by the Inflammatory Cytokine GM-CSF

Lemaire

Falzoni

Zhang

et al. 2011

“…It is tempting to speculate that MGC formation may be associated with the reversible pseudoapoptotic signaling pathway induced by brief activation of P2X 7 receptors (44). In support of this, experimental evidence has demonstrated that intercellular fusion leading to multinucleation may occur through mechanisms that are independent of apoptosis (45,46). In contrast, overexpression of P2X 7 has been associated with cell death (24), and PMB has been shown to augment P2X 7 -dependent cytotoxicity (36).…”

Section: Discussionmentioning

confidence: 99%

Involvement of the Purinergic P2X7 Receptor in the Formation of Multinucleated Giant Cells

Lemaire

Falzoni

Leduc

et al. 2006

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.

“…In addition, externalization of PS characterizes early placental development during cytotrophoblast differentiation and occurs independently of apoptosis (47). Therefore, exposure of PS may occur by several mechanisms in patients with APS.…”

Section: Discussionmentioning

confidence: 99%

Arginine Residues Are Important in Determining the Binding of Human Monoclonal Antiphospholipid Antibodies to Clinically Relevant Antigens

Giles

Lambrianides

Pattni

et al. 2006

In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially β2-glycoprotein I (β2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic β2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different VH and VL sequences. VH sequences were derived from IS4 by altering the number of Arg residues in CDR3. VL sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (β2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, β2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to β2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and β2GPI.