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• W1992121470 abstract "Viral DNA induces potent antiviral immunity by activating dendritic cells; however, the mechanism governing viral DNA-mediated triggering or aggravation of glomerulonephritis is unknown. Glomerular endothelial cells (GEnCs) do not express toll-like receptor (TLR)9, the only DNA-specific TLR. We therefore hypothesized that DNA could activate GEnCs via the recently discovered TLR-independent viral DNA recognition pathway. Indeed, double-stranded non-CpG (B-) DNA activated GEnCs to produce interleukin-6, CCL5/RANTES, CCL2/MCP-1, CXCL10/IP10, interferon (IFN)-α, and IFN-β when cationic lipids facilitated intracellular DNA uptake. This cytokine production was inhibited by chlorpromazine, suggesting that clathrin-dependent endocytosis is required for B-DNA entry. However, chloroquine and MyD88 inhibition did not affect GEnC activation, suggesting TLR-independent DNA recognition. In addition, IFN-β activated cytokine and chemokine mRNA expression, although only CXCL10/IP10 was induced at the protein level, and type I IFN did not activate GEnC in an autocrine-paracrine auto-activation loop. B-DNA complexes induced intercellular adhesion molecule-1 expression at the GEnC surface and increased intercellular adhesion molecule-1-dependent leukocyte adhesion and microvascular extravasation in vivo. Furthermore, B-DNA complexes increased albumin permeability of GEnC monolayers in culture or microvascular dextran leakage in vivo. In addition, B-DNA complexes impaired GEnC proliferation. Thus, complexed B-DNA activates GEnC to produce cytokines, chemokines, and type I IFNs, increases leukocyte adhesion and microvascular permeability, and reduces GEnC proliferation via a MyD88-independent cytosolic DNA recognition pathway. This innate antiviral response program suggests a novel pathomechanism regulating DNA virus-mediated induction or aggravation of glomerulonephritis. Viral DNA induces potent antiviral immunity by activating dendritic cells; however, the mechanism governing viral DNA-mediated triggering or aggravation of glomerulonephritis is unknown. Glomerular endothelial cells (GEnCs) do not express toll-like receptor (TLR)9, the only DNA-specific TLR. We therefore hypothesized that DNA could activate GEnCs via the recently discovered TLR-independent viral DNA recognition pathway. Indeed, double-stranded non-CpG (B-) DNA activated GEnCs to produce interleukin-6, CCL5/RANTES, CCL2/MCP-1, CXCL10/IP10, interferon (IFN)-α, and IFN-β when cationic lipids facilitated intracellular DNA uptake. This cytokine production was inhibited by chlorpromazine, suggesting that clathrin-dependent endocytosis is required for B-DNA entry. However, chloroquine and MyD88 inhibition did not affect GEnC activation, suggesting TLR-independent DNA recognition. In addition, IFN-β activated cytokine and chemokine mRNA expression, although only CXCL10/IP10 was induced at the protein level, and type I IFN did not activate GEnC in an autocrine-paracrine auto-activation loop. B-DNA complexes induced intercellular adhesion molecule-1 expression at the GEnC surface and increased intercellular adhesion molecule-1-dependent leukocyte adhesion and microvascular extravasation in vivo. Furthermore, B-DNA complexes increased albumin permeability of GEnC monolayers in culture or microvascular dextran leakage in vivo. In addition, B-DNA complexes impaired GEnC proliferation. Thus, complexed B-DNA activates GEnC to produce cytokines, chemokines, and type I IFNs, increases leukocyte adhesion and microvascular permeability, and reduces GEnC proliferation via a MyD88-independent cytosolic DNA recognition pathway. This innate antiviral response program suggests a novel pathomechanism regulating DNA virus-mediated induction or aggravation of glomerulonephritis. Extrarenal viral infections can trigger de novo immune complex glomerulonephritis, eg, hepatitis C virus-associated glomerulonephritis, but more frequently, acute viral infections trigger disease activity of pre-existing glomerulonephritis, as in IgA nephropathy, lupus nephritis, or renal vasculitis.1Lai AS Lai KN Viral nephropathy.Nat Clin Pract Nephrol. 2006; 2: 254-262Crossref PubMed Scopus (77) Google Scholar Viral entry activates systemic antiviral immune responses, which may trigger disease flares of glomerulonephritis by enhancing autoantibody production, immune complex formation or by systemic interferon (IFN) release.2Theofilopoulos AN Baccala R Beutler B Kono DH Type I interferons (alpha/beta) in immunity and autoimmunity.Annu Rev Immunol. 2005; 23: 307-336Crossref PubMed Scopus (1025) Google Scholar Rapid production of type I IFN is a central element of antiviral immunity, since type I IFNs inhibit viral replication in infected cells and have pleiotrophic immunomodulatory effects on macrophages, T cells, and natural killer cells.3Stark GR Kerr IM Williams BR Silverman RH Schreiber RD How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3398) Google Scholar In the intravascular compartment, plasmacytoid dendritic cells are the main type I IFN-producing cell type on recognition of viral proteins via Toll-like receptors (TLRs) 2 and 4 at the cell surface.2Theofilopoulos AN Baccala R Beutler B Kono DH Type I interferons (alpha/beta) in immunity and autoimmunity.Annu Rev Immunol. 2005; 23: 307-336Crossref PubMed Scopus (1025) Google Scholar, 4Shortman K Liu YJ Mouse and human dendritic cell subtypes.Nat Rev Immunol. 2002; 2: 151-161Crossref PubMed Scopus (1919) Google Scholar, 5Diebold SS Montoya M Unger H Alexopoulou L Roy P Haswell LE Al-Shamkhani A Flavell R Borrow P Reis e Sousa C Viral infection switches non-plasmacytoid dendritic cells into high interferon producers.Nature. 2003; 424: 324-328Crossref PubMed Scopus (509) Google Scholar, 6Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4986) Google Scholar Viral nucleic acids activate type I IFN production not before being taken up into intracellular endosomes where they activate TLR3 (double-stranded [ds]RNA), TLR7/8 (single-stranded RNA), or TLR9 (CpG-DNA).6Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4986) Google Scholar, 7Pichlmair A Reis e Sousa C Innate recognition of viruses.Immunity. 2007; 27: 370-383Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar DAI/ZBP1 has been proposed as one of potentially more cytosolic sensors of viral DNA but little is yet known about their structure, expression and cell type-specific function.8Ishii KJ Coban C Kato H Takahashi K Torii Y Takeshita F Ludwig H Sutter G Suzuki K Hemmi H Sato S Yamamoto M Uematsu S Kawai T Takeuchi O Akira S A toll-like receptor-independent antiviral response induced by double-stranded B-form DNA.Nat Immunol. 2006; 7: 40-48Crossref PubMed Scopus (647) Google Scholar, 9Takaoka A Wang Z Choi MK Yanai H Negishi H Ban T Lu Y Miyagishi M Kodama T Honda K Ohba Y Taniguchi T DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.Nature. 2007; 448: 501-505Crossref PubMed Scopus (1307) Google Scholar HIN-200 proteins also contribute to cytosolic DNA signaling, however, predominantly activate the inflammasome caspase-dependent release of interleukin (IL)-1β.10Roberts TL Idris A Dunn JA Kelly GM Burnton CM Hodgson S Hardy LL Garceau V Sweet MJ Ross IL Hume DA Stacey KJ HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.Science. 2009; Crossref Scopus (680) Google Scholar, 11Hornung V Ablasser A Charrel-Dennis M Bauernfeind F Horvath G Caffrey DR Latz E Fitzgerald KA AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.Nature. 2009; Crossref PubMed Scopus (1845) Google Scholar, 12Muruve DA Petrilli V Zaiss AK White LR Clark SA Ross PJ Parks RJ Tschopp J The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response.Nature. 2008; 452: 103-107Crossref PubMed Scopus (767) Google Scholar Despite 50 years of IFN research and the clinical association of viral infection and glomerulonephritis, little is known about local production of type I IFN in glomerulonephritis.13Borden EC Sen GC Uze G Silverman RH Ransohoff RM Foster GR Stark GR Interferons at age 50: past, current, and future impact on biomedicine.Nat Rev Drug Discov. 2007; 6: 975-990Crossref PubMed Scopus (891) Google Scholar We hypothesized that glomerular cells like glomerular endothelial cells (GEnCs) would also produce type I IFN on viral DNA exposure. GEnCs line the intraglomerular capillaries, acting as a barrier, but also contribute to fluid filtration, and therefore interact with circulating viral particles.14Kriz W Fenestrated glomerular capillaries are unique.J Am Soc Nephrol. 2008; 19: 1439-1440Crossref PubMed Scopus (12) Google Scholar, 15Haraldsson B Nystrom J Deen WM Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (629) Google Scholar Similar to other microvascular endothelia, GEnCs can contribute to local inflammation by secreting pro-inflammatory cytokines and by fostering glomerular leukocyte recruitment via presenting chemokines and adhesion molecules at the luminal GEnC membrane.16Pober JS Sessa WC Evolving functions of endothelial cells in inflammation.Nat Rev Immunol. 2007; 7: 803-815Crossref PubMed Scopus (1279) Google Scholar GEnCs express a limited pattern of TLRs, ie, TLR1-6, but lack the expression of the only DNA-specific TLR, ie, TLR9.17Allam R Pawar RD Kulkarni OP Hornung V Hartmann G Segerer S Akira S Endres S Anders HJ Viral 5′-triphosphate RNA and non-CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR-independent immune responses.Eur J Immunol. 2008; 38: 3487-3498Crossref PubMed Scopus (57) Google Scholar We hypothesized that GEnCs harbor a TLR-independent cytosolic DNA recognition pathway and that viral ds(non-CpG) DNA can trigger antiviral immunity in GEnC. GEnCs, prepared from ts A58 immorto mice,18Akis N Madaio MP Isolation, culture, and characterization of endothelial cells from mouse glomeruli.Kidney Int. 2004; 65: 2223-2227Crossref PubMed Scopus (74) Google Scholar were grown in medium RPMI 1640 (Invitrogen, Paisley, UK) with 10% heat-inactivated fetal calf serum. GEnCs were stimulated with endotoxin-free poly dA/dT (B-DNA, Sigma-Aldrich, St. Louis, MO) either pure or complexed with the cationic lipid (CL) lipofectamine 2000 (Invitrogen) for 24 hours in medium containing 2% fetal calf serum. B-DNA and CL were pre-incubated with polymyxin B (Invivogen, San Diego, CA) before use to block any residual lipopolysaccharaide (LPS) contamination. Murine IFN-α was purchased from Serotec (Oxford, UK), IFN-β from PBL (Piscataway, NJ), IFN-γ from PeproTech (Rocky Hill, NJ), chloroquine and methyl-β-cyclodextrin from Sigma-Aldrich (Steinheim, Germany), chlorpromazine from Merck (Darmstadt, Germany), cytochalasin D from BIOMOL (Plymouth Meeting, PA), MyD88 homodimerization inhibitory peptide or control peptide from Imgenex (San Diego, CA),19Loiarro M Sette C Gallo G Ciacci A Fanto N Mastroianni D Carminati P Ruggiero V Peptide-mediated interference of TIR domain dimerization in MyD88 inhibits interleukin-1-dependent activation of NF-{kappa}B.J Biol Chem. 2005; 280: 15809-15814Crossref PubMed Scopus (153) Google Scholar and Pam3CSK from Invitrogen.20Aliprantis AO Yang RB Mark MR Suggett S Devaux B Radolf JD Klimpel GR Godowski P Zychlinsky A Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.Science. 1999; 285: 736-739Crossref PubMed Scopus (1279) Google Scholar Rat monoclonal antibodies against mouse IFN-α and IFN-β (32100-1 and 32400-1, PBL Interferon Source, Piscataway, NJ) were used to neutralize GEnC-derived IFN in vitro. They both were added simultaneously with different concentrations of B-DNA, as indicated. Cytokine levels were measured in cell supernatants using commercial enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (OptEiA, BD, Mannheim Germany), IFN-α (PBL), IFN-β (PBL), IFN-γ (BD), CCL2/MCP-1 (BD), CCL5/RANTES, and CXCL10/IP-10 (R&D Systems, Minneapolis, MN). GEnC proliferation was determined after 72 hours using CellTiter 96 cell proliferation assay (Promega, Madison, WI) reading absorbance at 492 nm. For cellular uptake studies GEnCs were stimulated with 5′-rhodamine-labeled B-DNA. After 2 hours the cells were fixed with 2% paraformaldehyde, 10 mmol/L Pipes, 15% saturated picric acid at pH 6.0. Cells were incubated overnight with fluorescein isothiocyanate (FITC)-phalloidin (1:200, Invitrogen) before scanning with a LSM510 laser microscope (Carl Zeiss, Jena, Germany). Complementary DNA was generated from total RNA using random priming and MMLV reverse transcriptase (Invitrogen). Real-time PCR was performed using SYBRGreen PCR master mix or on Roche Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). All values were normalized to 18s rRNA. The oligonucleotide primer sequences are listed in table 1.Table 1Murine Probes Used for Real-Time RT-PCRGeneSequence18S Forward5′-GCAATTATTCCCCATGAACG-3′ Reverse5′-AGGGCCTCACTAAACCATCC-3′ZBP1 Left5′-TATGACGGACAGACGTGGAA-3′ Right5′-TGCTGACAAATAATCGCAGG-3′CCL2 Left5′-CCTGCTGTTCACAGTTGCC-3′ Right5′-ATTGGGATCATCTTGCTGGT-3′CCL5 Left5′-CCACTTCTTCTCTGGGTTGG-3′ Right5′-GTGCCCACGTCAAGGAGTAT-3′CXCL10 Left5′-GGCTGGTCACCTTTCAGAAG-3′ Right5′-ATGGATGGACAGCAGAGAGC-3′MX1 Left5′-TCTGAGGAGAGCCAGACGAT-3′ Right5′-CTCAGGGTGTCGATGAGGTC-3′IFNB1 Left5′-CCCTATGGAGATGACGGAGA-3′ Right5′-CCCAGTGCTGGAGAAATTGT-3′ Open table in a new tab GEnCs were washed and lysed in lysis buffer (1% Triton, 10 mmol/L Tris, 10 mmol/L NaCl, 10 mmol/L EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmol/L phenylmethylsulfonylfluoride, pH 7.4). Total protein aliquots of 25 to 50 μg of lysate was separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (Millipore, Eschborn, Germany). The blots were blocked in 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 for 1 hour and then incubated with primary antibodies overnight at 4°C as follows: phosphorylated interferon-related factor (pIRF)-3, 1:2500; β-actin, 1:5000 (all from Cell Signaling, Danvers, MA); pIRF-3, 3 μg/ml (Abcam, Cambridge, MA). Horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling) and anti-goat IgG (Jackson ImmunoResearch Laboratories, Suffolk, UK) were diluted to 1:20,000 in blocking solution (5% nonfat dry milk in buffer containing 0.1% Tween 20. Blots were developed with enhanced chemiluminescence (Amersham Pharmacia, Freiburg, Germany). B-DNA-stimulated GEnCs were washed with PBS and incubated with binding buffer containing either FITC-anti-annexin V (BD, Franklin Lakes, NJ) or propidium iodide (PI, BD) for 15 minutes at room temperature. In other experiments, cells were then washed with PBS and incubated with PBS containing FITC-anti-CD106 (VCAM-1) from BioLegend (San Diego, CA) or FITC-anti-CD54 (ICAM-1) from BioLegend for 45 minutes at room temperature The cells were analyzed by flow cytometry (FACS Calibur, BD) with acquisition of 30,000 events/sample. GEnCs were grown to confluent monolayers hanging on 1-μm pore size inserts (Millipore, Billerica, MA) in 24-well plates with 0.5 ml media on both sides of the membrane.17Allam R Pawar RD Kulkarni OP Hornung V Hartmann G Segerer S Akira S Endres S Anders HJ Viral 5′-triphosphate RNA and non-CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR-independent immune responses.Eur J Immunol. 2008; 38: 3487-3498Crossref PubMed Scopus (57) Google Scholar, 21Satchell SC Buchatska O Khan SB Bhangal G Tasman CH Saleem MA Baker DP Lobb RR Smith J Cook HT Mathieson PW Pusey CD Interferon-beta reduces proteinuria in experimental glomerulonephritis.J Am Soc Nephrol. 2007; 18: 2875-2884Crossref PubMed Scopus (30) Google Scholar FITC-labeled bovine serum albumin (Invitrogen) was added to inserts and the cells were stimulated with 10 μg/ml B-DNA/CL or 6 μg/ml CpG-DNA. The filtrate was sampled at different time points and OD was measured at 485 nm and emission at 535 nm. DAI/ZBP1 small interfering (si)RNA and negative control siRNA (Ambion/Applied Biosystems, Darmstadt, Germany) sequences were as follows: DAI/ZBP1, 5′-ACAGUCCAGACAGUCCACAUCAAAU-3′; control, 5′-GGCAACAAGAUGACCAUCCACCUUA-3′, 5′-GGAAGACACAGGUACAA GCUCUGAA-3′; control, 5′-GAAGAGCACGAGAUAGCAAUU-3′. GEnCs (1 × 105) were plated in 12-well plates in 2% fetal calf serum-RPMI medium. Forty nmol/L siRNA was transfected twice with CL as above; 24 hours later, GEnCs were stimulated with 6 μg of B-DNA/CL. Knock-down efficacy of DAI/ZBP1, as well as CXCL10, IL-6, and IFN-β mRNA expression were determined by real-time reverse transcription-PCR after 6 hours and by Western blot after 24 hours. In vivo microscopy was performed as described.22Reichel CA Khandoga A Anders HJ Schlondorff D Luckow B Krombach F Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue.J Leukoc Biol. 2006; 79: 114-122Crossref PubMed Scopus (99) Google Scholar, 23Reichel CA Rehberg M Bihari P Moser CM Linder S Khandoga A Krombach F Gelatinases mediate neutrophil recruitment in vivo: evidence for stimulus specificity and a critical role in collagen IV remodeling.J Leukoc Biol. 2008; 83: 864-874Crossref PubMed Scopus (52) Google Scholar In brief, 6-week-old male C57BL/6 mice (Charles River, Sulzfeld, Germany) received an intrascrotal injection of PBS, CL, or 10 μg B-DNA/CL (n = 4 in each group) 6 hours before microscopic analysis. Some mice received 100 μg of an anti-CD54/ICAM-1 antibody (clone YN1/1.7.4, BioLegend, San Diego, CA) or isotype control antibody by intravenous injection, 5 minutes before intrascrotal injection of DNA. Images were obtained with water immersion lenses (×20/numerical aperture 0.5 and ×40/numerical aperture 0.8). For measurement of centerline blood flow velocity, green fluorescent microspheres (2 μm diameter; Molecular Probes, Leiden, The Netherlands) were injected via a arterial catheter, and their passage through the vessels of interest was recorded using the FITC filter cube under appropriate stroboscopic illumination (exposure 1 ms, cycle time 10 ms, λ = 488 nm). From measured vessel diameters and centerline blood flow velocity, apparent wall shear stress was calculated, assuming a parabolic flow velocity profile over the vessel cross section. The quantitative analysis of leukocyte migration was performed using Cap-Image software (Dr. Zeintl, Heidelberg, Germany). Firmly adherent cells were determined as those resting in the associated blood flow for more than 30 seconds and related to the luminal surface per 100 μm vessel length. Transmigrated cells were counted in regions of interest covering 75 μm on both sides of a vessel over 100-μm vessel length. Dextran permeability was determined as described.23Reichel CA Rehberg M Bihari P Moser CM Linder S Khandoga A Krombach F Gelatinases mediate neutrophil recruitment in vivo: evidence for stimulus specificity and a critical role in collagen IV remodeling.J Leukoc Biol. 2008; 83: 864-874Crossref PubMed Scopus (52) Google Scholar In brief, FITC-dextran (5 mg in 0.1 ml saline, Mr150,000, Sigma-Aldrich) was infused intra-arterially after DNA stimulation. Five postcapillary vessel segments, as well as the surrounding perivascular tissue were excited at 488 nm, and emission >515 nm was recorded by a CCD camera (Sensicam, PCO, Kelheim, Germany) 30 minutes after injection of FITC-dextran using an appropriate emission filter (LP 515). Mean gray values of fluorescence intensity were measured by digital image analysis (TILLvisION 4.0, TILL Photonics) in six randomly selected regions of interest (50 × 50 μm2), localized ∼50 μm distant from the postcapillary venule under investigation. The average of mean gray values was calculated. Data were expressed as mean ± SEM. Groups were compared by two-tailed t-test or one-way analysis of variance. A value of P < 0.05 was considered to be statistically significant. As GEnCs lack the only DNA-specific TLR (TLR9),17Allam R Pawar RD Kulkarni OP Hornung V Hartmann G Segerer S Akira S Endres S Anders HJ Viral 5′-triphosphate RNA and non-CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR-independent immune responses.Eur J Immunol. 2008; 38: 3487-3498Crossref PubMed Scopus (57) Google Scholar it is questionable whether DNA can at all activate GEnCs. In fact, B-DNA and CpG-DNA both did neither induce IL-6, CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, IFN-α, nor IFN-β secretion in cultured GEnCs (Figure 1, A and B). However, when being complexed with CL, low concentrations of B-DNA activated GEnCs to secrete all of these factors in a dose-dependent manner (Figure 1, A and B). CL alone had no stimulatory effect on GEnCs. B-DNA induced higher IL-6, CCL5/RANTES, and CXCL10/IP-10 levels as 3 μg/ml LPS, a potent endothelial cells (EC) activator (Figure 1, A and B). GEnCs did not produce IFN-γ (not shown). Thus, low amounts of B-DNA activate GEnCs to secrete cytokines, chemokines, and type I (but not type II) IFN only when being complexed with CL. In dendritic cells and embryonic fibroblasts, dsDNA needs to reach the cytosol before it can interact with their putative cytosolic DNA receptors.9Takaoka A Wang Z Choi MK Yanai H Negishi H Ban T Lu Y Miyagishi M Kodama T Honda K Ohba Y Taniguchi T DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.Nature. 2007; 448: 501-505Crossref PubMed Scopus (1307) Google Scholar, 10Roberts TL Idris A Dunn JA Kelly GM Burnton CM Hodgson S Hardy LL Garceau V Sweet MJ Ross IL Hume DA Stacey KJ HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.Science. 2009; Crossref Scopus (680) Google Scholar, 11Hornung V Ablasser A Charrel-Dennis M Bauernfeind F Horvath G Caffrey DR Latz E Fitzgerald KA AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.Nature. 2009; Crossref PubMed Scopus (1845) Google Scholar, 24Stetson DB Medzhitov R Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.Immunity. 2006; 24: 93-103Abstract Full Text Full Text PDF PubMed Scopus (816) Google Scholar Consistently, rhodamine-labeled dsDNA was not detectable in the intracellular cytosol of GEnCs, unless complexed with CL, as assessed by confocal microscopy (Figure 2A). To identify the possible intracellular uptake mechanism, we stimulated GEnCs with B-DNA/CL in the presence or absence of chlorpromazine, known to specifically block clathrin-dependent endocytosis25Saleh MC van Rij RP Hekele A Gillis A Foley E O'Farrell PH Andino R The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.Nat Cell Biol. 2006; 8: 793-802Crossref PubMed Scopus (400) Google Scholar, 26Itoh K Watanabe A Funami K Seya T Matsumoto M The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-beta production.J Immunol. 2008; 181: 5522-5529Crossref PubMed Scopus (69) Google Scholar; methyl-β cyclodextrin, blocking caveolae-dependent endocytosis27Okamoto Y Ninomiya H Miwa S Masaki T Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.J Biol Chem. 2000; 275: 6439-6446Crossref PubMed Scopus (149) Google Scholar; and cytochalasin D, blocking phagocytosis.26Itoh K Watanabe A Funami K Seya T Matsumoto M The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-beta production.J Immunol. 2008; 181: 5522-5529Crossref PubMed Scopus (69) Google Scholar The B-DNA-induced production of CXCL10/IP-10 was inhibited by chlorpromazine, but not by any of the other compounds (Figure 2B), suggesting a role for clathrin in the endocytosis of complexed B-DNA. The endosomal dsRNA receptor TLR3 is the only nucleic acid-specific TLR expressed by GEnCs and should not be involved in innate B-DNA recognition. In fact, B-DNA-induced CXCL10/IP10 secretion was not affected by chloroquine (Figure 2B), a base that inhibits the acidification of endosomes, which is a mandatory requirement for TLR3 signaling. In addition, GEnC activation by B-DNA did not involve MyD88, the signaling adaptor of all other TLR, because GEnC activation was not affected by 100 μmol/L of a MyD88 inhibiting peptide, which entirely prevented GEnC activation by the TLR2/MyD88 pathway agonist Pam3Cys (Figure 2C). Together, complexed B-DNA enters GEnCs via clathrin-dependent endocytosis and activates GEnCs independent of TLR signaling. TLR-independent DNA recognition triggering cytokine and type I interferon release may involve DAI/ZBP1 but other, yet unknown, receptors may exist.9Takaoka A Wang Z Choi MK Yanai H Negishi H Ban T Lu Y Miyagishi M Kodama T Honda K Ohba Y Taniguchi T DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.Nature. 2007; 448: 501-505Crossref PubMed Scopus (1307) Google Scholar The mRNA expression levels of DAI/ZBP1 were low under basal culture conditions but rapidly increased on IFN-β stimulation (Figure 3A). IFN-β-induced expression of receptor mRNAs was rapid and returned to baseline levels at 12 hours, in the case of IFN-β promoter stimulator-1 and type I interferon receptor (IFNAR) (Figure 3A). DAI/ZBP1 mRNA remained elevated, but only at 3000 U of IFN-β. Immunoblotting of GEnC protein extracts taken after 24 hours of stimulation confirmed that IFN-β induced the expression of DAI/ZBP1 (Figure 3B). IFN-β was also found to trigger the phosphorylation of IRF-3 (Figure 3B), a central transcription factor for type 1 IFN induction and signaling.24Stetson DB Medzhitov R Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.Immunity. 2006; 24: 93-103Abstract Full Text Full Text PDF PubMed Scopus (816) Google Scholar, 28Tamura T Yanai H Savitsky D Taniguchi T The IRF family transcription factors in immunity and oncogenesis.Annu Rev Immunol. 2008; 26: 535-584Crossref PubMed Scopus (927) Google Scholar By contrast, GEnCs constitutively expressed IFNAR mRNA and interferon-β promoter stimulator-1 mRNA, the latter being a mitochondrial adaptor involved in sensing of viral nucleic acids in the cytosol (Figure 3A). IFN-β stimulation did not affect IFN-β promoter stimulator-1 and IFNAR mRNA levels at 6 and 12 hours with a trend to lower mRNA levels with high doses of IFN-β(Figure 3A). We conclude that IFN-β induces DAI/ZBP1 expression and IRF3 phosphorylation both suggestive of the TLR-independent B-DNA recognition pathway. In dendritic cells, the viral nucleic acid–induced release of type I IFN enhances subsequent nucleic acid recognition via autocrine-paracrine type I IFN signaling involving the IFNAR and signal transducer and activator of transcription 1 phosphorylation.29Gautier G Humbert M Deauvieau F Scuiller M Hiscott J Bates EE Trinchieri G Caux C Garrone P A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells.J Exp Med. 2005; 201: 1435-1446Crossref PubMed Scopus (451) Google Scholar We have recently shown that viral RNA triggers the same phenomenon in mesangial cells,30Flür K Zecher D Kulkarni O Allam R Lichtnekert J Schwarz M Beutler B Anders HJ Viral RNA sensors induce type I interferon-dependent cytokine release and cell death in glomerular mesangial cells. Implications for viral infection-induced glomerulonephritis.Am J Pathol. 2009; 175: 2008-2016Abstract Full Text Full Text PDF Scopus (61) Google Scholar hence, we hypothesized the same for B-DNA in GEnCs. IFN-β stimulation induced the mRNA expression of CCL2/MCP-1, CCL5/RANTES, MX-1, and CXCL10/IP-10 (but not IFN-α and IL-6; not shown) in GEnCs in a dose-dependent manner (Figure 4A). However, at the protein level, IFN-β induced only the secretion of CXCL10/IP-10, while CCL2/MCP-1 and CCL5/RANTES levels remained unaffected by IFN-β stimulation (Figure 4B). The CXCL10/IP-10 levels induced by 3000 U IFN-β were low (<6 ng/ml, Figure 4B) as compared with the levels induced by B-DNA (50 to 100 ng/ml, Figure 1B). To examine whether type I IFN produced by the GEnCs contributes to DNA-induced secretion of CXCL10/IP-10 in an autocrine-paracrine manner, we stimulated GEnCs with B-DNA in the absence or presence of increasing doses of antibodies that neutralize the functions of IFN-α and IFN-β. By blocking both type I IFN the DNA-induced CXCL10/IP-10 production of GEnCs remained unaffected (Figure 4C). These data show that cytosolic DNA signaling activates GEnCs to secrete type I IFN but this does not contribute to GENC activation in autocrine-paracrine activation loop. EC activation can induce luminal expression of adhesion molecules, which facilitate the firm adhesion of rolling leukocytes.16Pober JS Sessa WC Evolving functions of endothelial cells in inflammation.Nat Rev Immunol. 2007; 7: 803-815Crossref PubMed Scopus (1279) Google Scholar We used flow cytometry for intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) to test whether B-DNA induces GEnC adhesion molecule expression. The basal GEnC surface expression of VCAM-1 was almost double as high as that of ICAM-1 (Figure 5A). ICAM-1 and VCAM-1 24 hours after B-DNA exposure to GEnCs significantly increased the percentage of GEnCs with ICAM-1 surface expression, but VCAM-1 expression was not induced (Figure 5A). ICAM-1 induction depended on complex formation with CL. However, the B-DNA-related induction of ICAM-1 was lower, as compared with that of LPS (Figure 5A). What is the functional significance of B-DNA-induced ICAM-1 induction in vivo? To answer this quest" @default.
• W1992121470 created "2016-06-24" @default.
• W1992121470 creator A5042798641 @default.
• W1992121470 creator A5046223338 @default.
• W1992121470 creator A5046528033 @default.
• W1992121470 creator A5072125974 @default.
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• W1992121470 date "2009-11-01" @default.
• W1992121470 modified "2023-10-18" @default.
• W1992121470 title "Double-Stranded DNA Activates Glomerular Endothelial Cells and Enhances Albumin Permeability via a Toll-Like Receptor-Independent Cytosolic DNA Recognition Pathway" @default.
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