Nitrosative Stress Inhibits the Aminophospholipid Translocase Resulting in Phosphatidylserine Externalization and Macrophage Engulfment

Abstract

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/ oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO ⅐ scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.

A Functional NADPH Oxidase Prevents Caspase Involvement in the Clearance of Phagocytic Neutrophils

“…This implies that caspases are not involved in the clearance of phagocytic neutrophils but that an oxidant-dependent event is crucial. This is consistent with the observation of Kagan and colleagues that oxidation of PS is necessary for its externalization (16,22,30); more recently, Tyurina et al showed that in HL-60 cells exposed to nitrosative stress PS exposure was dissociated from the common apoptotic pathway (29). We have also recently reported that 24-h ascorbate-deficient neutrophils fail to undergo PS exposure and uptake by macrophages despite activation of caspases (33).…”

Microglial dysfunction as a key pathological change in adrenomyeloneuropathy

“…The first is activated microglia with upregulated microglial receptors and opsonins like MFGE8, which can recognize a stressed neuron and tag it for engulfment. The second is PS exposure on the neuronal cell surface, which can occur as a result of oxidative stress, an increase in calcium levels, or adenosine triphosphate depletion, among other possible mechanisms 29, 30, 31. PS exposure is not itself toxic to neurons, but rather marks the neuron for selective removal by microglia.…”

The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system

“…The percentage of NBD-PS internalized was determined as the percentile of dithionite-resistant NBD-PS fluorescence from the total NBD-PS fluorescence as described earlier. 26 …”