Elsevier

Molecular Immunology

Volume 44, Issue 9, March 2007, Pages 2370-2377
Molecular Immunology

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

https://doi.org/10.1016/j.molimm.2006.10.011Get rights and content

Abstract

Vitamin D binding protein (DBP) is a multifunctional plasma transport protein that is also found on the surface of many cell types. Cell surface DBP significantly enhances chemotactic activity of complement (C) peptides C5a and C5a des Arg. However, both DBP binding and C5a chemotaxis enhancement can vary among neutrophil donors. To test if activation during cell purification is responsible for this variability, neutrophils were isolated using both standard and lipopolysaccharide (LPS)-free protocols. Cells isolated by the LPS-free method had no DBP-enhanced chemotaxis to C5a or DBP binding to plasma membranes. Moreover, neutrophils treated with LPS bound more avidity to immobilized DBP than sham-treated cells. Subcellular fractionation of neutrophils (standard protocol) revealed a heavy plasma membrane (HM) band that contained components of light plasma membranes and all three granules. The HM band possessed most of the DBP binding activity (58%), and activation of cells with ionomycin greatly increased DBP binding to HM. Azurophil granules contained 33% of the total DBP binding sites and there was a highly significant positive correlation (r = 0.988) between release of the granule marker myeloperoxidase and DBP binding. These results indicate that fusion of granules with the plasma membrane forms HM that contains DBP binding sites.

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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    Present address: Rheumatology Division, Hospital for Special Surgery, New York, NY 10021, United States.

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