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

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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 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 , 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 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 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). Samples of DBP to neutrophils. Specific elastase inhibitors disrupt steady were prepared for electrophoresis by boiling (100°C) for 7 min with an state binding, which causes DBP to accumulate on the plasma equal volume of electrophoresis sample buffer (0.125 M Tris, pH 6.8, 20% membrane and suppresses co-chemotactic activity for C5a. glycerol, 4% SDS) containing 0.2 M DTT as the reducing agent. After electrophoresis, the gels were stained, destained, dried, and exposed to x-ray film at Ϫ80°C. Materials and Methods Downloaded from Reagents Data analysis and statistics Human rC5a was a generous gift from Karl Mollison of Abbott Laboratories A minimum of three experiments was performed for each assay. Results of (Abbott Park, IL), and was prepared using an Escherichia coli expression several experiments were analyzed for significant differences among group system, as previously described (38). Purified human DBP was purchased means using the Newman-Keul’s Multiple Comparisons test using a sta- from Biodesign International (Kennebunkport, ME). The detergent Triton tistical software program (InSTAT). X-100 was purchased from Sigma (St. Louis, MO). Protease inhibitors were purchased from the following sources: 3,4-dichloroisocoumarin, PMSF, 1,10- http://www.jimmunol.org/ phenanthroline, N-methoxysuccinyl-ala-ala-pro-ala-chloromethyl ketone Results (AAPA-CMK), N-methoxysuccinyl-ala-ala-pro-val-chloromethyl ketone In an earlier report, we observed that neutrophils (at 37°C) shed (AAPV-CMK), trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane previously bound DBP from the plasma membrane into the extra- (E-64), leupeptin, and pepstatin A were purchased from Sigma; tosyl-L- cellular media (26). To determine whether a specific class of cell lysine-chloromethyl ketone and tosyl-L-phenylalanine-chloromethyl ketone surface proteases mediated this effect, neutrophils were treated were from Bachem (Torrance, CA); D-phenylalanine-L-proline-L-arginine- chloromethyl ketone (FPR-CMK) and D-phenylalanine-L-phenylalanine- with inhibitors specific to either metallo (1,10-phenanthroline)-, L-arginine-chloromethyl ketone (FFR-CMK) were purchased from Calbio- thiol (E-64)-, or serine proteases (PMSF, Pefabloc SC), and the chem (San Diego, CA); chymostatin and 4-(2-aminoethyl)-benzenesulfonyl amount of previously bound radioiodinated DBP was measured in fluoride (Pefabloc SC) were from Roche Molecular (Indianapolis, IN); carbo- the cell-free supernatant. Fig. 1A shows that ϳ70% of previously by guest on September 26, 2021 benzoxy-glycine-leucine-phenylalanine-chloromethyl ketone (Z-GLF-CMK) was from Enzyme Systems Products (Livermore, CA); and recombinant hu- bound DBP is released from cells during a 60-min incubation at man secretory leukocyte protease inhibitor (SLPI) was obtained from R&D 37°C. Treatment of cells with thiol- or metalloprotease inhibitors Systems (Minneapolis, MN). had no effect on the amount of DBP shed. In contrast, the serine Isolation of human neutrophils and neutrophil plasma protease inhibitor PMSF, or its water soluble analogue Pefabloc membranes SC, significantly suppressed the amount of DBP released by neu- trophils (Fig. 1A). Radiolabeled DBP released into the cell-free Neutrophils were isolated from the venous blood of healthy, medication- supernatant also was examined by SDS-PAGE to determine free, paid volunteers (who gave informed consent) using a standard three- whether the protein was cleaved. Fig. 1B shows that 125I-DBP shed step isolation procedure described previously (26). Subcellular fraction- ation of neutrophils and isolation of purified plasma membranes have been from neutrophils has no detectable alteration in its m.w. These described in detail previously (27). results demonstrate that a serine protease probably cleaves the binding site, releasing DBP in an unaltered form. Radioiodination of DBP Because the serine protease inhibitor PMSF was very effective Purified DBP (200 ␮g) was labeled using one Iodobead (Pierce, Rockford, at preventing neutrophils from shedding DBP into the extracellular 125 IL) and 1 mCi of Na I (DuPont-NEN, Wilmington, DE) for 5 min. The media, it follows that cells pretreated with PMSF should accumu- reaction was terminated by removing the solution from the Iodobead. Free Na125I was separated from 125I-labeled DBP (125I-DBP) by gel filtration on late radiolabeled DBP over time. Fig. 2 demonstrates that neutro- 125 a PD-10 (Sephadex G-25; Pharmacia-LKB, Piscataway, NJ) desalting col- phils pretreated with 0.5 mM PMSF and then incubated with I- umn. The 125I-DBP was concentrated using a Centricon 30 microconcen- DBP accumulate the protein in a linear manner vs time. In contrast, trator (molecular mass cutoff 30 kDa; Millipore, Bedford, MA). TCA at the level of cell-associated 125I-DBP in sham-treated (control) neu- 10% was used to determine the percentage of protein-associated counts. trophil plateaus between 30 and 60 min at 35–40 fmol DBP/106 Radioiodinated DBP preparations generally had a sp. act. of 0.5 ␮Ci/␮g (Ϯ10% among the different preparations) and had greater than 99% of the cells, very similar to what has been reported previously (26). Fur- total counts precipitable with 10% TCA. thermore, subcellular fractionation revealed that almost 90% of the cell-associated DBP remained with the plasma membrane fraction Quantitative binding assay in both PMSF-treated and control cells (data not shown) (27). At The binding of radioiodinated DBP to intact neutrophils has been described the concentrations of PMSF used (0.5 mM), greater than 95% of previously (26). Radiolabel binding to purified plasma membranes was cells were viable, as determined by trypan blue dye exclusion and measured using a vacuum filtration manifold and 0.1-␮m pore-size Du- rapore type VV filters (Millipore), as described previously (27). the release of lactate dehydrogenase (data not shown), consistent with our previous findings (40). Preparation of neutrophil detergent lysates The neutrophil DBP binding site, a chondroitin sulfate proteo- Detergent lysates of neutrophils were prepared by adding 100 ␮lof1% glycan, localizes to the detergent-insoluble fraction of cells solu- Triton X-100, 50 mM HEPES (pH 7.4) containing 20 mM benzamidine, 10 bilized using Triton X-100 (27). Therefore, when DBP is bound to 2690 ELASTASE REGULATES DBP BINDING TO NEUTROPHILS

FIGURE 2. Neutrophils treated with PMSF accumulate DBP vs time. Neutrophils (107 cells) were pretreated with either buffer (HBSS) or 0.5 mM PMSF for 10 min at 22°C. Cells then were incubated in HBSS with 100 nM 125I-DBP for the designated time at 37°C. The cells were washed three times in ice-cold HBSS, then counted for total cell-associated radio- activity. Data are expressed as fmol of DBP associated per million neu- trophils. The data represent the mean ϩ SEM of four to seven separate experiments performed in duplicate using cells from different donors. Val- Downloaded from ues for 30-, 60-, 120-, and 180-min PMSF-treated cells were significantly greater than control cells (p Ͻ 0.01 for 30-min sample; p Ͻ 0.001 for 60-, 120-, and 180-min samples).

FIGURE 1. Inhibition of serine proteases causes a decrease in the per- with intact cells, demonstrating that membrane preparations are a centage of DBP that is shed from neutrophils. Purified neutrophils (107)in HBSS were incubated with 100 nM 125I-DBP for 45 min at 37°C in buffer good model system to investigate DBP binding (27). Plasma mem- http://www.jimmunol.org/ (HBSS). After 45 min, cells were pelleted, washed, and resuspended in branes should have a fixed number of DBP binding sites and serine HBSS containing E-64 (0.5 mM), 1,10-phenanthroline (1 mM), Pefabloc protease activity, and thus, one would predict that there would be (0.5 mM), or PMSF (0.5 mM), and were incubated another 60 min at 37°C. no steady state binding. Indeed, Fig. 4A shows that the binding of A, After this second incubation, the cells were pelleted and both the cellular 125I-DBP to neutrophil plasma membrane (2°C) plateaus at 60 min pellet and the cell-free supernatant were counted for radioactivity. Data are between 17 and 18 fmol/␮g membrane protein. In contrast, the expressed as the percentage of total counts in the cell-free supernatant. maximal amount of DBP bound to membranes at 37°C peaks at 20 6 After the first incubation, cells contained 38 fmol DBP/10 neutrophils. min and only was 65% of the maximal amount bound at 2°C. Following the second incubation with protease inhibitors, the cells con- 6 Continued incubation at 37°C resulted in a rapid diminution of

tained the following amounts of DBP/10 neutrophils: control (12.5 fmol), by guest on September 26, 2021 125I-DBP binding, with a complete loss in the binding capacity of E-64 (13 fmol), 1,10-phenanthroline (11.5 fmol), Pefabloc (27.5 fmol), and PMSF (30 fmol). The results represent the mean ϩ SEM of three experi- membranes at 120 min (Fig. 4A). Addition of PMSF to the mem- ments performed in duplicate using different donors. Values for Pefabloc- branes prevents the loss in DBP-binding capacity at 37°C by more and PMSF-treated cells were significantly less (p Ͻ 0.001) than all other than 90% (data not shown). The foregoing data raise the question, groups. B, After the second incubation, the cell-free supernatant was col- does DBP need to be bound in order for a serine protease to in- lected and analyzed by SDS-PAGE under reducing conditions. Approxi- activate its binding site? To address this question, plasma mem- mately equal counts were added to each lane, and a representative autora- branes were preincubated for various times at 37°C, after which 125 diogram is shown. Lane 1, I-DBP that was not incubated with cells radiolabeled DBP was added and the binding assay was performed (control); lane 2, cell-free supernatant from untreated cells; lane 3, cell-free on ice (2°C). Fig. 4B clearly shows that degradation of the DBP- supernatant from PMSF-treated neutrophils. binding capacity of plasma membranes is constitutive and does not require DBP bound to its binding site. These results demonstrate its proteoglycan binding site, the complex partitions with the Tri- that the constitutive activity of a cell surface serine protease re- ton X-100-insoluble fraction. Fig. 3 shows that neutrophils accu- duces the binding capacity of plasma membranes for DBP. mulate DBP in the detergent-insoluble fraction with increasing Neutrophils possess several serine esterases, including elastase, concentrations of PMSF, indicating that inhibition of a serine pro- G, , and -type plasminogen acti- tease results in the accumulation of DBP binding site complexes. vator (41). In addition, plasma-derived serine proteases such as Previously, we reported that neutrophils bind 5-fold less radioio- , , and can bind to neutrophils, and their dinated human albumin than DBP (26). Treatment of neutrophils proteolytic activity can be detected on the cell surface (42–44). with 0.5 mM PMSF does not permit neutrophils to accumulate Thus, there are several serine esterases that potentially could albumin, and there is no shifting of the protein to the detergent- cleave the plasma membrane DBP binding site. To identify the insoluble pellet, indicating that the effect on DBP binding is not a responsible serine protease, plasma membranes were treated with generalized consequence of PMSF treatment (data not shown). several selective inhibitors and then were assessed for their capac- Figs. 2 and 3 indicate that inhibition of a neutrophil serine protease ity to bind radioiodinated DBP. Fig. 5 clearly shows that inhibitors disrupts the steady state balance of DBP binding/shedding, which of neutrophil elastase (AAPA-CMK and AAPV-CMK), neutrophil results in its accumulation on the cell surface. serine proteases (SLPI), as well as the serine class-specific inhib- The role of serine proteases in the regulation of cellular DBP itors PMSF and Pefabloc SC significantly increase the binding of binding was characterized further using purified neutrophil plasma DBP to plasma membranes over the untreated controls. In addi- membranes. Plasma membranes were used instead of intact cells to tion, similar treatment of intact neutrophils produced almost iden- avoid the potential problem of up-regulation of from in- tical results (data not shown). These results implicate membrane- tracellular granules. Previously, we have shown that the binding of bound neutrophil elastase as the protease that cleaves the DBP 125I-DBP to neutrophil plasma membranes essentially is identical binding site. The Journal of Immunology 2691

FIGURE 3. PMSF causes DBP to accumulate in the Triton X-100-in- soluble fraction. Purified neutrophils were incubated in HBSS with 100 nM 125I-DBP and the designated concentration of PMSF for 45 min at 37°C. The cells were washed in ice-cold HBSS, then lysed in 1% Triton X-100

(containing a complete protease inhibitor cocktail) for1hat37°C. The Downloaded from soluble and insoluble fractions were separated by centrifugation. Data are expressed as fmol of DBP associated per million neutrophils. The data represent the mean ϩ SEM of three to five separate experiments performed in duplicate using cells from different donors. Percentage of counts in the FIGURE 4. Binding of DBP to neutrophil plasma membranes. A,A Triton X-100-insoluble pellet was significantly greater than control (p Ͻ total of 10 ␮g of plasma membranes was incubated in HBSS-0.1% BSA 0.001) for 0.2, 0.3, and 0.5 mM PMSF-treated cells. containing 100 nM 125I-DBP for the designated times, either at 37°C or at

2°C (ice). The samples then were separated by vacuum filtration and http://www.jimmunol.org/ washed, and the filters were counted for radioactivity. Data are expressed Finally, the effect of elastase inhibitors on the ability of neutro- as fmol DBP bound per ␮g membrane protein. Data represent the mean of phils to generate C5a co-chemotactic activity from DBP was ex- seven separate experiments using membrane preparations from different Ͻ ␮ amined. Previously, we reported that cell surface binding of DBP neutrophil donors. The SEM of all samples was 1 fmol/ g; therefore, the ␮ is temporally correlated with the generation of C5a co-chemotactic error bars are not noticeable in the figure. B, A total of 10 g of membranes in HBSS-0.1% BSA was preincubated for the designated times at 37°C. activity (26). Therefore, if the cell surface binding of DBP is per- After the preincubation period, 100 nM 125I-DBP was added and the sam- turbed, it follows that the co-chemotactic activity should be al- ples were incubated for 20 min on ice (2°C). The samples then were sep- tered. Fig. 6 demonstrates that pretreatment of neutrophils with arated by vacuum filtration and washed, and the filters were counted for either small synthetic chloromethyl ketone-based inhibitors radioactivity. Data are expressed as a percentage of DBP bound to the by guest on September 26, 2021 (AAPA-CMK, AAPV-CMK), or an endogenous protein inhibitor control membranes (15 Ϯ 2.4 fmol DBP bound per ␮g membrane protein), (SLPI) prevents the generation of C5a co-chemotactic activity which were not preincubated at 37°C. Data represent the mean ϩ SEM of from DBP. In contrast, inhibitors of neutrophil (Z- three experiments using membrane preparations from different neutrophil ␣ donors. GLF-CMK and 1-antichymotrypsin) had no effect on co-chemo- tactic activity. None of the inhibitors altered neutrophil chemotaxis to an optimal concentration of C5a (1 nM); both control and in- hibitor-treated cells migrated an average of 65 Ϯ 4 ␮m/30 min. (26, 49). Furthermore, neutrophils (as well as monocytes, U937 These results indicate that the steady state binding of DBP is es- cells, and HL-60 cells) not only bind DBP, but spontaneously shed sential for co-chemotactic activity, and further suggest that neu- the protein into the extracellular media (26) (R.R.K., unpublished trophil elastase may play a critical role in the C5a co-chemotactic observations). Therefore, the aim of the present study was to in- mechanism. vestigate the regulation of DBP binding to human neutrophils. The results reported in this study show that neutrophil elastase Discussion controls the amount of DBP bound to cells by shedding its binding The current level of understanding of extracellular chemoattractant site. A previous report has shown that the binding of 125I-DBP (at regulatory factors has lagged far behind that of the chemotactic 2°C) to intact neutrophils or plasma membranes is nonsaturable vs factors they regulate. Moreover, the physiological significance of increasing concentration of the ligand up to 5 ␮M (27). However, these factors is not widely appreciated. Indeed, the initial descrip- at 37°C, the cellular levels of DBP, at any single concentration, tions of a C5a co-chemotactic factor in serum were reported more will plateau with time (Fig. 2) (26), probably reflecting a steady than 20 years ago (45–47). Identification of DBP as the serum- state between binding and shedding of DBP on the plasma mem- derived C5a co-chemotactic factor was described in 1988 (11, 12), brane. Moreover, inhibition of a serine protease (i.e., elastase), by and subsequently confirmed by several other groups (13–16). inhibitors or low temperature, disrupts the balance, allowing DBP However, the mechanism by which DBP enhances chemotaxis to to accumulate on the cell surface bound to a chondroitin sulfate C5a is still unknown. DBP does not alter neutrophil C5a receptor proteoglycan (Figs. 2 and 3) (27). There are several pieces of ev-

number or Kd for C5a (16, 48) (R.R.K., unpublished observations), idence to indicate that elastase cleaves the binding site rather than thereby discounting the most obvious explanation for its co-che- DBP. First, Fig. 1B demonstrates that DBP shed from the cell motactic effect. We believe that the key to uncovering the co- surface does not have a reduction in its m.w., suggesting that it is chemotactic mechanism first lies in understanding how DBP in- not degraded by cell surface proteases. Second, we often have teracts with its plasma membrane binding site. It is clear that DBP observed that DBP (radiolabeled or unlabeled) is remarkably re- needs to be bound to its cell surface binding site (a chondroitin sistant to neutrophil-mediated proteolysis (R.R.K., unpublished sulfate proteoglycan) to mediate the co-chemotactic effect for C5a observations), despite the fact that its primary sequence contains 2692 ELASTASE REGULATES DBP BINDING TO NEUTROPHILS

FIGURE 5. Binding of DBP to plasma membranes in the presence of FIGURE 6. Effect of protease inhibitor pretreatment on the generation various protease inhibitors. A total of 10 ␮g of plasma membranes was of C5a co-chemotactic activity from DBP. Neutrophils (4 ϫ 106/ml) in incubated in HBSS-0.1% BSA containing 100 nM 125I-DBP with either no chemotaxis buffer were pretreated for 15 min at 22°C with either buffer protease inhibitors or one of the following: PMSF (0.5 mM), Pefabloc SC ␮ ␮ ␮ ␮ (control); 50 M AAPV-CMK, AAPA-CMK, or Z-GLF-CMK; or 3 M (0.5 mM), AAPV-CMK (50 M), AAPA-CMK (50 M), Z-GLF-CMK ␣ SLPI or 1-antichymotrypsin (ACT). Cells then were added to the upper (50 ␮M), tosyl-L-phenylalanine-chloromethyl ketone (50 ␮M), tosyl-L-ly- compartments of the chemotaxis chamber in either the presence or absence sine-chloromethyl ketone (50 ␮M), FPR-CMK (50 ␮M), FFR-CMK (50 (control) of 50 nM DBP. Cell movement to 5 pM C5a alone then was ␮M), chymostatin (50 ␮M), 1,10-phenanthroline (200 ␮M), E-64 (50 ␮M), Downloaded from assayed for 30 min at 37°C. Numbers represent mean Ϯ SEM of three and SLPI (3 ␮M). Incubations were for 20 min at 37°C. The samples then experiments using cells from different donors. The C5a ϩ DBP values for were separated by vacuum filtration and washed, and the filters were control, ␣ ACT, and Z-GLF-CMK are significantly greater (p Ͻ 0.01) counted for radioactivity. Data are expressed as a percentage increase of 1 than all other samples. DBP bound over the control membranes (11 Ϯ 2.1 fmol DBP bound per ␮g membrane protein), which were not treated with inhibitors. Data represent the mean ϩ SEM of three to five separate experiments using membrane

substrate specificity as elastase and is inhibited by many of the http://www.jimmunol.org/ preparations from different neutrophil donors. Values for SLPI, PMSF, Pefabloc, AAPA-CMK, and AAPV-CMK are significantly greater (p Ͻ same reagents. To discriminate between these two proteases, SLPI 0.01) than all other samples. and AAPA-CMK were employed. Both are effective inhibitors of elastase, but do not inhibit proteinase 3 (54, 55). Therefore, if proteinase 3 were responsible for degrading the DBP binding site, several potential elastase and cathepsin G cleavage sites (50). SLPI and AAPA-CMK should have shown no increase in binding Third, Fig. 4B verifies that elastase constitutively degrades the over the untreated control. SLPI was the most effective inhibitor at binding site. Finally, we have reported earlier that purified elastase preventing degradation of the DBP-binding capacity of plasma treatment of neutrophil plasma membranes could reduce the DBP- membranes. Perhaps this is because the low molecular mass SLPI binding capacity by Ͼ90%; however, purified cathepsin G had no (11.7 kDa) is able to inhibit membrane-bound elastase (35, 56). by guest on September 26, 2021 effect on DBP binding (27). Clearly, DBP binding to cells is not mediated by a specific high Several reports have demonstrated that the shedding of cell sur- affinity receptor, but rather by nonselective low affinity binding face molecules is often mediated by more than one protease, in- sites, such as proteoglycans (27) and clearance/scavenger receptors cluding a combination of serine and metalloenzymes (32, 31). Fig. (28). Furthermore, multiple DBP molecules probably bind per 1A shows that PMSF or Pefabloc treatment does not completely mole of binding site (proteoglycan), and we have shown that DBP prevent DBP shedding, perhaps suggesting other enzymes act on bound to neutrophil plasma membranes can oligomerize (27). the binding site. However, we feel that this incomplete inhibition However, there is some selectivity in the interaction of DBP with of shedding is due to the nature of the protease inhibitors (Pefabloc neutrophils because its binding characteristics are distinctly differ- and PMSF) and the use on intact neutrophils. Pefabloc SC is a ent from that of human albumin (data not shown) (26), even water-soluble molecule that inhibits proteases in aqueous solution though DBP is part of the albumin gene family and both proteins much more effectively than membrane-bound enzymes. Con- share considerable amino acid and structural similarity (50). Neu- versely, PMSF is unstable in aqueous solutions at pH 7.4 and 37°C trophils bind 5-fold less albumin than DBP (26). Moreover, PMSF (51). Perturbation of neutrophils, such as the purification protocol treatment has no effect on the total amount of albumin bound to followed by a 60-min incubation at 37°C, can induce a fusion of cells and does not cause a redistribution of the protein into the intracellular granules with the plasma membrane, which releases detergent-insoluble fraction (Figs. 2 and 3, and data not shown). proteases from their internal stores (52, 53). Thus, during the 60- The precise identity of the chondroitin sulfate proteoglycan that min incubation at 37°C, there was probably an up-regulation of binds DBP is not known, although it is possible that DBP could surface-bound elastase from intracellular stores that was not in- bind to several different chondroitin sulfate proteoglycans or per- hibited and subsequently degraded the DBP binding site. haps any glycosaminoglycan-containing macromolecule. Cur- The panel of protease inhibitors employed in Fig. 5 clearly im- rently, this is an area of active investigation in our laboratory. plicates elastase as the serine protease mediating the shedding of It is not clear how DBP may induce a co-chemotactic response the DBP binding site. Selective elastase inhibitors AAPA-CMK to C5a, but it may involve proteoglycan-mediated clustering sig- and AAPV-CMK as well as SLPI were effective in preventing the naling components on the cytosolic side of the plasma membranes loss of DBP-binding capacity of neutrophil plasma membranes. (57). DBP binding and shedding on the neutrophil plasma mem- Moreover, the inhibitors that were not effective at deterring DBP brane correlate temporally with generation (binding) and decay shedding also do not inhibit elastase. Inhibitors specific for cathep- (shedding) of C5a co-chemotactic activity (26). One might spec- sin G (Z-GLF-CMK), kallikrein (FFR-CMK), and thrombin (FPR- ulate that DBP binds to elastase-rich, proteoglycan microdomains CMK) could not prevent shedding. Aprotinin, an effective plasmin on the extracellular face of the plasma membrane and triggers an and kallikrein inhibitor, also was used, but had no effect (data not assembly of intracellular signaling components that facilitates shown). Proteinase 3 (also known as myeloblastin) has a similar C5a-induced chemotaxis. Elastase may function to terminate the The Journal of Immunology 2693 signal by shedding the DBP binding site complex, and thus permit C5a-mediated chemotaxis, degranulation and respiratory burst. Mol. Immunol. a constant dynamic interaction between DBP and its cell surface 36:885. 17. Zwahlen, R. D., and D. R. Roth. 1990. Chemotactic competence of neutrophils binding site. 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Vitamin D binding protein (Gc-globulin). strated recently that elastase localizes to the migrating front (pseu- Endocr. Rev. 10:294. 21. Cleve, H., and J. Constans. 1988. The mutants of the vitamin-D-binding protein: dopod) of neutrophils responding to a gradient of platelet-activat- more than 120 variants of the GC/DBP system. Vox Sang. 54:215. ing factor (37). Chemoattractant receptors, including the C5a 22. Haddad, J. G. 1995. Plasma vitamin D-binding protein (Gc-globulin): multiple receptor, have been shown to cluster in the migrating front of tasks. J. Steroid Biochem. Mol. Biol. 53:579. 23. McLeod, J. F., M. A. Kowalski, and J. G. Haddad. 1986. Characterization of a leukocytes (61–63). However, we have never observed DBP-C5a monoclonal antibody to human serum vitamin D binding protein (Gc globulin): receptor complexes by either co-immunoprecipitation or chemical recognition of an epitope hidden in membranes of circulating monocytes. Endo- crinology 119:77. cross-linking (R.R.K., unpublished observations), suggesting that 24. Gouth, M., A. Murgia, R. M. Smith, M. B. Prystowsky, N. E. Cooke, and the two proteins do not interact. Nevertheless, the possibility that J. G. Haddad. 1990. Cell surface vitamin D-binding protein (Gc-globulin) is DBP induces membrane clustering, without interacting with the acquired from plasma. Endocrinology 127:2313. 25. Kew, R. R., M. A. Sibug, J. P. Liuzzo, and R. O. Webster. 1993. Localization and C5a receptor, remains to be tested. quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils. Downloaded from DBP is a ubiquitous protein in vivo; it has been detected in Blood 82:274. almost all body fluids at levels capable of inducing co-chemotaxis 26. Kew, R. R., J. A. Fisher, and R. O. Webster. 1995. Co-chemotactic effect of Gc-globulin (vitamin D binding protein) for C5a: transient conversion into an to C5a (20). 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