Resist Immune-Mediated Injury IL-11 Activates Human Endothelial Cells To
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IL-11 Activates Human Endothelial Cells to Resist Immune-Mediated Injury Keyvan Mahboubi, Barbara C. Biedermann, Joseph M. Carroll and Jordan S. Pober This information is current as of October 2, 2021. J Immunol 2000; 164:3837-3846; ; doi: 10.4049/jimmunol.164.7.3837 http://www.jimmunol.org/content/164/7/3837 Downloaded from References This article cites 53 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/164/7/3837.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 2, 2021 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IL-11 Activates Human Endothelial Cells to Resist Immune-Mediated Injury Keyvan Mahboubi,1* Barbara C. Biedermann,1,2* Joseph M. Carroll,† and Jordan S. Pober3* IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor ␣-chain and gp130. Human IL-11 causes rapid (2–10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phos- phorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15–20 min and largely disappears by 40 min. IL-11 does not activate NF-B nor does it inhibit NF-B activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by Downloaded from allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune- mediated injury in cultured HUVECs without inhibiting proinflammatory responses. The Journal of Immunology, 2000, 164: 3837–3846. http://www.jimmunol.org/ nterleukin-11 is a 20-kDa stromal cell-derived pleiotropic cy- parameters of lung injury including alveolar-capillary protein leak, tokine that interacts with a variety of hemopoietic and non- endothelial cell (EC)4 and epithelial cell membrane injury, lipid I hemopoietic cell types. Recombinant human IL-11 stimulates peroxidation, pulmonary neutrophil recruitment, IL-12 and TNF megakaryocytopoiesis in vitro (1) and in vivo (2). It also stimulates production, and DNA fragmentation. erythropoiesis and regulates macrophage proliferation and differ- The mechanism(s) by which IL-11 protects mucosae are not entiation (3). Due to its thrombopoietic activities in vivo, IL-11 is completely understood. Both antiinflammatory and direct cytopro- used to treat chemotherapy-induced thrombocytopenia (4). tective effects of IL-11 could contribute to the reduction of injury. In addition to its hemopoietic activities, IL-11 protects against IL-11 may exert antiinflammatory effects by reducing cytokine by guest on October 2, 2021 various forms of mucosal injury. These effects are most exten- production by macrophages (12–14). It also may promote immune sively described in the gastrointestinal tract of rodents where IL-11 deviation from a TH1-like to a TH2-like phenotype, which may protects small intestinal cells from combined radiation, chemother- ameliorate some types of immune-mediated injury (9). To date, apy, and ischemia in mice (5–7); reduces experimental colitis in- there have been no reports of direct cytoprotection in cultured duced by trinitrobenzenesulfonic acid in rats (8); and ameliorates cells. inflammatory bowel disease in mice (6). In these studies, treatment Direct cytoprotection would likely involve the induction of spe- with IL-11 not only decreased mucosal damage but also acceler- cific gene products. IL-11 belongs to the IL-6 family of cytokines, ated healing and improved survival. IL-11 also reduces immune- all of which use gp130 as a critical component for signal trans- mediated small bowel injury in acute graft-vs-host disease after duction (15–17). IL-11 initiates signaling via binding to a unique murine allogeneic bone marrow transplantation (9). IL-11-receptor-␣ (IL-11R␣) chain (18, 19). The IL-11/IL-11R␣ The protective effects of IL-11 are not restricted to the intestine complex is thought to bind to and induce clustering of gp130, because IL-11 has also been shown to improve survival after tho- leading to the activation, via transphosphorylation, of associated racic irradiation (10). Human IL-11, expressed as a transgene in Janus kinases (JAKs) (20, 21). Activated JAKs phosphorylate ty- bronchial mucosa, reduces mortality associated with hyperoxia in rosine residues within the cytoplasmic region of gp130 which then mice (11). This effect was associated with a reduction in multiple serve as docking sites for STAT3 and STAT1 proteins (22, 23). The activated JAKs subsequently phosphorylate tyrosine residues within the bound STAT proteins, causing the STATs to dissociate *Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510; and †Genetics Institute, from gp130, dimerize (24), and enter the nucleus to act as tran- Andover, MA 01810 scriptional activators of target genes (25, 26). STAT dimers may Received for publication June 4, 1999. Accepted for publication January 27, 2000. be additionally phosphorylated on serine or threonine residues by The costs of publication of this article were defrayed in part by the payment of page mitogen-activated protein kinases (MAPKs) (27, 28) that are also charges. This article must therefore be hereby marked advertisement in accordance activated in response to cytokine binding to the receptor (29, 30). with 18 U.S.C. Section 1734 solely to indicate this fact. 1 K.M. and B.C.B. contributed equally to this work. 2 Current address: Medinische Universitaetsklinik, Bruderholzspital, CH-4101 Brud- erholz/Basel, Switzerland. 4 Abbreviations used in this paper: EC, endothelial cell(s); ECGS, endothelial cell 3 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center growth supplement; IL-11R␣, IL-11-receptor ␣-chain; JAK, Janus kinase; MAPKs, for Molecular Medicine, Room 454, 295 Congress Avenue, New Haven, CT 06510. mitogen-activated protein kinases; MEK, mitogen-activated protein/extracellular sig- E-mail address: [email protected] nal-related kinase kinase; ECL, enhanced chemiluminescence. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 3838 IL-11 PROTECTS HUMAN EC FROM CTL AND Ab PLUS C This additional phosphorylation may potentiate STAT function as Immunoprecipitation and immunoblotting an activator of transcription. To analyze protein by immunoblotting of cultured cell lysates, cells (ECs Immune and inflammatory processes depend on cytokine-medi- and K562) were washed twice with ice-cold PBS containing 1 mM sodium ated activation of vascular endothelium (e.g., new adhesion mol- orthovanadate and 1 mM sodium fluoride and were then lysed in 100 l ecule expression via an NF-B-dependent process) (31) and often cold RIPA lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxy- injure vascular endothelium, resulting in additional epithelial in- cholate, 0.1% SDS, 1 mM PMSF, 10 g/ml leupeptin, 1 mM sodium or- thovanadate). Cell lysates were clarified by centrifugation at 10,000 ϫ g jury as a consequence of ischemia which follows endothelial dam- for 15 min, and protein concentrations of the supernatant were determined age. Therefore, we investigated whether IL-11 could act on cul- by using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Lysates were pre- tured HUVECs to inhibit proinflammatory functions or to induce pared for SDS-PAGE by adding an equal volume of 2ϫ SDS-PAGE sam- cytoprotection. We report here that HUVECs express functional ple buffer (100 mM Tris-Cl (pH 6.8), 200 mM DTT, 4% SDS, 0.2% brom- phenol blue, 20% glycerol) and heating the mixture in a boiling water bath IL-11 receptors and respond to IL-11 by activating both STAT and for 3 min. Aliquots (10 g) of cell lysate were resolved by SDS-PAGE MAPK signaling pathways. Pretreatment of HUVECs with low using 8% acrylamide gels and a Tris-glycine electrophoresis buffer system dose IL-11 can protect HUVECs from immune-mediated injury, (25 mM Tris, 250 mM glycine, 0.1% SDS, pH 8.3), and separated proteins measured as cytolytic CTL- or Ab plus C-mediated lysis, but did were transferred to a polyvinylidene difluoride membrane by electrophore- not inhibit TNF-induced activation of NF-B or expression of leu- sis (Immobilon P, Millipore, Bedford, MA). Membranes were incubated with blocking solution containing 5% nonfat dry milk in Tris-buffered sa- kocyte adhesion molecules.