Function of Liver Activation-Regulated Chemokine/CC Chemokine Ligand 20 Is Differently Affected by Cathepsin B and Cathepsin D Processing This information is current as of September 28, 2021. Lara Hasan, Luca Mazzucchelli, Mark Liebi, Maddalena Lis, Robert E. Hunger, Angus Tester, Christopher M. Overall and Marlene Wolf J Immunol 2006; 176:6512-6522; ; doi: 10.4049/jimmunol.176.11.6512 Downloaded from http://www.jimmunol.org/content/176/11/6512 References This article cites 58 articles, 27 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/176/11/6512.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Function of Liver Activation-Regulated Chemokine/CC Chemokine Ligand 20 Is Differently Affected by Cathepsin B and Cathepsin D Processing1 Lara Hasan,* Luca Mazzucchelli,*§ Mark Liebi,† Maddalena Lis,* Robert E. Hunger,‡ Angus Tester,†¶ Christopher M. Overall,†¶ and Marlene Wolf2† Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL201–66 isoform Downloaded from with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL201–55, CCL201–52, and a 12-aa C-terminal peptide CCL2059–70. Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1␣ and TNF-␣ in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular mem- branes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by http://www.jimmunol.org/ cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas. The Journal of Immunology, 2006, 176: 6512–6522. hemokines contribute to a variety of immunoregulatory mokines showing that matrix metalloproteases (MMPs)3 and also and proinflammatory responses by control of leukocyte dipeptidylpeptidase IV/CD26 are potent modifiers of chemokine C recruitment during inflammation (1–3). Beyond this pri- function in addition to their roles in inflammation and wound heal- mary function, chemokines also affect angiogenesis, angiostatic ing (1). MMP activity activates the CXCR2 murine chemokine regulation mechanisms, hemopoiesis, lymphatic organogenesis, CXCL5/LIX in vitro and in vivo (12), inactivates CXCL12 (13, and, through the migration of cancer cells expressing chemokine 14), and generates antagonists from all MCP chemokines (15). by guest on September 28, 2021 receptors to chemokine-secreting organs, tumor metastasis (3, 4). A causal link between chemokine function and organ trophism Recently, several chemokines were described to possess also an- in breast cancer was shown for CXCR4 and its cognate ligand timicrobial activities (5). Chemokine activity is modulated by sev- CXCL12 (16). Thus, the interrelationship between chemokines eral cytokines that directly or indirectly regulate chemokine ex- and members of the cathepsin family, such as cathepsin D pression and/or expression of their receptors (1). An increasing (Cath-D) and cathepsin B (Cath-B), may play an important role in body of evidence suggests that chemokine clearing by proteases tumor biology. Indeed, both cathepsins not only facilitate tumor may also strongly affect their activity (1). In this context, a specific progression (17) but may have a prognostic value in patients with role was recently suggested for cathepsins that cleave and inacti- breast cancer (17, 18) and malignant melanoma (19). We therefore vate CXCL12/stromal cell-derived factor-1␣ (6) and CCL3/MIP- reasoned that the generation of an antitumoral immune response or 1␣, CCL4/MIP-1␤ and CCL21/SLC (7). Conversely, the activity cancer cell motility may be modulated by Cath-D and Cath-B gen- of CXCL5/epithelial cell-derived neutrophil-activating protein-78 erated by the tumor or reactive stroma. Among the novel substrates (8) and CXCL8/IL-8 (9, 10), and CXCL7/neutrophil-activating identified, we focused on CCL20 (also known as liver and activa- peptide 2 (11) is potentiated upon interaction with cathepsins. This tion-regulated chemokine (LARC); MIP-3␣ or exodus-1), which is work builds on initial studies on the effects of proteases and che- a 9-kDa CC-type chemokine expressed constitutively in lung and liver (20), in keratinocytes in the epidermal layer of the skin (21), and in the intestinal mucosa (22–24). Several arguments indicate *Institute of Pathology, †Theodor-Kocher Institute, and ‡Department of Dermatology, CCL20 as a potential important player in the chemokine network. § University of Bern, Bern, Switzerland; Istituto di Patologia, Locarno, Switzerland; First, CCL20 is the unique chemokine ligand of CCR6 and does and ¶Department of Biochemistry and Molecular Biology, Centre for Blood Research and the Canadian Institutes for Health Research Group in Matrix Dynamics, Univer- not show the promiscuity of receptor binding characteristic of sity of British Columbia, Vancouver, British Columbia, Canada other chemokines (25, 26). Second, CCL20 not only triggers an Received for publication May 31, 2005. Accepted for publication March 21, 2006. innate immune response through the attraction of NK cells (27) but The costs of publication of this article were defrayed in part by the payment of page is also involved in adaptative immune responses, primarily by at- charges. This article must therefore be hereby marked advertisement in accordance tracting immature dendritic cells, allowing them to take up foreign with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Swiss National Science Foundation (Grant 3100-066804) and the Bernische Krebsliga. 3 Abbreviations used in this paper: MMP, matrix metalloprotease; Cath-D, cathepsin 2 Address correspondence and reprint requests to Dr. Marlene Wolf, Theodor-Kocher D; Cath-B, cathepsin B; Cath-H, cathepsin H; CS, chondroitin sulfate; GAG, glycos- Institute, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland. E-mail address: aminoglycan; EIA/RIA, enzyme immunoassay/radioimmunoassay; pAB, 4-amino- [email protected] benzoic acid; AMC, 7-amino-4-methylcoumarin. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6513 Ags and to mature (28, 29), and by attracting memory T and mem- CCL20 in PBSϩ containing 0.05% Tween 20 and 1% BSA was added to ory B cells to the site of inflammation (20, 30, 31). Furthermore, the GAG-coated wells and incubated for 2 h. After washing, the wells were ␮ CCL20 has antimicrobial activities, similarly to defensins, which incubated overnight at 37°C with 0.25 g/ml Cath-D or Cath-B, and washed again, and CCL20 was probed with biotinylated goat anti-human also bind to CCR6, and can therefore contribute to direct antimi- CCL20. Bound CCL20 was then measured using streptavidin-conjugated crobial defenses (5). Lastly, CCL20 expression has been described alkaline phosphatase and Blue Phos as described above. in several leukemia/lymphoma cell lines, and in a variety of human ϩ Intracellular calcium ([Ca2 ] ) mobilization neoplasms, such as colorectal adenocarcinoma, lung carcinoma, i 2ϩ and malignant glioma (32, 33). In this study, we demonstrate for [Ca ]i changes were measured in CCR6-expressing mouse 300-19 pre-B 6 the first time chemokines as substrates for Cath-B, and we char- cells (35) loaded with 0.1 nmol of fura 2-AM per 10 cells as described previously (36). acterize the processing of CCL20 by Cath-D and Cath-B. We fur- ther demonstrate that cleavage of CCL20 by these proteases is Cell migration retained when the chemokine is bound to extracellular matrix pro- Chemotaxis was assayed in 48-well chambers (Neuro Probe) using 3-␮m teins, and we characterized in functional assays the activity of pore size polycarbonate filters (Osmonics). Briefly, chemokines in chemo- degradation products. Using human malignant melanoma as a taxis buffer (RPMI 1640, 20 mM HEPES (pH 7.2), containing 1% pas- 5 model, we propose that neoplastic cells potentially exert autocrine teurized human plasma protein) were added to the lower wells, and 10 cells were resuspended in the same buffer to the upper wells and incubated regulatory mechanisms in the context of chemokine-protease in- for 90 min at 37°C. Cells migrating to the lower side of the filter were teractions by modulating the migratory activities of CCL20 toward stained and counted in five high-power fields. Cells migrating into the responsive cells of the antitumoral immune response.