Histidine-rich Glycoprotein Specifically Binds to Necrotic Cells via Its Amino-terminal Domain and Facilitates Necrotic Cell Phagocytosis

Abstract

Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidinerich glycoprotein (HRG), an abundant (ϳ100 g/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes.Cell death is vital for the morphological shaping of tissues during development and for the sculpting of functionally appropriate cellular repertoires as well as for protecting an individual from viral infections and pathogenic microorganisms (1, 2). Selective cell death continues to play a role in the homeostasis of mature tissues, such as the deletion of immune cells in the attenuation of an immune response (3) and the elimination of cells that have become functionally inappropriate, including virally infected and transformed cells (4). Apoptosis and necrosis represent two different forms of cell death and are characterized by distinct morphologies. Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis, whereas the role and extent of phagocytosis in the clearance of necrotic cells is not well documented. It is thought, however, that by engulfing necrotic and apoptotic cells phagocytes of the innate immune system not only provide a first line of defense against microbial pathogens but also dispose of self-antigens that are released from dying cells (5).Apoptosis is characterized by an orderly sequence of internal events, including chromatin condensation that precedes the loss of cellular integrity (6). Apoptotic cells also display phosphatidylserine and altered membrane carbohydrates on their surface. Multiple ligands and receptors have been implicated in the recognition and uptake of apoptotic cells by phagocytes prior to membrane lysis, thus preventing release of potentially toxic and immunogenic intracellular substances into tissues. In addition, the binding and/or uptake of apoptotic cells inhibits proinflammatory cytokine production (7). When there are disturbances in either apoptosis or the phagocytosis of apoptotic cells, antibodies against subsequently exp...

Molecular mechanisms of late apoptotic/necrotic cell clearance

“…92 Furthermore, HRG binds to FcgRI on HMDMs and functions as a bridging molecule to enhance the uptake of a mixture of early and late apoptotic cells through a FcgRI-dependent mechanism. 92 Similarly, studies by Jones et al 93 also showed that besides cell-surface HS, HRG binds strongly through its N-terminal domains to cytoplasmic ligand(s) exposed on heat-killed necrotic Jurkat T cells and enhances their phagocytic removal by THP-1 monocytic cells. 93 Interestingly, HRG has also been shown recently to have a minor role in regulating complement activation on heat-killed necrotic Jurkat T cells, possibly by interacting with complement components such as C1q, factor H and C8.…”

“…92 Similarly, studies by Jones et al 93 also showed that besides cell-surface HS, HRG binds strongly through its N-terminal domains to cytoplasmic ligand(s) exposed on heat-killed necrotic Jurkat T cells and enhances their phagocytic removal by THP-1 monocytic cells. 93 Interestingly, HRG has also been shown recently to have a minor role in regulating complement activation on heat-killed necrotic Jurkat T cells, possibly by interacting with complement components such as C1q, factor H and C8. 94 As the role of HRG in late apoptotic and necrotic cell clearance in vivo has yet to be examined, further studies using the recently generated HRG-deficient mouse would be of great importance.…”

Histidine-rich glycoprotein: the Swiss Army knife of mammalian plasma

“…1 HRG can also interact with cell-associated molecules, including Fc␥ receptors (Fc␥R), 20 heparan sulfate (HS), 21 phospholipids, 22 tropomysin, 23 ATP synthase, 24 DNA, 25 and cytoplasmic ligand(s) exposed on necrotic cells. 26 HRG domains and the predicted ligand binding sites are depicted in Figure 1. On the basis of the modular architecture of HRG, it has been proposed that HRG may act as an adaptor molecule that interacts with multiple ligands simultaneously through several independent binding sites.…”

“…32 HRG was shown to facilitate the removal of late (ie, plasma membrane permeabilized) apoptotic 25 and necrotic cells. 22,26,33 Studies by Gorgani et al 25 showed that HRG binds strongly to late apoptotic cells compared with viable or early (ie, plasma membrane intact) apoptotic cells, possibly by recognizing naked DNA. Consistent with previous studies, 20 HRG was suggested to …”

“…37,40 Recently, the antimicrobial role of HRG was validated in vivo by the use of HRG-deficient (HRG Ϫ/Ϫ ) mice, with HRG Ϫ/Ϫ mice being found to be more susceptible to Candida albicans and Streptococcus pyogenes infections than their wildtype counterparts (Table 1). 6,[17][18][19][20]22,25,26,28,33,38,40,[42][43][44][45][46][47][48][49][50] The cationicity of many antimicrobial peptides is known to be critical for the initial electrostatic attraction of peptides to the highly electronegative surface of bacteria and fungi, as well as the ability of peptides to traverse the microbial plasma membrane to aid pathogen killing via either membrane-or nonmembranedisruptive mechanisms. Although no bacterial ligand(s) of HRG have yet been identified, one could speculate that the cationicity of the HRR may play an important role because the antimicrobial effects of HRG and the HRR-derived peptides were highly dependent on low pH or the presence of Zn 2ϩ , 37,40 both of which impose a net positive charge on the molecule.…”