ИСТИНА

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response. Human ceruloplasmin (CP, ferro:O2-oxidoreductase) is abounding in plasma glycosylated multi-copper ferroxidase that is synthesized primarily in the liver. Its physiological role is not limited to copper deposition and transport. Being the positive acute-phase protein, ceruloplasmin plays a significant role in the development of immune responses. Previously we reported that CP inhibits 5-lipoxygenase and a number of serine proteases of leukocyte origin related to inflammatory and septic processes. This work is a comparative study of the in vitro effects of ceruloplasmin and its derivatives, both physiologic and synthetic, on neutrophil oxidative status and lifespan. Holo-ceruloplasmin, its demetallized (apo-) and partially proteolyzed forms, and synthetic free peptides RPYLKVFNPR (883–892) and RRPYLKVFNPRR (882–893) were investigated. CP was isolated from heparin-stabilized plasma of healthy donors. Proteolyzed CP (CPprot) was obtained after limited proteolysis with human thrombin. Apo-CP was prepared by treating the holo enzyme with copper-chelating agents. It was shown that in contrast to other CP derivatives investigated, the intact enzyme and the product of its partial hydrolysis have a pronounced effect on PMNL oxidative status. Our results indicate, that being effective superoxide radical scavenger, ceruloplasmin induces an immediate sharp increase in other intracellular oxidants, at least in experimental conditions. The ability of CP to exhibit both anti-oxidant and pro-oxidant properties persists after its partial proteolysis. Indeed, we observed a significant increase in the fluorescence emission of the DCF oxidation product when CP or CPprot were added to PMNLs. At the same time, the synthetic peptides weakly inhibited the generation of intracellular oxidants. Contrary to the unidirectional effect on PMNL oxidant status, intact and partially proteolyzed CP acted differently on delayed apoptosis. The inhibition of apoptosis we observed in the presence of CP occurred whether PMNLs were exposed to TNF-α or not. The mode of apoptosis regulation by CPprot, as well as other products of enzyme hydrolysis, switches from inhibition to activation in the presence of TNF-α. The mechanisms of superoxide elimination by CP are not completely clear, but could be linked to the oxidative activity of copper atoms, even it was shown by us that inorganic copper, when added to the cells in relevant concentrations, did not exert the same influence on PMNL oxidative or apoptotic status. TNF-α plays a critical role in modulating acute and chronic inflammation, largely due to its ability to stimulate myeloperoxidase (MPO) synthesis and secretion. CP and its derivatives differ in their ability to inhibit MPO peroxidase activity. It was found that partial proteolysis of CP dramatically reduces the inhibitory function of MPO on CP without disturbing its ferroxidase activity. CPprot cannot inhibit the peroxidase activity of MPO, but retains ferroxidase activity. Hereby we presented a potential mechanism associated with the change of oxidative status and activation of apoptosis in human neutrophils by CP and its derivatives.