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-α, 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 HRG-induced 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. Key Points HRG induces the phagocytic activity of human neutrophils against E. coli and S. aureus. HRG induces shape change of human neutrophils and prolongs the survival time. The effect of HRG on human neutrophils is mediated by CLEC1A receptor.

Epidural and subdural hematomas are commonly associated with traumatic brain injury. While surgical removal is the primary intervention for these hematomas, it is also critical to prevent and reduce complications such as post-traumatic epilepsy, which may result from inflammatory responses in the injured brain areas. In the present study, we observed that high mobility group box-1 (HMGB1) decreased in the injured brain area beneath the epidural hematoma (EDH) in rats, concurrent with elevated plasma levels of HMGB1. Anti-HMGB1 monoclonal antibody therapy strongly inhibited both HMGB1 release and the subsequent increase in plasma levels. Moreover, this treatment suppressed the up-regulation of inflammatory cytokines and related molecules such as interleukin-1-beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) in the injured areas. Our in vitro experiments using SH-SY5Y demonstrated that hematoma components—thrombin, heme, and ferrous ion— prompted HMGB1 translocation from the nuclei to the cytoplasm, a process inhibited by the addition of the anti-HMGB1 mAb. These findings suggest that anti-HMGB1 mAb treatment not only inhibits HMGB1 translocation but also curtails inflammation in injured areas, thereby protecting the neural tissue. Thus, anti-HMGB1 mAb therapy could serve as a complementary therapy for an EDH before/after surgery.

[Background]Histidine-rich glycoprotein (HRG) is 75 kDa plasma glycoprotein produced from the liver. In previous study, we reported that HRG treatment prevents lethality of sepsis model mice and HRG regulated spherical shape change, passage of microcapillary and production of extracellular ROS on the human neutrophils. Next, we analyzed functional evaluation of neutrophils spheroidized by HRG.[Method]Phagocytosis analysis: We quantified the area of fluorescence-labeled bacteria by pHrodo in the neutrophils. Viability analysis: The number of intact neutrophils were counted by the staining with calcein-AM. Determination of extracellular ROS production: After adding isoluminol, HRP and each test reagent to neutrophils, the intensity of luminescence at 30 minutes were measured.[Result]Neutrophils treated with HRG showed increased activity of phagocytosis in a dose-dependent manner. HRG also induced high survival rate. When Zymosan A was added to neutrophils, the increased ROS production was observed in the presence of HRG. [Discussion] The neutrophils treated with HBSS or HSA are firmly attached to the bottom of the plate and being stimulated with regard to ROS production. In contrast, HRG maintained the spherical shape of neutrophils, phagocytic activity and responsiveness to Zymosan A. These results suggested that HRG may act on neutrophils to suppress excessive adhesion to vascular endothelium under normal condition and induce the functional activation when neutrophils meet bacteria.