Elastase Controls the Binding of the Vitamin D-Binding Protein (Gc-Globulin) to Neutrophils: A Potential Role in the Regulation of C5a Co-Chemotactic Activity

Abstract

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37°C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.

Upregulation of Vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules

“…In addition, azurophil granules are the last granule population to be mobilized and fuse with the plasma membrane during phagocytosis and/or cell activation (Faurschou and Borregaard, 2003). Azurophil granules also are the storage compartment for elastase (Faurschou and Borregaard, 2003), and we have demonstrated that this protease cleaves the DBP binding site (but not DBP) and sheds it into the extracellular milieu (DiMartino et al, 2001). Inhibition of elastase causes large amounts of DBP to accumulate on the cell surface (a 5-fold increase after 60 min) and also inhibits C5a chemotactic cofactor activity (DiMartino et al, 2001).…”

“…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).…”

Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3

“…However, very little mechanistic information is available concerning how DBP enhances chemotaxis to the C5 derived peptides. Several studies from our laboratory have demonstrated that the binding of DBP to the neutrophil plasma membrane, and subsequent protease-mediated shedding of the binding site, are essential for the chemotaxis enhancement of C5a (DiMartino and Kew, 1999;DiMartino et al, 2001;Kew et al, 1995a;Kew et al, 1995b) More recently, we have demonstrated that DBP requires platelet-derived thrombospondin-1 for maximal cochemotactic activity (Trujillo and Kew, 2004). In addition, cell surface CD44 and annexin A2 are part of a DBP binding site complex and mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005).…”

“…However, previous studies have shown that neutrophils transiently generate co-chemotactic activity for C5a/C5a des Arg 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 into the extracellular milieu (DiMartino et al, 2001). Both plasma membrane binding and subsequent shedding of DBP are essential for the protein to function as a chemotactic cofactor for C5a.…”