TLR9 Signaling and Promotes Neutrophil Adherence Through
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Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling This information is current as Driss El Kebir, Levente József, Wanling Pan, Lili Wang and of September 26, 2021. János G. Filep J Immunol 2009; 182:4386-4394; ; doi: 10.4049/jimmunol.0803044 http://www.jimmunol.org/content/182/7/4386 Downloaded from References This article cites 52 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/182/7/4386.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling1 Driss El Kebir,2 Levente Jo´zsef,2 Wanling Pan, Lili Wang, and Ja´nos G. Filep3 TLR9 detects bacterial DNA (CpG DNA) and elicits both innate and adoptive immunity. Recent evidence indicates that TLR9 is expressed in more diverse cell types than initially thought. In this study, we report that HUVECs constitutively express TLR9 and selectively recognize unmethylated CpG motifs in bacterial DNA and synthetic immune stimulatory CpG oligodeoxynucleotides. HUVECs respond to CpG DNA with rapid phosphorylation of IB-␣ and NF-B-mediated gene transcription and surface ex- pression of the adhesion molecules ICAM-1 and E-selectin independent of MAPK signaling. The telomere-derived TLR9 inhib- itory oligonucleotide 5-TTT AGG GTT AGG GTT AGG G-3, agents that block endosomal acidification such as chloroquine and bafilomycin A, and NF-B inhibitors abrogated CpG DNA-induced signaling. HUVEC activation by CpG DNA led to markedly enhanced neutrophil adhesion under nonstatic conditions that was further enhanced when neutrophils were stimulated with CpG Downloaded from DNA. The adhesive interactions were blocked by Abs against CD18 and, to a lesser degree, by anti-E-selectin and anti-L-selectin  Abs. Our findings demonstrate that bacterial DNA promotes 2 integrin and E-selectin-mediated HUVEC-neutrophil adherence, and indicate the ability of CpG DNA to initiate and/or maintain the inflammatory response. The Journal of Immunology, 2009, 182: 4386–4394. oll-like receptors detect microbial molecular patterns and Accumulation of neutrophils at sites of infection or tissue injury http://www.jimmunol.org/ initiate innate and adoptive immune responses (1). By is a critical component of the inflammatory response. Neutrophil T eliciting potent inflammatory responses (2, 3), TLRs are recruitment is tightly regulated by coordinated expression of ad- also involved in cardiovascular diseases (4). Sustained inflamma- hesion molecules on both neutrophils and endothelial cells and tion due to chronic or recurrent infections has been linked to in- production of cytokines/chemokines (19). Accumulating evidence creased risk of cardiovascular diseases (5). TLR9, localized to in- indicates a role for bacterial DNA in the regulation of these events. tracellular compartments, recognizes viral and bacterial DNA, In mice, bacterial DNA or synthetic oligodeoxynucleotides which contains unmethylated CpG dinucleotides in certain base (ODNs)4 containing unmethylated CpG motifs promotes neutro- contexts (6, 7). Bacterial DNA (CpG DNA) released from prolif- phil accumulation at the primary sites of infection (8, 20–23). erating bacteria or following killing of bacteria may persist in tis- Consistent with enhanced neutrophil trafficking in vivo, CpG DNA by guest on September 26, 2021 sues in the absence of bacteria and participate in ongoing inflam- enhances neutrophil chemotaxis (12), induces IL-8 release (11, mation. Thus, bacterial DNA has been detected in the lung of 24), triggers L-selectin shedding and up-regulation of CD11b/ cystic fibrosis patients (8), coronary artery specimens (9), and in CD18 (Mac-1) (11, 12), and delays neutrophil apoptosis (13). the blood of critically ill patients who have had negative blood However, it is not known whether bacterial DNA could promote culture (10). The function of TLR9 in APCs has extensively been neutrophil adhesion to endothelial cells, a critical step in neutrophil studied, consistent with the role of these cells in immune surveil- trafficking into tissues. lance. TLR9 expression is, however, not restricted to APCs. For In this study, we investigated the impact of CpG DNA on neu- instance, human neutrophils (11–13), murine endothelial cells (14, trophil adherence to HUVECs. Here, we report that HUVECs con- 15), human dermal microvascular (16) and lymphatic endothelial stitutively express TLR9 and selectively recognize unmethylated cells (17), and endothelial cells of human atherosclerotic plaques CpG motifs in bacterial DNA and synthetic immune stimulatory express TLR9 (18). CpG DNA was reported to stimulate ICAM-1 CpG ODNs. CpG DNA activates HUVEC as well as neutrophils  mRNA and protein expression and IL-8 release from murine en- and promotes 2 integrin and E-selectin-mediated neutrophil ad- dothelial cells (14, 15), whereas it failed to affect human lymphatic hesion, indicating its ability to initiate and/or maintain the inflam- endothelial cells (17) and even suppressed IL-8 production in hu- matory response in the absence of other bacterial constituents. man dermal microvascular endothelial cells (16). Materials and Methods Research Center, Maisonneuve-Rosemont Hospital and Department of Pathology and Bacterial and mammalian DNA Cell Biology, University of Montreal, Montreal, Quebec, Canada Escherichia coli DNA (strain B) and calf thymus DNA (Sigma-Aldrich) Received for publication September 15, 2008. Accepted for publication January were purified by extraction with phenol:chloroform:isoamyl alcohol (25: 26, 2009. 24:1, v/v/v) and ethanol precipitation. Heat-denaturated (single-stranded) The costs of publication of this article were defrayed in part by the payment of page genomic DNA was used in all experiments. For some experiments, E. coli charges. This article must therefore be hereby marked advertisement in accordance DNA was treated for 16 h at 37°C with CpG methylase SssI(2U/g DNA) with 18 U.S.C. Section 1734 solely to indicate this fact. in NE buffer 2 supplemented with 160 M S-adenosylmethionine (New 1 This work was supported by Grant MOP-67054 (to J.G.F.) and a Doctoral Research Award (to J.L.) from the Canadian Institutes of Health Research. 2 D.E.K. and L.J. contributed equally to this work. 4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; iODN, TLR9 inhib- 3 Address correspondence and reprint requests to Dr. Ja´nos G. Filep, Research Center, itory oligodeoxynucleotide; ctrlODN, control ODN. Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montreal, Que- bec, Canada H1T 2M4. E-mail address: janos.g.fi[email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803044 The Journal of Immunology 4387 England Biolabs). Methylated DNA was purified as above. All DNA prep- 15-g nuclear extracts. Binding is expressed as OD following correction arations contained Ͻ5 ng of LPS/mg of DNA by Limulus assay. with binding to the mutant sequence GGCATAGGTCC. Phosphorylation of IB-␣ at Ser32 and Ser36 relative to total IB-␣ protein was monitored Culture of HUVECs using 20 g of cytosolic protein with the CASE kit for IB-␣ S32/S36 (SuperArray). The amounts of phosphorylated IB-␣ are expressed as OD. HUVECs (cryopreserved primary cells; Cascade Biologics) were cultured in Medium 200 containing Low Serum Growth Supplement (Cascade RNase protection assay Biologics). For multiple RNase protection assays, HUVECs were lysed with 50 lof TLR9 expression lysis/denaturation solution (Ambion). 32P-Labeled antisense RNA probes were generated using templates for IL-8, MCP-1, ICAM-1, E-selectin, L32, Nonpassaged primary culture of HUVECs and untreated confluent mono- and GAPDH (RiboQuant; BD Pharmingen), and the assays were performed layers of HUVECs (passages two and three) were lysed in situ. Protein with the direct protect kit (Ambion) as described previously (28). extracts were subjected to Western blotting using mouse anti-human TLR9 mAb 26C593 (Calbiochem). The results were confirmed using rat anti- Isolation and culture of neutrophils human TLR9 Ab eB72-1665 (eBioscience). In additional experiments, un- treated HUVECs were detached, permeabilized with Permeabilization Venous blood (anticoagulated with sodium heparin, 50 U/ml) was obtained Buffer (eBioscience), and stained with R-PE-conjugated anti-TLR9 Ab from healthy volunteers who had denied taking any medication for at least eB72-1665 or a class-matched irrelevant Ab (eBioscience). Fluorescence 2 wk. The Clinical Research Committee of the Maisonneuve-Rosemont was