Histidine rich glycoprotein HRG is a kDa plasma glycoprotein

Histidine-rich glycoprotein (HRG) is a 75kDa plasma glycoprotein produced in and secreted from the liver (Koide et al., 1986). The plasma levels of HRG in healthy human were reported to be constant around 1μM (Poon et al., 2011). HRG\'s primary amino monooxygenase sequence has an extraordinary number of histidine residues and typical GHHPH repeats (Borza et al., 1996). Since HRG binds to a diverse range of ligands including heparin, heparan sulfate, fibrin, fibrinogen, plasminogen, thrombospondin, divalent metal cations, heme, and complement C1q, it is suggested to be involved in the regulation of coagulation/fibrinolysis (Lijnen et al., 1983; Leung, 1986; Peterson et al., 1987; Silverstein et al., 1985), immune response (Gorgani et al., 1997; Morgan, 1985; Poon et al., 2010), and angiogenesis (Doñate et al., 2004; Juarez et al., 2002; Wake et al., 2009). Moreover, HRG is reported to have the antibacterial and antifungal activities in acidic pH and high Zn condition (Rydengård et al., 2007; Kacprzyk et al., 2007; Rydengård et al., 2008) as well as the neutralizing effect on lipopolysaccharide (LPS) (Bosshart and Heinzelmann, 2003). Actually, Shannon et al. demonstrated that HRG decreases mortality of septic mice model with S. pyogenes-induced abscess by killing and trapping of bacteria in abscess site (Shannon et al., 2010). However, the effects of HRG on the circulating leukocytes, especially neutrophils in systemic septic condition, are poorly understood.
Materials and Methods
Results
Discussion In the present study, we clearly showed a novel and important role of HRG in controlling the shape, adhesiveness, passage, and basal ROS-producing activity of neutrophils. No plasma protein has ever been reported to have such activity. Judging from the plasma HRG levels (around 1μM) in healthy human (Poon et al., 2011; Saigo et al., 1990), the effects of HRG on neutrophil shape in the circulation under normal condition are speculated to be maximal. A marked decrease in plasma levels of HRG in septic conditions due to rapid reduction in HRG gene expression, deposition of HRG on immunothrombi and degradation of HRG by thrombin (Fig 1E and F; Figs. S1G, S2A and S2B) may lead to increased adhesion of neutrophils to VECs and retardation of neutrophil passage in the microvasculature demonstrated by in vivo imaging and in vitro experiments; the changes are associated with the enhancement of ROS production. Thus, the neutrophil shape maintained by HRG should be suitable for their passage through the capillary vessels, sustaining rheological stability, and preventing unnecessary activaton of VECs. The spherical shape probably minimizes the surface attachment area of neutrophils to VECs and reduces physical contact between neutrophils and endothelial cells in the microvasculature, easing the passage of neutrophils through capillary vessels as observed by in vivo imaging of circulating neutrophils in CLP mice treated with HRG. In addition, it was revealed that HRG slightly inhibited the expression of activated but not inactivated form of CD11b, irrespective of the presence of neutrophil activation agonists. This effect of HRG may also limit the unnecessary tight interaction between circulating neutrophils and VECs. In contrast, the deformed neutrophils in septic condition sometimes occupy the flow of microcirculation by forming a cell cluster, implying enhanced interaction between deformed neutrophils and VECs or deformed cells themselves. This attachment pattern seems quite different from that observed during the migration of neutrophils infiltrating inflamed sites (Amulic et al., 2012; DiStasi and Ley, 2009; Phillipson and Kubes, 2011). Immunohistochemical analysis of septic ARDS in CLP mice clearly showed the existence of immunothrombi in lung vasculatures. The marked inhibitory effects of HRG on immunothrombosis in pulmonary vasculatures (Fig. 2C and E) were presumably due to suppression of initial neutrophil attachment by keeping morphological and functional quiescence of circulating neutrophils and inhibition of the formation of fibrin clot on neutrophil and platelets. In addition, almost complete inhibition of the increase in plasma IL-6 and TNF-α by HRG administration could contribute to cancellation of activation of VECs that facilitates immunothrombosis. The finding that HRG antagonized the pro-DIC state together with the anti-immunothrombotic effects as well as strong inhibition of lung inflammation implies that HRG administration suppressed the major and fatal responses in sepsis; DIC, multiple organ failure due to microthrombus formation, and severe inflammatory injuries in plural organs. It was reported that NET formation (Brinkmann et al., 2004; Yipp and Kubes, 2013) was involved in different types of immunothrombosis. In the present model, we confirmed NETosis occurring in the lung vasculatures leading to immunothrombosis (Fig. 2F and G).