LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages This information is current as Kristin Schubert, Laura E. Collins, Patricia Green, Hideaki of September 25, 2021. Nagase and Linda Troeberg J Immunol published online 18 January 2019 http://www.jimmunol.org/content/early/2019/01/17/jimmun ol.1800834 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/01/17/jimmunol.180083 Material 4.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 18, 2019, doi:10.4049/jimmunol.1800834 The Journal of Immunology LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages Kristin Schubert,1 Laura E. Collins, Patricia Green, Hideaki Nagase, and Linda Troeberg The metalloproteinase ADAM17 plays a pivotal role in initiating inflammation by releasing TNF from its precursor. Prolonged TNF release causes many chronic inflammatory diseases, indicating that tight regulation of ADAM17 activity is essential for resolution of inflammation. In this study, we report that the endogenous ADAM17 inhibitor TIMP-3 inhibits ADAM17 activity only when it is bound to the cell surface and that cell surface levels of TIMP-3 in endotoxin-activated human macrophages are dynamically con- trolled by the endocytic receptor LRP1. Pharmacological blockade of LRP1 inhibited endocytic clearance of TIMP-3, leading to an increase in cell surface levels of the inhibitor that blocked TNF release. Following LPS stimulation, TIMP-3 levels on the surface of macrophages increased 4-fold within 4 h and continued to accumulate at 6 h, before a return to baseline levels at 8 h. This dynamic Downloaded from regulation of cell surface TIMP-3 levels was independent of changes in TIMP-3 mRNA levels, but correlated with shedding of LRP1. These results shed light on the basic mechanisms that maintain a regulated inflammatory response and ensure its timely resolution. The Journal of Immunology, 2019, 202: 000–000. disintegrin and metalloproteinase 17 (ADAM17) reg- by substrate availability, either by localization (7), dimerization ulates paracrine and autocrine immune signaling, cell (18), intracellular domain phosphorylation (19), or glycosylation http://www.jimmunol.org/ A proliferation, tissue regeneration, and cancer progression (20). Regulation of ADAM17 intracellular trafficking by iRhoms through its ability to cleave or “shed” the transmembrane pre- (21–23) and the sorting protein PACS2 (24) also contribute to cursors of multiple cytokine and growth factor proteins and re- the interrelated network of molecular mechanisms regulating ceptors. Key ADAM17 substrates include the precursor form of ADAM17 activation. the inflammatory cytokine TNF, giving the enzyme its alternative Prolonged or increased TNF release is associated with numerous name of TNF-a–converting enzyme (TACE) (1–4). Although the inflammatory conditions, such as septic shock (25), rheumatoid transmembrane TNF precursor has biological activity, in vivo arthritis (26), colitis (27, 28), and Crohn’s disease (29), indicating studies indicate that the soluble form of the cytokine plays in- that ADAM17 activity must be tightly regulated. Although acti- by guest on September 25, 2021 dispensable roles in initiating paracrine signaling events central to vation of ADAM17 has been studied in detail, mechanisms con- acute inflammatory responses (5, 6). trolling resolution of ADAM17 activity are less well understood. ADAM17 activity is rapidly induced in response to LPS or PMA Tissue inhibitor of metalloproteinases 3 (TIMP-3) inhibits ADAM17 stimulation, without any increase in its cell surface expression. activity by forming a high affinity 1:1 stoichiometric complex with the Many mechanisms have been proposed to explain this phenome- enzyme (30). Timp3-null mice exhibit increased TNF release (5, 31– non, including compartmentalization of the enzyme in membrane 33) and increased susceptibility to TNF-associated tissue damage in microdomains (7, 8) and conformational changes in the ADAM17 models of diabetes (31), atherosclerosis (34), kidney fibrosis (33), lung ectodomain (9, 10), postulated to involve protein disulfide isom- injury (35) and liver regeneration (5). These studies indicate that erase (10, 11) and/or interaction of juxtamembrane regions of TIMP-3 is the primary physiological inhibitor of ADAM17 and that ADAM17 with the lipid bilayer (12). Phosphorylation of the in- TIMP-3 is critical for termination of TNF release. tracellular domain of ADAM17 affects activity in some cases We have previously demonstrated that extracellular levels of (13–17), but not all (9). ADAM17 activity may also be regulated TIMP-3 in chondrocytes are primarily regulated posttranslationally, by endocytosis via the endocytic scavenger receptor low-density Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute of lipoprotein receptor-related protein 1 (LRP1) and subsequent deg- Rheumatology, University of Oxford, Oxford OX3 7FY, United Kingdom radation in lysosomes (36–38). Because LRP1 is also highly 1 Current address: Department for Molecular Systems Biology, Helmholtz Centre for expressed in macrophages (39, 40), we investigated whether LRP1 Environmental Research UFZ, Leipzig, Germany. also controls TIMP-3 levels in these cells and whether LRP1 has an ORCIDs: 0000-0003-4365-084X (K.S.); 0000-0003-0939-4651 (L.T.). impact on the duration of ADAM17 activity and TNF release. By Received for publication June 14, 2018. Accepted for publication December 16, 2018. studying primary human macrophages stimulated with LPS, we were able to gain insight into physiological TIMP-3 and ADAM17 This work was supported by the Kennedy Trust for Rheumatology Research. regulation that has not been observed in previous studies using Address correspondence and reprint requests to Dr. Linda Troeberg at the current address: Norwich Medical School, Bob Champion Research and Education Building, macrophage-like cell lines (e.g., THP-1 or U937) or nonphysiological University of East Anglia, Norwich NR4 7UQ, U.K. E-mail address: [email protected] stimuli (e.g., PMA). We found that LPS-induced shedding of LRP1 The online version of this article contains supplemental material. from activated macrophages led to an accumulation of TIMP-3 on Abbreviations used in this article: ADAM17, a disintegrin and metalloproteinase 17; the cell surface, and consequent inhibition of TNF release. LRP1 HSPG, heparan sulfate proteoglycan; LRP1, low-density lipoprotein receptor-related pro- thus controls the duration of ADAM17 activity by regulating the tein 1; RAP, receptor-associated protein; TIMP-3, tissue inhibitor of metalloproteinases 3. abundance of its inhibitor, and contributes to the regulated resolution Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 of inflammation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800834 2 LRP1 REGULATES MACROPHAGE TNF RELEASE Materials and Methods Change in TNF concentration per hour Materials ½TNF concentration at x 2 ½TNF concentration at y ¼ ; FCS was from Life Technologies, RPMI 1640 medium was from PAA x 2 y Laboratories, and recombinant human GM-CSF, M-CSF, and IL-4 were where x and y represent successive time points. from PeproTech. Ultra-pure LPS from Escherichia coli 0111:B4 was from InvivoGen. Recombinant human TIMP-3, TIMP-3 blocking Ab (catalog Flow cytometry number MAB973, clone number 183551), and IL-6R ELISA kit were from R&D Systems. For immunoblotting, the TIMP-3 Ab (AB6000) was from Macrophages were detached by incubating in 2 mM EDTA/PBS for 10 min Merck Millipore, and the actin Ab (ab3280) was from Abcam. LRP1 at 37˚C. Fc receptors were blocked using 10% human AB serum. Cells blocking Ab (01-05) was purchased from BioMac. Porcine mucosal hep- were stained with fixable viability dye (eBioscience), conjugated Abs arin, PMA, and ionomycin were from Sigma-Aldrich (St Louis, MO). against ADAM17 (FAB9301F; R&D Systems), LRP-1 a-chain (12-0919- Receptor-associated protein (RAP) was expressed in E. coli and purified as 42; eBioscience), LRP1 b-chain (FAB6360A; R&D Systems), membrane *previously described (41), with addition of a Triton X-114 washing step TNF (HM2024F; Hycult Biotech), or matching isotope controls for 30 min to remove LPS (42). Removal of LPS was confirmed by the Limulus at 4˚C, washed, and fixed. The Ab against the LRP1 b-chain recognizes the Amebocyte Lysate assay (Pierce). intracellular portion of the b-chain, and cells were thus permeabilized using the Cytofix/Cytoperm Kit (BD Bioscience) according to manufacturer’s in- Cell isolation and culture structions. Samples were then analyzed on an LSR II (BD Bioscience),