Histidine-rich glycoprotein (HRG) is a multifunctional plasma protein and maintains the homeostasis of blood cells and vascular endothelial cells. In the current study, we demonstrate that HRG and recombinant HRG concentration dependently induced the phagocytic activity of isolated human neutrophils against fluorescence-labeled Escherichia coli and Staphylococcus aureus through the stimulation of CLEC1A receptors, maintaining their spherical round shape. The phagocytosis-inducing effects of HRG were inhibited by a specific anti-HRG Ab and enhanced by opsonization of bacteria with diluted serum. HRG and C5a prolonged the survival time of isolated human neutrophils, in association with a reduction in the spontaneous production of extracellular ROS. In contrast, HRG maintained the responsiveness of neutrophils to TNF-a, zymosan, and E. coli with regard to reactive oxygen species production. The blocking Ab for CLEC1A and recombinant CLEC1A-Fc fusion protein significantly inhibited the HRGinduced neutrophil rounding, phagocytic activity, and prolongation of survival time, suggesting the involvement of the CLEC1A receptor in the action of HRG on human neutrophils. These results as a whole indicated that HRG facilitated the clearance of E. coli and S. aureus by maintaining the neutrophil morphology and phagocytosis, contributing to the antiseptic effects of HRG in vivo.

Nishibori (2020): Histidine-rich glycoprotein possesses antioxidant activity through self-oxidation and inhibition of hydroxyl radical production via chelating divalent metal ions in Fenton's reaction, Free Radical Research,

Background: Histidine-rich glycoprotein (HRG) treatment ameliorated the survival rate of septic mice by suppressing excess immunothrombus formation. Although such findings suggested that HRG may be one of the most useful drugs for sepsis, obtaining a stable experimental system to standardize the HRG drug product is difficult to achieve using neutrophils isolated from volunteers. This is due to the short survival time and individual differences of human neutrophils. In the present study, we determined whether the differentiated neutrophil-like cell lines exhibited similar responses to HRG compared to human purified neutrophils. Method: All-trans retinoic acid (ATRA) was employed to induce the differentiation of the human myeloid leukemia cell lines, HL-60 and NB-4. Thereafter, the cells were treated with Hank's balanced salt solution, human serum albumin, or HRG. The effects of HRG on these cells were evaluated according to cell shape, microcapillary passage, ROS production, neutrophil extracellular traps (NETs) formation, the expression of activated CD11b, and cell viability. Result: HRG maintained the round shape of differentiated neutrophil-like cells, decreased the time required by cells to pass through the microcapillaries, and inhibited ROS production, NETs formation, and the expression of activated CD11b on the cell surface. Moreover, the cells could survive longer in the presence of HRG than the control. Conclusion: The ATRA-induced differentiated cell lines could be used as alternatives to neutrophils to investigate the effects of HRG on neutrophils. This method can thus be used as This article has not been copyedited and formatted. The final version may differ from this version.

Background: Excess immunothrombus formation induced by neutrophil adhesion on vascular wall, neutrophil extracellular traps (NETs) release and ROS production causes the multiple organ failure (MOF) in septic condition, leading to high mortality. Our previous study revealed that HRG treatment ameliorated the survival rate of septic mice due to suppressing the neutrophil adhesion, NETs release and immunothronbosis. These results suggested that HRG may be one of the most useful strategy for the treatment of septic patients, however, the effects of HRG on human purified neutrophil are not understood in detail. Human purified neutrophil can not survive for long time in vitro condition, and there are individual differences in the neutrophil response. Therefore, human neutrophils isolated from peripheral blood are not suitable for the long-term experiments. Thus, it's difficult to obtain the stable experiment system using neutrophils. All trans retinoic acid (ATRA) induces differentiation of the human myeloid leukemia cell lines HL-60 and NB-4. In the present study, we examined whether the differentiated myeloid cell line HL-60 and NB-4 showed the similar responses to human neutrophils purified from peripheral blood. Also, we investigate the effects of HRG on differentiated neutrophil-like cells in this study. Method: HL-60 and NB-4 differentiated into a neutrophil lineage by ATRA. These cells were treated with Hank's Balanced Salt Solution (HBSS), human serum albumin (HSA), or HRG. The effects of HRG on these cells were evaluated on the basis of cell shape, extracellular ROS production, and microcapillary passage. Result: HRG-treatoment kept the rounding shape of differentiated neutrophil-like cells and decreased the time to pass the cells through microcapillaries. These data indicated that HRG inhibits neutrophil adhesion on microcapillaries by maintaining the rounding cell shape. Moreover, this treatment suppressed the ROS production induced by TNF- and LPS stimulations. Conclusion: We showed that the ATRA-induced differentiated cell lines could be used as the alternative cells to investigate the effect of HRG on neutrophils.