Extracellular Polyphosphate Promotes Macrophage and Fibrocyte Differentiation, Inhibits Leukocyte Proliferation, and Acts as a Chemotactic Agent for Neutrophils This information is current as of October 1, 2021. Patrick M. Suess, Luis E. Chinea, Darrell Pilling and Richard H. Gomer J Immunol published online 3 June 2019 http://www.jimmunol.org/content/early/2019/05/31/jimmun ol.1801559 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/05/31/jimmunol.180155 Material 9.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 October 1, 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 June 3, 2019, doi:10.4049/jimmunol.1801559 The Journal of Immunology Extracellular Polyphosphate Promotes Macrophage and Fibrocyte Differentiation, Inhibits Leukocyte Proliferation, and Acts as a Chemotactic Agent for Neutrophils Patrick M. Suess,1 Luis E. Chinea, Darrell Pilling, and Richard H. Gomer Fibrocytes are monocyte-derived fibroblast like cells that participate in wound healing, but little is known about what initiates fibrocyte differentiation. Blood platelets contain 60–100-mer polymers of phosphate groups called polyphosphate, and when activated, platelets induce blood clotting (the first step in wound healing) in part by the release of polyphosphate. We find that activated platelets release a factor that promotes fibrocyte differentiation. The factor is abolished by treating the crude platelet factor with the polyphosphate-degrading enzyme polyphosphatase, and polyphosphate promotes fibrocyte differentiation. Mac- rophages and recruited neutrophils also potentiate wound healing, and polyphosphate also promotes macrophage differentiation and induces chemoattraction of neutrophils. In support of the hypothesis that polyphosphate is a signal that affects leukocytes, we Downloaded from observe saturable binding of polyphosphate to these cells. Polyphosphate also inhibits leukocyte proliferation and proteasome activity. These results suggest new roles for extracellular polyphosphate as a mediator of wound healing and inflammation and also provide a potential link between platelet activation and the progression of fibrosing diseases. The Journal of Immunology, 2019, 203: 000–000. ibrocytes are monocyte-derived cells that have properties recruitment, activation, and proliferation of a variety of leuko- http://www.jimmunol.org/ of both inflammatory macrophages and tissue remodeling cytes including neutrophils, lymphocytes, and monocyte-derived F fibroblasts (1). Fibrocytes participate in wound healing by macrophages to a site of tissue damage, and these events are secreting inflammatory cytokines and ECM proteins (2). Fibro- important steps in the process of tissue repair (12–14). cytes are also found at fibrotic lesions and have been implicated in In this article, we report that polyphosphate has multiple effects the progression of fibrosing diseases (2). What initiates fibrocyte on leukocyte function, including inducing monocytes to differ- differentiation in a wound remains poorly understood (3–5). entiate into fibrocytes and macrophages, inhibits leukocyte pro- Blood clotting is an early and critical component of wound liferation and proteasome activity, and acting as a chemoattractant healing, which is induced upon the activation of platelets at the for neutrophils. We found that the incubation of PBMC with ac- site of tissue damage (6). Numerous factors released from acti- tivated releasates from platelets also induces fibrocyte differenti- by guest on October 1, 2021 vated platelets interact with leukocytes and elicit various func- ation, and this activity is lost upon treatment of releasates with the tions (7, 8); however, the role of platelet-derived factors on polyphosphate-degrading enzyme exopolyphosphatase, suggesting fibrocyte differentiation have not been well investigated. One a new role for polyphosphate as a mediator of innate immunity and factor released by activated platelets is polyphosphate, a highly wound healing. anionic, linear polymer of phosphates bound by phosphoanhydride bonds (9). Platelet-derived extracellular polyphosphate contrib- Materials and Methods utes to wound healing and inflammation by binding to and en- Platelet, neutrophil, and PBMC isolation and culture hancing activation of the plasma protease factor XII, which Human peripheral blood was collected from healthy volunteers who gave triggers a response leading to fibrin and bradykinin generation written consent, with specific approval from the institutional review board (10). Topical application of polyphosphate enhances wound of Texas A&M University. Whole blood was collected in vacutainers healing in vivo, leading to an increase in collagen and a-smooth containing acid–citrate–dextrose to prevent clotting. Blood was gently muscle actin (11). The inflammatory response also drives the transferred to 15-ml conical tubes using plastic transfer pipettes and clarified by centrifugation at 200 3 g for 20 min. The upper two thirds of the straw-colored platelet-rich plasma was transferred to a fresh tube con- Department of Biology, Texas A&M University, College Station, TX 77843 taining HEP buffer (140 mM NaCl, 2.7 mM KCL, 3.8 mM HEPES, 5 mM 1 EGTA, pH 7.4) containing 1 mM PG E1 (Sigma-Aldrich, St Louis, MO) at Current address: University of Michigan Medical School, Ann Arbor, MI. a 1:1 ratio. Samples were mixed gently and clarified by centrifugation at ORCIDs: 0000-0002-9805-9005 (P.M.S.); 0000-0003-3511-2830 (L.E.C.); 0000- 100 3 g for 20 min. The supernatant was transferred to a new tube, and 0002-7413-2773 (D.P.). platelets were collected by centrifugation at 800 3 g for 20 min. The Received for publication November 28, 2018. Accepted for publication May 8, 2019. platelet pellet was washed two times in platelet wash buffer (10 mM so- dium citrate, 150 mM NaCl, 1 mM EDTA, 1% [w/v] dextrose, pH 7.4) This work was supported by National Institutes of Health Grant R01 GM102280. without resuspension. The platelet pellet was gently resuspended in Address correspondence and reprint requests to Dr. Richard H. Gomer, Department Tyrode’s buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM of Biology, Texas A&M University, ILSB, 301 Old Main Drive, College Station, TX Na2HPO4, 1 mM MgCl2, 10 mM HEPES, pH 7.4) containing 5 mM glu- 77843-3474. E-mail address: [email protected] cose and 3 mg/ml BSA (Amresco, Solon, OH). Platelets were counted with The online version of this article contains supplemental material. a hemocytometer, and the platelet concentration was adjusted in Tyrode’s Abbreviations used in this article: FMI, forward migration index; FSC, forward buffer. To activate platelets, samples were incubated with 1.2 U/ml of scatter; PPX1, yeast exopolyphosphatase; RPMI-BSA, RPMI 1640 containing 2% human thrombin (Sigma-Aldrich) for 1 h, and secreted material was iso- BSA; SFM, serum-free medium. lated by passage through a 0.22-mm type 25-243 syringe filter (Genesee Scientific, San Diego, CA). Exopolyphosphatase treatment of platelet Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 releasates was performed as previously described (15). To remove www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801559 2 EXTRACELLULAR POLYPHOSPHATE EFFECT ON LEUKOCYTES thrombin, exopolyphosphatase, and large proteins, samples were clarified filter as described above. After centrifugation, 10 ml of the diluted by centrifugation for 10 min at 14,000 3 g through a 30-kDa cutoff Spin-X thrombin, flow through, or Tyrode’s buffer were mixed with 90 mlof UF size exclusion filter (Corning, Tewksbury, MA). Platelets were also 0.2 mM D-FPIPR-pNA chromogenic thrombin substrate (Molecular In- activated by incubation on ice or at 37˚C with continual mixing (16, 17). novations, Novi, MI) in PBS in the well of a 96-well plate, and the ab- Platelets at 37˚C were also activated in the presence of 200 mg human sorbance at 405 nM was read every 30 s for 10 min. Fits to a line were used collagen-I–coated beads (SphereCol; Advanced BioMatrix, Carlsbad, CA) to determine the change in absorbance with time. per 1 3 108 platelets (16). After 60 min, platelet releasates were clarified by centrifugation for 10 min at 14,000 3 g. Releasates from platelets Statistics incubated with collagen beads were further clarified by centrifugation for Statistical analysis was performed using Prism (GraphPad, San Diego, CA). 10 min at 14,000 3 g through a 30-kDa cutoff Spin-X UF size exclusion Differences between two groups were assessed by Student t test. Differ- filter. ences between multiple groups were assessed by one-way ANOVA using PBMC and neutrophils were isolated as previously described (18, 19), Tukey posttest. Significance was defined as p , 0.05. with the exception that neutrophils