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 This information is current as of September 26, 2021. Stephen J. DiMartino, Anisha B. Shah, Glenda Trujillo and Richard R. Kew J Immunol 2001; 166:2688-2694; ; doi: 10.4049/jimmunol.166.4.2688 http://www.jimmunol.org/content/166/4/2688 Downloaded from References This article cites 64 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/166/4/2688.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Elastase Controls the Binding of the Vitamin D-Binding Protein (Gc-Globulin) to Neutrophils: A Potential Role in the Regulation of C5a Co-Chemotactic Activity1 Stephen J. DiMartino, Anisha B. Shah, Glenda Trujillo, and Richard R. Kew2 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 Downloaded from 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 http://www.jimmunol.org/ elastase may play a critical role in the C5a co-chemotactic mechanism. The Journal of Immunology, 2001, 166: 2688–2694. omplement activation and cleavage of C5 generate the significantly enhance the chemotactic activity (i.e., co-chemotactic potent chemoattractants C5a, and its derivative, C5a des activity) of C5-derived peptides for human (11–16) and bovine C Arg. C5-derived peptides are considered to be among the neutrophils (17). DBP also has been shown to augment monocyte most physiologically important leukocyte chemotactic factors (1, and fibroblast chemotaxis to C5-derived peptides (18, 19). The 2). During the past several years, the use of molecular approaches chemotactic enhancing properties of DBP appear to be restricted to has substantially enhanced the knowledge about several major che- C5a/C5a des Arg because this protein cannot enhance the chemo- moattractants and their receptors (3–6). However, much less at- tactic activity of formylated peptides, IL-8, leukotriene B4,or by guest on September 26, 2021 tention has been paid to extracellular inhibitory and enhancing platelet-activating factor (11–17). Although DBP appears to be a factors that modulate chemoattractant function, several of which physiologically important regulator of leukocyte chemotactic ac- have been described over the last 25 years (7, 8). Identification of tivity for activated complement, the mechanism of chemotactic specific regulatory molecules and/or their mechanisms of action enhancement is not yet known. largely have remained obscure, although there are a couple of no- DBP is a multifunctional 56-kDa plasma protein that can bind table exceptions (9, 10). The vitamin D-binding protein (DBP),3 several diverse ligands (20, 21). In addition to functioning as a also known as Gc-globulin, is one protein that has been shown to co-chemotactic factor for C5-derived peptides, DBP functions to transport vitamin D sterols and acts as a scavenger protein to clear extracellular G-actin released from necrotic cells, and a deglyco- Department of Pathology, School of Medicine, State University of New York, Stony Brook, NY 11794 sylated form of DBP has been shown to be a potent macrophage and osteoclast-activating factor (22). Plasma-derived DBP also Received for publication August 16, 2000. Accepted for publication December 1, 2000. binds to the surface of many cell types including neutrophils (23– The costs of publication of this article were defrayed in part by the payment of page 25). DBP bound to the plasma membrane of neutrophils appears to charges. This article must therefore be hereby marked advertisement in accordance play an essential role in enhancing chemotaxis to C5-derived pep- with 18 U.S.C. Section 1734 solely to indicate this fact. tides (26). Recently, we have demonstrated that a cell surface 1 This investigation was supported in part by grants to R.R.K. from the Smokeless chondroitin sulfate proteoglycan serves as the DBP binding site on Tobacco Research Council (0548) and the Scleroderma Foundation (001099). S.J.D. was supported in part by a Medical Scientist Training Program Grant from the Na- neutrophils (27). Moreover, another recent report has shown that tional Institutes of Health. A.B.S. was supported in part by the M.D. with Distinction ␮ DBP binds with low affinity (Kd of 111 M) to megalin (600-kDa in Research Program, School of Medicine, State University of New York at Stony Brook. G.T. was supported by a W. Burghardt Turner Fellowship and a National multiligand clearance receptor) on the surface of renal proximal Institute of Health Training Grant (GM 08468). tubule cells (28). However, megalin is not expressed on neutro- 2 Address correspondence and reprint requests to Dr. Richard R. Kew, Department of phils or other cells of the myeloid lineage (29, 30). Identification Pathology, State University of New York, Stony Brook, NY 11794-8691. E-mail of cell surface DBP binding sites is a major step for our under- address: [email protected] standing of the C5a co-chemotactic mechanism. However, little is 3 Abbreviations used in this paper: DBP, vitamin D-binding protein; AAPA-CMK, known concerning the regulation of DBP binding to cells. N-methoxysuccinyl-ala-ala-pro-ala-chloromethyl ketone; AAPV-CMK, N-methoxy- succinyl-ala-ala-pro-val-chloromethyl ketone; E-64, trans-epoxysuccinyl-L-leucyl- Protease-mediated cleavage and shedding of plasma membrane amido-(4-guanidino)butane; FFR-CMK, D-phenylalanine-L-phenylalanine-L-arginine- macromolecules is a well-established negative regulatory mecha- chloromethyl ketone; FPR-CMK, D-phenylalanine-L-proline-L-arginine-chloromethyl ketone; 125I-DBP, 125I-labeled DBP; SLPI, secretory leukocyte protease inhibitor; nism (31). Neutrophils in particular employ cell surface proteases Z-GLF-CMK, carbobenzoxy-glycine-leucine-phenylalanine-chloromethyl ketone. to rapidly change their profile of cell surface macromolecules. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 2689 Neutrophil elastase, also known as human leukocyte elastase mM EDTA, 10 mM NaN3, as well as the following inhibitors added fresh (E.C.3.4.21.37), has been shown to cleave several CD Ags (CD14, immediately before lysis: 2 mM PMSF, 2 mM 1,10-phenanthroline, 0.5 CD16, CD43, CD44), releasing a soluble form into the extracel- mM E-64, 0.2 mM 3,4-dichloroisocoumarin, 0.1 mM leupeptin, and 0.1 mM pepstatin. Lysates were vortexed thoroughly until all particulate matter lular media (32, 33). Although the majority of neutrophil elastase was solubilized (usually 5–10 s) and then placed at 37°C for 60 min. The is stored in azurophil granules, several reports have demonstrated detergent-insoluble material was then pelleted by centrifuging the lysates active enzyme on the cell surface (34–37). An earlier report from in a microfuge for 10 min at 15,000 ϫ g at 4°C. our laboratory has shown that, at 37°C, neutrophils shed DBP from Chemotaxis assay the plasma membrane into the extracellular media (26). Moreover, the loss of DBP from the cell surface is correlated temporally Cell movement was quantitated using a 48-well microchemotaxis chamber with the decay in C5a co-chemotactic activity (26). These results (Neuroprobe, Cabin John, MD) and 5-␮m pore-size cellulose nitrate filters suggested that either DBP or its binding site is proteolytically pro- (Toyo, purchased from Neuroprobe), as previously described (26). cessed by neutrophils. The goal of the present study was to char- PAGE and autoradiography acterize the regulation of DBP binding to human neutrophils. The results demonstrate that elastase is needed for steady state binding Samples were separated by PAGE in the presence of SDS (SDS-PAGE) using the discontinuous buffer system described by Laemmli (39).