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Circulating Soluble IL-6R but Not ADAM17 Activation Drives Mononuclear Cell Migration in Tissue Inflammation

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Circulating Soluble IL-6R but Not ADAM17 Activation Drives Mononuclear Cell Migration in Tissue Inflammation Circulating Soluble IL-6R but Not ADAM17 Activation Drives Mononuclear Cell Migration in Tissue Inflammation This information is current as Neele Schumacher, Stefanie Schmidt, Jeanette Schwarz, of October 2, 2021. Dana Dohr, Juliane Lokau, Jürgen Scheller, Christoph Garbers, Athena Chalaris, Stefan Rose-John and Björn Rabe J Immunol 2016; 197:3705-3715; Prepublished online 3 October 2016; doi: 10.4049/jimmunol.1600909 http://www.jimmunol.org/content/197/9/3705 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/10/01/jimmunol.160090 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 52 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/197/9/3705.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Circulating Soluble IL-6R but Not ADAM17 Activation Drives Mononuclear Cell Migration in Tissue Inflammation Neele Schumacher,* Stefanie Schmidt,* Jeanette Schwarz,*,1 Dana Dohr,* Juliane Lokau,* Jurgen€ Scheller,† Christoph Garbers,* Athena Chalaris,* Stefan Rose-John,* and Bjo¨rn Rabe* Neutrophil and mononuclear cell infiltration during inflammatory processes is highly regulated. The first cells at the site of infection or inflammation are neutrophils, followed by mononuclear cells. IL-6 plays an important role during inflammatory states. It has been shown in several models that the soluble form of IL-6R (sIL-6R) is involved in the recruitment of mononuclear cells by a mechanism called IL-6 trans-signaling. It had been speculated that sIL-6R was generated at the site of inflammation by shedding from neutrophils via activation of the metalloprotease ADAM17. Attempts to genetically delete the floxed ADAM17 gene selec- cre tively in myeloid cells infiltrating an air pouch cavity upon injection of carrageenan failed because in transgenic mice, LysM did Downloaded from not lead to appreciable loss of the ADAM17 protein in these cells. We therefore used ADAM17 hypomorphic mice, which only express ∼5% of ADAM17 wild-type levels in all tissues and show virtually no shedding of all tested ADAM17 substrates, to clarify the role of ADAM17 during local inflammation in the murine air pouch model. In the present study, we demonstrate that although IL-6 and the trans-signaling mechanism is mandatory for cellular infiltration in this model, it is not ADAM17-mediated shedding of IL-6R within the pouch that orchestrates this inflammatory process. Instead, we demonstrate that sIL-6R is infiltrating from the circulation in an ADAM17-independent process. Our data suggest that this infiltrating sIL-6R, which is needed for IL-6 trans- http://www.jimmunol.org/ signaling, is involved in the controlled resolution of an acute inflammatory episode. The Journal of Immunology, 2016, 197: 3705– 3715. eutrophils are the most abundant cells in human blood and this complex associates with the ubiquitously expressed re- from where they rapidly infiltrate tissues upon wounding, ceptor gp130 (5). IL-6 only shows measurable affinity to IL-6R N infection, and inflammation (1). Resolution of the neu- but not to gp130. Also, IL-6R alone does not bind to gp130. Only trophil infiltrate is associated with subsequent recruitment of the complex of IL-6 and IL-6R binds to and activates the gp130 mononuclear cells (2). We have shown earlier that in acute bac- protein. Because IL-6R is only expressed on few cell types of the by guest on October 2, 2021 terial peritonitis, IL-6 signaling orchestrates the switch from the human body, only these cell types can be stimulated by IL-6 (6). neutrophilic to the mononuclear phase of the inflammatory pro- However, a soluble form of IL-6R (sIL-6R) can bind to IL-6 and cess and thereby contributes to the resolution of the inflammatory associate with gp130 on cells not expressing the membrane-bound process (2). IL-6R. Such cells in the absence of sIL-6R would not be able to IL-6 is an inflammatory cytokine, the level of which is elevated respond to IL-6. This paradigm has been called IL-6 trans- in most if not all inflammatory states (3, 4). IL-6 binds to IL-6R, signaling (7). Furthermore, a soluble form of gp130 (sgp130) generated via *Institute of Biochemistry, Medical Faculty, Christian Albrechts University, 24098 alternative splicing (8) is present in the circulation (9) and represents Kiel, Germany; and †Institute of Biochemistry and Molecular Biology II, Medical the natural inhibitor of IL-6 trans-signaling (10). A recombinant Faculty, Heinrich Heine University, 40225 Dusseldorf,€ Germany form of sgp130 dimerized by a human IgG1 Fc (sgp130Fc) was 1Current address: Department of Biomedical Sciences and Biotech Research and shown to be an even 10- to 50-fold more effective inhibitor of IL-6 Innovation Center, University of Copenhagen, Copenhagen, Denmark. trans-signaling than was monomeric sgp130 (10, 11). ORCID: 0000-0003-4939-6950 (C.G.); 0000-0002-7519-3279 (S.R.-J.). Using sgp130Fc as a molecular tool, it has been demonstrated Received for publication May 25, 2016. Accepted for publication August 31, 2016. that IL-6 responses via the membrane-bound IL-6R are rather This work was supported by Deutsche Forschungsgemeinschaft (Bonn, Germany) protective and regenerative whereas IL-6 trans-signaling appears to be Grant CRC877 (Proteolysis as a Regulatory Event in Pathophysiology, projects A1 and A10, and the Cluster of Excellence “Inflammation at Interfaces”). involved in the proinflammatory activities of this cytokine (6, 12). We have previously shown that IL-6 trans-signaling plays a N.S., A.C., S.R.-J., and B.R. designed the study, collected data, performed the sta- tistical analysis, and interpreted the data; S.R.-J. wrote the manuscript; and S.S., J.S., decisive role in the switch from the neutrophilic to the mononu- D.D., J.L., J.S., and C.G. performed measurements and analyzed data. All coauthors clear phase (13, 14) in the mouse air pouch model (15, 16). Upon critically reviewed all versions of the manuscript. injection of the irritant carrageenan, local sIL-6R levels increased Address correspondence and reprint requests to Prof. Stefan Rose-John, Institute of during the course of inflammation, whereas neutralizing sIL-6R Biochemistry, Christian Albrechts University, Olshausenstrasse 40, 24098 Kiel, Ger- many. E-mail address: [email protected] activity with a specific Ab significantly reduced the recruitment of The online version of this article contains supplemental material. mononuclear cells (13). Furthermore, we generated mice in which Abbreviations used in this article: DppI, dipeptidyl peptidase I; PR3, proteinase 3; transgenic overexpression of sgp130Fc resulted in blockade of IL-6 sgp130, soluble form of gp130; sgp130Fc, recombinant form of sgp130 dimerized by trans-signaling. Consequently, invasion of immune cells into the 2/2 a human IgG1 Fc; sIL-6R, soluble form of IL-6R; sTNFRI, soluble form of TNFRI; inflamed air pouch was impaired as seen in IL-6 animals, in- wt, wild-type. dicating that IL-6 trans-signaling is essential for correct cellular Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 infiltration (14). As depletion of neutrophils led to decreased sIL-6R www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600909 3706 IL-6 TRANS-SIGNALING AND INFLAMMATION levels within the air pouch, it has been speculated that sIL-6R CD45+CD11b+Ly6G2Ly6Clow cells, and inflammatory monocytes as + + 2 high generation takes place locally via ADAM17-mediated shedding CD45 CD11b Ly6G Ly6C cells (23, 30). from infiltrating neutrophils (17). ELISAs In humans, sIL-6R levels of 40–75 ng/ml are found, which together with sgp130 levels of ∼400 ng/ml constitute a buffer for Cytokines and cytokine receptors (murine sIL-6R, IL-6, KC, MCP-1, and a soluble form of TNFRI [sTNFRI]) were measured by ELISA (R&D Sys- IL-6 in the blood (3). Interestingly, a single nucleotide polymor- tems, Wiesbaden-Nordenstadt, Germany). Human sIL-6R was measured phism has been identified, which leads to 50% higher sIL-6R by ELISA as described (13). levels (18). This IL-6R single nucleotide polymorphism was as- Western blot analysis sociated with a significant decreased risk of coronary heart disease events (19, 20), which was explained with the higher capacity of Cells were lysed in 50 mM Tris, 150 mM NaCl2,2mMEDTA,and1% 3 the IL-6 buffer in the blood (21). IGEPAL (v/v) with 1 cOmplete protease inhibitors (Roche Diagnos- ex/ex tics, Mannheim, Germany) and 10 mM 1,10-phenanthroline (Sigma- We have recently developed ADAM17 hypomorphic (ADAM17 ) Aldrich, St. Louis, MO). Proteins were separated by SDS-PAGE, mice, which in all investigated tissues expressed only ∼5% of transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, ADAM17 levels (22). Using these mice, we could show that Germany), and detected with Abs against ADAM17 (18.2 or 10.1) during bacterial infection, IL-6R cleavage by ADAM17 is induced (23, 31), ADAM10 (Pin1) (23), and b-actin (Sigma-Aldrich, Deisenhofen, on the surface of different leukocyte populations.
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