CXC Chemokine Ligand 16 Regulates the Cell Surface Expression of 10

A Disintegrin and Metalloproteinase 10-Mediated Cleavage and Shedding Regulates the Cell Surface Expression of CXC Chemokine Ligand 16 This information is current as of October 2, 2021. Peter J. Gough, Kyle J. Garton, Paul T. Wille, Marcin Rychlewski, Peter J. Dempsey and Elaine W. Raines J Immunol 2004; 172:3678-3685; ; doi: 10.4049/jimmunol.172.6.3678

http://www.jimmunol.org/content/172/6/3678 Downloaded from

References This article cites 35 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/172/6/3678.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 October 2, 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Disintegrin and Metalloproteinase 10-Mediated Cleavage and Shedding Regulates the Cell Surface Expression of CXC Chemokine Ligand 16

Peter J. Gough,2* Kyle J. Garton,* Paul T. Wille,* Marcin Rychlewski,* Peter J. Dempsey,† and Elaine W. Raines*

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identiﬁed as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and ,bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4؉ and CD8؉ T cells through binding its receptor

CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms Downloaded from of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression

of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through http://www.jimmunol.org/ the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identiﬁcation of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule. The Journal of Immunology, 2004, 172: 3678–3685.

he scavenger receptor for phosphatidylserine and oxi- PSOX is expressed by macrophages in vitro and in atherosclerotic dized low-density lipoprotein (SR-PSOX)3 and CXC che- lesions in vivo (1, 5), suggesting that SR-PSOX activity may be mokine ligand (CXCL)16 were independently identified involved in the massive accumulation of cellular cholesterol dur- T by guest on October 2, 2021 as a scavenger receptor and chemokine, respectively, but have ing the generation of macrophage foam cells associated with ath- since been shown to be identical (1Ð3). SR-PSOX was identified erosclerotic lesion development (6). Similar to other members of through an expression cloning strategy designed to identify recep- the scavenger receptor family, SR-PSOX has recently been shown tors that could mediate cell adhesion to phosphatidylserine-coated to mediate the uptake of Gram-positive and -negative bacteria surfaces (1). It was subsequently shown to bind and internalize when expressed by macrophages and dendritic cells, indicating oxidized low-density lipoprotein (OxLDL) (1), making it a mem- that this receptor may play a role in innate immunity and initiation ber of the structurally diverse scavenger receptor family of cell of the acquired immune response (7, 8). surface receptors that is defined by the ability to recognize mod- CXCL16 was independently identified by two groups as the li- ified low-density lipoprotein (4). Further analysis showed that SR- gand for the orphan G-protein-coupled chemokine receptor Bonzo/ CXCR6 (2, 3). CXCL16 is the second transmembrane chemokine identified to date, and bears significant structural homology to frac- *Department of Pathology, University of Washington, Harborview Medical Center, talkine/CX3C chemokine ligand (CX3CL)1 (9, 10). A combination Seattle, WA 98104; and †Pacific Northwest Research Institute, Seattle, WA 98122 of immunohistochemistry and FACS analysis showed that Received for publication October 8, 2003. Accepted for publication January 13, 2003. CXCL16 is selectively expressed by APCs, including DCs, mac- The costs of publication of this article were defrayed in part by the payment of page rophages, and B cells (2, 3). When expressed by macrophages, charges. This article must therefore be hereby marked advertisement in accordance soluble CXCL16 is released into the medium and has chemoat- with 18 U.S.C. Section 1734 solely to indicate this fact. tractant activity that is mediated solely through the CXCR6 recep- 1 This work was supported by National Institutes of Health Grants HL18645 and tor. CXCR6 is expressed by many cell types including naive CD8ϩ HL67267 (to E.W.R.) and DK59778 and DK63363 (to P.J.D.), a postdoctoral fel- T cells, NK T cells, and a subset of memory CD4ϩ T cells, al- lowship from the American Heart Association (to P.J.G.), and the Paul G. Allen ϩ ϩ Foundation for Medical Research (to K.J.G.). though only activated CD4 and CD8 T cells appear to migrate 2 Address correspondence and reprint requests to Dr. Peter J. Gough at the current ad- strongly to the soluble chemokine (2, 11). Furthermore, CXCL16- ϩ dress: Atherosclerosis Department, Medicines Research Centre, GlaxoSmithKline, Gun- positive cells in the spleen were seen in close opposition to CD8 nels Wood Road, Stevenage SG1 2NY, U.K. E-mail address: [email protected] T cells, suggesting that, similar to CX3CL1, CXCL16 may act as 3 Abbreviations used in this paper: SR-PSOX, scavenger receptor for phosphatidyl- an intercellular adhesion molecule when expressed on the cell sur- serine and oxidized low-density lipoprotein; CXCL, CXC chemokine ligand; OxLDL, face (2, 12, 13). oxidized low-density lipoprotein; CX3CL, CX3C chemokine ligand; ADAM, a disintegrin and metalloproteinase; ThioM␾, thioglycolate-elicited peritoneal macro- Given the potential distinct functional activities of membrane- phage; BMDM, bone marrow-derived macrophage; m, murine; HA, hemagglutinin; bound CXCL16 as a scavenger receptor, and soluble CXCL16 as siRNA, small interfering RNA; RIPA, radioimmunoprecipitation assay; SIN, self inactivating; LPA, lysophosphatidic acid; GPCR, G-protein-coupled receptor; EGF, a chemokine, the mechanisms that regulate the conversion between epidermal growth factor. these two forms would appear to be important for determining the

role played by this molecule in vivo. We have previously shown transduction, 2 ϫ 105 cells were plated per well of a six-well plate 24 h before a 10-h incubation with retroviral supernatants containing 4 ␮g/ml Polybrene that membrane-bound CX3CL1 can be proteolytically cleaved from the cell surface by at least two distinct metalloproteinases (Sigma-Aldrich). Transduction efﬁciency was enhanced by centrifuging plates at 1700 ϫ g for2hat37¡C at the beginning of the 10-h incubation period. (14). We identiﬁed a disintegrin and metalloproteinase (ADAM) After transduction, retroviral supernatant was replaced with fresh medium, and family member ADAM17 as the protease responsible for stimu- cells were allowed to recover for at least 48 h before use in subsequent ex-

lated shedding of CX3CL1 (14, 15), whereas constitutive release of periments. For puromycin selection, cells were cultured in the presence of 15 ␮g/ml puromycin for 48 h following recovery from transduction. CX3CL1 has subsequently been shown to be mediated by ADAM10 (16). Given the structural similarity between CX3CL1 and CXCL16, we have examined whether similar proteolytic CXCL16 shedding assays mechanisms are responsible for generating soluble CXCL16. In Macrophages (RAW-264, BMDM, and ThioM␾) were plated at a density this study, we demonstrate that CXCL16 is synthesized as an in- of 2.5 ϫ 106 cells per 60-mm dish, and dermal fibroblasts at a density of tracellular precursor that is rapidly transported to the cell surface 5 ϫ 105 cells per 60-mm dish in complete growth medium 24 h before where it undergoes metalloproteinase-dependent cleavage. By ma- stimulation. Cells were pretreated for 20 min by addition of GM6001 (50 ␮ nipulating the functional expression of ADAM10, we show that it M final) or DMSO vehicle control directly to the culture medium. Cells were washed with serum-free medium and stimulated with 2 ml of serum- is responsible for the constitutive shedding of CXCL16, and that free medium with or without PMA (100 ng/ml), GM6001 (50 ␮M), or this proteolytic activity is a key regulator of CXCL16 cell surface DMSO control, followed by incubation at 37¡C for 30 min or the indicated expression. This identification of ADAM10 as a major protease time. Following stimulation, supernatants were removed, and cells were responsible for the conversion of CXCL16 from a membrane- washed once with cold PBS and subsequently lysed with 1 ml of radio- bound scavenger receptor to a soluble chemoattractant should help immunoprecipitation assay (RIPA) buffer (PBS, 1% Nonidet P-40, 0.5%

sodium deoxycholate, 0.1% SDS, 2 ␮g/ml aprotinin, 2 ␮g/ml leupeptin, 1 Downloaded from elucidate the physiological function of this molecule. ␮g/ml pepstatin, and 100 ␮g/ml PMSF). Resulting cell supernatants and lysates were cleared by centrifugation at 15,000 ϫ g and stored at Ϫ20¡C Materials and Methods until analysis. CXCL16 concentrations in cellular lysates and supernatants Cell culture and reagents were determined by ELISA using a rat monoclonal capture Ab, a biotinylated goat polyclonal detection Ab, and an rCXCL16 standard (R&D Sys- The murine macrophage cell line RAW-264.7 was maintained in RPMI tems). The levels of CXCL16 present in medium or in detergent extracts 1640 supplemented with 10% FCS. Thioglycolate-elicited peritoneal mac- were determined for triplicate dishes and are reported as the average Ϯ SD. rophages (ThioM␾) were isolated by peritoneal lavage with PBS from http://www.jimmunol.org/ C57BL/6 mice injected 4 days previously with 1 ml of 3% thioglycolate i.p., and were cultured in RPMI 1640 plus 10% FCS. Bone marrow-derived Protein analysis, metabolic labeling, and immunoprecipitation macrophages (BMDMs) were generated by culturing bone marrow cells in RMPI 1640 containing 10% FCS and 2000 U/ml recombinant human M- Cells were plated and stimulated as described above. Poststimulation, cells were washed twice with PBS and lysed in 350 ␮l of RIPA buffer for 30 min CSF (R&D Systems, Minneapolis, MN) as previously described (17). Der- ϫ mal ﬁbroblasts expressing an IFN-␥-inducible, temperature-sensitive SV40 on ice. Cell lysates were cleared by centrifugation at 15,000 g for 10 large T Ag transgene, and lacking functional expression of ADAM9 or min, and protein concentrations were determined using the bicinchoninic ADAM17, were isolated as previously described (18). These cells were acid protein assay (Pierce, Rockford, IL). Lysates were separated by SDS- maintained at 32¡C in DMEM plus 10% FCS plus 5 U/ml recombinant PAGE under reducing conditions, transferred to Immobilon PVDF mem- ␥ branes (Millipore, Bedford, MA), and subsequently immunoblotted with

murine (rm)IFN- (R&D Systems). For experiments, dermal fibroblasts by guest on October 2, 2021 were plated in the absence of IFN-␥ and grown at 37¡C in DMEM plus specific Abs, before visualization by ECL (Amersham Pharmacia Biotech, 10% FCS for 24 h to arrest immortalization by the large T Ag. The fol- Piscataway, NJ). For metabolic labeling, cells were washed twice with PBS lowing Abs were used: polyclonal rabbit anti-hemagglutinin (HA) epitope and then incubated with RPMI 1640 lacking cysteine and methionine, supplemented with 10% dialyzed calf serum for 1 h. Cells were subsequently tag (Zymed Laboratories, San Francisco, CA), rat monoclonal and biotin- ␮ 35 35 ylated goat polyclonal anti-mCXCL16, rat monoclonal anti-mADAM10 labeled for 30 min with 500 Ci/ml Translabel [ S]Met and [ S]Cys (R&D Systems), and PE- and peroxidase-conjugated streptavidin, anti- (ICN, Irvine, CA), and chased for the indicated times in RPMI 1640 plus rabbit and anti-rat IgG (Jackson ImmunoResearch, West Grove, PA). PMA 10% FCS. Cells were washed twice with PBS and lysed as above, and CXCL16 was immunoprecipitated by overnight incubation with 5 ␮gof and all other chemicals not specified were from Sigma-Aldrich (St. Louis, ␮ MO). GM6001 was purchased from Elastin Products (Owensville, MO). anti-HA Ab (Zymed Laboratories) and 30 l of a 50% slurry of protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipi- Generation of retroviral expression and small interfering RNA tates were separated by 12% SDS-PAGE under reducing conditions and (siRNA) constructs and retroviral transduction visualized by autoradiography. For cell surface protein biotinylation, cells were washed in cold PBS and incubated where indicated with 1 mg/ml All constructs were generated using standard molecular biology tech- NHS-LC-biotin (Pierce) in PBS for 45 min on ice. Labeling reagent was niques, and were verified by DNA sequencing. A cDNA-encoding murine quenched with 0.1 M glycine, and cells were lysed in RIPA buffer as CXCL16 with a cytoplasmic, C-terminal HA epitope tag was amplified by above. Where indicated, cell extracts were incubated with 100 ␮lofa50% RT-PCR using primers containing a 5Ј BamHI site and a 3Ј HA epitope tag slurry of agarose-streptavidin for1hat4¡C followed by centrifugation at and a NotI site, and cloned into the retroviral expression vectors pBM- 2500 ϫ g to remove biotinylated proteins. Equal volumes of the resulting IRES-EGFP (19) and pBM-IRES-PURO (20). An expression construct for cell extracts were separated by SDS-PAGE and immunoblotted with an murine ADAM10 was generated by cloning an RT-PCR-amplified cDNA anti-HA Ab as above. containing 5Ј BamHI and 3Ј NotI sites into the pBM-IRES-PURO retroviral expression vector. PCR mutagenesis was subsequently used to mutate Glu385ϾAla, generating a catalytically inactive ADAM10 mutant FACS analysis (ADAM10 EϾA). A 315-bp fragment of the mouse U6 gene promoter was Cell surface levels of CXCL16 were determined by staining cells stimu- PCR amplified from genomic DNA using primers to incorporate 5Ј XhoI lated with PMA in the presence or absence of GM6001 as described above, and BamHI sites, a PmeI site at the transcriptional start site and 3Ј EcoRI with a rat monoclonal anti-CXCL16 Ab followed by PE-conjugated anti- and HindIII sites. The fragment was cloned into the vector LZRS-SIN- rat IgG secondary Ab. CXCL16 expression was measured by flow cytom- CD68L-HA-EGFP (21), which had been digested with XhoI and HindIII to etry using a FACScan (BD Biosciences, San Jose, CA) flow cytometer and remove the CD68-HA-EGFP expression cassette, generating the plasmid data analysis with CellQuest software. Relative CXCL16 cell surface ex- SIN-U6. Complementary oligonucleotides encoding an siRNA hairpin pression is reported as the average mean fluorescence intensity of triplicate with a 9-bp loop against nt 609Ð627 of mouse ADAM10 cDNA were samples Ϯ SD. annealed and cloned via the PmeI and EcoRI sites of SIN-U6 to generate the plasmid SIN-U6 ADAM10. Further details of the sequences of the oligonucleotides and plasmids used for cloning are available on request. High-titer RT-PCR analysis of ADAM expression retroviral supernatants were prepared by calcium phosphate-mediated trans- fection of Phoenix amphotropic packaging cells (generously provided by G. Total RNA was isolated from macrophage populations (RAW-264, Nolan (Stanford University, Stanford, CA)) as previously described (19). For BMDM, and ThioM␾) using TRIzol reagent (Invitrogen, Carlsbad, CA) 3680 SHEDDING OF CXCL16 IS MEDIATED BY ADAM10 according to the manufacturer’s recommended protocol. RNA was reverse detected by the Ab against the cytoplasmic tail HA epitope tag, but transcribed using Moloney murine leukemia virus reverse transcriptase and not by the Ab against the CXCL16 extracellular domain, suggest- an oligo(dT) primer (Invitrogen), with reactions set up in the presence and ing that it was generated following proteolytic cleavage of full- absence of reverse transcriptase to control for genomic DNA contamina- tion. The cDNA obtained served as a template for PCR using the oligo- length CXCL16 (Fig. 1B). Conditioned cell medium contained a nucleotide pairs specific for murine ADAM2, -8, -9, -10, -12, -15, and -17 single ϳ40-kDa CXCL16 species that could be detected only with (sequence of primers are available on request). PCR products were sepa- an Ab against the extracellular domain of CXCL16, again indicat- rated by electrophoresis on a 1.2% agarose gel and visualized by ethidium bromide staining. ing that it was generated through the juxtamembrane cleavage of full-length CXCL16 (Fig. 1B). Results To characterize further the maturation and processing of Rapid maturation, processing, and release of murine CXCL16 CXCL16, we performed pulse chase analysis of RAW-264 cells expressing murine CXCL16 (Fig. 1D). CXCL16 is synthesized as CXCL16 can exist as two functionally distinct forms: a cell-asso- 28- and 35-kDa precursors that undergo rapid maturation, presum- ciated form and a soluble form that has been proposed to arise ably by glycosylation, to yield 55-kDa mature CXCL16 (Fig. 1D). from processing of the membrane-bound protein (2, 3). To study the relationship between these two forms, we expressed murine These precursors can also be detected under steady-state condi- CXCL16 containing a cytoplasmic, C-terminal HA epitope tag tions through Western blotting of cell lysates (Fig. 1B). Mature (Fig. 1A) in the murine macrophage cell line RAW-264.7, which CXCL16 disappears very rapidly from the cell, with a half-life of ϳ does not express endogenous CXCL16 (B). Western blot analysis 45 min. The disappearance of CXCL16 is associated with the gen- of detergent cell extracts using Abs recognizing either the eration of a 15-kDa fragment that can be immunoprecipitated with an CXCL16 extracellular domain or the HA epitope tag revealed mul- Ab against the C-terminal HA epitope tag, although this fragment Downloaded from tiple CXCL16 protein species (Fig. 1B). Both Abs recognized a does not accumulate within the cell (Fig. 1D). These data suggest that ϳ55-kDa form that has previously been suggested to represent CXCL16 is initially synthesized as an intracellular precursor that un- mature CXCL16 protein (2). Similar Western blots performed after dergoes very rapid glycosylation and transport to the cell surface as a depletion of cell surface proteins through their biotinylation and 55-kDa glycoprotein. This mature CXCL16 protein can then be incubation with agarose-streptavidin confirmed that this 55-kDa released from the cell surface, yielding a soluble 40-kDa fragment that form was the only CXCL16 species present at the cell surface (Fig. likely contains the majority of the glycosylated ectodomain and a http://www.jimmunol.org/ 1C). A band of ϳ15 kDa that migrated close to the gel front was 15-kDa transmembrane cytoplasmic tail fragment. by guest on October 2, 2021

FIGURE 1. CXCL16 is rapidly processed and released by murine macrophages. A, Schematic representation of cytoplasmic, C-terminally HA epitope- tagged CXCL16 used in this study. B, RAW-264 cells expressing HA epitope-tagged CXCL16 (RAW-CXCL16) or uninfected cells (RAW-WT) were cultured for2hinserum-free RPMI 1640. Conditioned medium (SUP) and cell lysates (LYS) were analyzed by Western blotting with Abs against the cytoplasmic HA epitope tag (␣-HA) or the extracellular domain (␣-CXCL16). Mature (MAT), precursor (PRE), a C-terminal fragment (CTF), and soluble (SOL) species were detected as shown. C, Cell surface proteins of RAW-CXCL16 cells were biotinylated with NHS-LC-biotin where indicated, lysed with RIPA buffer, and incubated in the presence or absence of agarose-streptavidin to remove biotinylated proteins. A total of 25 ␮l of the resulting cleared lysates were separated by SDS-PAGE and immunoblotted with an anti-HA Ab. D, Cells were labeled for 30 min with 500 ␮Ci/ml [35S]Met,Cys and chased for the indicated times in RPMI 1640 plus 10% FCS. Cells were lysed, and CXCL16 was immunoprecipitated with an anti-HA Ab and analyzed by SDS-PAGE and autoradiography. The Journal of Immunology 3681

Metalloproteinase-mediated shedding of CXCL16 regulates its of this increase was somewhat variable, and was always signifi- cell surface expression cantly less than we have previously observed for CX3CL1 (14). To examine the consequences of both constitutive and PMA- It has recently been shown that the constitutive release of CX3CL1 is mediated by ADAM10, and this can be inhibited by broad- induced shedding on the expression of CXCL16, we used flow spectrum metalloproteinase inhibitors (14Ð16). Given the struc- cytometry to measure the levels of cell surface CXCL16 (Fig. 2C). PMA stimulation caused a modest, but reproducible, decrease in tural similarity of CX3CL1 and CXCL16, we sought to determine whether the constitutive shedding of CXCL16 was also mediated the cell surface expression of CXCL16. In contrast, inhibition of by a metalloproteinase. When expressed by RAW-264 cells, constitutive CXCL16 shedding, through incubation of cells with CXCL16 is constitutively released at a constant rate as determined GM6001, led to an almost doubling of CXCL16 expression on the by ELISA measurements of CXCL16 present in cell supernatants cell surface. This increase was also seen in cells stimulated with (Fig. 2A). Constitutive CXCL16 release could be efficiently inhib- PMA in the presence of GM6001. The significance of constitutive ited by a broad-spectrum zinc-dependent metalloproteinase inhib- shedding on the expression of CXCL16 was confirmed by Western itor, GM6001 (Fig. 2B), and this led to a corresponding increase in blot analysis of detergent cell extracts using Abs against extracel- the amount of cell-associated CXCL16 and a decrease in the ratio lular and intracellular CXCL16 epitopes (Fig. 2D). As seen with of soluble/cellular CXCL16 consistent with decreased CXCL16 FACS analysis, PMA stimulation slightly decreased levels of ma- shedding (Fig. 2B). ture, 55-kDa CXCL16, whereas pretreatment with GM6001 caused a significant increase in the amount of this form of the In addition to ADAM10-mediated constitutive release, CX3CL1 shedding can be markedly stimulated by phorbol esters such as protein. Taken together, these results support a model in which

PMA, a process that is mediated by another ADAM family pro- full-length mature CXCL16 at the cell surface is constitutively Downloaded from tease, ADAM17 (14, 15). To examine whether shedding of cleaved by a metalloproteinase to release the majority of the CXCL16 could be similarly enhanced, we measured CXCL16 in CXCL16 ectodomain, leaving a cell-associated cytoplasmic tail lysates and supernatants of RAW-264 cells expressing CXCL16 fragment. Furthermore, this metalloproteinase-mediated constitu- following stimulation with PMA in the presence or absence of tive cleavage appears to play an important role in determining the GM6001 (Fig. 2B). PMA stimulation led to a modest increase in cell surface expression of CXCL16. soluble CXCL16 levels, a concomitant slight decrease in cell-as- The data presented so far show that CXCL16 can be constitu- http://www.jimmunol.org/ sociated CXCL16, leading to an increase in the soluble/cellular tively released by a metalloproteinase when overexpressed in CXCL16 ratio (Fig. 2B). This enhanced shedding of CXCL16 in RAW-264 cells, and this shedding can be moderately enhanced by response to PMA stimulation could be effectively inhibited by stimulation with PMA. To determine whether this shedding was a GM6001 pretreatment. Although the enhanced shedding of consequence of high-level overexpression, we examined the re- CXCL16 induced by PMA was highly reproducible, the magnitude lease of endogenous CXCL16 in primary macrophages. ThioM␾ by guest on October 2, 2021

FIGURE 2. Metalloproteinase-mediated cleavage and shedding of CXCL16 regulates its cell surface expression in RAW-264 cells. A, The kinetics of the release of soluble CXCL16 from RAW-264 cells was examined by ELISA. Each point represents the mean Ϯ SD obtained from duplicate dishes, and the data are representative of two independent experiments. B, RAW-264 cells expressing CXCL16 were cultured for 30 min in RPMI 1640 with or without PMA (100 ng/ml) or GM6001 (50 ␮M) as indicated. CXCL16 concentrations in conditioned medium or detergent cell extracts were determined by ELISA. Data represent averaged values of duplicate dishes Ϯ SD, and are representative of three independent experiments. C, CXCL16 cell surface levels were measured by ﬂow cytometry in RAW-CXCL16 cells stimulated with PMA (100 ng/ml) in the presence or absence of GM6001 (50 ␮M). Data represent averaged values of triplicate samples Ϯ SD, and are representative of three independent experiments. D, RAW-264 cells expressing HA epitope-tagged CXCL16 were stimulated with PMA (100 ng/ml) in the presence or absence of GM6001 (50 ␮M). Cell lysates were analyzed by Western blotting with Abs against the cytoplasmic tail HA epitope tag (␣-HA) and the CXCL16 extracellular domain (␣-CXCL16). The blot shown is underexposed relative to Fig. 1B to highlight differences in the levels of expression of mature (MAT) CXCL16 protein. The positions of precursor (PRE) and C-terminal cleavage fragment (CTF) CXCL16 protein species are indicated based on their appearance in longer exposures of the same blot (data not shown). 3682 SHEDDING OF CXCL16 IS MEDIATED BY ADAM10

and BMDM both shed CXCL16 constitutively at a rate similar to that seen in RAW-264 cells, and this release can be inhibited by GM6001 (Fig. 3). PMA stimulation caused an increase in the shedding of CXCL16 by ThioM␾, and a more modest increase in BMDMs, although this was largely attributable to the differences in the rate of constitutive CXCL16 shedding. In both cell types, the PMA-induced component could be inhibited by GM6001, indicating that it was also mediated by metalloproteinase activity. Hence, shedding of endogenous and overexpressed CXCL16 appears to be mediated by similar mechanisms.

Macrophages express multiple protease domain-containing ADAMs

Previous studies examining the shedding of CX3CL1, in addition to the inhibition studies above, suggested that ADAM proteases were good candidates for the cleavage and shedding of CXCL16 FIGURE 4. Macrophages express multiple protease domain-containing ␾ (14Ð16). To examine which ADAM proteases were expressed by ADAMs. Total RNA was isolated from RAW-264, BMDM, and ThioM , macrophages that have the ability to shed CXCL16, we performed and reverse transcribed using Moloney murine leukemia virus reverse transcriptase and an oligo(dT) primer. The resulting cDNA served as a template RT-PCR analysis using primer pairs specific for multiple ADAM for PCR using primer pairs specific for ADAM2, -8, -9, -10, -12, -15, and -17. family members (Fig. 4). ThioM␾, BMDM, and RAW-264 cells Reaction products were separated on a 1.2% agarose gel and visualized by Downloaded from show a similar profile of ADAM expression, including no or low- ethidium bromide staining. No products were detectable from PCR using level expression of ADAM2 and -12, but significant expression of cDNA generated in the absence of reverse transcriptase (data not shown). ADAM8, -9, -10, -15, and -17, making these good candidates for the shedding of CXCL16. mal fibroblasts was also metalloproteinase mediated, as determined ADAM9 and ADAM17 do not appear to play a role in CXCL16 by experiments using GM6001 (data not shown). Despite some vari- shedding ability between experiments, we never observed any significant dif- http://www.jimmunol.org/ To begin to examine the role played by specific ADAM proteases, ferences in the constitutive or PMA-induced shedding of CXCL16 in we analyzed CXCL16 shedding in cells derived from ADAM-de- dermal fibroblasts lacking functional expression of ADAM9 or ficient mice. For these studies, we used immortalized dermal fi- ADAM17 (Fig. 5). This indicates that, unlike CX3CL1, ADAM17 broblasts isolated from mice obtained by crossing ADAM-defi- does not play a major role in the generation of soluble CXCL16. cient mice with transgenic mice expressing an IFN-␥-inducible Knockdown of ADAM10 expression by siRNA reduces and temperature-sensitive SV40 large T Ag allele (14, 18). Dermal constitutive shedding of CXCL16 fibroblasts derived from wild-type (DF-WT), or ADAM9 (DF- Given that the constitutive release of CX CL1 is mediated by 9KO)- and ADAM17 (DF-17KO)-deficient mice were transduced 3 by guest on October 2, 2021 with a retrovirus encoding CXCL16 with a C-terminal HA epitope ADAM10 (16), we next examined whether this protease also plays tag, and constitutive and PMA-inducible CXCL16 shedding was a role in the shedding of CXCL16. To modulate the expression of determined by ELISA. Wild-type dermal fibroblasts shed CXCL16 ADAM10, we took advantage of the ability of siRNAs to effi- constitutively at a rate similar to that seen in RAW-264 cells, and ciently reduce the expression of a target gene in mammalian cells this shedding could be modestly enhanced by stimulation with (22). To drive the expression of siRNAs, we developed a novel PMA (Fig. 5). Similar to macrophages, CXCL16 shedding by der- retroviral vector, SIN-U6, containing the RNA polymerase III U6 gene promoter in a self-inactivating (SIN) vector backbone, such that only the U6 promoter has transcriptional activity in transduced cells (Fig. 6A) (23). To generate an siRNA specific for ADAM10, we cloned in a DNA fragment encoding a dsRNA hairpin with a 9-bp loop targeting nt 609Ð627 of the ADAM10 coding sequence, which would be predicted to be processed by the RNase III enzyme Dicer to generate an active siRNA (24). To test the function of this retroviral vector, we used it to transduce dermal fibroblasts expressing CXCL16, and examined the expression of ADAM10 by Western blotting. Cells transduced with the empty SIN-U6 vector showed a similar level of ADAM10 expression to that of mock- transduced control cells (Fig. 6B). In contrast, cells transduced with the SIN-U6 ADAM10 vector showed a Ͼ90% reduction in ADAM10 protein levels (Fig. 6B), and this appeared specific for ADAM10, because the expression of ADAM17 remained unchanged (data not shown). The constitutive and PMA-induced shedding of CXCL16 in FIGURE 3. Shedding of endogenous CXCL16 by primary macrophages cells with reduced expression of ADAM10 was determined by is also mediated by metalloproteinases. ThioM␾ (A) or BMDM (B) were ELISA. Mock- and SIN-U6 vector-transduced cells showed a sim- cultured for 30 min in RPMI 1640 in the presence or absence of PMA (100 ng/ml) or GM6001 (50 ␮M) as indicated. CXCL16 concentrations in con- ilar constitutive and PMA-induced shedding of CXCL16, but there ditioned medium or detergent cell extracts were determined by ELISA, and was a significant reduction of 60% in the basal shedding of expressed as a ratio of CXCL16 present in the supernatants and lysates. CXCL16 by cells expressing the ADAM10 siRNA (Fig. 6C). Sim- Data are calculated from mean values derived from duplicate dishes, and ilar to other cell types, PMA-induced shedding of CXCL16 in cells are representative of three independent experiments. transduced with the SIN-U6 ADAM10 vector was variable, but The Journal of Immunology 3683

FIGURE 5. The absence of ADAM9 or ADAM17 does not affect the shedding of CXCL16. Dermal fibroblasts (DF) isolated from ADAM9Ϫ/Ϫ (DF-9KO) and ADAM17 ⌬Zn/⌬Zn mice (DF-17KO) crossed with transgenic mice overexpressing an IFN-␥-inducible, temperature-sensitive SV40 large T Ag allele (14, 18) were transduced with a CXCL16-IRES- PURO retrovirus. CXCL16 shedding was determined with and without Downloaded from PMA stimulation (100 ng/ml) by ELISA measurements of conditioned medium and detergent cell extracts, and is expressed as a ratio of CXCL16 present in the supernatants and lysates. Data are calculated from mean FIGURE 6. Knockdown of ADAM10 expression by siRNA signifi- values derived from duplicate dishes, and are representative of three inde- cantly reduces the constitutive shedding of CXCL16. A, Schematic repre- pendent experiments. sentation of the SIN retroviral construct (SIN-U6) used to drive expression of the siRNA hairpin specific for ADAM10. B, Wild-type dermal fibroblasts transduced with a CXCL16-IRES-EGFP retrovirus (DF CXCL16-I- http://www.jimmunol.org/ EGFP) were retransduced with unconditioned medium (Mock) or super- was still detected (Fig. 6D). To characterize the CXCL16 shedding natant containing the SIN-U6 retrovirus without an siRNA hairpin activity that remains in cells expressing the ADAM10 siRNA, we (SIN-U6 EMPTY) or with a hairpin specific for ADAM10 (SIN-U6 examined its sensitivity to GM6001. Both constitutive and PMA- ADAM10). Samples containing 30 ␮g of cell lysate were analyzed by inducible shedding of CXCL16 could be completely abolished by Western blotting using an Ab against murine ADAM10. Positions of the GM6001, suggesting that there was either incomplete inhibition of prodomain-containing (PRO) and mature (MAT) ADAM10 protein species ADAM10 function, or that there is/are other metalloproteinase(s) are indicated. C, CXCL16 shedding by the cells described in B was deter- mediating CXCL16 shedding (Fig. 6D). mined following a 30-min stimulation with and without PMA (100 ng/ml) by ELISA measurements of conditioned medium and detergent cell ex- Overexpression of catalytically active or inactive ADAM10 tracts, and is expressed as a ratio of CXCL16 present in the supernatants by guest on October 2, 2021 significantly alters the shedding and cell surface expression of and lysates. Data are calculated from mean values derived from duplicate CXCL16 dishes, and are representative of three independent experiments. D, CXCL16 shedding by DF CXCL16-I-EGFP cells that had been secondarily transduced To confirm the data obtained through the expression of an siRNA with SIN-U6 ADAM10 retrovirus was analyzed by ELISA following a 30-min against ADAM10, we examined CXCL16 shedding in dermal fi- stimulation in the presence or absence of PMA (100 ng/ml) or GM6001 (50 broblasts overexpressing wild-type ADAM10 or a catalytically in- ␮M) as indicated. Data are presented as described in C. active Glu385ϾAla ADAM10 mutant. We have previously shown that a similar catalytically inactive ADAM17 mutant has dominant-negative properties, inhibiting the shedding of CX3CL1 and control (Fig. 7C). These results were confirmed by Western blot VCAM-1, presumably through competition with endogenous en- analysis of detergent cell extracts using an Ab against the CXCL16 zyme for substrate, or key regulatory cytoplasmic factors (14, 18). extracellular domain (Fig. 7D). As seen with FACS analysis, over- Western blotting of transduced cells revealed significant overex- expression of ADAM10 decreased levels of mature, 55-kDa pression of both ADAM10 proteins when expressed using a pBM- CXCL16, whereas overexpression of the catalytically inactive IRES-PURO retroviral vector (Fig. 7A). Overexpression of wild- ADAM10 mutant caused a significant increase in the amount of type ADAM10 led to a Ͼ2-fold increase in the constitutive release this form of the protein. Taken together, these results demonstrate of CXCL16, but the relative increase in CXCL16 shedding in re- that ADAM10 plays a significant role in the constitutive cleavage sponse to PMA stimulation remained unchanged (Fig. 7B). In con- and shedding of CXCL16, and its activity is a key determinant of trast, overexpression of the catalytically inactive ADAM10 mutant CXCL16 cell surface levels. inhibited CXCL16 constitutive shedding by Ͼ50%, without affecting the relative magnitude of the PMA-induced increase in Discussion CXCL16 shedding. These results confirm the data from cells ex- It has previously been shown that CXCL16 can exist as either a pressing an siRNA against ADAM10, namely that ADAM10 con- membrane-anchored cell surface protein or as a soluble chemokine tributes significantly to the constitutive shedding of CXCL16. (1Ð3). In this study, we have shown that CXCL16 is synthesized as Having already shown that metalloproteinase activity regulates an intracellular precursor, undergoes rapid maturation, presumably the expression of CXCL16 on the cell surface, we wanted to ex- by glycosylation, followed by transport to the cell surface where it plore whether ADAM10-mediated shedding was responsible for quickly becomes a substrate for metalloproteinase-mediated cleav- these observations. Flow-cytometric analysis showed that overex- age and shedding. This high rate of basal shedding can be modestly pression of ADAM10 resulted in a significant decrease, whereas enhanced by the phorbol ester PMA through the activity of a met- catalytically inactive ADAM10 increased the cell surface levels of alloproteinase. Using a novel retroviral vector to drive the expres- CXCL16 compared with cells transduced with the empty vector sion of siRNAs, and overexpression of wild-type and catalytically 3684 SHEDDING OF CXCL16 IS MEDIATED BY ADAM10

down or the expression of dominant-negative ADAM10 (Figs. 6C and 7B). Indeed, the enhancement of CXCL16 cell surface expression seen in cells stimulated with PMA in the presence of GM6001 (Fig. 2C) suggests that the enhanced shedding seen in response to PMA may reflect an increase in intracellular protein trafficking rather than stimulation of a specific enzymatic activity. These apparent dif-

ferences in proteolytic specificity between CXCL16 and CX3CL1 means that domain-swap experiments of their juxtamembrane regions may prove valuable in determining the sequence and structural epitopes required for generating ADAM10 and ADAM17 substrates. Determining the physiological relevance of ADAM10-mediated shedding of CXCL16 will prove difficult given the current tools available. ADAM10-deficient mice die at day 9.5 of embryonic development with multiple defects of both the central nervous and cardiovascular systems (25), and this therefore precludes studies addressing the role of ADAM10-mediated shedding of CXCL16 in vivo. In an attempt to examine the significance of ADAM10-mediated CXCL16 shedding in macrophages in vitro, we used the various retroviral constructs described in this paper to enhance and inhibit ADAM10 function in the RAW-264 cell line. Despite the Downloaded from ability of these tools to modulate ADAM10 function in dermal fibroblasts, expression of either wild-type or catalytically inactive ADAM10 enzyme, or ADAM10 siRNA led to significant cell FIGURE 7. Overexpression of catalytically active or inactive ADAM10 death, and only a 10Ð20% increase or decrease in ADAM10 ex- significantly alters the shedding and cell surface expression of CXCL16. A, pression in surviving cells (data not shown). These observations Wild-type dermal fibroblasts transduced with a CXCL16-IRES-EGFP ret- were identical independent of the expression of CXCL16. We are http://www.jimmunol.org/ rovirus (DF CXCL16-I-EGFP) were retransduced with an IRES-PURO currently investigating the significance and mechanism of this ob- retrovirus encoding either wild-type ADAM10 (ADAM10), a catalytically servation, but it suggests that the precise level of ADAM10 activ- inactive Glu385ϾAla ADAM10 mutant (ADAM10 EϾA), or an empty ity is critical for survival in this macrophage cell line. vector control. Following selection in puromycin, samples containing 30 ␮g of cell lysate were analyzed by Western blotting using an Ab against Recent data have suggested that constitutive ectodomain shed- murine ADAM10. Positions of the prodomain-containing (PRO) and ma- ding observed in vitro may actually represent a form of induced ture (MAT) ADAM10 protein species are indicated. B, CXCL16 shedding shedding stimulated by the serum component lysophosphatidic by the cells described in A was determined following a 30-min stimulation acid (LPA) (26). Although definitive evidence linking LPA bind- with and without PMA (100 ng/ml) by ELISA measurements of condi- ing by its G-protein-coupled receptor (GPCR) to activation of by guest on October 2, 2021 tioned medium and detergent cell extracts, and is expressed as a ratio of ADAM10 proteolytic activity is lacking, LPA has been shown to CXCL16 present in the supernatants and lysates. Data are calculated from activate the shedding of known ADAM10 substrates including epi- mean values derived from duplicate dishes, and are representative of three dermal growth factor (EGF) and heparin-binding EGF (27Ð29). independent experiments. C, Cell surface levels of CXCL16 expressed by Other GPCR agonists have been shown to induce activation of the cells described in A were measured by flow cytometry following stain- ADAM10 proteolytic activity, including bombesin and platelet- ing with an anti-CXCL16 Ab. Data represent averaged values of triplicate samples Ϯ SD, and are representative of three independent experiments. D, activating factor, mediating receptor transactivation through the Expression of CXCL16 by the cells described in A was examined by West- shedding of EGF receptor ligands (30, 31). In addition to GPCR- ern blot analysis of 15 ␮g of cell lysate using an Ab against CXCL16. The mediated activation, ADAM10 proteolytic activity has also been mature form of CXCL16 is highlighted (MAT). shown to be regulated by cellular cholesterol levels. Depletion of plasma membrane cholesterol using the cholesterol acceptor methyl-␤-cyclodextrin, has been shown to stimulate ADAM10-medi- inactive enzyme, we identify ADAM10 as the predominant pro- ated shedding of the L1 adhesion molecule, the IL-6R, and the tease responsible for constitutive shedding of CXCL16. Further- amyloid precursor protein (32Ð34). Furthermore, inhibition of en- more, we show that ADAM10-mediated cleavage is a key regula- dogenous cholesterol biosynthesis by the 3-hydroxy-3-methylglu- tor of CXCL16 cell surface expression. taryl-CoA reductase inhibitor lovastatin, also stimulated ADAM10 Cleavage of CXCL16 by ADAM10 is consistent with its struc- proteolytic activity (33). We have also seen enhanced shedding of tural similarity to the other transmembrane chemokine identified to CXCL16 in response to treatment of RAW-264 cells with methyl-␤- date, CX3CL1, whose constitutive cleavage and shedding is also cyclodextrin (data not shown). Given the ability of CXCL16 to act as mediated by ADAM10 (16). However, unlike CX3CL1 where a scavenger receptor and internalize cholesterol from OxLDL, we are PMA stimulation induces a very robust increase in shedding, in- currently examining the intriguing possibility that CXCL16 cell sur- cubation of cells expressing CXCL16 with PMA only produced a face levels may be up-regulated by a positive-feedback loop due to the modest and variable enhancement of basal CXCL16 release (14). inhibition of ADAM10-mediated shedding caused by increased cel- Indeed, we could not detect any differences in constitutive or lular cholesterol during macrophage foam cell formation. PMA-induced shedding of CXCL16 in cells lacking functional We have shown that ADAM10 proteolytic activity is a key de-

ADAM17 expression, unlike our studies looking at CX3CL1 shed- terminant of CXCL16 cell surface expression, and therefore of its ding (14). Given the higher rate of basal, ADAM10-mediated ability to act as a scavenger receptor for modified low-density shedding of CXCL16 when compared with CX3CL1, it may be lipoprotein and bacteria. To attempt to address this issue, we have that any role for ADAM17 in CXCL16 shedding may be masked. examined the ability of RAW-264 cells expressing CXCL16 to However, PMA-stimulated shedding remained weak when internalize fluorescently labeled OxLDL in the presence or ab- ADAM10 function was inhibited through siRNA-mediated knock- sence of GM6001. To our surprise, overexpression of CXCL16 did The Journal of Immunology 3685 not augment the ability of these cells to endocytose OxLDL (data 11. Johnston, B., C. H. Kim, D. Soler, M. Emoto, and E. C. Butcher. 2003. Differ- ␣␤ not shown). Similar experiments performed using dermal fibro- ential chemokine responses and homing patterns of murine TCR NKT cell subsets. J. Immunol. 171:2960. blasts, which do not show detectable endogenous scavenger recep- 12. Haskell, C. A., M. D. Cleary, and I. F. Charo. 1999. Molecular uncoupling of tor activity, also failed to reveal any significant uptake of OxLDL fractalkine-mediated cell adhesion and signal transduction: rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation. J. Biol. Chem. by cells expressing CXCL16 (data not shown). In addition to the 274:10053. uptake of OxLDL, experiments looking at the phagocytosis of 13. Haskell, C. A., M. D. Cleary, and I. F. Charo. 2000. Unique role of the chemokine fluorescently labeled Escherichia coli by both RAW-264 cells and domain of fractalkine in cell capture: kinetics of receptor dissociation correlate with cell adhesion. J. Biol. Chem. 275:34183. dermal fibroblasts failed to show any enhanced uptake by cells 14. Garton, K. J., P. J. Gough, C. P. Blobel, G. Murphy, D. R. Greaves, overexpressing CXCL16, even when assays were done in the pres- P. J. Dempsey, and E. W. Raines. 2001. Tumor necrosis factor-␣-converting ence of GM6001 to maximize the cell surface expression of enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J. Biol. Chem. 276:37993. CXCL16 (data not shown). We are currently examining the rea- 15. Tsou, C. L., C. A. Haskell, and I. F. Charo. 2001. Tumor necrosis factor-␣- sons for the differences between our results and published data (1, converting enzyme mediates the inducible cleavage of fractalkine. J. Biol. Chem. 276:44622. 8), but it could represent variations in experimental conditions, the 16. Hundhausen, C., D. Misztela, T. A. Berkhout, N. Broadway, P. Saftig, K. Reiss, cell types used, and the species of CXCL16 being used. D. Hartmann, F. Fahrenholz, R. Postina, V. Matthews, et al. 2003. The disintegrin- In addition to affecting scavenger receptor activity, ADAM10- like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion. Blood 102:1186. mediated cleavage would be predicted to affect the function of 17. Gough, P. J., S. Gordon, and D. R. Greaves. 2001. The use of human CD68 CXCL16 as a chemokine at multiple levels. As a soluble form, transcriptional regulatory sequences to direct high-level expression of class A CXCL16 can act as a chemoattractant for CD4ϩ and CD8ϩ T cells scavenger receptor in macrophages in vitro and in vivo. Immunology 103:351. 18. Garton, K. J., P. J. Gough, J. Philalay, P. T. Wille, C. P. Blobel, R. H. Whitehead, bearing the receptor CXCR6 (2, 3), and therefore ADAM10-me- P. J. Dempsey, and E. W. Raines. 2003. Stimulated shedding of vascular cell Downloaded from diated shedding of membrane-bound CXCL16 would be predicted adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-␣-convert- to act as a regulator of CXCL16 chemoattractant bioactivity. Mem- ing enzyme (ADAM 17). J. Biol. Chem. 278:37459. 19. Hitoshi, Y., J. Lorens, S. I. Kitada, J. Fisher, M. LaBarge, H. Z. Ring, U. Francke, brane-bound CX3CL1 has been shown to act as an intercellular J. C. Reed, S. Kinoshita, and G. P. Nolan. 1998. Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells. Immunity 8:461. adhesion molecule for cells expressing the CX3CR1 receptor (12, 20. Garton, K. J., N. Ferri, and E. W. Raines. 2002. Efficient expression of exogenous 13). During the course of the review of this manuscript, Shimaoka genes in primary vascular cells using IRES-based retroviral vectors. BioTech- et al. (35) reported that CXCL16-CXCR6 interactions can simi- niques 32:830. larly mediate intercellular adhesion. Furthermore, they show that 21. Gough, P. J., and E. W. Raines. 2003. Gene therapy of apolipoprotein E-deficient http://www.jimmunol.org/ mice using a novel macrophage-specific retroviral vector. Blood 101:485. these adhesive interactions can be strengthened by increasing the cell 22. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. surface expression of CXCL16, suggesting that ADAM10-mediated 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured CXCL16 shedding may regulate these adhesive properties as is seen mammalian cells. Nature 411:494. 23. Yu, S. F., T. von Ruden, P. W. Kantoff, C. Garber, M. Seiberg, U. Ruther, for CX3CL1 (16). Definitive answers regarding these in vivo func- W. F. Anderson, E. F. Wagner, and E. Gilboa. 1986. Self-inactivating retroviral tions of CXCL16 as a scavenger receptor and chemokine will be vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. provided by analyses of CXCL16-deficient mice. Irrespective of the Acad. Sci. USA 83:3194. 24. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. A system for stable true in vivo role of CXCL16, ADAM10-mediated cleavage is likely expression of short interfering RNAs in mammalian cells. Science 296:550. to act as an important regulator of CXCL16 function. 25. Hartmann, D., B. de Strooper, L. Serneels, K. Craessaerts, A. Herreman, W. Annaert, L. Umans, T. Lubke, A. Lena Illert, K. von Figura, and P. Saftig. by guest on October 2, 2021 2002. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling Acknowledgments but not for ␣-secretase activity in fibroblasts. Hum. Mol. Genet. 11:2615. We thank Julie Philalay and Cindy Chang for technical assistance, and 26. Hirata, M., T. Umata, T. Takahashi, M. Ohnuma, Y. Miura, R. Iwamoto, and Garry Nolan for providing retroviral expression plasmids. E. Mekada. 2001. Identification of serum factor inducing ectodomain shedding of proHB-EGF and studies of noncleavable mutants of proHB-EGF. Biochem. Bio- phys. Res. Commun. 283:915. References 27. Casas-Gonzalez, P., A. Ruiz-Martinez, and J. A. Garcia-Sainz. 2003. Lysophos- ␣ ␤␥ 1. Shimaoka, T., N. Kume, M. Minami, K. Hayashida, H. Kataoka, T. Kita, and phatidic acid induces 1B-adrenergic receptor phosphorylation through G , S. Yonehara. 2000. Molecular cloning of a novel scavenger receptor for oxidized phosphoinositide 3-kinase, protein kinase C and epidermal growth factor receptor low density lipoprotein, SR-PSOX, on macrophages. J. Biol. Chem. 275:40663. transactivation. Biochim. Biophys. Acta 1633:75. 2. Matloubian, M., A. David, S. Engel, J. E. Ryan, and J. G. Cyster. 2000. A 28. Kue, P. F., J. S. Taub, L. B. Harrington, R. D. Polakiewicz, A. Ullrich, and transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nat. Im- Y. Daaka. 2002. Lysophosphatidic acid-regulated mitogenic ERK signaling in munol. 1:298. androgen-insensitive prostate cancer PC-3 cells. Int. J. Cancer. 102:572. 3. Wilbanks, A., S. C. Zondlo, K. Murphy, S. Mak, D. Soler, P. Langdon, 29. Umata, T., M. Hirata, T. Takahashi, F. Ryu, S. Shida, Y. Takahashi, D. P. Andrew, L. Wu, and M. Briskin. 2001. Expression cloning of the STRL33/ M. Tsuneoka, Y. Miura, M. Masuda, Y. Horiguchi, and E. Mekada. 2001. A dual BONZO/TYMSTR ligand reveals elements of CC, CXC, and CX3C chemokines. signaling cascade that regulates the ectodomain shedding of heparin-binding epi- J. Immunol. 166:5145. dermal growth factor-like growth factor. J. Biol. Chem. 276:30475. 4. Greaves, D. R., P. J. Gough, and S. Gordon. 1998. Recent progress in defining the 30. Lemjabbar, H., and C. Basbaum. 2002. Platelet-activating factor receptor and role of scavenger receptors in lipid transport, atherosclerosis and host defence. ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nat. Curr. Opin. Lipidol. 9:425. Med. 8:41. 5. Minami, M., N. Kume, T. Shimaoka, H. Kataoka, K. Hayashida, Y. Akiyama, 31. Yan, Y., K. Shirakabe, and Z. Werb. 2002. The metalloprotease Kuzbanian I. Nagata, K. Ando, M. Nobuyoshi, M. Hanyuu, et al. 2001. Expression of SR-PSOX, (ADAM10) mediates the transactivation of EGF receptor by G protein-coupled a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in receptors. J. Cell Biol. 158:221. human atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 21:1796. 32. Matthews, V., B. Schuster, S. Schutze, I. Bussmeyer, A. Ludwig, C. Hundhausen, 6. Li, A. C., and C. K. Glass. 2002. The macrophage foam cell as a target for T. Sadowski, P. Saftig, D. Hartmann, K. J. Kallen, and S. Rose-John. 2003. therapeutic intervention. Nat. Med. 8:1235. Cellular cholesterol depletion triggers shedding of the human interleukin-6 re- 7. Gough, P. J., and S. Gordon. 2000. The role of scavenger receptors in the innate ceptor by ADAM10 and ADAM17 (TACE). J. Biol. Chem. 278:38829. immune system. Microbes Infect. 2:305. 33. Kojro, E., G. Gimpl, S. Lammich, W. Marz, and F. Fahrenholz. 2001. Low 8. Shimaoka, T., T. Nakayama, N. Kume, S. Takahashi, J. Yamaguchi, M. Minami, cholesterol stimulates the nonamyloidogenic pathway by its effect on the ␣-secre- K. Hayashida, T. Kita, J. Ohsumi, O. Yoshie, and S. Yonehara. 2003. Cutting tase ADAM 10. Proc. Natl. Acad. Sci. USA 98:5815. edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by 34. Gutwein, P., S. Mechtersheimer, S. Riedle, A. Stoeck, D. Gast, S. Joumaa, APCs through its chemokine domain. J. Immunol. 171:1647. H. Zentgraf, M. Fogel, and D. P. Altevogt. 2003. ADAM10-mediated cleavage of 9. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, L1 adhesion molecule at the cell surface and in released membrane vesicles. D. R. Greaves, A. Zlotnik, and T. J. Schall. 1997. A new class of membrane- FASEB J. 17:292. bound chemokine with a CX3C motif. Nature 385:640. 35. Shimaoka, T., T. Nakayama, N. Fukumoto, N. Kume, S. Takahashi, 10. Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath, J. Yamaguchi, M. Minami, K. Hayashida, T. Kita, J. Ohsumi, et al. 2004. Cell M. Gosselin, J. Ma, B. Dussault, et al. 1997. Neurotactin, a membrane-anchored surface-anchored SR-PSOX/CXC chemokine ligand 16 mediates firm adhesion chemokine upregulated in brain inflammation. Nature 387:611. of CXC chemokine receptor 6-expressing cells. J. Leukocyte Biol. 75:267.