ADAM17 Activity and Other Mechanisms of Soluble L- Production during Death Receptor-Induced Leukocyte Apoptosis

This information is current as Yue Wang, Adam C. Zhang, Zhenya Ni, Amy Herrera and of October 2, 2021. Bruce Walcheck J Immunol 2010; 184:4447-4454; Prepublished online 10 March 2010; doi: 10.4049/jimmunol.0902925 http://www.jimmunol.org/content/184/8/4447 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/03/11/jimmunol.090292 Material 5.DC1 http://www.jimmunol.org/ References This article cites 63 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/184/8/4447.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ADAM17 Activity and Other Mechanisms of Soluble L-selectin Production during Death Receptor-Induced Leukocyte Apoptosis

Yue Wang, Adam C. Zhang, Zhenya Ni, Amy Herrera, and Bruce Walcheck

L-selectin is an adhesion molecule expressed by neutrophils that broadly directs their infiltration in to sites of inflammation. It is also present at relatively high levels in the serum of normal individuals. It is well established that L-selectin is efficiently shed from the surface of neutrophils upon their activation, a process that regulates its density and binding activity. Neutrophil programmed cell death is critical for the resolution of inflammation, and L-selectin downregulation is induced during this process as well. The mech- anisms underpinning this latter process are much less understood, and were investigated in this study. Using a and metal- loprotease (ADAM)-17 radiation chimeric mice, we demonstrate for the first time that during early events of death receptor-mediated neutrophil apoptosis, L-selectin downregulation occurs primarily by ADAM17-mediated shedding. This was observed as well upon Downloaded from using shRNA to knock down ADAM17 expression in Jurkat cells, a well-studied cell line in terms of the molecular processes involved in the induction of apoptosis. These findings directly reveal that ADAM17 activity occurs during programmed cell death. Hence, the cleavage of particular ADAM17 substrates may be an additional component of the anti-inflammatory program initiated by apoptotic neutrophils. Of interest was that during later stages of induced leukocyte apoptosis, soluble L-selectin production occurred indepen- dent of ADAM17, as well as membrane events, such as blebbing and microparticle production. This process may provide an expla- nation for the lack of diminished serum L-selectin levels in ADAM17-null mice, and suggests a mechanism for the homeostatic http://www.jimmunol.org/ maintenance of soluble L-selectin levels in the blood of healthy individuals. The Journal of Immunology, 2010, 184: 4447–4454.

ctodomain shedding is a regulated proteolytic process that therefore, it is an excellent model for studying ADAM17 directs the cleavage of cell surface , typically at activity in leukocytes. L-selectin is constitutively expressed at E a juxta-membrane site, resulting in the release of a soluble high levels by resting neutrophils, and essentially all molecules extracellular domain fragment (1–3). The functional implications of are cleaved within minutes of neutrophil activation by a variety of ectodomain shedding are diverse, because it can promote an im- physiological stimuli (19). mediate and prolonged formation of soluble agonists and antago- In addition to their proinflammatory role during activation, nists, as well as regulate the density of receptors and adhesion neutrophils mediate an anti-inflammatory program upon pro- by guest on October 2, 2021 molecules. The majority of a growing list of shed proteins are grammed cell death (20, 21), which includes an efficient down- cleaved by a disintegrin and metalloprotease (ADAM)-17 (4). regulation of cell surface L-selectin expression (22, 23). In our Leukocytes express ADAM17, and a number of their products study, we directly examined the role of ADAM17 in this process undergo ectodomain shedding (5). By directly examining leuko- by using leukocytes that lack expression of the sheddase. Our cytes that are deficient in functional ADAM17, it has been reported findings show that during early events of induced neutrophil ap- that this sheddase cleaves various inflammation regulating factors, optosis, L-selectin downregulation occurs primarily by ADAM17- including the pleiotropic cytokine TNFa, its two receptors TNFRI mediated ectodomain shedding, indicating ADAM17 activity (CD120a) and TNFRII (CD120b), and L-selectin (CD62L) (6–11). during the anti-inflammatory program. We also report that at later The enzymatic activity of ADAM17 is inducible, and its function time points of induced neutrophil apoptosis, soluble L-selectin has been primarily studied in the context of cell activation (12–14). production can occur in a manner independent of ADAM17. This The leukocyte-expressed adhesion molecule L-selectin is perhaps process may be important for regulating the high levels of soluble one of the best characterized shed molecules in terms of structure L-selectin in the blood of healthy individuals, thus providing and function analyses and its targeting by ADAM17 (8, 14–18); a potential anti-adhesive environment.

Materials and Methods Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Abs and other reagents Paul, MN 55108 Received for publication September 3, 2009. Accepted for publication February 3, The anti-human L-selectin mAb DREG-200 has been previously described 2010. (24). The anti–L-selectin mAb LAM1-116 (detects both murine and human L-selectin) conjugated to PE, biotin, or allophycocyanin, and recombinant The work was supported by National Institutes of Health Grant R01HL61613. human were purchased from Ancell (Bayport, MN). PE-conjugated Address correspondence and reprint requests to Dr. Bruce Walcheck, Department of anti-mouse LFA-1 (CD11a/CD18) mAb, PE- and allophycocyanin-conjugated Veterinary and Biomedical Sciences, University of Minnesota, 295j AS/VM Build- anti-mouse L-selectin (Ly-22) mAb, and Alexa Fluor-488 anti-mouse Ly-6G ing, 1988 Fitch Avenue, St. Paul, MN 55108. E-mail address: [email protected] (Gr-1) were purchased from eBioscience (San Diego, CA). The anti-human The online version of this article contains supplemental material. LFA-1 (CD11a/CD18) mAb R3.1 has been previously described (25). The Abbreviations used in this paper: ADAM, a disintegrin and metalloprotease; CHX, anti-Fas IgM mAb CH-11 was purchased from Upstate (Lake Placid, NY). cycloheximide; FLICA, fluorescent-labeled inhibitor of caspases; rhFasL, recombi- The anti-ADAM10 and ADAM17 mAbs (clones 163003 and 111623, re- nant human Fas ligand; SSC, laser side scatter. spectively) and TNFa were purchased from R&D Systems (Minneapolis, MN). Biotin, FITC-, PE-, and allophycocyanin-conjugated F(ab9)2 goat anti- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 mouse IgG secondary Abs and allophycocyanin-conjugated streptavidin www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902925 4448 ADAM17 ACTIVITY DURING INDUCED APOPTOSIS were purchased from Jackson Immunoresearch (West Grove, PA). DMEM, conjugated streptavidin. Isotype-matched negative control mAbs were used RPMI 1640, PBS, HBSS, HEPES, and molecular-grade water were pur- to evaluate levels of nonspecific staining. All stained cells were analyzed chased from Mediatech (Hevdon, VA). FBS was purchased from Atlas on a FACSCanto instrument (Becton Dickinson, San Jose, CA). Biologicals (Fort Collins, CO). Penicillin-streptomycin solution was pur- Soluble murine or human L-selectin in tissue culture media supernatants chased from Cellgro (Manassas, VA). PMA was purchased from Sigma- were quantified by ELISA, as previously described (8, 17). In some cases, Aldrich (St. Louis, MO). TAPI-1 was purchased from Peptides International media supernatants were filtered (0.22 mm pore size) then centrifuged at (Louisville, KY). Leupeptin, 6-aminocaproic acid, APMSF-HCl, aprotinin, 100,000 3 g before ELISA. pepstatin A, and chymostatin were all purchased from Sigma-Aldrich. For cell apoptosis detection, a fluorescent-labeled inhibitor of caspases (FLICA) Results Poly Caspase (FAM-VAD-FMK)was purchased from ImmunoChemistry ADAM17 cleaves L-selectin upon the induction of apoptosis Technologies (Bloomington, MN), and FITC- and PE-conjugated annexin-V was purchased from BD Pharmingen (San Diego, CA). Upon induction of apoptosis, human neutrophils downregulate their surface L-selectin in a manner involving (23). Mice L-selectin is shed by ADAM17 following neutrophil activation (8); All experimental procedures were performed in accordance with protocols however, recent studies have revealed that other sheddases can also approved by the Animal Care and Use Committee of the University of cleave L-selectin (28–30). To directly establish the role of ADAM17 Minnesota. ADAM17 deficiency in mice is lethal between embryonic day in L-selectin shedding during death receptor-induced apoptosis, we 17.5 and soon after birth (7), and therefore radiation chimeric mice were generated by reconstitution with embryonic day 15–16, liver hematopoietic examined murine neutrophils lacking functional ADAM17. Because cells that either lacked or expressed functional ADAM17, as previously important differences can occur between human and murine neu- described (8, 9). These mice are referred to as either ADAM17 chimeric or trophils (31), we initially established the effects of induced apoptosis wild-type chimeric mice, respectively. ADAM8 knockout mice were pro- by murine neutrophils on their expression of L-selectin. Our primary vided by Dr. Andy J.P. Docherty (Celltech R&D, Slough, U.K.) via Dr. Downloaded from Carl P. Blobel (Hospital for Special Surgery, Weill Medical College of means of inducing apoptosis by isolated human neutrophils has been Cornell University) (26). Age-matched mice were used for all experiments. through the (23), because of its well established sig- naling cascades and its direct manner of caspase activation (32). Cell culture, transduction, and induction of cellular apoptosis However, our Fas-binding reagents for human leukocytes (see below) All reagents used for cell isolation and incubations were sterile and tested demonstrated little activity with murine neutrophils (data not shown). for endotoxin. Cell viabilities were assessed by exclusion of the vital dye Instead, TNFa in the presence of cycloheximide was used, which is trypan blue. Mouse bone marrow neutrophils were isolated as previously a reproducible method for inducing neutrophil apoptosis (33–36). http://www.jimmunol.org/ described (8, 9). For neutrophil treatments, cells were suspended in PBS at Murine TNFa was initially titered down to a concentration that in- the indicated concentrations and initially equilibrated to culture conditions duced nominal L-selectin shedding via neutrophil activation during at 37˚C in 5% CO2. Some cell samples were incubated with the broad- spectrum zinc metalloprotease inhibitor TAPI (50 nM) for 30 min, and the timeframe of our assays. We observed that our source of TNFa at then were treated with TNFa (40 pg/ml) with or without cycloheximide 40 pg/ml had little effect on phosphatidylserine exposure and L-se- m (35 M) for the indicated time points. Jurkat cells (an acute T cell leu- lectin shedding by murine neutrophils, whereas when combined with kemia cell line) were maintained in DMEM/F12 supplemented with 8% cycloheximide, both events were markedly enhanced (Fig. 1). heat-inactivated FBS and antibiotics at 37˚C in 5% CO2. For Jurkat cell treatments, cells were suspended in RPMI 1640 at the indicated concen- L-selectin surface expression was downregulated in a relatively rapid trations and initially equilibrated to culture conditions at 37˚C in 5% CO2. manner and typically occurred before appreciable reactivity by an- by guest on October 2, 2021 Some cell samples were incubated with TAPI (50 nM) for 30 min and nexin V with the neutrophils, as determined by flow cytometry. In treated with PMA (25 ng/ml), recombinant human FasL, or anti-Fas Ab Figs. 1 and 2, the two events were evaluated at different time points. CH-11 for the indicated time points (both of these reagents were used at a final concentration of 0.5 mg/ml). In additional experiments, Jurkat cells The downregulation in L-selectin surface expression and production were incubated with TAPI and/or various broad-spectrum inhibitors of of its soluble extracellular fragment were consistently observed in serine, cysteine, or aspartate (leupeptin, 100 mM; 6-amino- our assays by 90 min after the induction of apoptosis (Fig. 1); and at m m caproic acid, 10 mM; APMSF-HCl, 50 M; aprotinin, 20 g/ml; pepstatin this time point, soluble L-selectin production was essentially abol- A, 3 mM; chymostatin, 20 mM) for 30 min, and then treated with anti-Fas Ab CH-11 for the indicated time points. The working concentrations of ished by broad-spectrum zinc metalloprotease inhibitors, such as these inhibitors were based on those frequently used in the literature TAPI (Fig. 1). Such inhibitors, however, did not prevent the cells (http://sciencegateway.org/resources/protease.htm). from undergoing apoptosis, as previously described for human For expression of shRNA in Jurkat cells, the human ADAM17 sequence 59- neutrophils (23). 9 GCTTGATTCTTTGCTCTCA-3 , which was determined to knock down Targeting the Adam17 in all cells of mice is lethal, and most ADAM17 expression, or the ADAM17 sequence 59-CAGCAGCTGGAGT- CCTGTG-39, which did not knock-down ADAM17 expression and was used die soon after birth (7). However, radiation chimeric mice recon- as a control to assess off-target effects, were followed by a 9 nucleotide stituted with hematopoietic cells lacking functional ADAM17 noncomplementary spacer (TTCAAGAGA). With their respective reverse (ADAM17 chimeras) are viable (8, 9). An assessment of neutrophils complement sequences, the dsDNA constructs were inserted into the pLL3.7 from wild-type chimeras (reconstituted with wild-type hematopoi- lentiviral vector (Addgene, Cambridge, MA), per the manufacture’s in- structions, after vector digestion with XhoIandHpaI (New England Biolabs, etic cells) revealed that they also shed their L-selectin upon the Ipswich, MA). Lentivirus generation and transduction were performed as induction of apoptosis by TNFa/cycloheximide, as determined by previously described (14, 17). flow cytometry and ELISA (Fig. 2A). In contrast to these cells, the shedding of L-selectin by neutrophils from ADAM17 chimeras was Flow cytometry and ELISA quite minimal at early time points of induced apoptosis (Fig. 2A). For cell staining, Fc receptor and nonspecific Ab binding sites were blocked, Neutrophils from both chimeras demonstrated similar levels of and cells were stained with particular mAbs as previously described (8, 27). phophatidylserine exposure after their treatment with TNFa/cy- Mouse bone marrow leukocytes were triple stained with anti–Gr-1, anti- cloheximide (Fig. 2A), indicating that a lack of functional ADAM17 mouse L-selectin or anti–LFA-1, and annexin V. For cell staining that involved annexin V (for labeling of externalized phosphatidylinositol) or did not prevent the apoptotic process. FLICA (labeling of active caspases), nonfixed cells were used; after their ADAM8 has also been implicated in L-selectin shedding by staining with Abs, the cells were treated with either reagent (per the re- activated neutrophils (29); therefore, we examined its contribution spective manufactures’ instructions) and immediately examined by flow in the shedding of L-selectin by apoptotic neutrophils as well. This cytometry, as previously described (23). ADAM17 and ADAM10 are typically expressed at low levels; therefore, so staining on Jurkat cells was was done in a direct manner by using neutrophils obtained from amplified by treating cells with an anti-ADAM17 or anti-ADAM10 mAb, ADAM8 knockout mice. In contrast to ADAM17-deficient neu- biotin-conjugated F(ab9)2 goat anti-mouse IgG, and then allophycocyanin- trophils, however, we observed that ADAM8-deficient neutrophils The Journal of Immunology 4449 Downloaded from

FIGURE 1. L-selectin shedding occurs by murine neutrophils upon the induction of apoptosis. A, Mouse bone marrow-derived neutrophils (2 3 106 /ml) were either untreated or treated with TNFa,TNFa and cycloheximide (CHX), or TNFa, CHX, and TAPI. The cells were cultured at 37˚C for 90 min before L-selectin detection and for 180 min before phosphatidylserine de- tection. Relative L-selectin surface expression levels and annexin V reactivity FIGURE 2. ADAM17, but not ADAM8, mediates L-selectin shedding at http://www.jimmunol.org/ were determined by flow cytometry, as described in Materials and Methods. early time points of induced apoptosis. A, Mouse bone marrow-derived Data shown are representative of at least three independent experiments using neutrophils (2 3 106 per ml) from wild-type chimeras or ADAM17 chi- neutrophils isolated from separate animals. Negative control Ab staining of meras were either untreated or treated with TNFa or TNFa and CHX for untreated cells is indicated (Isotype). The x-axis = Log 10 fluorescence. To 90 min (panels marked L-selectin) or for 180 min (panels marked annexin- 3 6 assess soluble L-selectin levels, neutrophils (4 10 per ml) were treated as V) at 37˚C. B, Mouse bone marrow-derived neutrophils (2 3 106 per ml) described above for 90 min at 37˚C, and media supernatants from the re- from ADAM8 knockout mice were treated as described above. Relative L- spective samples were subjected to ELISA, as described in Materials and selectin surface expression levels and annexin-V reactivity were de- 6 Methods. The ELISA data are the mean SD of three independent experi- termined by flow cytometry, and data shown are representative of at least ments performed in duplicate. three independent experiments using neutrophils isolated from separate by guest on October 2, 2021 animals. Negative control Ab staining of untreated cells is indicated efficiently downregulated their surface L-selectin upon their (Isotype). The x-axis = Log 10 fluorescence. Soluble L-selectin levels in treatment with TNFa/cycloheximide (Fig. 2B). the media supernatants were determined by ELISA, and data shown are the 6 We also directly examined the role of ADAM17 in L-selectin mean SD of three independent experiments performed in duplicate. shedding by human leukocytes. This was done by using shRNA specific for ADAM17 to knock-down its expression. Targeting ADAM17 (38), was equivalent for both populations (Fig. 3A). ADAM17 in human neutrophils is problematic considering that they Control ADAM17 shRNA was observed to have no effect on the are short-lived and that their manipulation by transfection or surface levels of ADAM17 expression (Fig. 3A). Following the transduction consistently induced some level of activation, which treatment of Jurkat cells transduced with active ADAM17 shRNA may confound our results. Instead, Jurkat cells—a human T cell with rhFasL, L-selectin surface expression downregulated to line—were used. The advantages of these cells are that they express a greater extent by GFP-negative cells than by GFP-positive cells high levels of endogenous L-selectin, unlike most human myeloid (Fig. 3B). When the transduced cells were treated with TAPI be- cell lines, and Jurkat cells are one of the best-studied cell types in fore the induction of apoptosis, L-selectin shedding by the GFP- regard to Fas-induced apoptosis (37). We found that the engage- negative cells was blocked, demonstrating that the downregulation ment of their Fas receptor led to efficient L-selectin shedding, of L-selectin occurred by ectodomain shedding (Fig. 3B). These which was performed by using either soluble recombinant human findings reveal ADAM17 activity upon the induction of leukocyte FasL (rhFasL) or the anti-Fas IgM mAb CH-11 (Supplemental Fig. apoptosis, and that it is the primary sheddase of L-selectin during 1A–C). As with neutrophils, the induction of L-selectin shedding by this process. Jurkat cells occurred as an early apoptotic event before phospha- tidylserine exposure (Supplemental Fig. 1A). The treatment of Soluble L-selectin is produced at later stages of induced Jurkat cells with rhFasL consistently induced L-selectin shedding leukocyte apoptosis independent of ADAM17 and phosphatidylserine exposure more rapidly than did CH-11 When monitoring L-selectin shedding by Jurkat cells undergoing treatment (data not shown), which may be related to different induced apoptosis in the presence of TAPI over an extended period, manners of Fas engagement. we noted that although initially blocked, considerable production Jurkat cells were transduced with a bicistronic lentivirus vector of soluble L-selectin did occur at later time points. As shown in Fig. that expressed GFP and ADAM17 shRNA in a proportional 4A, soluble L-selectin production was assayed for up to 4 h by manner. Greater than 50% of the transduced Jurkat cells expressed Jurkat cells that were either untreated, treated with CH-11, or GFP, and the GFP-positive cells expressed ∼50% less cell surface treated with CH-11 and TAPI. Two hours after the induction of ADAM17 than the GFP-negative cells (Fig. 3A), whereas surface apoptosis, high levels of soluble L-selectin were detected, which ADAM10 expression, the ADAM family member most similar to was essentially abolished by the presence of TAPI. At the 3 and 4450 ADAM17 ACTIVITY DURING INDUCED APOPTOSIS

FIGURE 3. shRNA specific for ADAM17 diminishes L-selectin shedding upon Fas engagement. A, Jurkat cells were transduced with a bicistronic lentivirus vector expressing GFP and shRNA directed against human ADAM17 or control shRNA, as described in Materials and Methods. Transduction efficiency was .50%, as indicated by GFP expression. A representative dot plot indicating the proportion of GFP expressing cells is shown in panel 1. GFP-negative and -positive cells (left and right boxes, respectively, panel 1) were electronically gated, and each population was analyzed for their relative expression levels of ADAM17 and ADAM10 by flow cytometry. Transduced Jurkats cells expressing ADAM17 shRNA were stained for cell surface

ADAM17 (panel 2) or ADAM10 (panel 4). Transduced Jurkats cells expressing control shRNA were stained for ADAM17 expression (panel 3). B, The Downloaded from transduced Jurkats cells were treated with rhFasL in the presence or absence of TAPI, as indicated, at 37˚C for 30 min. Relative L-selectin surface ex- pression levels were determined by flow cytometry. Negative control Ab staining of all cells is indicated (Isotype). The x-axis = Log 10 fluorescence. The data are representative of three independent experiments. SSC, laser light side scatter.

4 h time points, however, apoptotic Jurkat cells in the presence of various other broad-spectrum inhibitors produced levels TAPI produced significantly higher levels of soluble L-selectin of soluble L-selectin similar to those in cells treated with TAPI http://www.jimmunol.org/ than time-matched, untreated cells (Fig. 4A). At these later time alone. points of apoptosis, cell surface L-selectin also underwent a cor- Membrane blebbing and microparticle production are additional responding downregulation in expression in the presence of TAPI mechanisms by which certain surface proteins can be down- (Fig. 4B). This late apoptotic process did not result in a general regulated during leukocyte apoptosis. Interestingly, CD43 and downregulation in the expression of cell surface molecules, as the CD16, which undergo ectodomain shedding upon neutrophil ac- surface levels of LFA-1 on the same cells were only modestly tivation (42), can be downregulated in expression during neutro- decreased (Fig. 4B). We also monitored ADAM17 surface ex- phil apoptosis by these processes (43, 44). To determine whether pression on Jurkat cells at various time points of apoptosis. Jurkat L-selectin released from the surface of Jurkat cells treated with by guest on October 2, 2021 cells treated with CH-11 for 2 h demonstrated surface ADAM17 CH-11 and TAPI was associated with membrane particles, the expression levels equivalent to time-matched, untreated cells, particles were removed from the media supernatant by filtration whereas cells treated for with CH-11 for 4 h demonstrated and ultracentrifugation. For example, media supernatant from a marginal decrease in ADAM17 staining when compared with Jurkat cells treated with CH-11 and TAPI for 4 h was divided into time-matched, untreated cells. Hence, soluble L-selectin pro- two equal volumes, and one of the aliquots was filtered (0.22 mm duction occurred at later stages of apoptosis in the presence of pore size) then centrifuged at 100,000 3 g for 1 h. Soluble a broad-spectrum metalloprotease inhibitor and during the L-selectin levels in the two aliquots were then compared by downregulation of ADAM17 surface expression. ELISA. If soluble L-selectin produced by apoptotic Jurkat cells in An obvious consideration for assays involving TAPI is its sta- the presence of TAPI was primarily associated with membrane bility during extended cell incubation periods. We examined this in particles, we would expect that its levels would be significantly a cell-based assay by collecting media supernatants from Jurkat decreased after the described treatments; however, this was not the cells treated with CH-11 for 4 h in the presence or absence of TAPI, case (Fig. 4D). and using the media supernatants to suspend untreated Jurkat cells, Soluble L-selectin occurs at high levels in the blood of healthy which were then overtly activated with PMA to induce L-selectin humans and mice (45, 46). Interestingly, the levels of soluble shedding. We found that activated Jurkat cells incubated in media L-selectin in the blood of ADAM17 and wild-type chimeric mice supernatant from the CH-11/TAPI-treated cells did not down- are not significantly different (8), suggesting another mechanism regulate their expression of surface L-selectin, whereas Jurkat cells for its production. As with Jurkat cells, we observed that murine incubated in media supernatant from the CH-11–treated cells did neutrophils also downregulated their surface L-selectin at later time (Fig. 4C), indicating activity by the metalloprotease inhibitor. We points of induced apoptosis in the presence of TAPI. For instance, have also added TAPI to Jurkat cells in culture prior to and at by 3 h of induced apoptosis, neutrophils from unmanipulated mice regular time points after the induction of apoptosis, and again treated with TNFa/cycloheximide in the presence of TAPI dem- noted a significant downregulation in L-selectin expression at the onstrated an appreciable downregulation in L-selectin expression, later time points of apoptosis (data not shown). as determined by flow cytometry (Fig. 5A). Again, surface levels In addition to a broad-spectrum metalloprotease inhibitor, we of LFA-1 on the same cells decreased only marginally at this examined the effects of other protease inhibitors on the late apo- time point (Fig. 5A). In contrast to early time points following the ptosis release of soluble L-selectin, in particular inhibitors of serine induction of apoptosis, neutrophils isolated from ADAM17 chi- proteases, which can also cleave L-selectin and have been im- meras also demonstrated a downregulation in surface L-selectin plicated in ectodomain shedding (39–41). As shown in Supple- expression as apoptosis progressed (Fig. 5B), resulting in a cor- mental Fig. 2, Jurkat cells treated with TAPI to block the initial responding increase in soluble L-selectin levels (Fig. 5C). The shedding of L-selectin during the induction of apoptosis with above findings reveal that soluble L-selectin production can The Journal of Immunology 4451 Downloaded from

FIGURE 4. Soluble L-selectin is generated by Jurkat cells in the presence of TAPI at later time points of induced apoptosis. A, Jurkat cells (2 3 106 per http://www.jimmunol.org/ ml) were incubated with the anti-Fas Ab CH-11 in the presence or absence of TAPI for the indicated time points at 37˚C. The presence of soluble L-selectin in the cell supernatants was determined by ELISA. B, Jurkat cells (2 3 106 per ml) were incubated with the anti-Fas Ab CH-11 in the presence or absence of TAPI, or were left untreated, as indicated, for 4 h at 37˚C. Relative L-selectin or LFA-1 surface expression levels and caspase activity (FLICA reactivity) were determined by flow cytometry. C, Jurkat cells (2 3 106 per ml) were incubated with the anti-Fas Ab CH-11 or were left untreated, as indicated, for 2 h (left panel)or4h(right panel) at 37˚C. Relative ADAM17 surface expression levels were determined by flow cytometry. D, Jurkat cells (2 3 106 per ml) were incubated with the anti-Fas Ab CH-11 in the presence or absence of TAPI, as indicated (TAPI, CH-11 or CH-11, respectively) for 4 h at 37˚C. Tissue culture media supernatants from the two cell treatments were removed of cells by centrifugation, filtered (0.22 mm), and used to suspend previously untreated Jurkat cells (2 3 106 per ml). These cells were then incubated with PMA to induce their overt activation. Relative L-selectin surface expression levels were determined by flow cytometry. Negative control Ab (Isotype) staining of untreated cells is indicated (B–D). The x-axis = Log 10 fluorescence. E, Jurkat cells were incubated in the absence or presence of the anti-Fas Ab CH-11 plus TAPI, as indicated, for 4 h at 37˚C. The tissue culture media supernatant from both by guest on October 2, 2021 cell treatments was divided into two equal aliquots, and one of the aliquots was subjected to filtration (0.22 mm) then ultracentrifugation (100,000 3 g)for1h. The levels of soluble L-selectin in the two aliquots for both cell treatments were determined by ELISA. B–D, data are representative of at least three in- dependent experiments. Data in A and E are the mean 6 SD of three independent experiments performed in duplicate. Statistical significance was determined by an unpaired Student t test. pp , 0.001 versus untreated; ppp , 0.05 versus untreated. occur by distinct mechanisms invoked at different stages of in- apoptosis by using two distinct genetic approaches. Fas signaling of duced apoptosis. apoptosis in Jurkat cells has been extensively studied (37), and these cells, which express high levels of surface L-selectin, were Discussion used to knock down ADAM17 expression using shRNA. This ap- Emerging evidence indicates that ectodomain shedding by leuko- proach decreased the expression of cell surface ADAM17, but not cytes is a key posttranslational mechanism for regulating in- the expression of its most similar family member ADAM10, and flammation. In part, this process modulates the activity of various also reduced L-selectin shedding upon Fas engagement when cytokines, cytokine receptors, and adhesion molecules (5). compared with control cells. We also examined the shedding of ADAM17 induction occurs upon cell activation, and its biological L-selectin by neutrophils lacking functional ADAM17 following functions have been mainly examined in this context. The fate of the induction of apoptosis. In contrast to wild-type neutrophils, neutrophils following their infiltration into sites of inflammation is ADAM17-deficient neutrophils demonstrated a near complete ab- programmed cell death, which is an effector activity that plays rogation of L-selectin shedding at early time points of induced a vital role in resolving acute inflammation (20, 21). In this study, we apoptosis. ADAM8 has been implicated in L-selectin shedding provide the first direct evidence that ADAM17 cleaves L-selectin by neutrophils as well (29), and we also directly examined its role upon the induction of neutrophil apoptosis, which appears to be an by using neutrophils from ADAM8 knockout mice. We observed early apoptotic event. We also report that as the apoptotic process that these cells efficiently downregulated their surface L-selectin progresses, L-selectin can be released from the surface of leuko- expression soon after the induction of apoptosis. Hence, our find- cytes by a mechanism distinct from ADAM17-mediated shedding ings indicate that in addition to neutrophil activation, ADAM17 and the membrane events of blebbing and microparticle production. is a primary sheddase of L-selectin upon death receptor-induced We have previously reported that L-selectin undergoes efficient apoptosis. shedding upon the induction of human neutrophil apoptosis by Activation-induced L-selectin shedding regulates the receptor’s a mechanism that involves metalloproteases (23). L-selectin cell surface density and neutrophil adhesiveness (18, 47). The shedding has been reported to occur in an ADAM17-dependent and purpose of L-selectin shedding during neutrophil apoptosis is less independent manner (8, 28–30); therefore, we directly examined its clear at this time, although receptor downregulation by apoptotic role in L-selectin shedding upon death receptor-induced leukocyte neutrophils likely contributes to a general diminution in their 4452 ADAM17 ACTIVITY DURING INDUCED APOPTOSIS

during this cellular process. We have recently reported that redox modifications of cysteinyl sulfhydryl groups in the ectodomain of mature ADAM17 upregulates its enzymatic activity (14). It will be interesting to determine whether this inducer mechanism might also regulate the activity of ADAM17 upon death receptor-induced neutrophil apoptosis. In addition, increased conversion of ADAM17’s proform to mature form has also been reported during leukocyte apoptosis (53). Our data also reveal that during the late stages of neutrophil ap- optosis,L-selectinexpressioncanbedownregulatedbyamechanism distinct from ADAM17, which results in the production of soluble L-selectin. CD16 and CD43 can undergo ectodomain shedding as well upon neutrophil activation, and they have been reported to be released from the surface of neutrophils by membrane blebbing and microparticle production during apoptosis (43, 44). An approach used to discern soluble adhesion molecules that are cleaved from those associated with membrane particles is by sedimentation of the latter using ultracentrifugation (44, 54). We found that a combina- tion of filtration ($0.22 mm particle size) as well as ultracentrifu- gation (100,000 3 g) did not significantly reduce the levels of Downloaded from soluble L-selectin in the tissue culture media supernatant from cells undergoing prolonged apoptosis in the presence of the broad spec- trum zinc metalloprotease inhibitor TAPI, suggesting that it may have been produced by a proteolytic process. FIGURE 5. ADAM17-deficient neutrophils release L-selectin at later ADAM10 has been reported to cleave L-selectin in the absence time points of induced apoptosis. A, Mouse bone marrow-derived neu- of its primary sheddase ADAM17 (30). However, it would seem http://www.jimmunol.org/ 6 trophils (2 3 10 per ml) were either untreated or treated with TNFa or unlikely that ADAM10 is playing a major role in soluble L-se- a TNF /CHX in the presence or absence of TAPI for 180 min at 37˚C. B, lectin production by apoptotic leukocytes, considering that it has 3 6 Mouse bone marrow-derived neutrophils (2 10 per ml) from ADAM17 been shown to downregulate in expression during this process chimeras were either untreated or treated with TNFa or TNFa/CHX for (23), and that soluble L-selectin production occurred in the pres- 180 min at 37˚C. A and B, Relative L-selectin or LFA-1 surface expression levels and annexin-V reactivity were determined by flow cytometry. ence of TAPI, which is known to block ADAM10 activity (55–57). Negative control Ab staining of untreated cells is indicated (Isotype). The Serine proteases have been implicated as sheddases and can also x-axis = Log 10 fluorescence. Data are representative of at least three cleave L-selectin (39–41). However, we show that various broad- independent experiments using neutrophils isolated from separate animals. spectrum serine protease inhibitors, as well as inhibitors of as- by guest on October 2, 2021 C, Mouse bone marrow-derived neutrophils (2 3 106 per ml) from partic and cysteine proteases, also did not block soluble L-selectin ADAM17 chimeras were either untreated or treated with TNFa/CHX at production by late apoptotic leukocytes. Thus, the precise pro- 37˚C for the indicated time points. The presence of soluble L-selectin in teolytic process by which soluble L-selectin production occurs the cell supernatants was determined by ELISA. Data are represented as during later stages of leukocyte apoptosis remains to be de- 6 the mean SD of three independent experiments performed in duplicate. termined. In addition, it will be interesting to determine whether this putative protease targets other cell surface determinants. For responsiveness to inflammatory cues (22). Neutrophils that have example, surface expression of PSGL-1, a ligand of L-selectin recently infiltrated sites of inflammation already express low levels (58), can also be downregulated during neutrophil apoptosis, of L-selectin because of their activated state (8, 18, 48); therefore, it which appears to involve a non-ADAM17 proteolytic process as would seem that nominal levels of cell surface L-selectin would be well (54). available for further ADAM17-mediated cleavage upon the in- The current study is the first to establish mechanisms of soluble duction of neutrophil apoptosis. However, activated neutrophils L-selectin production upon death receptor-induced, neutrophil ap- express L-selectin mRNA, actively synthesize the adhesion mole- optosis. We have previously reported that ADAM17-deficient cule, and can re-express appreciable levels of surface L-selectin neutrophils can generate soluble L-selectin upon spontaneous ap- following their activation, but prior to undergoing apoptosis (23) optosis, which appears to involve other metalloproteases (8). The (49, 50). Consequently, ADAM17-mediated shedding during neu- biologic significance of ADAM17-independent processes that trophil apoptosis at sites of inflammation may produce physiolog- mediate the release of surface L-selectin from apoptotic neutrophils ically relevant levels of localized soluble L-selectin, among is unclear at this time. Soluble L-selectin is maintained at high additional substrates. Other settings in which ADAM17-dependent levels in the blood of healthy individuals and mice (45, 46). We shedding may occur upon leukocyte apoptosis is during the clear- have reported that the levels of soluble L-selectin in the blood of ance of senescent neutrophils from the blood and the turnover of ADAM17 and wild-type chimeric mice are not significantly dif- lymphocytes during negative selection, which may be a mechanism ferent (8), suggesting that ADAM17 might not be the principle for the downregulation of various receptors and/or the production means of maintaining blood levels of soluble L-selectin. However, of assorted soluble agonists and antagonists. The enzymatic activity Venturi et al. (18) have reported that soluble L-selectin in the blood of ADAM17 is robustly induced upon cell activation with various is primarily derived by proteolytic cleavage, as gene-targeted mice stimuli, which occurs in part through an intrinsic process (51, 52). We expressing noncleavable L-selectin have considerably lower levels speculate that the enzymatic activity of ADAM17 is enhanced upon of serum L-selectin. In conjunction with our findings, it is tempting the induction of leukocyte apoptosis as well, although at this time we to speculate that soluble L-selectin levels in the blood of normal cannot rule out that changes in the conformation of L-selectin and/or individuals may be maintained by a proteolytic mechanism other its cell surface distribution might also contribute to its shedding than ADAM17, perhaps occurring during leukocyte apoptosis. The Journal of Immunology 4453

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