Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells This information is current as Ingrid U. Schraufstatter, Khanh Trieu, Lyudmila Sikora, P. of September 26, 2021. Sriramarao and Richard DiScipio J Immunol 2002; 169:2102-2110; ; doi: 10.4049/jimmunol.169.4.2102 http://www.jimmunol.org/content/169/4/2102 Downloaded from References This article cites 79 articles, 40 of which you can access for free at: http://www.jimmunol.org/content/169/4/2102.full#ref-list-1 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 26, 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells1 Ingrid U. Schraufstatter,2* Khanh Trieu,* Lyudmila Sikora,† P. Sriramarao,† and Richard DiScipio† In leukocytes, C3a and C5a cause chemotaxis in a Gi-dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress fibers, which was not PT-sensitive, but depended on rho activation implying coupling to G␣12 or G␣13. Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and Downloaded from of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade–transactivation of the EGFR–that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably G␣12/13. The Journal of Immunology, 2002, 169: 2102–2110. omplement activation leads to the production of the ana- tion of these receptors on cell types other than leukocytes is http://www.jimmunol.org/ phylatoxins C3a and C5a, which are basic polypeptides under active investigation. C of 74–77 aa derived from the ␣-chains of their parent Both the C3aR and the C5aR are GPCRs, which couple to the proteins. C3a and C5a are known for their stimulatory effect on pertussis toxin (PT)-sensitive Gi␣ (21–24) and to the PT-insensi- leukocytes including chemotaxis of neutrophils, eosinophils, and tive G␣16 (25, 26) in leukocytes. Leukocytes are very rich in Gi␣, monocytes (1–5). Furthermore, the anaphylatoxins can also induce and G␣16 expression is limited to cells of hemopoietic lineage (27). smooth muscle contraction (6) and can evoke an increase in vas- Because G protein usage is not absolute, it is conceivable that other cular permeability (7, 8). cell types use additional G proteins and/or signal transduction However, it has been appreciated only recently that the expres- cascades. by guest on September 26, 2021 sion of anaphylatoxin receptors is not limited to leukocytes. In In this study, we report that cultured human endothelial cells particular, expression of the C5aR is widespread and has been express C3aRs and C5aRs, and that the two receptors use different shown in liver, lung, kidney, and the CNS (9–12). Although it was signal transduction cascades to activate cellular responses distinct reported that expression of C5aRs is low on endothelial cells (13, from those on leukocytes. 14), it is incorrect to conclude that activation of these receptors is functionally irrelevant, because engagement of a few hundred G Materials and Methods protein-coupled receptors (GPCRs)3 per cell is sufficient for Purification of C3a and C5a maximal cell activation (15). Indeed, it has been suggested that Human C3a and C5a were produced after cleavage of their parent proteins, endothelial cell-derived superoxide generation played a role in C3 and C5, respectively, using a fluid phase C3/C5 convertase, CVF, and C5a-mediated pulmonary injury (16). Still less is known about Bb (28). C3a and C5a were collected after direct passage through DEAE- C3aRs on nonhemopoietic cells. Their expression has been de- Sephacel equilibrated in 20 mM imidazole HCl (pH 7)/0.075 M NaCl. Then, the anaphylatoxins were adsorbed onto CM-Sephadex (Amersham scribed in the CNS (17, 18), on epithelial cells (19), and on Pharmacia Biotech, Piscataway, NJ), and eluted with 0.25 M NaCl. The endothelial cells following ischemia/reperfusion (20). The func- synthetic polypeptide consisting of the 21 terminal amino acids of C3a, C3a (57–77), was a kind gift of Dr. T. E. Hugli (La Jolla Institute for Molecular Medicine, San Diego, CA). Cell culture Departments of *Cancer Biology and †Vascular Biology, La Jolla Institute for Mo- lecular Medicine, San Diego, CA 92121 HUVECs were obtained from Clonetics (San Diego, CA) and used between passage 5 and 7. Human immortalized dermal microvascular endothelial Received for publication December 19, 2001. Accepted for publication June 19, 2002. cells (HMECs; Ref. 29) were obtained from the Center for Disease Control The costs of publication of this article were defrayed in part by the payment of page (Atlanta, GA). The cells were grown in endothelial growth medium as charges. This article must therefore be hereby marked advertisement in accordance recommended by the supplier (Clonetics). with 18 U.S.C. Section 1734 solely to indicate this fact. HMECs or HUVECs were seeded at low density on collagen-coated 1 This work was supported by National Institute Health Grants HL55657 (to I.U.S.) coverslips and grown in endothelial growth medium (Clonetics) containing and AI35796 (to P.S.). 10% FCS. On the day of the experiment, cells were serum starved for 2 h 2 Address correspondence and reprint requests to Dr. Ingrid U. Schraufstatter, De- (HUVECs) or 3–4 h (HMECs) and stimulated with C3a or C5a for the time partment of Cancer Biology, La Jolla Institute for Molecular Medicine, 4570 Exec- indicated for each experiment. For immuneprecipitations, HMECs were utive Drive, #100, San Diego, CA 92121. E-mail address: [email protected] serum starved for 16 h. All experiments were performed at 37°C in a tissue 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; HMEC, hu- culture incubator. man immortalized dermal microvascular endothelial cell; EGF, epidermal growth To block Gi␣, endothelial cells were incubated for 16 h with 100 ng/ml factor; EGFR, EGF receptor; PT, pertussis toxin; LPA, lysophosphatidic acid; HB- of PT (List Biological Laboratories, Campbell, CA). Rho was inhibited EGF, heparin-binding EGF-like factor; F-actin, filamentous actin. with C3 botulinum toxin (10 ␮g/ml; List Biological Laboratories) for 24 h, Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 2103 which leads to inactivation of rho in ϳ90% of endothelial cells (30). Rho Immuneprecipitations of the EGFR ␮ kinase, a downstream target of rho, was inhibited with 10 M Y27632 (gift 2 of Yoshitomi Pharmaceuticals, Osaka, Japan) added 30 min before the HMECs were grown to confluence on 100-mm tissue culture plates, serum addition of the stimulus (31). Similarly, tyrphostin AG 1478 (1 ␮Min starved for 18 h, incubated with 5 ml fresh serum-free media, stimulated DMSO; Calbiochem, La Jolla, CA), a specific inhibitor of the epithelial with 50 nM C5a, 500 nM C3a, or 30 ng/ml of EGF for the indicated times ␮ growth factor (EGF) receptor (EGFR; Ref. 32), and PP2 (Calbiochem), a at 37°C, placed on ice, washed once with PBS, and lysed in 400 l lysis specific inhibitor of src kinase, (33) were added for 30 min before the buffer (150 mM NaCl, 25 mM Tris (pH 7.5), 1 mM EDTA, 2 mM sodium addition of stimulus. All blocking Abs were used at 10 ␮g/ml. vanadate, 10 mM NaF, 2 mM sodium pyrophosphate, 1% Nonidet P-40, 2 ␮g/ml aprotinine, 2 ␮g/ml leupeptin, 2 mM PMSF, 10% glycerol). Fol- lowing centrifugation for 10 min at 10,000 ϫ g, the protein content in the FACS analysis supernatants was determined using the bicinchoninic acid reagents (Pierce, Rockford, IL). Ab (rabbit polyclonal anti-EGFR Ab, 5 ␮g/sample; Upstate For FACS analysis, HMECs or HUVECs were trypsinized shortly, placed Biotechnology, Lake Placid, NY) was added to each sample containing 0.8 in PBS containing 2% FCS, incubated for 30 min on ice with anti-C5aR or mg protein, and the samples were rotated for2hat4°C. Protein A/G anti-C3aR Ab (1/200 dilution; both from BD PharMingen, San Diego, CA), Sepharose (30 ␮l of a 50% slurry; Amersham Pharmacia Biotech) was washed three times with PBS/FCS, and labeled with mouse- or rabbit- added for 30 min, and the samples were washed three times in lysis buffer biotinylated IgG for 30 min.