Involvement of ecto‐ATPase and extracellular ATP in polymorphonuclear granulocyte‐endothelial interactions

Abstract

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Soluble Mediators Regulating Immunity in Early Life

“…Extracellular ATP (eATP) influences T-cell activation (224, 225) and proliferation (226, 227), promotes neutrophil/endothelial cell adhesion (228), degranulation (229, 230), and reactive oxygen species (ROS) production (231), as well as other pro-inflammatory immune cell functions (232). eATP is transported to the extracellular space by vesicular trafficking, secreted via pannexin-1 channels (233), or released in large quantities from necrotic cells.…”

Nucleotide receptors: an emerging family of regulatory molecules in blood cells

“…187,188 Of course, it cannot be excluded that the ATP effect could be at least in part mediated by its hydrolysis to adenosine, 189 but recent data suggest that ecto-ATPase activity has an inhibitory rather than stimulatory effect on granulocyte-endothelium interaction. 190 Dubyak and coworkers reported that chronic stimulation with ATP␥S or UTP could drive differentiation of myelomonocytic progenitor cells (HL-60 and U937). 191 Later studies showed that myeloid progenitors express P2Y 1 at earlier and P2Y 2 at later stages of differentiation.…”

Neutrophil Cellular Responses to Various Salmonella typhimurium LPS Chemotypes

“…After 30 min of incubation with or without the additives in a CO 2 incubator at 37°C to allow neutrophil adherence, wells were washed twice with 500 µl of PBS solution for removal of nonadherent PMNLs. The extent of adherence was measured after the addition of detergent and a myeloperoxidase substrate, as described (Schierwagen et al, 1990;Sud'ina et al, 1998). A solution (300µl) of 5.5mM o-phenylenediamine and 4mM H 2 O 2 in buffer (67mM Na 2 HPO 4 , 35mM citric acid and 0.1% Triton X-100, pH5) was added to each well, and after 5 min the reaction was stopped by the addition of an equal volume of 1M H 2 SO 4 .…”