Upregulation of Vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules
Introduction
The Vitamin D binding protein (DBP), also known as Gc-globulin, is a multifunctional, albumin-like 56 kDa plasma protein that can bind several diverse ligands (White and Cooke, 2000). DBP functions to transport Vitamin D sterols and fatty acids, acts as a scavenger protein to clear extracellular G-actin released from necrotic cells, and a deglycoslated form of DBP has been shown to be a macrophage and osteoclast activating factor (White and Cooke, 2000). The chemotactic activity of the complement (C) activation peptides C5a and C5a des Arg also can be enhanced significantly by DBP (Kew and Webster, 1988, Perez et al., 1988), an observation independently verified by several different groups (Binder et al., 1999, Metcalf et al., 1991, Petrini et al., 1991, Piquette et al., 1994, Senior et al., 1988, Zwahlen and Roth, 1990). This positive chemotactic cofactor function of DBP (i.e., co-chemotactic activity) is most readily observed using either suboptimal or non-chemotactic concentrations of the C5-derived peptides and is very specific for C5a/C5a des Arg (Kew et al., 1995a, Kew et al., 1995b, Kew and Webster, 1988, Perez et al., 1988). However, DBP by itself lacks chemotactic activity (Kew et al., 1995a, Kew and Webster, 1988). Plasma-derived DBP also binds to the surface of many cell types including neutrophils (DiMartino and Kew, 1999, DiMartino et al., 2001, White and Cooke, 2000). DBP appears to bind with low affinity to multiple cell surface ligands such as chondroitin sulfate proteoglycans (DiMartino and Kew, 1999), megalin (Nykjaer et al., 1999, Nykjaer et al., 2001), cubulin (Nykjaer et al., 2001), CD44 and annexin A2 (McVoy and Kew, 2005). Neutrophils transiently generate co-chemotactic activity for C5a on the cell surface within 15–20 min of DBP binding (Kew et al., 1995a). These cells also utilize membrane-bound elastase to shed the DBP binding site complex into the extracellular milieu (DiMartino et al., 2001). Both plasma membrane binding and subsequent shedding of DBP are essential in order for the protein to function as a chemotactic cofactor for C5a (DiMartino et al., 2001, Kew et al., 1995a).
The binding of DBP to cells is required for the protein to mediate its numerous functions: a chemotactic cofactor for C5a, a macrophage or osteoclast activating factor, clearance of DBP–actin complexes by the liver and delivery of Vitamin D sterols and free fatty acids to cells. Thus, expression of cell surface DBP binding sites is a key factor that limits the functions of this protein. We have noted that the DBP-enhanced neutrophil chemotactic response to C5a can vary considerably among different blood donors, and have speculated that neutrophils may need to be activated in order to bind DBP and show a co-chemotactic response to C5a. The objective of this study was to compare DBP binding and chemotactic enhancement to C5a in neutrophils prepared using a lipopolysaccharide (LPS)-free method versus a standard cell isolation protocol. Results clearly show that activated neutrophils bind DBP and display enhanced chemotaxis to C5a. Furthermore, DBP binds to a unique “activated” region of the plasma membrane that contains contents of intracellular granules.
Section snippets
Reagents
Purified recombinant human C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP), cytochalasin D, monensin and phorbol myristate acetate (PMA) were purchased from Sigma–Aldrich (St. Louis, MO). Vitamin D binding protein was purified from human plasma and purchased from Athens Research and Technology (Athens, GA). Lipopolysaccharide from E. coli 0111:B4 was obtained from Sigma. The protease inhibitors PMSF, 1,10-phenanthroline, E-64, leupeptin and pepstatin A were purchased from Sigma while Pefabloc
Results
The DBP-enhanced neutrophil chemotactic response to C5a can be variable, and it has been speculated that neutrophils need to be activated to some extent in order to bind DBP and show a co-chemotactic response to C5a (McVoy and Kew, 2005). This may explain why it often takes 15–20 min after purified DBP is added to neutrophils before C5a co-chemotactic activity is detected in vitro (Kew et al., 1995a). Indeed, we have observed a dramatic difference in both DBP binding and C5a chemotactic
Discussion
The results presented in this paper help to clarify how DBP interacts with neutrophils and mediate enhanced chemotaxis to C5a. The objective of this study was to ascertain if neutrophil activation correlates with an increase in DBP binding and chemotactic enhancement to C5a. This question arose from observations that both parameters are variable among neutrophils obtained from healthy human blood donors, even in cells derived from the same individual but on different days. A reasonable
Acknowledgments
This investigation was supported in part by a grant to R.R.K. from the National Institutes of Health (GM 63769). S.J.D. and L.A.M. were supported in part by a Medical Scientist Training Program (MSTP) grant from the National Institutes of Health. G.T. was supported by a W. Burghardt Turner Fellowship and the NSF-funded AGEP Program (both at Stony Brook University), and by an N.I.H. training grant (GM 08468).
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2014, Advances in Clinical ChemistryCitation Excerpt :Quiescent neutrophils do not bind DBP nor display an enhanced chemotaxis to C5a. Azurophil granules are a latent reservoir of DBP binding sites and their fusion with the plasma membranes greatly increases the neutrophils capacity to bind DBP [78]. By binding to its receptors, CD36 and CD47, platelet-derived thrombospondin-1 (TSP-1) facilitates the augmentation of C5a-induced chemotaxis by DBP.
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2011, Molecular ImmunologyCitation Excerpt :Although the protein by itself lacks chemotactic activity, it associates with the plasma membrane of many cell types and appears to bind with low avidity to multiple cell surface ligands such as chondroitin sulfate proteoglycans (DiMartino and Kew, 1999), CD44 (McVoy and Kew, 2005) megalin (Nykjaer et al., 1999), and cubulin (Nykjaer et al., 2001). A cell surface DBP binding site complex has been inferred by functional, structural and kinetic cell binding studies and its interaction with DBP is essential for chemotaxis enhancement of C5a (DiMartino and Kew, 1999; DiMartino et al., 2001, 2007; Kew et al., 1995; Trujillo and Kew, 2004; McVoy and Kew, 2005). Formation of a DBP binding site complex in leukocytes is a dynamic, multi-step and transient process requiring cell activation (DiMartino et al., 2007) and perhaps several distinct macromolecules.
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Present address: Rheumatology Division, Hospital for Special Surgery, New York, NY 10021, United States.